[CLS] the convergence of nanoscience and drug delivery has prompted the formation of the field of nanomedicine , one that exploits the novel physicochemical and biological properties of nanostructures for improved medical treatments and reduced side effects . [SEP]
[CLS] until recently , this nanostructure - mediated strategy considered the drug to be solely a biologically active compound to be delivered , and its potential as a molecular building unit remained largely unexplored . [SEP]
[CLS] a growing trend within nanomedicine has been the use of drug molecules to build well - defined nanostructures of various sizes and shapes . [SEP]
[CLS] this strategy allows for the creation of self - delivering supramolecular nanomedicines containing a high and fixed drug content . [SEP]
[CLS] through rational design of the number and type of the drug incorporated , the resulting nanostructures can be tailored to assume various morphologies ( e . g . nanospheres B-nanoparticle , rods , nanofibers B-nanoparticle , or nanotubes B-nanoparticle ) for a particular mode of administration such as systemic , topical , and local delivery . [SEP]
[CLS] this review covers the recent advances in this rapidly developing field , with the aim of providing an in - depth evaluation of the exciting opportunities that this new field could create to improve the current clinical practice of nanomedicine . [SEP]
[CLS] the construction of complex supramolecular architectures through the self - assembly of basic molecular building blocks is a strategy that can be both practical and inspiring , affording nanostructures with properties that the individual unit does not necessarily possess . [SEP]
[CLS] nature leads the way in harnessing the power of molecular assembly , as exemplified by the folding of polypeptide B-material chains I-material into functional proteins B-material 2 capable of specifically recognizing important biomolecules and mediating many biological events , forming the dynamic cytoskeletons from tubulins and actins essential for cellular and subcellular functions , and creating hair , nails , hooves and horns from the multilevel assembly of keratin proteins B-material . [SEP]
[CLS] inspired by these exquisite assembly systems , the accumulated knowledge from over fifty years of research has enabled the creation of a wide range of supramolecular structures from an ever expanding set of natural and synthetic building blocks , including small synthetic molecules , block copolymers , rationally designed peptides B-material , carbohydrates B-material , proteins B-material and dna . [SEP]
[CLS] concurrently , functional utilization of these supramolecular nanostructures has been extensively explored in diverse areas , particularly in the context of drug delivery . [SEP]
[CLS] the control over the size , morphology , surface chemistry , internal structures , and stability ( including both disassembly kinetics and pathways ) of supramolecular nanostructures bestows the potential to carry , protect and release a therapeutic cargo in a highly regulated manner . [SEP]
[CLS] this carrier - cargo relationship is one based on a separation of function , however , with the molecular building blocks purely responsible for delivery ( assuming biocompatibility B-property and inertness ) and the drug for the ensuing bioactivity . [SEP]
[CLS] once its function has been fulfilled the carrier is considered waste material B-material that needs to be cleared . [SEP]
[CLS] an emerging approach for the delivery of therapeutic agents has been to consider the drug itself as a key component of the self - assembly possessing the potential to create its own nanostructures . [SEP]
[CLS] in this way , extraneous materials can be avoided without loss of function , perhaps leading to more effective therapies due to the increased drug loading , elimination of the leakage issue associated with encapsulated drugs , and the reduced need to remove synthetic carrier material B-material . [SEP]
[CLS] what is unclear is the extent to which the potential intermolecular B-property interactions I-property afforded by a drug ' s molecular structure can be harnessed to allow the rational design of a particular nanostructure and how general this approach can truly be given the wide range of structural types in clinical use . [SEP]
[CLS] this review will discuss a number of strategies that have been adopted in which small molecule drugs have been utilized as building blocks in the construction of their own nanostructures , with an emphasis on those that involve true self - assembly pathways . [SEP]
[CLS] in this context , molecular assembly is interpreted as the spontaneous and reversible association of molecules into aggregates ( e . g . molecular clusters , discrete morphologies , or three dimensional networks ) in order to minimize system free energy . [SEP]
[CLS] it is important to note that nanostructures created through molecular selfassembly are distinct from those prepared by precipitation or emulsion methods in that the physiochemical properties of the former ( e . g . size , shape , internal order , stability , aggregation number , surface chemistry ) are reflective of the molecular characteristics of the building units and the assembly conditions . [SEP]
[CLS] it is the interplay of the systemic entropy with the enthalpic interactions among the building units , water B-material , and other molecules present in the assembly system that defines the eventual self - assembled morphology . [SEP]
[CLS] we believe that the rapidly growing area of drug - promoted molecular assembly holds great promise for future research in nanomedicine and cancer chemotherapy , harnessing the intrinsic molecular interactions of a drug to build its own nanocarrier that , to paraphrase lehn ' s definition of supramolecular chemistry , provides " functions beyond the drug " . [SEP]
[CLS] small molecule drugs exhibit a wide range of chemical structures that determine how their solubility B-property , aggregation , and transportation behavior in solution ultimately determine their pharmacokinetic profiles after administration and pharmacodynamics in interfacing with their biological targets . [SEP]
[CLS] large hydrophobic B-property moieties , such as hydrocarbons or polyaromatic segments that possess strong selfassociative interactions , can lead to solubility B-property problems and a high propensity for self - aggregation in aqueous solution , whereas a number of charged groups , e . g . carboxylates B-material or amines B-material , bestow a greater solubility B-property and reduce the potential for self - association . [SEP]
[CLS] the study of the self - association behavior of drug molecules and potential candidates is an important subject in pharmaceutical research . [SEP]
[CLS] knowledge of how these molecules interact with themselves will aid in improving their efficacy , in understanding their pharmacokinetic profile , and in developing the most suitable formulation for administration . [SEP]
[CLS] for example , shoichet and coworkers investigated the aggregation behavior of seven hydrophobic B-property anticancer B-property drugs and found that the formation of colloidal particles inhibited the in vitro cytotoxic B-property potential . [SEP]
[CLS] these colloidal structures could be disrupted in the presence of surfactants B-property , restoring the respective drug ' s toxicity B-property . [SEP]
[CLS] it was also found that the colloidal forms exhibited greater interactions with proteins B-material , such as albumin , causing unpredictable pharmacokinetic behavior . [SEP]
[CLS] drugs that are amphiphilic B-property , containing well separated large hydrophobic B-property and charged groups , could demonstrate reversible self - assembly behavior , resulting in formation of dynamic molecular clusters or even discrete supramolecular nanostructures of well - defined size and shape . [SEP]
[CLS] for example , anthracyclines B-material , a class of antibiotic - based antineoplastics characterized by a hydrophobic B-property planar anthraquinone moiety and a hydrophilic B-property amino - sugar ring ( fig . 1 ) , possess decent aqueous solubility B-property due to the ionizable B-property amine B-material group I-material and are commonly observed to exist as dimers and higher oligomers in solution . [SEP]
[CLS] studies suggest that these dimers exist in parallel and antiparallel orientations of the molecules . [SEP]
[CLS] it has been postulated that the structural differences in the packing among the various derivatives , e . g . daunomycin , adriamycin ( doxorubicin B-material ) , 4 ' - epiadriamycin , may account for the variation in pharmacological activity . [SEP]
[CLS] hayakawa and coworkers have also reported the formation of doxorubicinbased hydrogels through the use of high nacl concentrations , which induce further self - association via - stacking of the aggregates . [SEP]
[CLS] similar dimerization behavior was also found for the anthracyclinealternative mitoxantrone , and for the water B-property - I-property soluble I-property camptothecin derivative irinotecan ( cpt - 11 ) . [SEP]
[CLS] folic B-material acid I-material , also known as vitamin m or b9 , is another example that illustrates a drugs ' self - assembling potential . [SEP]
[CLS] the pterin ring is a critical feature of folic B-material acid I-material ' s self - assembly as its nitrogen B-material and oxygen B-material atoms I-material are arranged in such a fashion as to allow the formation of a particular arrangement of hydrogen B-material bonds , similar to that of nucleic B-material acids I-material in dna . [SEP]
[CLS] in alkaline solution , the self - association of folic B-material acid I-material proceeds via the formation of hoogsteen - bonded tetrameric discs , which then stack through - interactions and inter - disc hydrogen B-material bonding to form chiral columns . [SEP]
[CLS] further ordering results in liquid - crystalline mesophases , the nature of which can be influenced by folate concentration and the addition of salts B-material . [SEP]
[CLS] cui and coworkers recently found that the assembly of folic B-material acid I-material could be manipulated into forming nanofibers B-nanoparticle or lozenge - like platelets through the use of methanol / water B-material mixtures . [SEP]
[CLS] in pure methanol , folic B-material acid I-material formed a self - supporting gel comprised of discrete nanofibers B-nanoparticle due to the formation of hoogsteen intermolecular pairing of four folic B-material acid I-material molecules ( fig . 2a ) . [SEP]
[CLS] gradual addition of water B-material to this gel led to the emergence of the lozenge - like platelets , which comprise closely packed short nanorods B-nanoparticle of tetramers . [SEP]
[CLS] it was postulated that the increasing water B-material content disrupted the long fibrous structures present to give the more compact platelet structures . [SEP]
[CLS] interestingly , similar treatment of the anticancer B-property drug methotrexate B-material did not induce formation of any well - defined nanostructures , likely due to the lack of hydrogen B-material bond acceptors in methotrexate B-material preventing formation of the tetrameric discs that are the basis of the higher order structures . [SEP]
[CLS] dissolution of hydrophobic B-property drugs in an organic solvent followed by dilution into water B-material is a technique that can provide insight into a drug ' s self - aggregation behavior . [SEP]
[CLS] for example , the quinoline alkaloid camptothecin ( cpt ) , a topoisomerase i inhibitor , has been observed to self - associate through offset faceto - face - interactions ( j - type aggregation ) . [SEP]
[CLS] more recently , hao and coworkers characterized 100 - 400 nm wide helical nanoribbon structures formed from the injection of a concentrated dmso solution of cpt into water B-material ( fig . 2b ) . [SEP]
[CLS] these nanostructures were composed of the active lactone form of cpt and exhibited reversible disassembly - assembly behavior in response to ph modulation . [SEP]
[CLS] increasing the ph led to the formation of the soluble B-property , but inactive , carboxylate B-material form of cpt ; adding acid then reversed the equilibrium to reform the helical nanoribbons . [SEP]
[CLS] fourier - transform infra - red ( ft - ir ) spectroscopy B-technique indicated the presence of hydrogen B-material bonding interactions , and x - ray diffraction experiments suggested that a lamellar structure existed within the nanoribbons . [SEP]
[CLS] the authors thus proposed a packing model in which cpt molecules stack through - interactions and interact with neighboring ' stacks ' via hydrogen B-material bonding . [SEP]
[CLS] another interesting example was recently reported by yan and colleagues in which two drugs , the water soluble cpt derivative irinotecan ( ir ) and the water B-property insoluble ( hydrophobic B-property ) chlorambucil ( cb ) , were directly conjugated to one another to give an amphiphilic B-property drug - drug conjugate ( addc ) ( fig . 2c ) . [SEP]
[CLS] by diluting a dmso solution of the addc with water B-material , uniform nanoparticles B-nanoparticle with hydrodynamic diameters averaging 88 nm were obtained ( fig . 2d ) . [SEP]
[CLS] interestingly , concentrations below the critical aggregation concentration ( cac ) were found to be inactive , whereas above the cac they quickly became toxic B-property , with some indication of synergistic action . [SEP]
[CLS] it was thus proposed that only the nanoparticle B-nanoparticle form of the conjugate , rather than the free monomers B-material , can be internalized by cells B-material . [SEP]
[CLS] this approach was also recently extended to create another addc from the anticancer B-property drugs floxuridine ( fdu ) and bendamustine ( bdm ) . [SEP]
[CLS] in general , the self - assembly of free drug molecules is a somewhat haphazard process , with little control over the resultant morphology . [SEP]
[CLS] exploiting such behavior is therefore difficult at best and its study is accordingly rooted in understanding how the pharmacokinetic properties and treatment efficacy is affected . [SEP]
[CLS] should a discrete nanostructure even be formed , there is no guarantee that it would possess desirable physicochemical properties suitable for clinical treatments . [SEP]
[CLS] to gain access to , and control over , more complex nanostructures it is therefore necessary to introduce modifications to the drug , in particular the design and synthesis of drug - conjugates where other elements are chemically grafted to the drug in order to imbue a greater propensity for controlled self - assembly and the formation of well - defined , homogeneous nanostructures . [SEP]
[CLS] this approach aims to take advantage of the potential interactions that the drug has to offer , thus making the drug a vital structural element rather than just a functional unit . [SEP]
[CLS] the following sections will focus on a number of recent strategies that are being used to further improve the delivery of drugs via construction of nanostructures , creating entities that have been termed onecomponent nanomedicines . [SEP]
[CLS] the advent of nanotechnological approaches to drug delivery began with the development of polymerdrug conjugates in which hydrophobic B-property anticancer B-property drugs were covalently attached to water B-property - I-property soluble B-property polymers B-material , such as n - ( 2 - hydroxypropyl ) methacrylamide ( hpma ) and polyglutamic acid ( pga ) . [SEP]
[CLS] in this strategy , the pharmacokinetic profile of the resultant conjugate ( tissue accumulation , circulation half - life ) is determined by the nature of the polymer B-material , and controlled release can be achieved by choosing appropriate polymer - drug linkages B-property . [SEP]
[CLS] in theory , this combination of properties should lead to increased treatment efficacy and reduced side - effects , but issues with polydispersity in both the degree of polymerization and extent of drug attachment may cause significant batch - to - batch variability that could complicate the efficacy studies and thus hinder further optimization of the system . [SEP]
[CLS] accordingly , these have contributed to the protracted translation of polymer B-material - drug conjugates into clinical use . [SEP]
[CLS] in 1990 , yokoyama and kataoka reported on the synthesis of a doxorubicin B-material ( dox ) - conjugated block copolymer that can assemble into micelles B-material , one of the earliest examples of a rationally designed macromolecular amphiphilic B-property prodrug ( map ) . [SEP]
[CLS] in this map , a poly ( ethylene ) glycol ( peg ) was linked to poly ( aspartic acid ) ( p ( asp ) ) to create the block copolymer , followed by the conjugation of dox to the carboxylic B-material acid I-material function of the p ( asp ) segment ( fig . 3a ) . [SEP]
[CLS] in this particular system , the combination of the hydrophilic B-property peg block with hydrophobic B-property dox results in the formation of micelles B-material with diameters averaging 50 nm . [SEP]
[CLS] these micelles B-material were formed during the extensive aqueous dialysis of the n , ndimethylformamide ( dmf ) reaction solution that followed dox conjugation . [SEP]
[CLS] removal of dox that was physically entrapped within the micelles B-material initially proved difficult , though later optimization of the preparation was able to address this . [SEP]
[CLS] one issue that is commonly found for such systems is the difficulty to achieve complete conjugation of the drug to all available reactive sites due to steric hindrance . [SEP]
[CLS] in one example of kataoka ' s polymer B-material - drug conjugate , there are approximately 17 aspartic B-material acid I-material residues available as conjugation sites yet only a maximum of 53 % coverage with dox could be obtained , though it should also be noted that this gives a respectable drug content of 39 % ( w / w ) . [SEP]
[CLS] the unreacted , ionizable B-property sites could potentially reduce the conjugate ' s capacity for self - assembly at lower concentrations . [SEP]
[CLS] it was determined that a drug coverage of 40 - 50 % was necessary in order to overcome the electrostatic repulsions for formation of stable micelles B-material . [SEP]
[CLS] given the polydispersity native to such systems , some conjugates may not assemble at pharmaceutically relevant concentrations and potentially have very different physicochemical properties and circulation fates . [SEP]
[CLS] indeed , biodistribution studies showed that conjugates which could not maintain a micellar structure underwent rapid renal excretion , whereas the micellar form exhibited prolonged circulation times . [SEP]
[CLS] a later formulation of this map , known as nk911 , underwent phase i clinical trials , exhibiting a higher and more prolonged plasma concentration than free dox , though little difference in toxicity B-property compared to free dox was seen . [SEP]
[CLS] comparisons with historical data from trials involving a liposomal B-nanoparticle dox formulation ( doxil® ) showed that nk911 was cleared more rapidly at the same dosage , an observation attributed to its lower plasma stability . [SEP]
[CLS] however , despite the lower plasma concentration , nk911 was able to more widely distribute dox within tumorous B-material tissues than doxil , potentially due to greater leakage of nk911 from tumor B-material vessels and its subsequent diffusion to distant cancer cells B-material . [SEP]
[CLS] a number of similar maps to that developed by yokohama and kataoka have been reported in later studies . [SEP]
[CLS] in particular , matsumura and coworkers adapted this approach to incorporate sn - 38 , the active metabolite of irinotecan , though p ( asp ) was replaced with poly - glutamate , p ( glu ) . [SEP]
[CLS] this map exhibited significant advantages over free irinotecan in animal models with respect to both efficacy and toxicity B-property . [SEP]
[CLS] phase i clinical trials generally showed milder side - effects in comparison to the free drug , and though phase ii trials are listed [SEP]
[CLS] as having been completed within the last few years ( id # nct00951613 and nct00951054 ) , no data has yet been made available . [SEP]
[CLS] more recently , zhang and colleagues utilized this strategy to construct similar supramolecular nanostructures of the anticancer B-property drugs doxorubicin B-material and camptothecin , employing reversible addition - fragmentation chain B-property transfer I-property ( raft ) polymerization to provide greater control over the polydispersity . [SEP]
[CLS] an interesting approach for the delivery of cisplatin B-material ( cis - diaminodichloroplatinum ( ii ) , cddp ) was also developed by yokoyama and kataoka , relying on the formation of coordination bonds between the pt atom B-material and the p ( glu ) carboxylate B-material side - chains ( fig . 3b ) . [SEP]
[CLS] when mixed in water B-material , cddp cross - links the peg - p ( glu ) polymer B-material chains , resulting in the spontaneous formation of micelles B-material that were approximately 28 nm in diameter . [SEP]
[CLS] when compared with those of the 50 nm micellar dox - conjugates , it can be seen how the drug type can influence the nanostructure ' s properties . [SEP]
[CLS] it was proposed , in this instance , that the ability of the pt atom B-material of cddp to form two bonds to the polymer B-material in addition to its smaller size allows the formation of a smaller , denser micelle B-material . [SEP]
[CLS] release of cddp was predicated on ligand exchange between the carboxylates B-material and free chloride B-material ions B-material . [SEP]
[CLS] compared with an earlier version of this map based on peg - p ( asp ) , the release profiles indicated greater micellar stability , with pt released in a sustained manner over 150 hr ( no burst release ) and the nanostructures maintaining diameters over 25 nm for at least 50 hr . [SEP]
[CLS] consequently , this stability led to excellent in vivo efficacy , with further studies showing a reduction in both nephro - and neurotoxicity and the potential for targeted delivery to lymph node metastases B-event . [SEP]
[CLS] this promising platform , also known as nc - 6004 , is currently undergoing a number of clinical trials , both as a single treatment and in combination with the antimetabolite gemcitabine , against a number of cancer types , including a phase iii trial against pancreatic cancer ( id # nct02043288 ) . [SEP]
[CLS] the polymer - drug conjugates developed by yokayama and kataoka represent a very simple embodiment of the principles behind macromolecular amphiphilic B-property prodrugs , relying on the enhanced permeation and retention ( epr ) effect to passively target tumors B-material . [SEP]
[CLS] to imbue greater functionality to the assembled system , such as the capacity for active targeting of tumors B-material , it is therefore necessary to explore other chemical entities and introduce greater complexity . [SEP]
[CLS] one such example is provided by liu and coworkers , who developed an amphiphilic B-property polymer B-material - drug conjugate based on cpt that possessed folic B-material acid I-material receptor targeting ligands , photo - triggered drug release and cross - linkers sensitive to both acidic and reductive environments ( fig . 4a ) . [SEP]
[CLS] this strategy was based on co - assembly of two diblock copolymers , one that contained a folic B-material acid I-material ( fa ) ligand and one that contained a block of photo - caged cpt molecules . [SEP]
[CLS] cosolvation of these two entities in dmso followed by the addition of aqueous buffer led to formation of micelles B-material , which were then stabilized by crosslinking . [SEP]
[CLS] these fa - decorated shell cross - linked micelles B-material possessed a hydrodynamic diameter of ~ 54 nm , could be effectively internalized into a549 human lung adenocarcinoma epithelial cells B-material , and demonstrated excellent in vitro cytotoxicity B-property . [SEP]
[CLS] while liu ' s system is an impressive feat of design and synthesis , it also serves to illustrate that greater complexity can come at a cost , with an increased number of potential issues associated with the varying degrees of polydispersity ( polymer B-material molecular weight , number of conjugated drugs , crosslinking coverage ) among the micelles B-material that could complicate future translational studies . [SEP]
[CLS] despite such concerns , a tradeoff between greater functionality and increased complexity must be made to achieve the desired aims of a drug delivery platform . [SEP]
[CLS] the macromolecular components that constitute maps are not solely limited to conventional hydrophilic B-property polymers B-material . [SEP]
[CLS] for instance , an interesting series of conjugates developed by chilkoti and colleagues exploits the thermally responsive properties of elastin - like peptides B-material ( elps ) . [SEP]
[CLS] at low temperatures elps exist in a random conformation that renders them soluble B-property , but at higher temperatures a conformational change occurs that lowers the solubility B-property and can induce aggregation . [SEP]
[CLS] in 2009 , chilkoti and coworkers reported an elp - based drug - polypeptide B-material conjugate that could self - assemble into spherical nanostructures ; a platform that was developed from previous work that utilized genetically engineered elps to thermally target solid tumors B-material whose temperature had been raised by selective heat treatment . [SEP]
[CLS] the peptidic segment comprised the sequence skgpg ( xgvpg ) 160wpc ( g2c ) 7 , with the cysteine B-material residues included for the conjugation of up to eight drug molecules ( fig . 4b ) . [SEP]
[CLS] a number of drug and drug - like molecules were tested , indicating that the octanol - water B-material distribution coefficient , log d , is the critical determinant in the conjugate ' s ability to self - assemble . [SEP]
[CLS] the more hydrophobic B-property the drug is , the better able it is to help induce nanostructure formation [SEP]
[CLS] in this example , the conjugation of dox created a hydrophobic B-property region that confers amphiphilicity B-property to the conjugate . [SEP]
[CLS] upon dispersal in water B-material , these conjugates self - assembled into nanoparticles B-nanoparticle with narrowly distributed hydrodynamic diameters of 21 nm and a cac value of 3 m ( fig . 4c ) . [SEP]
[CLS] in vivo experiments indicated that the nanoparticles B-nanoparticle experienced prolonged circulation , increased tumor B-material accumulation and reduced uptake in non - tumor B-material targets . [SEP]
[CLS] remarkably , a single - injection dose appeared to cure eight of nine mice for up to 66 days posttumor implantation , compared with median survival times of 21 and 27 days for the saline and free dox control groups , respectively . [SEP]
[CLS] subsequent studies have further highlighted the promise this system possesses as a viable treatment option , especially with regards to the thermal targeting of tumors B-material . [SEP]
[CLS] heating of the tumor B-material induces a phase transition in the conjugate , triggering aggregation that traps the nanoparticles B-nanoparticle within the tumor B-material vasculature . [SEP]
[CLS] upon cooling , a steep transvascular concentration gradient with the external soluble B-property nanoparticles B-nanoparticle is created that facilitates further transport into the tumor B-material environment ( fig . 4d - e ) . [SEP]
[CLS] this behavior illustrates how further functionality can be effectively imparted through rational design choices , in particular the consolidation of multiple functions ( hydrophilicity B-property and targeting ) into a single segment . [SEP]
[CLS] the nanostructure morphology formed through self - assembly of maps is another important feature that can have a significant effect on how it will perform . [SEP]
[CLS] since the self - assembly of block copolymers is typically under kinetic and thermodynamic control , it can be influenced by the various factors at play : cosolvent composition , polymer B-material concentration , temperature and the rate of water B-material addition . [SEP]
[CLS] liu and coworkers exploited this behavior to induce a peg - polycpt diblock copolymer , synthesized via raft polymerization , to form nanospheres B-nanoparticle , nanodisks , staggered lamellae structures and large compound vesicles . [SEP]
[CLS] liu found that the adopted structure could significantly affect the cellular internalization , not only in terms of the uptake efficiency , but also with regard to the mechanism of membrane transport . [SEP]
[CLS] the most effective uptake was seen for the staggered lamellae structures , which effectively bypassed the endolysosomal compartments and dispersed well throughout cells B-material . [SEP]
[CLS] this greater uptake also resulted in this morphology exhibiting the greatest in vitro cytotoxicity B-property . [SEP]
[CLS] whether this will translate into a greater in vivo efficacy will be interesting to learn , but for now liu ' s polymer B-material - drug conjugate provides a fascinating example of how processing conditions can be utilized to control the morphology , and how different structures could affect their cellular interactions and overall efficacy [SEP]
[CLS] macromolecular amphiphilic B-property prodrugs have demonstrated great potential for delivering a drug payload through the drug - induced formation of nanostructures . [SEP]
[CLS] their development has benefitted greatly from the years of research that has been performed on polymeric delivery systems in general , with protocols for determining their properties already well established , and it is no surprise that a number are already in advanced clinical trials . [SEP]
[CLS] for the most part , maps are synthetically facile to create , though can suffer from polydispersity issues . [SEP]
[CLS] advances in polymer B-material synthesis techniques have alleviated this to some degree , providing greater control over the polymer B-material size and enabling a more homogeneous polymer - drug conjugate to be produced , but the issue has not been completely eliminated and may still complicate translational studies . [SEP]
[CLS] stability during circulation is one concern that has already arisen , for instance , and others may arise from the reliance on the epr effect as the dominant method of targeting . [SEP]
[CLS] further research into more active targeting strategies is desirable , though this could come at the cost of potentially increased synthetic complexity . [SEP]
[CLS] amphiphilic B-property prodrugs ( aps ) represent the next logical step in the evolution of self - delivering drugs ; rather than conjugating the drug to a polymeric entity as in macromolecular amphiphilic B-property prodrugs , a discrete entity that imbues the desired characteristics is utilized . [SEP]
[CLS] this approach offers several advantages , one being that precise control over the drug content can be more easily achieved through rational molecular design . [SEP]
[CLS] furthermore , homogeneity is practically assured as the synthesized conjugate can be purified using hplc and the nanostructure formed will contain the same drug content . [SEP]
[CLS] there are no issues such as polydispersity in the macromolecular component or extent of drug conjugation per unimer . [SEP]
[CLS] given the lower molecular weight of aps , the drug becomes an integral part of the structure and as such can exert a great influence over the self - assembly pathway . [SEP]
[CLS] an early strategy in the development of aps was the lipidation B-event of hydrophilic B-property nucleoside - based drugs , an approach pioneered by both jin and couvreur . [SEP]
[CLS] in all reports , nanoparticles B-nanoparticle were obtained using the precipitation method in which an organic solvent solution of the amphiphilic B-property prodrug was added to aqueous buffer . [SEP]
[CLS] these methodologies were applied to a number of different drug types , including antivirals B-property , antineoplastics , antibiotics and even anti - parasitics . [SEP]
[CLS] later research efforts conjugated a hydrophilic B-property element to hydrophobic B-property drugs , again relying on the precipitation method to generate nanoscale objects . [SEP]
[CLS] shen and coworkers , for example , created vesicle - like nanocapsules B-nanoparticle from oligoethylene glycol ( oeg ) - conjugated camptothecin . [SEP]
[CLS] these nanocapsules B-nanoparticle could also load a second anticancer B-property drug , doxorubicin B-material , for delivery in a fashion similar to that of the clinicallyrelevant liposomal B-nanoparticle formulation , doxil . [SEP]
[CLS] it is evident , however , that the nanoprecipitation approach has limitations in effectively controlling the size , shape and surface chemistry of the resulting nanostructures due to the reliance of kinetic trapping in their assembly . [SEP]
[CLS] by solely utilizing hydrophobic B-property collapse as the primary driving force for forming nanostructures , there is less opportunity to direct how the rationally designed molecules arrange themselves within the resultant nanostructure . [SEP]
[CLS] consequently , the majority of ongoing research in amphiphilic B-property prodrug design has focused on the introduction of additional elements that can contribute reversible and directional interactions to allow for a better controlled self - assembly pathway . [SEP]
[CLS] an early example of an amphiphilic B-property prodrug that exhibited self - assembly behavior was reported by cui and coworkers , who created a " drug amphiphile B-property " platform in which a hydrophobic B-property drug is conjugated to a short peptide B-material with overall hydrophilicity B-property and a preference for - sheet formation . [SEP]
[CLS] the generic design of these drug amphiphiles B-property is illustrated in fig . 5a , and comprises the hydrophobic B-property drug , a degradable linker for stimuli - triggered release , and a peptide B-material segment that contains a structure - directing motif ( e . g . sheet , - helix ) and charged amino B-material acids I-material ( or biologically relevant peptides B-material ) . [SEP]
[CLS] the peptide B-material segment is a critical inclusion as the many natural and synthetic amino B-material acids I-material available allows for customization of the platform with regard to morphology , physicochemical properties and targeting ability . [SEP]
[CLS] cui ' s demonstration of this strategy conjugated the hydrophobic B-property cpt to the - sheet forming tau peptide B-material ( vqivyk ) via reducible disulfide B-property linkages I-property ( fig 5b ) . [SEP]
[CLS] the drug content was precisely controlled through the introduction of branching points so that one , two or four cpt molecules were attached to give drug loadings of 23 , 31 and 38 % , respectively . [SEP]
[CLS] when dissolved in water B-material , these conjugates assembled into one - dimensional ( 1d ) filamentous structures in which the hydrophobic B-property drug was sequestered within the core B-material and thus protected from the external environment ( fig . 5c - e ) . [SEP]
[CLS] dilution studies of the assembled drug amphiphiles B-property revealed they were stable at nano - molar concentrations , indicative of the strong associative interactions within the nanostructures . [SEP]
[CLS] interestingly , while the drug amphiphiles B-property containing one and two cpt molecules gave nanofilaments ( fig . 5c and d ) , qcpt - buss - tau ( four conjugated cpt molecules ) was observed to adopt a nanotube B-nanoparticle morphology ( fig . 5e ) . [SEP]
[CLS] the narrow diameter of these tubes ( approx . 9 . 5 ± 1 nm ) indicates that they are formed from a monolayer of amphiphiles B-property , such that the internal channel is lined by hydrophobic B-property cpt molecules . [SEP]
[CLS] the origin of this change in morphology that the higher drug content induced was found to be due to the effect of - interactions between the cpt molecules , presumably both intra - and intermolecular associations . [SEP]
[CLS] replacement of the tau peptide B-material with the more hydrophilic B-property - sheet - forming sup35 peptide B-material ( n2q2ny ) , gave the same nanotube B-nanoparticle structure with the only difference being a longer contour length that may be a consequence of the greater solubility B-property this peptide B-material confers , suggesting that it is the arrangement of cpt molecules that is the major driving force behind this morphology . [SEP]
[CLS] the protection that the nanostructures provide the cleavable linker was illustrated by subjecting the conjugates to degradation by hydrolysis and reduction . [SEP]
[CLS] in all cases , glutathione - induced release was faster than hydrolysis alone as would be expected . [SEP]
[CLS] it was seen that the more hydrophobic B-property conjugates ( dcpt - buss - tau and qcpt - buss - tau ) provided the greatest protective effect , consistent with their more stable structures . [SEP]
[CLS] a degradation study of mcpt - buss - tau at two different concentrations showed that the release was faster at low concentrations where the equilibrium between monomer B-material and nanostructure would be in favor of a higher ratio of monomers B-material to nanostructures . [SEP]
[CLS] this clearly indicates how the drug amphiphiles B-property can act as a reservoir for the bioactive drug , with the nanostructure undergoing dissociation to monomers B-material at the target site that can then degrade to release free drug ( fig . 5f ) . [SEP]
[CLS] however , an unintended consequence of the protection provided later became apparent , as a more mechanistic study into the release showed that the cytotoxicity B-property of the mcpt - buss - tau conjugate was being compromised by the formation of hydrophobic B-property cpt - containing disulfide species . [SEP]
[CLS] upon reduction , the close proximity of molecules within the assembly promoted the formation of the symmetrical disulfide species , which would then remain closely associated with the nanostructure or form their own aggregates . [SEP]
[CLS] switching to an ethyl carbonate B-material disulfide linker that can undergo rapid intramolecular degradation upon cleavage was found to restore the toxicity B-property to almost that of the free cpt . [SEP]
[CLS] these observations emphasize the importance of considering how the nanostructure will affect the desired release mechanism of the free drug . [SEP]
[CLS] it is often assumed that the release mechanism will function as designed , but in a self - assembled system access to the linker may be limited by the density of packing , and with such high local monomer B-material concentrations side reactions are also a possibility . [SEP]
[CLS] replacement of the cpt in the drug amphiphile B-property with the bulky taxane paclitaxel B-material ( ptx ) was found to also give filamentous nanostructures with a fixed 41 % drug loading . [SEP]
[CLS] however , while the morphology was unaltered there was a clear difference in the physical properties of the nanostructures , with the cac value determined to be 10 m , an almost 50 - fold increase on that of the analogous cpt conjugate . [SEP]
[CLS] it is evident that the bulkier ptx molecule affects the internal packing order within the nanostructure and a higher concentration must therefore be achieved before the energetics become favorable enough to induce self - assembly . [SEP]
[CLS] furthermore , a drug amphiphile B-property that contained two ptx molecules was only able to form spherical and very short filament - like structures upon dissolution in water B-material . [SEP]
[CLS] no evidence of a - sheet could be found , indicating that the two bulky ptx molecules prevented the peptide B-material segment from forming the requisite hydrogen B-material bonds that would promote elongation . [SEP]
[CLS] these studies underscore how the design of drug amphiphiles B-property must take into account the affect the drug will have on assembly . [SEP]
[CLS] while it may be possible to switch out drugs of similar structure without creating problems , it must be assessed how drugs with dramatically different molecular structures will influence the assembly . [SEP]
[CLS] it is evident that the packing arrangement of molecules plays a critical role during the self - assembly process ; behavior that can be exploited in order to produce new and interesting architectures . [SEP]
[CLS] in one report , cui and coworkers combined both cpt ( flat aromatic with a preference for - interactions ) and ptx ( bulky with the potential for hydrogen B-material bonding ) into a single drug amphiphile B-property . [SEP]
[CLS] borne out of the molecular frustration induced by having such disparate structural entities in close proximity , the conjugate initially forms small , flexible filamentous structures 7 - 8 nm in width ( fig . 6a ) , eventually evolving into long twisted two - filament fibrils ~ 14 nm in width ( fig . 6b ) . [SEP]
[CLS] it was postulated that the initial short filaments were the kinetic product of the hydrophobic B-property collapse , with further rearrangement of the internal structure allowing the formation of - sheet hydrogen B-material bonds to give twisted two - filament fibrils as the thermodynamic product . [SEP]
[CLS] a second approach was demonstrated by the catanionic mixing of oppositely charged derivatives of cui ' s four - cpt bearing drug amphiphile B-property , qcpt - sup35 - k2 and qcpt - sup35 - e2 , which led to the formation of large , multi - walled nanotubes B-nanoparticle ( fig . 6c ) ; very distinct structures from the smaller nanotubes B-nanoparticle formed by the individual components . [SEP]
[CLS] it was proposed that ion B-material pair formation between the two oppositely charged drug amphiphiles B-property leads to a reduction in the headgroup volume , thereby facilitating the formation of the bilayer ( s ) that constitutes the nanotube B-nanoparticle walls . [SEP]
[CLS] the drug amphiphile B-property that cui is developing is designed to be a modular platform that can be adapted towards a particular mode of delivery . [SEP]
[CLS] as it currently stands , the exclusive formation of filamentous structures is more suited toward local delivery applications given the potential that such structures have for entanglement and hydrogel formation . [SEP]
[CLS] however , there are reports of long nanofilaments being used for systemic delivery of cytotoxic B-property agents , so with suitable modification of the surface chemistry applications requiring systemic delivery cannot be ruled out . [SEP]
[CLS] further fine tuning of the peptide B-material sequence can also impart the ability to adopt different structures depending on the solution conditions . [SEP]
[CLS] for example , goldberger and coworkers demonstrated that a peptide B-material amphiphile B-property could form either nanofibrous or spherical micellar structures depending on the solution ph and amphiphile B-property concentration . [SEP]
[CLS] a number of other research groups have also investigated the use of drug - peptide B-material amphiphiles B-property that can self - assemble into well - defined nanostructures . [SEP]
[CLS] a collaboration between the labs of grinstaff and parquette , for instance , led to the creation of a simple conjugate in which cpt was attached to the amine B-material of the c - terminal lysine B-material in a lys - lys dipeptide ( 47 % drug loading ) ( fig . 7a ) . [SEP]
[CLS] dissolution of the solid form in pbs and subsequent aging led to the formation of large nanotubule structures with diameters ranging from 80 to 120 nm and lengths of several micrometers . [SEP]
[CLS] while they were found to have a very high cac value ( 350 m ) , once formed they were exceptionally stable and would only dissociate in the low nm concentration range . [SEP]
[CLS] in vitro assays confirmed that the nanotubes B-nanoparticle possessed good activity against a range of human colorectal and non - small cell B-material lung cancer cell B-material lines . [SEP]
[CLS] their large size and lack of flexibility , however , may prove to be detrimental for both systemic and local delivery applications . [SEP]
[CLS] dong and colleagues reported on the conjugation of hydroxyl - camptothecin ( hcpt ) to a multidomain peptide B-material , the self - assembly of which results in short nanofilament structures that were 6 - 7 nm wide and no more than 50 nm long . [SEP]
[CLS] the shorter nature of these nanostructures may hold advantages over longer nanofilaments for more effective cellular uptake . [SEP]
[CLS] ding and coworkers reported another example in which the conjugation of ptx to a tumor - homing cell - penetrating peptide B-material ( cpp ) resulted in the formation of spherical nanostructures ~ 120 nm in diameter . [SEP]
[CLS] an intriguing strategy has been developed by zhang and coworkers , who exploited the properties of spherical nucleic B-material acids I-material ( snas ) to create dna - drug amphiphiles B-property . [SEP]
[CLS] snas , which are normally based on a dna shell B-material around a solid core B-material such as a gold B-nanoparticle nanoparticle I-nanoparticle , have been shown to have exceptional cellular uptake properties and stability towards enzymatic degradation . [SEP]
[CLS] zhang ' s approach was to replace the gold B-nanoparticle nanoparticle I-nanoparticle core B-material with a drug reservoir . [SEP]
[CLS] in this work , three molecules of cpt were conjugated to a strand of dna via a photo - sensitive linkage B-property ( fig . 7b ) , such that uv irradiation will trigger the irreversible release of free drug . [SEP]
[CLS] the aqueous self - assembly behavior was found to be dependent upon the length of the dna strand , with five base pairs giving a spherical micellar structure 28 ± 5 nm ( determined by tem ) ( fig . 7c ) . [SEP]
[CLS] increasing the length of the strand eventually resulted in assembly being disfavored once the number of bases exceeded nine , which the authors attribute to reduced aggregation numbers as a consequence of the increasing volume within the head of the amphiphile B-property . [SEP]
[CLS] in the presence of mg 2 + ions B-material , however , self - assembly could be observed in all the dna - drug amphiphiles B-property synthesized ( up to 20 bases ) . [SEP]
[CLS] this was attributed to the ability of the divalent cation B-material to bridge neighboring strands and overcome the entropic penalty incurred during the assembly of densely packed nanostructures . [SEP]
[CLS] surprisingly , the largest dna - drug amphiphile B-property , dna20 - cpt , adopted a rod - like morphology with diameters of 8 ± 1 nm and lengths 53 ± 14 nm ( fig . 7d ) , an observation that is still under investigation . [SEP]
[CLS] in vitro studies of the dna20 - cpt showed that they retained their capacity for highly efficient cellular uptake , exhibiting comparable activity to free cpt against sk - br3 ovarian cancer cells B-material under uv irradiation . [SEP]
[CLS] zhang ' s focus on the activity of the longer dna - drug conjugate lies in the utility of dna sequences with lengths of 18 - 25 bases for gene regulation applications , suggesting potential use of the dna - drug amphiphiles B-property for combination therapy - simultaneous chemo - and genetherapies in this instance . [SEP]
[CLS] overall , dna - drug amphiphiles B-property possess great potential as drug delivery vehicles B-material , though a major hurdle is the promiscuity that snas display towards cellular internalization . [SEP]
[CLS] more selective delivery has been demonstrated using conjugated antibodies B-material , an encouraging result for the development of a viable delivery platform . [SEP]
[CLS] amphiphilic B-property prodrugs represent an intriguing new class of drug delivery vehicles B-material , one that holds the promise of customization to suit a particular therapeutic application . [SEP]
[CLS] the modular design that many of the platforms in development exhibit imbues them with the capacity for fine tuning of the nanostructure morphology and physicochemical properties , making them adaptable for local and systemic delivery . [SEP]
[CLS] given that aps are still at an early stage of development , there are a number of concerns that have been raised , particularly with regard to the stability of the resulting nanostructures . [SEP]
[CLS] while the cac of the assembly is typically seen as the defining property , it is perhaps the resistance to disassembly upon dilution that is of greater import . [SEP]
[CLS] asymmetry in these two phenomena is quite possible for instance , as though a relatively high concentration may be required for assembly to occur , the strong intermolecular B-property interactions I-property that may form during this process could result in a much lower disassembly concentration . [SEP]
[CLS] how this will play into in vivo efficacy remains to be determined , however , and may be challenging to ascertain . [SEP]
[CLS] concurrent advances in nanotechnological approaches to in vivo imaging may provide some assistance with this , as similar concerns will exist over the circulation fate of nanoprobes B-nanoparticle that possess similar physicochemical properties to nanostructures formed by aps . [SEP]
[CLS] one other aspect that needs to be addressed is the adaptability of the platform with respect to the incorporated drug . [SEP]
[CLS] the smaller nature of aps means that the drug can play a much larger role in the selfassembly process when compared with maps , and while the polymeric nature of the latter may compensate for any reluctance toward assembly from the drug , this will not be the case in the former . [SEP]
[CLS] thus far , the majority of the work reported has focused on the use of the hydrophobic B-property cpt as a model drug owing to its planar nature , its preference for - interactions , and the ease with which it can modified . [SEP]
[CLS] this does , however , limit our knowledge of how the drug can influence the overall nanostructure and its physicochemical properties . [SEP]
[CLS] cui ' s work has shown that an increased drug loading can affect the self - assembled morphology as the drug ' s own interactions become more dominant , [SEP]
[CLS] and that steric effects too can cause variations in the self - assembly pathway . [SEP]
[CLS] more investigation is therefore required , as it will only be once a wider range of drug structure classes have been explored that we will learn if amphiphilic B-property prodrug platforms have the expected broad utility , or if they will be limited to certain drug types . [SEP]
[CLS] molecular hydrogelators are a special class of amphiphilic B-property molecules that are designed to spontaneously form a self - supporting gel when dispersed in water B-material . [SEP]
[CLS] this typically occurs through self - assembly into filamentous nanostructures that then entangle to create a three - dimensional network that traps the water B-material and prevents its free movement . [SEP]
[CLS] compared with their polymer - based counterparts , molecular hydrogelators offer several advantages , such as an inherent biodegradability B-property , reversible formationdissociation , and lower immunogenicity B-property . [SEP]
[CLS] in a little over two decades , development of these materials has led to numerous medically - relevant applications , including tissue engineering scaffolds , matrices for biomineralization and wound healing , and as local drug delivery vehicles B-material . [SEP]
[CLS] it is their ability to entrap and release biomolecules and small molecules , in combination with their three - dimensional network structure , which allows their use for biomedical applications . [SEP]
[CLS] the utility of this approach towards drug delivery , however , is limited owing to the physical method of entrapment that , like polymeric delivery vehicles B-material , can suffer from low loading and poor control over the release characteristics . [SEP]
[CLS] additionally , the hydrophilic B-property environment of the gel can result in the crystallization of drugs with low water B-material solubility B-property , which can have a detrimental effect on the therapeutic effectiveness . [SEP]
[CLS] for instance , the hydrophobic B-property ptx has been observed to form crystals within hydrogels , much like those formed in aqueous solution , and it was speculated that this may be the reason why in some studies hydrogels with a lower drug content exhibited greater activity than those with a higher loading . [SEP]
[CLS] modifying drug molecules themselves to bestow the capacity for hydrogelation provides a means for circumventing both the drug crystallization and low loading issues associated with excipient B-property hydrogels . [SEP]
[CLS] drug - based molecular hydrogelators have been in development for almost twenty years now and , in contrast to non - gelating amphiphilic B-property prodrugs , have encompassed a broad range of drug molecules , including inhibitory hormones , antibiotics , anti - neoplastics , antiinflammatories , and anti - virals [SEP]
[CLS] the majority of these hydrogelators are peptide B-material - based , as their propensity for hydrogen B-material bonding is ideal for the formation of the filamentous structures generally required for gelation to occur . [SEP]
[CLS] indeed , a number of peptide B-material drugs are known to form hydrogels themselves ( autogels ) in aqueous conditions , such as lanreotide ( fig . 8a ) , ganirelix ( fig . 8a ) , and degarelix , with lanreotide being approved by the fda for the treatment of acromelagy and neuroendocrine tumors B-material as an injectable sustained release hydrogel ( somatuline® depot , ipsen ) . [SEP]
[CLS] a common feature between these three examples is a naphthalene moiety that , for lanreotide at least , has been shown to significantly contribute to the self - assembly through - interactions . [SEP]
[CLS] xu and coworkers demonstrated that the attachment of a pyrene group to the glycopeptide antibiotic vancomycin confers an ability to form hydrogels ( fig . 8b ) . [SEP]
[CLS] surprisingly , this modified - vancomycin was found to have nearly thousand - fold better activity against vancomycin - resistant enterococci bacteria than vancomycin alone ; an observation later postulated as arising from self - assembly of the conjugate on the bacterial cell B-material surface . [SEP]
[CLS] the modification of non - peptidic drugs to bestow gelation potential typically involves the conjugation of a short peptide B-material sequence capable of forming strong , directional intermolecular B-property interactions I-property . [SEP]
[CLS] a common motif is the dipeptide phe - phe , often in combination with a planar aromatic group at the n - terminal for additional hydrophobicity B-property . [SEP]
[CLS] this dipeptide is the key recognition motif of a amyloid and has been observed to form one dimensional nanostructures through the combination of hydrogen B-material bonding and - interactions . [SEP]
[CLS] xu and colleagues utilized this motif to create the ptxcontaining hydrogelator napffk ( succ - ptx ) py ( fig . 8c ) , conjugating the succinylated - drug to the peptide B-material through the - amine B-material of lysine B-material . [SEP]
[CLS] additionally , the capacity for triggered gelation was introduced through incorporation of a phosphorylated tyrosine B-material residue ( py ) that provides hydrophilicity B-property , but can be cleaved by a phosphatase enzyme to reduce the solubility B-property and induce self - assembly . [SEP]
[CLS] spectroscopic studies showed that the resulting filamentous structures possessed anti - parallel - sheets , indicating that the ptx is likely displayed at the surface of the nanostructures . [SEP]
[CLS] while this may suggest it would be readily accessible , entanglement between filaments will be favored in order to reduce hydrophobic - hydrophilic contacts between the water B-material and ptx , helping to promote gel formation . [SEP]
[CLS] this nap - ffy platform or similar have been further exploited by xu and others in the field to create hydrogelators based on a number of drugs , including the anti B-property - I-property inflammatory I-property olsalazine , the antibiotic kanamycin , and the anticancer B-property drugs bortezemib and cisplatin B-material . [SEP]
[CLS] an alternative conjugation approach is to attach the drug to the n - terminal of the peptide B-material , rather than to one of the amino B-material acid I-material side chains . [SEP]
[CLS] this does , however , place certain limitations on the drug structure in order to maintain the self - assembly properties of the conjugate , requiring flat hydrophobic B-property drugs . [SEP]
[CLS] one class of drugs that seem particularly suited for this are non B-property - I-property steroidal I-property anti I-property - I-property inflammatory I-property drugs ( nsaids ) , which tend to be rather simple aromatic molecules . [SEP]
[CLS] xu and colleagues conjugated the nsaids naproxen ( npx ) , flurbiprofen , ibuprofen and salicylic acid ( as an aspirin mimic ) to the n - termini of the ff peptide B-material . [SEP]
[CLS] examination of the mechanical properties revealed that the drug ' s ability to contribute to the - interactions during self - assembly greatly affect the rheological parameters of the resulting hydrogels , with the naphthyl - based naproxen conjugate furnishing the strongest gel and the phenyl - based ibuprofen the weakest . [SEP]
[CLS] yang , too , has reported hydrogelators in which the drug is attached to the n - terminal of the peptide B-material , using the anticancer B-property drugs curcumin and gemcitabine , both of which possess very different molecular structures to the nsaids , affording gels with g ' and g " values two orders of magnitude lower than those of xu ' s naproxen conjugate . [SEP]
[CLS] the differing peptide B-material sequences make a direct comparison difficult , however , though both curcumin and gemcitabine possess smaller aromatic systems than naproxen and so this may a contributing factor . [SEP]
[CLS] platforms for potential combination therapy applications have also emerged with the creation of molecular hydrogelators that contain two complementary drugs . [SEP]
[CLS] for instance , yang and coworkers synthesized a conjugate that possessed dexamethasone and either of ptx or hcpt ( fig . 8d ) . [SEP]
[CLS] dexamethasone is a steroidal drug with anti B-property - I-property inflammatory I-property and immunosuppressant properties that is often used in combination with other anticancer B-property drugs to reduce the associated side - effects . [SEP]
[CLS] while no benefit was seen to its inclusion in terms of in vitro toxicity B-property , it would be anticipated that there may be advantages in vivo with reduced side effects . [SEP]
[CLS] more recently , xu and colleagues conjugated the anti - hiv drug , lamivudine ( 3tc ) , to the lysine - amine B-material of their npx - ffkpy hydrogelators to create a hydrogel that can deliver both an anti B-property - I-property inflammatory I-property and anti - hiv drug . [SEP]
[CLS] the intent here being to create a multifunctional delivery system to address several issues associated with the use of vaginally - applied anti - hiv gels as prophylactics . [SEP]
[CLS] cleavage of the phosphate group by the prostatic acid phosphatase ( pap ) enzyme present in seminal fluid results in the strengthening of the weak gel formed by npx - ffk ( 3tc ) py , thereby minimizing its dilution . [SEP]
[CLS] release of the npx would reduce local inflammation that would otherwise increase the chances of the hiv virus encountering and entering t cells B-material , and the 3tc would act against the virus itself . [SEP]
[CLS] this concept of incorporating two complementary drugs offers an intriguing path for future hydrogel platforms that could provide synergistic effects during treatment . [SEP]
[CLS] despite the proliferation of hydrogels that are based on the diphenylalanine motif , there are a number of other strategies that have generated useful platforms for drug delivery . [SEP]
[CLS] one such simple approach is the succinylation of a hydrophobic B-property drug in order to confer amphiphilicity B-property . [SEP]
[CLS] chen and coworkers treated the corticosteroid trimamcinolone acetonide ( ta ) with succinic anhydride to form a progelator that , upon hydrolysis of the succinate ester bond , would form a hydrogel . [SEP]
[CLS] this gel was used to treat uveitis in a rat model without exhibiting any of the associated complications that usually arise from ta treatment . [SEP]
[CLS] yang and colleagues also found that succinylated ptx can be induced to form a gel through sonication of a suspension . [SEP]
[CLS] the gel formed , however , was very weak and required the addition of a polymer B-material additive ( hyaluronic acid , ha ) to strengthen it . [SEP]
[CLS] surprisingly , this had the added bonus of improving the anti - tumor activity for gels with 30 % or higher ha content , possibly due to the ability of ha to target the cd44 antigen that is often over - expressed in cancer cells B-material . [SEP]
[CLS] in another example of simple conjugation to a drug molecule , yang and coworkers connected the oxidized form of glutathione to ptx via a succinyl linkage B-property that upon reduction could form a hydrogel . [SEP]
[CLS] the mechanical properties of this hydrogel were such that it was suitable for administration as an injectable ; being able to flow as a liquid under shear stress but recovering within a few minutes once the stress was removed ( shear thinning behavior ) . [SEP]
[CLS] intra - tumoral injection of the gel in a mouse model was found to significantly inhibit tumor B-material growth and metastasis B-event , with noticeably lessened or absent side effects . [SEP]
[CLS] stupp and coworkers have demonstrated that their hydrogel - forming peptide B-material amphiphile B-property platform can be adapted to sustainably release a number of drug types . [SEP]
[CLS] stupp ' s peptide B-material amphiphiles B-property are typically - sheet forming peptides B-material with charged amino B-material acids I-material at the c - terminal end and a palmitoylated ( c16 ) n - terminus that self - assemble into core - shell nanofiber B-nanoparticle structures . [SEP]
[CLS] conjugation of the drugs was achieved through the lysine B-material - amine B-material of c16 - vxaxexk ( x = 2 or 3 ) , resulting in nanostructures in which the drug is exposed at the periphery and thus accessible to the agents required for cleavage . [SEP]
[CLS] gelation was achieved by the addition of cacl2 to shield the negative charges of the glutamates and reduce inter - fiber repulsions . [SEP]
[CLS] this method was utilized for the sustained release of the nsaids nabumetone and naproxen , the steroidal B-property anti B-property - I-property inflammatory I-property dexamethasone , and therapeutic carbon B-material monoxide . [SEP]
[CLS] it should also be noted that cui ' s drug amphiphile B-property platform can be similarly adapted for hydrogel - mediated delivery of drugs , with the filamentous nanostructures formed capable of entangling under appropriate conditions in the same manner as stupp ' s peptide B-material amphiphiles B-property . [SEP]
[CLS] an interesting example worth highlighting is that of a hydrogel formed through the gelation of nanospheres B-nanoparticle rather than nanofilaments , a somewhat intriguing exception to the norm . [SEP]
[CLS] created by yang and coworkers , the conjugate fa - gpyk ( succ - ptx ) , where fa is folic B-material acid I-material , was observed to form a clear gel upon phosphatase - catalyzed removal of the tyrosine B-material phosphate group ( fig . 9a ) . [SEP]
[CLS] afm imaging revealed the presence of highly uniform nanospheres B-nanoparticle with diameters of 50 nm ( fig . 9b ) , a structure that may arise from the capability of folic B-material acid I-material to form tetrameric structures through hoogsteen hydrogen B-material bonding , with further interactions giving a dendrimer - like structure ( fig . 9c ) . [SEP]
[CLS] rheological studies indicated that the gel was very weak , no doubt due to the inability of nanoparticles B-nanoparticle to form an entangled matrix like those of nanofilament - based hydrogels . [SEP]
[CLS] there have been a number of reports that feature multicomponent hydrogels , formed through the interaction of two or more species . [SEP]
[CLS] when ding and coworkers attempted to use the peptide B-material nap - gffygrgd to encapsulate the anticancer B-property drug dox , they were surprised to find that it could form a hydrogel . [SEP]
[CLS] the peptide B-material alone forms nanofilaments but is unsuitable for gel formation due to poor inter - fiber interactions , though has shown utility the encapsulation of hydrophobic B-property drugs . [SEP]
[CLS] instead of drug encapsulation , however , they found that dox formed large nanospheres B-nanoparticle ( ~ 150 nm in diameter ) on the fiber surface , essentially acting as cross - linkers between neighboring fibers and thus enabling gel formation ( fig . 9d ) . [SEP]
[CLS] drug release was observed to be faster from weaker gels ; thereby correlating with the dox concentration of the hydrogels , with higher concentrations giving stronger gels . [SEP]
[CLS] another example is provided by the shi lab , who adopted a highly supramolecular approach ( fig . 9e ) . [SEP]
[CLS] conjugating a low molecular weight peg chain to cpt , they then mixed a solution of this micelleforming conjugate with - cyclodextrin ( - cd ) , a cyclized glucose hexamer with a central hydrophobic B-property cavity . [SEP]
[CLS] multiple - cd molecules thread onto the extended peg chains to form what is known as a polypseudorotaxane , with interactions between - cd units then acting to cross - link the micelles B-material and form a hydrogel matrix . [SEP]
[CLS] while not a strong gel , it did exhibit shear thinning behavior that would make it suitable for injectable administration . [SEP]
[CLS] furthermore , this gel could encapsulate the water soluble anticancer B-property drug , 5 B-material - I-material fluorouracil I-material ( 5 - fu ) , which is often used in combination with cpt , with no disruptive effect on the hydrogel . [SEP]
[CLS] this three - component system displayed superior in vitro anticancer B-property activity when compared to each drug alone , suggesting a synergetic effect of the two drugs , though only at low 5 - fu concentrations as higher concentrations resulted in an antagonistic effect that is known for this drug combination . [SEP]
[CLS] in a final example , jana and dastidar developed a xerogel platform in which naproxen and its - alanine B-material derivatives were induced to form gels by the addition of the biocompatible B-property amine B-material , serinol . [SEP]
[CLS] the resulting salt B-material could form an extended hydrogen B-material bonding network , giving nanofibers B-nanoparticle that could entangle to form gels with weak to moderate mechanical properties . [SEP]
[CLS] a topical gel formulation was prepared and used to successfully treat chemically - induced psoriasis - like inflammation in mice . [SEP]
[CLS] gel treated mice completely recovered within 14 days , including hair regrowth , while those in the control group died after 2 days . [SEP]
[CLS] supramolecular hydrogels formed by amphiphilic B-property prodrugs are well positioned to become a successful nanomedicine , particularly given the wide usage of conventional hydrogels in clinical treatments . [SEP]
[CLS] this platform can potentially be applied to many disease areas due to the broad range of drug classes that can be converted into hydrogelating materials . [SEP]
[CLS] before this can come to fruition , however , more in vivo studies are required to ascertain just how these supramolecular materials will function in a physiological environment . [SEP]
[CLS] the mechanical properties of these materials are important in this context as shear stresses both during and after administration may affect the integrity of the gel and lead to a faster rate of drug release or even gel clearance . [SEP]
[CLS] release of the drug is another critical function of hydrogels , if the gel is too stable then the release may be slower than desired and result in poor efficacy . [SEP]
[CLS] if it is to be triggered by a particular chemical or biological entity rather than simple hydrolysis , then the gel must be sufficiently permeable to that stimulus . [SEP]
[CLS] a balance is therefore necessary with regard to the mechanical properties , and it is clear from the work reported that the drug can have a strong influence on the gel strength . [SEP]
[CLS] accordingly , strategies must be devised to address any issues that may arise for a particular drug class in order to develop the most effective therapeutic hydrogel possible . [SEP]
[CLS] utilizing drugs as building units for the construction of nanostructures represents a paradigm shift in the growing field of nanomedicine , one in which drugs are no longer considered as passive cargos but as active participants in their own delivery to therapeutic targets . [SEP]
[CLS] by exploiting the molecular properties that drug molecules possess in the context of self - assembly and combining them with complementary entities , it is now possible to construct discrete nanostructures that possess a high and precisely defined drug content through rational molecular design . [SEP]
[CLS] three main strategies have emerged to accomplish this , each having their own unique advantages and disadvantages . [SEP]
[CLS] for instance , while macromolecular amphiphilic B-property prodrugs are the most well - developed of the three due to their similarity with other polymer - based delivery systems , the prevalence of spherical nanostructures may limit their use and optimization to address the sophisticated challenges associated with systemic delivery applications . [SEP]
[CLS] conversely , supramolecular hydrogels formed by amphiphilic B-property prodrugs are ideally suited to local delivery for treating cancers or other diseases that can benefit from topical or local administration of therapeutics . [SEP]
[CLS] amphiphilic B-property prodrugs straddle the line between maps and hydrogelators , with their tunable morphologies enabling the adaptation for either route of administration . [SEP]
[CLS] however , it has yet to be established how adaptable this platform is due to the strong influence of the drug molecule as a structural element and the limited number of drug structures tested so far . [SEP]
[CLS] given the push towards personalized medicine that is currently underway , it is important that the principles underlying how a drug molecule can affect the assembly of a nanostructure are determined and understood . [SEP]
[CLS] the drug delivery community as a whole would benefit greatly from the availability of generic platforms that can deliver a particular drug type . [SEP]
[CLS] after all , even though medicines may become personalized , they will still need to be effectively delivered to target sites so as to exert the desired biological effects . [SEP]
[CLS] for all systems , a big question to be answered is how will the physiological environment impact the physicochemical properties and eventually the performance of these drug - containing nanostructures ? [SEP]
[CLS] how is the integrity of the nanostructures affected by interactions with proteins B-material and the body ' s natural defense systems ? [SEP]
[CLS] part of this lies with the critical aggregation concentration of the self - assembled nanostructure , for which a careful balance is required as too low a value may impede the drug release , while too high will lead to rapid dissociation . [SEP]
[CLS] the latter is clearly undesirable for the purpose of systemic delivery , but could be an advantageous property for local delivery , particularly if the dissociation kinetics can be used to tune the drug release rate . [SEP]
[CLS] for systemic delivery , if the goal is to reduce the cac as much as possible , then strategies to facilitate dissociation at the desired location must also be considered in parallel . [SEP]
[CLS] one potential method is the inclusion of self - immolating components that will breakdown in specific environments , thereby negating the need for equilibrium driven dissociation . [SEP]
[CLS] while many challenges are still ahead , the direct use of drugs to build well - defined nanostructures presents a significant extension from both fields of molecular assembly and medicine . [SEP]
[CLS] aside from a few examples formed from macromolecular amphiphilic B-property prodrugs that have been evaluated in clinical trials , those created by assembly of small molecular amphiphilic B-property prodrugs are still focused on fundamental studies of their assembly behavior and in vitro evaluation of their potency against cancer cell B-material lines . [SEP]
[CLS] clearly , the next logical step will be testing these supramolecular materials in animals to collect information on their circulation fate and therapeutic efficacy , which will prove essential for guiding the design of next generation of drug - containing nanostructures for improved performance . [SEP]
[CLS] introducing greater complexity into macromolecular amphiphile B-property prodrugs . [SEP]
[CLS] ( a ) liu ' s approach utilizes a photo - caged poly - cpt - containing map coassembled with a folic B-material acid I-material conjugated polymer B-material to form a micelle B-material that was then cross - linked with an acid - and reduction - sensitive linker . [SEP]
[CLS] reprinted with permission from ref , copyright 2013 american chemical society . [SEP]
[CLS] ( b ) chilkoti ' s elp - based delivery platform that assembles into micelles B-material ( c ) when conjugated to doxorubicin B-material . [SEP]
[CLS] in vivo visualization of the phase transition that occurs on heating of the tumor B-material vasculature from 37°c ( d ) to 42°c ( e ) , indicating the increased accumulation of doxorubicin B-material within the vasculature after thermal cycling . [SEP]
[CLS] panels b - c were adapted with permission from ref , copyright 2009 nature publishing group ; panels d - e were adapted with permission from ref , copyright 2014 american chemical society . [SEP]
[CLS] futher interactions between these nanospheres B-nanoparticle induces the formation of a weak gel . [SEP]
[CLS] adapted with permission from ref , copyright 2011 royal society of chemistry . [SEP]
[CLS] ( d ) schematic illustration of xue ' s platform , in which a non - gelating peptide B-material is induced to form a hydrogel through the formation of doxorubicin B-material nanospheres B-nanoparticle that act as cross - linkers between filaments . [SEP]
[CLS] reprinted with permission from ref , copyright 2015 nature publishing group . [SEP]
[CLS] ( e ) schematic illustration of the highly supramolecular approach adopted by shi , in which cpt - peg micelles B-material form polypseudorotaxane structures via threading of - cyclodextrin onto one or more peg chains , thereby cross - linking and triggering gel formation . [SEP]
[CLS] the drug 5 B-material - I-material fluorouracil I-material ( 5 - fu ) could also be encapsulated within the gel , with the slow dethreading of the psuedorotaxane breaking down the gel and releasing 5 - fu and peg - cpt micelles B-material , that then underwent hydrolysis to release cpt . [SEP]
[CLS] reprinted from ref , copyright 2013 royal society of chemistry . [SEP]
[CLS] figures [SEP]
[CLS] 1 molecular structures of some small molecule drugs with the potential for self - association into molecular clusters or discrete nanostructures . [SEP]
[CLS] 2 representative transmission B-technique electron I-technique microscopy I-technique ( tem ) images of discrete nanostructures formed by the assembly of pure drugs . [SEP]
[CLS] ( a ) cui and coworkers demonstrated how folic B-material acid I-material could be induced to form nanofilamentous nanostructures by the addition of water B-material to a solution in meoh . [SEP]
[CLS] adapted with permission from ref [ 43 ] , copyright 2013 royal society of chemistry . [SEP]
[CLS] ( b ) hao characterized helical nanoribbons formed by camptothecin . [SEP]
[CLS] adapted with permission from ref [ 45 ] , copyright 2014 royal society of chemistry . [SEP]
[CLS] ( c - d ) yan and coworkers developed an irinotecan - chlorambacil ( ir - cb ) drug - drug conjugate ( c ) that could assemble into nanoparticles B-nanoparticle ( d ) . [SEP]
[CLS] adapted with permission from ref [ 46 ] , copyright 2014 american chemical society . [SEP]
[CLS] 3 macromolecular amphiphilic B-property prodrug platform developed by yokoyama and kataoka . [SEP]
[CLS] ( a ) peg - p ( asp ( adr ) ) for the delivery of doxorubicin B-material and ( b ) pt cross - linked peg - p ( glu ) polymer B-material for the delivery of cisplatin B-material upon ligand exchange . [SEP]
[CLS] panel b was adapted with permission from ref [ 67 ] , copyright 1996 elsevier . [SEP]
[CLS] 4introducing greater complexity into macromolecular amphiphile B-property prodrugs . [SEP]
[CLS] ( a ) liu ' s approach utilizes a photo - caged poly - cpt - containing map coassembled with a folic B-material acid I-material conjugated polymer B-material to form a micelle B-material that was then cross - linked with an acid - and reduction - sensitive linker . [SEP]
[CLS] reprinted with permission from ref , copyright 2013 american chemical society . [SEP]
[CLS] ( b ) chilkoti ' s elp - based delivery platform that assembles into micelles B-material ( c ) when conjugated to doxorubicin B-material . [SEP]
[CLS] in vivo visualization of the phase transition that occurs on heating of the tumor B-material vasculature from 37°c ( d ) to 42°c ( e ) , indicating the increased accumulation of doxorubicin B-material within the vasculature after thermal cycling . [SEP]
[CLS] panels b - c were adapted with permission from ref , copyright 2009 nature publishing group ; panels d - e were adapted with permission from ref , copyright 2014 american chemical society . [SEP]
[CLS] 5 a self - assembling drug amphiphile B-property platform developed by cui and coworkers . [SEP]
[CLS] ( a ) the generic design of the drug amphiphile B-property , illustrating the essential features : a hydrophobic B-property drug , cleavable linker and a short peptide B-material with overall hydrophilicity B-property and a strong preference for a particular secondary structure . [SEP]
[CLS] a branching point allows attachment of multiple drug molecules . [SEP]
[CLS] ( b ) molecular structure of a cpt - containing drug amphiphile B-property , mcpt - buss - tau , comprising a reduction - sensitive linker and a sheet forming tau peptide B-material . [SEP]
[CLS] ( c - e ) representative tem images of the drug amphiphiles B-property mcpt - buss - tau ( c ) , dcpt - buss - tau ( d ) , and qcpt - buss - tau ( e ) . [SEP]
[CLS] white arrows represent the open ends of the nanotubes B-nanoparticle formed by qcpt - buss - tau . [SEP]
[CLS] ( f ) schematic illustration of how assembled drug amphiphiles B-property can act as drug reservoirs , with the assembled form protecting the linker from premature cleavage . [SEP]
[CLS] dissociation of the nanostructure releases the single molecule form which is vulnerable to [SEP]
[CLS] 6 construction of interesting architectures through the manipulation of the molecular packing . [SEP]
[CLS] ( a - b ) representative tem images of a dual drug amphiphile B-property containing both cpt and ptx , indicating the structural evolution of cpt - ptx - sup35 , which initially forms a mixture of small , flexible filaments and two - filament twisted fibrils ( a ) , but after 24 h the two - filament twisted fibril nanostructure is the only structure observed ( b ) . [SEP]
[CLS] adapted with permission from ref [ 106 ] , copyright 2014 royal society of chemistry . [SEP]
[CLS] ( c ) cryogenic tem image of a catanionic mixture of two oppositely charged drug amphiphiles B-property , showing the formation of form large multiwalled nanotubes B-nanoparticle . [SEP]
[CLS] adapted from ref [ 110 ] , copyright 2014 royal society of chemistry . [SEP]
[CLS] 7 alternate approaches to the creation of amphiphilic B-property prodrugs . [SEP]
[CLS] ( a ) grinstaff and parquette ' s di - lysine - based cpt conjugate that can assemble into large nanotubes B-nanoparticle via the formation and maturation of helical tape structures . [SEP]
[CLS] adapted with permission from ref [ 117 ] , copyright 2015 wiley - vch . [SEP]
[CLS] ( b - d ) dna - drug amphiphile B-property platform . [SEP]
[CLS] schematic illustration of the molecular structure , assembly and degradation of dna - drug amphiphile B-property ( b ) . [SEP]
[CLS] representative tem images of the spherical nanostructure formed by dna5 - cpt in phosphate - buffered saline ( pbs ) ( cb ) and the rod - like structure formed by dna20 - cpt in pbs with 5 mm mgcl2 ( d ) . [SEP]
[CLS] adapted from from ref [ 177 ] , copyright 2015 american chemical society . [SEP]
[CLS] 8 examples of peptide B-material - based molecular hydrogelators . [SEP]
[CLS] ( a - b ) peptide B-material - based drugs that are capable of forming hydrogels either by themselves ( autogels ) ( a ) or by the addition of a hydrophobic B-property moiety to induce gelation ( b ) . [SEP]
[CLS] panel b was adapted with permission from ref [ 141 ] , copyright 2002 american chemical society . [SEP]
[CLS] ( c ) xu ' s taxol - based hydrogelator , nap - ffk ( succ - ptx ) py , for which the phosphatase - catalysed removal of a tyrosine B-material phosphate group decreases the solubility B-property , triggering nanofilament B-nanoparticle formation and ultimately gelation via entanglement . [SEP]
[CLS] adapted with permission from ref , copyright 2009 american chemical society . [SEP]
[CLS] ( d ) yang ' s dual drug containing hydrogelator , in which an anticancer B-property drug ( ptx or hcpt ) is paired with the anti B-property - I-property inflammatory I-property / immunosuppressant , dexamethasome ( dex ) . [SEP]
[CLS] gelation is triggered by the reduction of the disulfide bond that is used to attach a hydrophilic B-property tail . [SEP]
[CLS] adapted with permission from ref , copyright 2012 royal society of chemistry . [SEP]
[CLS] 9 alternate approaches to the creation of molecular hydrogelators . [SEP]
[CLS] ( a - c ) yang ' s folic B-material acid I-material ( fa ) - taxol based hydrogelator ( a ) that forms nanospheres B-nanoparticle rather than nanofilaments , as indicated by atomic B-technique force I-technique microscopy I-technique ( afm ) ( b ) , through hoogsteen hydrogen B-material bonding between fa molecules ( c ) . [SEP]
[CLS] futher interactions between these nanospheres B-nanoparticle induces the formation of a weak gel . [SEP]
[CLS] adapted with permission from ref , copyright 2011 royal society of chemistry . [SEP]
[CLS] ( d ) schematic illustration of xue ' s platform , in which a non - gelating peptide B-material is induced to form a hydrogel through the formation of doxorubicin B-material nanospheres B-nanoparticle that act as cross - linkers between filaments . [SEP]
[CLS] reprinted with permission from ref , copyright 2015 nature publishing group . [SEP]
[CLS] ( e ) schematic illustration of the highly supramolecular approach adopted by shi , in which cpt - peg micelles B-material form [SEP]
[CLS] the surface properties of nanoparticles B-nanoparticle ( nps B-nanoparticle ) dictate their interaction with the outside world . [SEP]
[CLS] the use of precisely designed molecular ligands to control np B-nanoparticle surface properties provides an important toolkit for modulating their interaction with biological systems , facilitating their use in biomedicine . [SEP]
[CLS] in this review we will discuss the application of the atom - by - atom control provided by organic synthesis to the generation of engineered B-nanoparticle nanoparticles I-nanoparticle , with emphasis on how the functionalization of nps B-nanoparticle with these " small " organic molecules ( mw < 1 , 000 ) can be used to engineer nps B-nanoparticle for a wide range of applications . [SEP]
[CLS] fabricating nanoparticles B-nanoparticle ( nps B-nanoparticle ) with unique biological properties is a challenging but rewarding task . [SEP]
[CLS] the combination of multiple np B-nanoparticle features such as core B-material size , [SEP]
[CLS] shape , and surface chemistry [SEP]
[CLS] allows the regulation of the biological behavior . [SEP]
[CLS] the np B-nanoparticle surface is the interface with the outside world , and plays a prominent role in the interaction with biomolecules . [SEP]
[CLS] the relatively large surface area of nps B-nanoparticle facilitate the attachment of a wide range of biomacromolecules such as peptides B-material , proteins B-material , nucleic B-material acids I-material , and viruses to dictate np B-nanoparticle - protein or np B-nanoparticle - cell B-material interactions . [SEP]
[CLS] likewise , polymers B-material have been widely employed as np B-nanoparticle coverages . [SEP]
[CLS] the structural complexity and / or potential biodegradability B-property of these macromolecular systems , however , introduce complexity to the interactions between nps B-nanoparticle and biomolecules . [SEP]
[CLS] the use of non - polymeric " small " organic molecules provides a robust and scalable methodology to tailor the nano - bio interface . [SEP]
[CLS] the wide variety of moieties available through organic chemistry provides a rich toolkit to provide atom - by - atom control of the np B-nanoparticle - biomolecule interactions . [SEP]
[CLS] in this review we will present research focusing on controlling the interactions of nps B-nanoparticle with proteins B-material and cells B-material by using these " small " molecule ( mw < 1 , 000 ) ligands . [SEP]
[CLS] the interaction modes of nps B-nanoparticle with proteins B-material and cells B-material can be modified by designing the surface monolayer , concomitantly modulating biological functions . [SEP]
[CLS] 24 this fine - tuned control provides a finely - honed tool for a wide array of biological interactions . [SEP]
[CLS] modulating enzyme - nanoparticle B-nanoparticle interactions . [SEP]
[CLS] engineered interactions between nps B-nanoparticle and enzymes provide tools for both enhancing enzyme stability and regulating activity . [SEP]
[CLS] decorating nps B-nanoparticle with engineered ligands facilitates the ' dialing in ' of specific modes of interaction including electrostatic , hydrogen B-material bonding , and van der waals forces . [SEP]
[CLS] this capability has been demonstrated using anionic gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) and chymotrypsin ( cht ) , utilizing the positive " patch " around the cht active site . [SEP]
[CLS] the studies demonstrated that the anionic surface of nps B-nanoparticle can selectively recognize this cationic B-material patch , thus inhibiting cht activity . [SEP]
[CLS] similarly , a variety of amino acid - terminated ligands bearing tunable charge and hydrophobicity B-property ( fig . 1 ( a ) ) provided detailed information about np B-nanoparticle - cht interfacial recognition . [SEP]
[CLS] a thorough examination of the binding constants revealed that aunps B-nanoparticle bearing hydrophobic B-property groups bind more strongly to cht than aunps B-nanoparticle with hydrophilic B-property groups , indicating the importance of hydrophobic B-property interactions I-property at the interface ( fig . 1 ( b ) ) . [SEP]
[CLS] in addition to hydrophobicity B-property , the chirality of the amino B-material acids I-material is also an important factor for the affinity of nps B-nanoparticle and proteins B-material . [SEP]
[CLS] these results demonstrate that very specific chemical features can be employed to modulate protein B-material recognition . [SEP]
[CLS] furthermore , these studies also evidenced that unlike small molecule enzyme regulators , the binding affinity of nps B-nanoparticle to enzymes is not the only factor that modifies the catalytic activity . [SEP]
[CLS] the work from rotello et al . showed that the reduction of enzymatic activity mediated by nps B-nanoparticle was also observed when nps B-nanoparticle were functionalized with hydrophilic B-property ligands because of the denaturation of cht caused by these functional groups ( fig . 1 ( c ) and ( d ) ) . [SEP]
[CLS] likewise , hamad - schifferli et al . reported that irreversible denaturation of glucose oxidase ( gox ) caused by the interaction with nps B-nanoparticle had a negative effect on its enzymatic activity . [SEP]
[CLS] recently , das et al . reported that nps B-nanoparticle can also improve enzymatic activity . [SEP]
[CLS] their investigation on the activity of mitochondrial membrane cytochrome c ( cyt c ) bound with cationic B-material nps B-nanoparticle demonstrated the enhanced peroxidase activity by increasing hydrophobicity B-property of nps B-nanoparticle . [SEP]
[CLS] structural reorganization caused by hydrophobic B-property nps B-nanoparticle exposed the heme B-material group I-material of the enzyme , facilitating substrate access to the catalytic site . [SEP]
[CLS] in stark contrast to their ability to denature protein B-material , functionalized nps B-nanoparticle have been used as synthetic chaperones that assist in the refolding of denatured enzymatic proteins B-material . [SEP]
[CLS] for instance , fully recovery of enzymatic activity of thermally denatured cationic B-material proteins B-material such as cht and papain has been observed using malonic acid - functionalized anionic aunps B-nanoparticle followed by disruption of the protein - np B-nanoparticle complex with aqueous sodium B-material chloride I-material . [SEP]
[CLS] enzymatic activity can also be modified by using the nps B-nanoparticle functionalized with specific small molecule enzyme inhibitors or activators . [SEP]
[CLS] supuran and co - workers coated aunps B-nanoparticle with an inhibitor of carbonic anhydrase ( ca ) , and the np B-nanoparticle displayed higher selectivity toward tumor associated isoform ix over the cytosolic isozymes i and iiix . [SEP]
[CLS] likewise , ca showed increased enzymatic activity when mixed with nps B-nanoparticle bearing a ca activator . [SEP]
[CLS] these studies show functionalization on np B-nanoparticle surface can modulate the activity B-property of I-property small I-property molecule I-property enzyme regulators , providing a new platform for the enzyme regulation . [SEP]
[CLS] correlation between the denaturation rate constants ( k ) of cht and the hydrophobicity B-property index of amino B-material acid I-material side chains in nanoparticles B-nanoparticle . [SEP]
[CLS] reprinted with permission from . [SEP]
[CLS] modulation of protein - protein interactions . protein - protein interactions ( ppi ) play a central role in numerous cellular B-event processes I-event including apoptosis B-event and angiogenesis B-event , thus targeting theinterface between proteins B-material provides a potential way for treating diseases . [SEP]
[CLS] however , due to the large contact area between proteins B-material in these complexes , effective inhibition of ppis is quite challenging . [SEP]
[CLS] knapp and rotello reported that charged aunps B-nanoparticle can inhibit the recognition of cyt c with cytochrome c peroxidase ( cpp ) ( fig . 2 ) . [SEP]
[CLS] since this ppi is defined by saltbridges between the cationic B-material surface of cyt c and the anionic surface of cpp , they designed aunps B-nanoparticle with cationic B-material , neutral , or anionic aunps B-nanoparticle , and examined charge dependent inhibition of ppi . [SEP]
[CLS] as a result , effective disruption of the cyt c - cpp complex was observed only when charged nps B-nanoparticle were used , while neutral nps B-nanoparticle had no effect . [SEP]
[CLS] notably , because of the multivalency of nps B-nanoparticle , a single np B-nanoparticle could interact with more than one protein B-material , implying that nps B-nanoparticle could be effective modulators that can disrupt multiple ppis . [SEP]
[CLS] minimizing nonspecific np B-nanoparticle - protein B-material interactions . [SEP]
[CLS] minimizing interactions between nanoparticles B-nanoparticle and proteins B-material is also an important challenge for a diversity of biological applications . [SEP]
[CLS] for example , when nps B-nanoparticle are intended for biomedical imaging or as therapeutic agents , the adsorption and denaturation of proteins B-material over the np B-nanoparticle surface induces a rapid elimination of the from the body . [SEP]
[CLS] this phenomenon critically reduces the efficacy of the engineered delivery vehicles B-material and targeted nps B-nanoparticle . [SEP]
[CLS] the classical approach to provide " non - interacting " capabilities to the nps B-nanoparticle is the use of oligo ( ethylene glycol ) ( oeg ) functionalities , which reduces protein B-material denaturation over the np B-nanoparticle surface . [SEP]
[CLS] this is translated into longer blood circulation times comparative to other systems , achieving higher tumor B-material accumulation rates . [SEP]
[CLS] however , oeg functionalized nps B-nanoparticle undergo a process known as accelerated blood clearance ( abc ) , a phenomenon that increases the elimination of the nps B-nanoparticle after the second dose due to the formation of antibodies B-material against the probe . [SEP]
[CLS] an alternative to oeg functionalization is the use of zwitterionic molecules based principally on two chemical structures , namely betaines and amino B-material acids I-material . [SEP]
[CLS] the zwitterionic nature of those structures confers strong non - fouling properties to nps B-nanoparticle . [SEP]
[CLS] as an example , it has been reported that small aunps B-nanoparticle ( ~ 5 nm , high area / volume ratio ) coated with a mixture of citrate and cysteine B-material do not absorb proteins B-material even at physiological serum concentrations ( fig . 3 ) . [SEP]
[CLS] in addition , functionalized quantum B-nanoparticle dots I-nanoparticle ( 3 . 4 nm ) with cysteine B-material and tumor B-material targeting groups have exhibited significantly lower renal clearance rates as well as the high tumor B-material targeting efficiency ( fig . 4 ) . the derivatization of ligands using amino B-material acids I-material , however , is limited by the fact that neutral charge is desirable for the stealth capabilities , and the addition of more amino B-material acids I-material may modify this property . [SEP]
[CLS] betaines , on the other hand , come in a myriad of structures ( carboxybetaines , phosphobetaines , sulfobetaines ) and they have been shown to enhance the stealth properties of nps B-nanoparticle such as reduced protein B-material adsorption , longer blood circulation time and low clearance . [SEP]
[CLS] in addition , betaines are easily modified by changing the substituents over the ammonium group without affecting the overall neutral charge . [SEP]
[CLS] a final method to obtain zwitterionic properties at the np B-nanoparticle surface is the use of a mixed monolayer nps B-nanoparticle containing both cationic B-material and anionic ligands . [SEP]
[CLS] by adjusting the ratio of those ligands during the place exchange reaction , the zero net charge can be achieved . [SEP]
[CLS] although this technique has also rendered nps B-nanoparticle with strong non - fouling properties , reproducibility is still a challenge . [SEP]
[CLS] reprinted with permission from . [SEP]
[CLS] the cellular adsorption and internalization of nps B-nanoparticle are predominantly determined by their surface charge . [SEP]
[CLS] multiple reports demonstrate a significant difference on the cellular uptake rate between positively charged nps B-nanoparticle and other charged nps B-nanoparticle ( negative , neutral , and zwitterionic ) ( fig . 5 ) ] . [SEP]
[CLS] the strong electrostatic interaction between the negatively charged cell B-material membranes and cationic B-material nps B-nanoparticle is the underlying factor that increases the amount of nps B-nanoparticle adsorbed onto the cell B-material surface , thus enhancing cellular uptake . [SEP]
[CLS] however , in some case , the strong cellular uptake was observed with negatively charged nanoparticles B-nanoparticle . [SEP]
[CLS] the studies demonstrated that iron B-material oxide I-material ( fe2o3 ) nanoparticles B-nanoparticle functionalized with carboxylate B-material group I-material can be efficiently taken up by cancer cell B-material lines presumably because of the direct diffusion through membrane or the pinocytosis of anionic nanoparticles B-nanoparticle . [SEP]
[CLS] in addition to the surface charge , the hydrophobicity B-property of nps B-nanoparticle can also be employed to modify the cellular uptake of these materials . [SEP]
[CLS] a study using cationic B-material nps B-nanoparticle demonstrated that an increase of the aunp B-nanoparticle surface hydrophobicity B-property enhances cellular uptake in the absence of serum , while it decreases uptake in the presence of serum . [SEP]
[CLS] the binding B-event of I-event serum I-event proteins I-event to aunps B-nanoparticle is influenced by the surface charge and hydrophobicity B-property , compromising the np B-nanoparticle uptake . [SEP]
[CLS] there are some efforts to enhance the cellular uptake of zwitterionic aunps B-nanoparticle by using hydrophobicity B-property , demonstrating slightly higher cellular uptake of hydrophobic B-property aunps B-nanoparticle compared with hydrophilic B-property counterparts , either in the presence or absence of serum proteins B-material . [SEP]
[CLS] the binding mode between ligand and nanoparticle B-nanoparticle surface can affect the cellular interaction . [SEP]
[CLS] cell B-material experiments using cationic B-material quantum B-nanoparticle dots I-nanoparticle bearing monothiol or dithiol coordination groups revealed a significant difference on the cellular uptake of nanoparticles B-nanoparticle ( fig . 6 ) . [SEP]
[CLS] these results indicate the importance of molecular design that considers not only effects from the headgroups but also effects from the overall ligand design . [SEP]
[CLS] uptake of quantum B-nanoparticle dots I-nanoparticle after the incubation B-technique with hela cells B-material . [SEP]
[CLS] reprinted with permission from . [SEP]
[CLS] in addition to " permanent " physicochemical properties , a chemical designing approach can also construct " responsive " surfaces that can alter the physicochemical features of the nps B-nanoparticle by endogenous stimuli . [SEP]
[CLS] such chemical surfaces generate unique interaction modes with biological systems . [SEP]
[CLS] ph - responsive aggregate formation . [SEP]
[CLS] enhancing cellular uptake with stimuli responsive np B-nanoparticle surfaces was initially reported by the kim group . [SEP]
[CLS] negatively charged citraconic amide functionalized " smart " aunps B-nanoparticle form aggregates upon exposure to weakly acidic environments ( ph = 5 . 5 ) ( fig . 7 ( a ) and ( b ) ) . [SEP]
[CLS] acid induced partial cleavages of citraconic amide B-material group I-material and subsequent exposure of cationic B-material primary B-material amine I-material neutralize the surface charge of nps B-nanoparticle , inducing aggregation . [SEP]
[CLS] aunps B-nanoparticle are exposed to acidic endosomal environments during cellinternalization process . [SEP]
[CLS] when an aggregate is formed inside an endosome , the exocytosis of aunps B-nanoparticle is blocked , resulting in a larger amount of aunps B-nanoparticle accumulated in b16f10 cells B-material compared to negatively charged aunps B-nanoparticle used as controls ( fig . 7 ( c ) ) . [SEP]
[CLS] aunps B-nanoparticle bearing a mixed monolayer of quaternary ammonium and carboxylic B-material acid I-material ligands can provide high tunable ph - induced aggregation systems . [SEP]
[CLS] partial protonation of carboxylate B-material functionality at acidic ph can induce the aggregate formation by the neutralization of surface charge . [SEP]
[CLS] the ph value at which nps B-nanoparticle form aggregates can be tuned by changing the surface ligand coverage ratio and core B-material size . [SEP]
[CLS] ji et al . applied this system for tumor B-material targeting , utilizing the ph difference between tumor B-material tissue ( ph 6 . 0 - 6 . 6 ) and normal tissue ( ph 7 . 4 ) as a tumor B-material environment signal . [SEP]
[CLS] mixing the same mole ratio of cationic B-material and anionic ligands with 16 nm aunps B-nanoparticle , they constructed aunps B-nanoparticle that form aggregates at ph 6 . 5 ( fig . 8 ( a ) and ( b ) ) . [SEP]
[CLS] in vitro cellular uptake studies demonstrated that a ph decrease enhanced np B-nanoparticle internalization ( fig . 8 ( c ) ) due to the accelerated sedimentation of aunps B-nanoparticle . [SEP]
[CLS] subsequent in vivo studies indicated the enhancement of tumor B-material accumulation and retentions of these aunps B-nanoparticle ( fig . 8 responsive surface which can reversibly switch from zwitterionic into cationic B-material . [SEP]
[CLS] the chemical moiety at the surface features alkoxyphenyl acylsulfonamide groups that is protonated under weakly acidic conditions ( fig . 9 ( a ) ) . [SEP]
[CLS] aunps B-nanoparticle functionalized with this ph - responsive zwitterionic group exhibited neutral zeta B-property - potential I-property at ph 7 . 4 and showed a sharp response to ph decrease , switching into cationic B-material at ph 6 . 6 and 6 . 0 without forming aggregates ( fig . 9 ( b ) ) . [SEP]
[CLS] as discussed previously , aunps B-nanoparticle with cationic B-material ligands are prone to be adsorbed by the negatively charged cell B-material membranes , resulting in higher cellular uptake than that of zwitterionic aunps B-nanoparticle . [SEP]
[CLS] cell B-material studies indicated that the ph - responsive zwitterionic surface does induce an enhancement in cellular uptake of aunps B-nanoparticle by cancerous cells B-material at tumor B-material ph ( 6 . 6 - 6 . 0 ) , achieving a four - fold increase at ph 6 . 0 compared to the cellular uptake at ph 7 . 4 . [SEP]
[CLS] on the other hand , a nonresponsive alkyl analogue did not show differences over the ph range studied ( fig . 9 ( c ) ) . [SEP]
[CLS] this result indicates that subtle structural differences of zwitterionic moieties could generate a targeting function for selective accumulation of aunps B-nanoparticle in tumor B-material tissues . [SEP]
[CLS] esterase - induced cellular uptake of amphiphilic B-property aunp B-nanoparticle . [SEP]
[CLS] ph is not the only stimulus to enhance the interaction between nps B-nanoparticle and cells B-material . [SEP]
[CLS] niikura and coworkers demonstrated that an esterase - catalyzed hydrolysis of ester moieties can be used as to enhance the cellular uptake of amphiphilic B-property aunps B-nanoparticle . [SEP]
[CLS] 10 nm aunps B-nanoparticle were functionalized with ester or ether - headed amphiphilic B-property peg ligands which enable nps B-nanoparticle to dissolve both of polar B-material and nonpolar solvents I-material through systematic conformational changes ( fig . 10 ( a ) and ( b ) ) . [SEP]
[CLS] investigation of the cellular internalization of those aunps B-nanoparticle revealed that aunps B-nanoparticle with ester functionalities were taken up by cells B-material in a greater extent than the corresponding ether - aunps B-nanoparticle ( fig . 10 ( c ) ) . [SEP]
[CLS] the authors assumed that even though both ester and ether - headed aunps B-nanoparticle have a high affinity to hydrophobic B-property cell B-material membranes , the exposure of alkyl moieties during the interaction with cells B-material generated an esterase - mediated hydrolysis that enabled longer retention of aunps B-nanoparticle at the cell B-material membrane , accelerating endocytosis B-event ( fig . 10 ( d ) ) . [SEP]
[CLS] 10d is based on information obtained in the report . [SEP]
[CLS] as shown above , surface chemistry defines the interactions of nps B-nanoparticle with biological systems . [SEP]
[CLS] building the surface with proper chemical structures has the potential to overcome many challenges currently present in nanomedicine . [SEP]
[CLS] the examples highlighted in this review clearly show the power of chemical approaches for tailoring the physicochemical properties of nps B-nanoparticle , providing fruitful biological features . [SEP]
[CLS] organic chemistry can be utilized as a " palette " containing an unlimited range of functional groups for the modulation of the np B-nanoparticle functions . [SEP]
[CLS] desired features and functions can be constructed along with the synthesis of functional groups , providing an opportunity of the atom by atom level regulation . [SEP]
[CLS] this approach results in the information of structure - function relationships , providing a deeper understanding of the molecular recognition between nps B-nanoparticle and biological systems as well as bringing along clues on how to control desired effects at specific places . [SEP]
[CLS] such methods will help to maximize the potential of nanomaterials B-material for the creation of novel therapeutics , diagnostics and imaging agents . [SEP]
[CLS] 1 ( a ) structures of amino acid functionalized aunps B-nanoparticle . [SEP]
[CLS] ( b ) correlation between gibbs free energy changes and hydrophobicity B-property index of amino B-material acid I-material side chains . [SEP]
[CLS] ( c ) normalized activity of cht ( 3 . 2 m ) with nanoparticles B-nanoparticle ( 0 . 8 m ) bearing various amino B-material acid I-material side chains . [SEP]
[CLS] ( d ) [SEP]
[CLS] 2 schematic illustration of the interaction between cyt c , ccp , and aunps B-nanoparticle with different surface charge . [SEP]
[CLS] reprinted with permission from [ 42 ] . [SEP]
[CLS] 3 ( a ) chemical structure of citrate and lysine coated aunp B-nanoparticle . [SEP]
[CLS] ( b ) chemical structure of citrate and cysteine coated aunp B-nanoparticle . [SEP]
[CLS] ( c ) dls distributions of citrate and lysine coated aunp B-nanoparticle ( coverage ; lysine B-material / citrate = 1 . 4 ) in water B-material ( green curve ) and fbs ( red curve ) , and citrate aunp B-nanoparticle in fbs ( black curve ) . [SEP]
[CLS] ( d ) dls distributions of citrate and cysteine coated aunp B-nanoparticle ( cysteine B-material / citrate = 1 . 6 ) in water B-material ( green curve ) and fbs ( red curve ) . [SEP]
[CLS] reprinted with permission from [ 49 ] . [SEP]
[CLS] 4 ( a ) chemical structure of zwitterionic quantum B-nanoparticle dots I-nanoparticle bearing targeting groups . [SEP]
[CLS] ( b ) fluorescent B-property images of prostate - specific membrane antigen ( psma ) - positive lncap and psmanegative prostate cancer cells B-material after incubation B-technique with functionalized zwitterionic quantum B-nanoparticle dots I-nanoparticle . [SEP]
[CLS] 5 schematic illustration of the interaction between cell B-material membrane and aunps B-nanoparticle with different surface charge . [SEP]
[CLS] 6 ( a ) chemical structure of mono - and dithiol capped cdse / zns quantum B-nanoparticle dots I-nanoparticle . [SEP]
[CLS] ( b ) [SEP]
[CLS] 7 ( a ) chemical structure of the aunps B-nanoparticle capable of aggregating in acidic ph . [SEP]
[CLS] ( b ) tem images of aunps B-nanoparticle in ph 5 . 5 acetate buffer with different incubation B-technique times of ( i ) 0 , ( ii ) 10 , ( iii ) 30 , ( iv ) 60 , ( v ) 90 min . [SEP]
[CLS] ( c ) dark field microscope images of b16 f10 cells B-material incubated B-technique with " smart " aunps B-nanoparticle ( first column ) , citrate aunps B-nanoparticle ( second column ) , and mua ( 11 - mercaptoundecanoic acid ) - capped aunps B-nanoparticle ( last column ) . [SEP]
[CLS] reprinted with permission from [ 64 ] . [SEP]
[CLS] 3a is based on information obtained in the literature . [SEP]
[CLS] ( d ) and ( e ) ) . [SEP]
[CLS] 8 ( a ) chemical structure of mixed monolayer aunps B-nanoparticle capable of aggregating in acidic conditions . [SEP]
[CLS] ( b ) tem images of aunps B-nanoparticle at ph 7 . 4 and 6 . 5 [ scale bar 50 nm ( upper image ) , 20 nm ( lower image ) ] . [SEP]
[CLS] ( c ) effect of ph on the cellular uptake of aunps B-nanoparticle by hepg2 cells B-material . [SEP]
[CLS] ( d ) comparison of tumor B-material accumulation in kb tumor B-material in balb / c nude mice between ph - responsive aunp B-nanoparticle and peg - functionalized aunp B-nanoparticle . [SEP]
[CLS] ( e ) comparison of tumor B-material tissue retention pattern between ph - responsive aunp B-nanoparticle and peg - functionalized aunp B-nanoparticle . [SEP]
[CLS] reprinted with permission from [ 54 ] . [SEP]
[CLS] 3ais based on information obtained in the literature . [SEP]
[CLS] 9 ( a ) chemical structures of the acylsulfonamide - functionalized ph - responsive and nonresponsive zwitterionic aunps B-nanoparticle , and the mechanism of acidic ph enhanced cellular uptake . [SEP]
[CLS] ( b ) ph dependence of zeta B-property - potential I-property of acylsulfonamide functionalized aunp B-nanoparticle ( c ) effects of ph difference on cellular uptake of aunps B-nanoparticle [SEP]
[CLS] reprinted with permission from . [SEP]
[CLS] 10 ( a ) conformational change of the amphiphilic B-property peg ligands on aunps B-nanoparticle in polar B-material and nonpolar solvents I-material . [SEP]
[CLS] ( b ) chemical structures of the ester and ether - headed amphiphilic B-property peg ligands . [SEP]
[CLS] ( c ) difference of the number of aunps B-nanoparticle taken up by hela cells B-material ( 1 . 0 x 10 5 ) . [SEP]
[CLS] ( d ) esterase - [SEP]
[CLS] nanoparticles B-nanoparticle have been widely studied as versatile platforms for in vivo imaging and therapy . [SEP]
[CLS] however , their use to image and / or treat the brain is limited as they are often unable to cross the blood - brain barrier B-property ( bbb ) . [SEP]
[CLS] to overcome this problem , herein we report the use of focused ultrasound in vivo to successfully deliver dnacoated gold B-nanoparticle nanoparticles I-nanoparticle to specific locations in the brains of mice . [SEP]
[CLS] functionalised metal B-nanoparticle nanoparticles I-nanoparticle that are able to codeliver multiple drugs and / or imaging agents to a target location have attracted great interest in recent years . [SEP]
[CLS] in particular , spherical B-nanoparticle nanoparticles I-nanoparticle coated with a dense layer of oligonucleotides , first pioneered by mirkin et al . , have emerged as promising nanoplatforms for in vivo applications . [SEP]
[CLS] it has been shown that these nanoparticles B-nanoparticle can spontaneously enter over 50 different cell B-material types without the need for an external transfecting agent , have low immunogenicity B-property in cells B-material , good stability and have shown no toxicity B-property so far . [SEP]
[CLS] in addition to this , they are highly versatile , allowing them to be easily tailored for a wide range of applications . [SEP]
[CLS] a variety of core B-material materials can be used to prepare them , ranging from iron B-nanoparticle oxide I-nanoparticle nanoparticles I-nanoparticle to quantum B-nanoparticle dots I-nanoparticle , and almost any oligonucleotide sequence can be attached to the particle surface . [SEP]
[CLS] however , due to their size , the majority of functionalised nanoparticles B-nanoparticle show limited accumulation in the brain due to their inability to cross the blood - brain barrier B-property ( bbb ) . [SEP]
[CLS] this limits their potential application for the imaging and treatment of a range of diseases such as alzheimer ' s disease , parkinson ' s disease and brain tumours . [SEP]
[CLS] as of now , the only way to disrupt the bbb in a non - invasive , transient and targeted manner is with focused ultrasound and microbubbles . [SEP]
[CLS] in this method , microbubbles and the drug of choice are first injected intravenously into the bloodstream . [SEP]
[CLS] the target region is then exposed to ultrasound , which drives the microbubbles into radial oscillation . [SEP]
[CLS] by exerting mechanical stress on the blood vessel walls , an increase in bbb permeability is observed , resulting in enhanced drug delivery . [SEP]
[CLS] the exact biological mechanisms through which this occurs is still under investigation , but it has been linked to a widening of the tight junctions between endothelial cells B-material , increased vesicle activity and a suppression of the drug efflux pumps located at the surface of endothelial cells B-material . [SEP]
[CLS] to date , focused ultrasound - mediated bbb disruption has been successfully used to deliver a wide range of small molecules into the brains of rodents and nonhuman primates . [SEP]
[CLS] in contrast , there are significantly fewer examples where nanoparticles B-nanoparticle have been successfully delivered across the bbb in vivo using focused ultrasound . [SEP]
[CLS] a reason for this is that the dependency of focused ultrasound - mediated delivery on nanoparticle B-nanoparticle diameter has not been previously investigated , making it difficult to design nanoparticles B-nanoparticle for this application : larger nanoparticles B-nanoparticle allow for higher payloads of drug to be delivered to the target location per particle , but may not be delivered as efficiently across the bbb using this technology compared to smaller nanoparticles B-nanoparticle . [SEP]
[CLS] with the aim to address this , herein we report the synthesis of a range of dna - coated gold B-nanoparticle nanoparticles I-nanoparticle ( dna - au nps B-nanoparticle ) of different sizes and show that they can be successfully delivered to specific brain locations across the bbb in mice using focused ultrasound ( figure 1 ) . [SEP]
[CLS] the motivation to carry out this study is to establish a methodology for the future use of nanoparticles B-nanoparticle as delivery vehicles B-material for delivering drugs and / or imaging agents to specific locations in the brain . [SEP]
[CLS] the size - dependency of focused ultrasound - mediated nanoparticle B-nanoparticle delivery across the bbb has also been investigated in this study . [SEP]
[CLS] three sets of au nps B-nanoparticle of different sizes were synthesised via a seeded growth protocol involving the reduction of haucl4 with sodium B-material citrate and traces of tannic acid ( figure 2a ) . [SEP]
[CLS] au nps B-nanoparticle were characterised using dynamic B-technique light I-technique scattering I-technique ( dls ) , transmission B-technique electron I-technique microscopy I-technique ( tem ) and uv - vis spectroscopy B-technique . [SEP]
[CLS] dls measurements and tem images showed that the au nps B-nanoparticle were quasi - spherical in shape and narrowly dispersed , with core B-material diameters of 6 . 3 ± 0 . 7 nm , 9 . 5 ± 1 . 4 nm and 14 . 2 ± 1 . 2 nm ( figure 2b , c and table s1 ) . [SEP]
[CLS] the presence of the characteristic surface plasmon resonance ( spr ) band at ~ 515 nm was observed in the absorption spectra of all au nps B-nanoparticle synthesised ( figure s2 ) . [SEP]
[CLS] moreover , the absorbance and wavelength of the spr band was seen to increase with increasing np B-nanoparticle size as expected . [SEP]
[CLS] synthesised au nps B-nanoparticle were functionalised with cy5 - labelled oligonucleotides to give cy5 - dna - au nps B-nanoparticle a , b and c . [SEP]
[CLS] as previously described by li et al . , tween 20 was first added to stabilise the au nps B-nanoparticle and prevent their aggregation in high ionic strength environments . [SEP]
[CLS] thiolated oligonucleotides were then added to the reaction mixture in the presence of salt B-material to minimise electrostatic repulsions between oligonucleotide strands . a 25 base oligonucleotide sequence was chosen for this study as this is the typical length of oligonucleotides used for gene therapies . [SEP]
[CLS] dls measurements showed that the synthesised cy5 - dna - au nps B-nanoparticle had hydrodynamic diameters of 21 . 6 ± 3 . 3 nm ( a ) , 23 . 3 ± 2 . 6 nm ( b ) and 25 . 2 ± 1 . 9 nm ( c ) respectively ( table 1 ) . [SEP]
[CLS] as expected , changes in the hydrodynamic diameters and zeta B-property potentials I-property were observed after functionalisation with oligonucleotides . [SEP]
[CLS] moreover , the zeta B-property potentials I-property were found to vary between cy5 - dna - au nps B-nanoparticle a , b and c as the oligonucleotide loading was adjusted in each case so that the overall fluorescence B-property intensities were similar to allow for comparison after delivery across the bbb ( figure s3 ) . [SEP]
[CLS] the feasibility of delivering cy5 - dna - au nps B-nanoparticle a - c into the brain in vivo using ultrasound pulses was then assessed . [SEP]
[CLS] wild type c57lb / 6 mice were first injected with sonovue © microbubbles , followed by cy5 - dna - au nps B-nanoparticle a , b or c ( figure 3 ) . [SEP]
[CLS] the left thalamus of the brain was sonicated using ultrasound pulses ( frequency : 1 mhz ; peak - negative pressure : 530 kpa ; pulse length : 10 ms ; pulse repetition frequency : 0 . 5 hz ; sonication duration : 250 s ) , whilst the right thalamus acted as the non - sonicated control . [SEP]
[CLS] following sonication , the brain was extracted , sectioned into 30 μm horizontal sections and then imaged using brightfield and fluorescence B-technique microscopy I-technique . [SEP]
[CLS] evidence of successful bbb disruption in the left thalamus was observed in all experiments conducted . [SEP]
[CLS] due to the characteristic red colour of the au np B-nanoparticle cores B-material , their presence in the left thalamus could be detected in brightfield images ( figure 4 ) . [SEP]
[CLS] fluorescence B-property signal from the attached cy5labelled oligonucleotides was found at the exact same locations , indicating that the cy5 - dna - au nps B-nanoparticle remain intact as they cross the bbb ( figure 4 ) . [SEP]
[CLS] to the best of our knowledge , this is the first time that dna - coated gold B-nanoparticle nanoparticles I-nanoparticle have been delivered across the bbb in vivo using focused ultrasound . [SEP]
[CLS] moreover , in contrast to prior work by jensen et al . in which dna - au nps B-nanoparticle were reported to be able to cross the intact bbb in wild type mice by themselves , no evidence of cy5 - dna - au nps B-nanoparticle a - c was detected in the right thalamus ( control ) in our experiments ( figure 5a ) . [SEP]
[CLS] it should be noted , however , that a lack of fluorescence B-property signal does not indicate that there are no nps B-nanoparticle present ; instead , it might indicate that there are not enough nps B-nanoparticle present to generate a detectable signal . the data presented here therefore shows that focused ultrasound and microbubbles can be used to significantly enhance the delivery of cy5 - dna - au nps B-nanoparticle across the bbb ; furthermore , it allows the directed delivery of nanoparticles B-nanoparticle to specific locations within the brain . [SEP]
[CLS] to investigate the effect of varying np B-nanoparticle diameter on the efficiency of delivery using focused ultrasound - mediated bbb disruption , the normalised optical density - the average fluorescence B-property intensity in the targeted region normalised by the control region - was calculated from the fluorescence B-property images ( figure 5 ) . [SEP]
[CLS] a two - sided student ' s t - test was performed to assess statistical significance . [SEP]
[CLS] our results show that there is a sizedependency associated with the delivery of cy5 - dna - au nps B-nanoparticle across the bbb using focused ultrasound , with the smallest nps B-nanoparticle tested in this study ( a ) being delivered across the bbb six times more efficiently than the largest nps B-nanoparticle tested ( c ) . [SEP]
[CLS] as proposed in the past , the presence of a size - dependency suggests that the enhanced bbb permeability caused by focused ultrasoundmediated bbb disruption is primarily due to enhanced paracellular transport rather than increased vesicular transport as vesicles are much larger than the majority of therapeutic agents . [SEP]
[CLS] it should also be noted that the difference in diameter between cy5 - dna - au nps B-nanoparticle a and c is small ( less than 5 nm ) , suggesting that np B-nanoparticle diameter is an important design criterion to take into account if one wishes to deliver nps B-nanoparticle into the brain using focused ultrasound : whilst larger nps B-nanoparticle offer a greater surface area for the attachment of therapeutic and diagnostic B-property agents I-property , our results show that they are delivered less efficiently than smaller nps B-nanoparticle using the same ultrasound parameters . [SEP]
[CLS] to assess the safety of the focused ultrasound - mediated bbb disruption conducted in this study , brain sections covering the entirety of the focal volume were stained with haematoxylin and eosin ( h & e ) . [SEP]
[CLS] for these experiments , a texas red - labelled 3 kda dextran B-material was injected as a marker of bbb opening and the brain was sonicated using the same ultrasound parameters as above . [SEP]
[CLS] following sonication , mice were immediately sacrificed and brains were extracted for histological analysis . [SEP]
[CLS] in agreement with prior studies , minimal tissue effects were observed , with just a few sites of minor red blood cell B-material extravasation identified in the targeted region ( figure s4 ) . [SEP]
[CLS] in summary , we have shown that dna - au nps B-nanoparticle can be successfully delivered across the bbb using focused ultrasound in a non - invasive and targeted manner . [SEP]
[CLS] moreover , a sizedependency was observed , with smaller nps B-nanoparticle being delivered more efficiently across the bbb using this technology than larger nps B-nanoparticle . [SEP]
[CLS] 1 . schematic representation showing the use of microbubbles and focused ultrasound to deliver dna - coated gold B-nanoparticle nanoparticles I-nanoparticle across the bbb . [SEP]
[CLS] figure 2 . a ) three sets of au nps B-nanoparticle of core B-material diameters 6 . 3 ± 0 . 7 nm , 9 . 5 ± 1 . 4 nm and 14 . 2 ± 1 . 2 nm were synthesised via a seeded growth approach . [SEP]
[CLS] synthesised au nps B-nanoparticle were functionalised with cy5 - labelled oligonucleotides to give cy5 - dna - au nps B-nanoparticle a - c . b ) representative transmission B-technique electron I-technique microscopy I-technique images of au nps B-nanoparticle a - c . [SEP]
[CLS] nanoparticles B-nanoparticle were observed to be quasi - spherical and narrowly dispersed . [SEP]
[CLS] c ) size distribution of au nps B-nanoparticle determined from transmission B-technique electron I-technique microscopy I-technique images . [SEP]
[CLS] a total of 100 nps B-nanoparticle per set were counted using imagej . [SEP]
[CLS] cy5 - dna - au nps B-nanoparticle a - c were successfully delivered to the left thalamus of the brain using focused ultrasound . [SEP]
[CLS] fluorescence B-property signal corresponding to the attached cy5 - labelled oligonucleotides ( red ; cy5 channel ) was found at the exact same locations as signal from the au np B-nanoparticle cores B-material ( blue ; brightfield ) , indicating that the cy5 - dna - au nps B-nanoparticle remain intact as they cross the bbb via focused ultrasound - mediated bbb disruption . [SEP]
[CLS] experimental setup for focused ultrasound - mediated bbb disruption . [SEP]
[CLS] microbubbles are injected intravenously , followed by cy5 - dna - au nps B-nanoparticle a , b or c . [SEP]
[CLS] the left thalamus is sonicated using ultrasound pulses ( frequency : 1 mhz ; peak - negative pressure : 530 kpa ; pulse length : 10 ms ; pulse repetition frequency : 0 . 5 hz ; sonication duration : 250 s ) , whilst the right thalamus acts as the control . [SEP]
[CLS] figure 5 . a ) representative fluorescence B-property images from the delivery of cy5 - dna - au nps B-nanoparticle a - c across the bbb using focused ultrasound . [SEP]
[CLS] a spot - like distribution of fluorescence B-property is observed in the left thalamus , whilst no fluorescence B-property signal is detected in the right thalamus . [SEP]
[CLS] b ) normalised optical density was calculated from fluorescence B-property images as a measure of the nanoparticle B-nanoparticle dose delivered across the bbb ( n = 5 for each set of nps B-nanoparticle ) . [SEP]
[CLS] a size dependency is observed , with the smallest nps B-nanoparticle synthesised ( a ) being delivered six times more efficiently across the bbb using focused ultrasound than the largest nps B-nanoparticle ( c ) ( p < 0 . 05 ) . [SEP]
[CLS] measured [SEP]
[CLS] metal - based anion B-material receptors have several important applications in sensing , separation and transport of negatively charged species . [SEP]
[CLS] amongst these receptors , di - zinc ( ii ) complexes are of particular interest for the recognition of oxoanions , in particular phosphate derivatives . [SEP]
[CLS] herein we report the synthesis of a di - zinc ( ii ) receptor B-material and show that it has high affinity and selectivity for bisphosphonates such as alendronate and etidronatewhich are used to treat a number of skeletal disorders as well as showing interesting anticancer B-property properties . [SEP]
[CLS] the binding mode of the di - zinc ( ii ) receptor B-material with alendronate and etidronate has been unambiguously established by single crystal x - ray crystallography . [SEP]
[CLS] in addition , by modifying the backbone of the receptor B-material , we show that the drug - loaded receptor B-material can be attached onto gold B-nanoparticle nanoparticles I-nanoparticle as potential drug - delivery vehicles B-material . [SEP]
[CLS] over the past two decades , there has been great interest in the development of molecular receptors for oxoanions such as carboxylates B-material , phosphates and nitrates , amongst several others . [SEP]
[CLS] such receptors have been designed for a wide range of applications such as sensing , 2 removal of pollutants from solutions 3 and to transport anionic guests across biological membranes . [SEP]
[CLS] one large family of such receptors is based on metal B-material complexes with the ability to coordinate reversibly to the target anionic guest . [SEP]
[CLS] depending on the number and nature of the metal B-material ion B-material as well as its coordination environment , it is possible to tune the selectivity of metallo - receptors for a given oxoanion . [SEP]
[CLS] for example , di - zinc ( ii ) complexes have been shown to display high affinity for phosphate derivatives and , by tuning the coordination environment , display good selectivity for a given phosphate over other anions B-material . [SEP]
[CLS] one class of phosphate derivatives that attracted our attention are bisphosphonates such as etidronate and alendronate ( see scheme 1 ) . [SEP]
[CLS] these oxoanions act as inhibitors of osteoclastic bone resorption and are therefore used in the treatment of a variety of skeletal disorders such as paget´s disease and osteoporosis . [SEP]
[CLS] recent studies have also revealed that this type of bisphosphonates can induce apoptosis B-event in tumour cells B-material , are synergistic with antineoplastic drugs and can act as anti - angiogenic agents . [SEP]
[CLS] however , the use of bisphosphonates in the clinic has been limited since less than 1 % of an orally administered dose is taken up by the cell B-material . [SEP]
[CLS] this has been attributed to their negative electrostatic charge , which hinders their transport through the lipophilic B-property cell B-material membrane . [SEP]
[CLS] furthermore , bisphosphonates exhibit a short plasma half - life and are rapidly eliminated in urine . [SEP]
[CLS] to address these problems , liposomes B-nanoparticle , nanopeptides B-material , calcium B-material phosphate - dna nanoparticles B-nanoparticle and nanopolymers B-material have been explored as drug carriers . [SEP]
[CLS] with this in mind , the aim of the research herein presented was to study whether di - zinc ( ii ) complexes could act as good receptors for bisphosphonates and whether it would be possible to attach such host - guest systems to gold B-nanoparticle nanoparticles I-nanoparticle ( aunp B-nanoparticle ) as nanocarriers for future biological applications . [SEP]
[CLS] thus , we report the synthesis of the di - zinc ( ii ) molecular receptor B-material 2 ( see scheme 1 ) and its interactions with etidronate and alendronate . [SEP]
[CLS] the binding process has been studied by spectroscopic techniques ( i . e . uv - vis and 31 p nmr spectroscopy B-technique ) and the exact binding mode of both bisphosphonates to the receptor B-material has been established by x - ray crystallography . [SEP]
[CLS] furthermore , the drug - loaded metallo - receptor B-material was further functionalised with dithiocarbamate and successfully attached onto aunps B-nanoparticle ( see scheme 2 ) . [SEP]
[CLS] synthesis and spectroscopic characterization of di - zinc ( ii ) complex 2 . [SEP]
[CLS] the bis ( zinc ( ii ) dipicolylamine ) motif has been used extensively in receptors for several oxoanions such as phosphates and carboxylates B-material . [SEP]
[CLS] there is also one previous example where this type of complex has been incorporated into a receptor B-material for bisphosphonates . [SEP]
[CLS] therefore , we synthesised the di - zinc ( ii ) receptor B-material 2 ( see scheme 1 ) and explored its ability to selectively recognise alendronate and etidronate over several other anions B-material . [SEP]
[CLS] in addition to the di - zinc B-material recognition moiety , the complex also contains a benzylamine substituent since it can be readily converted into a dithiocarbamate which in turn would render the complex suitable for attachment to gold B-nanoparticle nanoparticles I-nanoparticle ( see below ) . [SEP]
[CLS] ligand 1 was obtained following a synthetic procedure previously reported for similar bisdipicolylamine compounds ( see scheme s1 ) . [SEP]
[CLS] metallo - receptor B-material 2 was synthesized by reacting 1 with two equivalents of zn ( oac ) 2 and one equivalent of nabf4 ( see scheme 1 ) . [SEP]
[CLS] 1 : synthetic route for the preparation di - zinc ( ii ) receptor B-material 2 and its complexes with bisphosphonate - based drugs ( i . e . etidronate and alendronate ) . [SEP]
[CLS] complex 3 is electrostatically neutral due to the protonation of the benzyl amine B-material group I-material while complex 4 is + 1 due to the protonation of both the benzylamine and the amine B-material of alendronate . [SEP]
[CLS] the 1 h and 13 c nmr spectrum of complex 2 ( as compared to 1 ) shows that the pyridine c / h are in two different chemical environments due to the coordination to the zinc ( ii ) centres ( see indicator B-property displacement assays ( ida ) and uv / vis titrations . [SEP]
[CLS] to study whether 2 could act as a molecular receptor B-material for bisphosphonate - based drugs , its interaction to etidronate and alendronate was investigated via ida . [SEP]
[CLS] the interaction of 2 with several other anions B-material which are physiologically relevant ( i . e . hp2o7 3 - , hpo4 2 - , so4 2 - , so3 2 - , co3 2 - , no3 - , oh - and cl - ) was also studied to establish its selectivity profile . [SEP]
[CLS] this was performed using pyrocatechol violet ( pv ) as an indicator B-property , which is yellow when free in solution but blue when coordinated to zinc ( ii ) . [SEP]
[CLS] compound 2 ( 50 µm ) was mixed with one molar equivalent of pv and the resulting 2 • pv complex was individually incubated B-technique with one molar equivalent of each anion B-material . [SEP]
[CLS] upon mixing with etidronate and alendronate , an instantaneous colour change was observed , indicating the displacement of the dye by the bisphosphonate drugs ( figure 1a ) . [SEP]
[CLS] in contrast , mixing 2 • pv with the other anions B-material under study did not generate a significant change in the colour of the original solution ( figure 1a ) . [SEP]
[CLS] the same experiment was repeated using 10 equivalents of each anion B-material ; in this case , besides etidronate and alendronate , pyrophosphate also induced a colour change ( consistent with previous reports showing that at high concentrations pyrophosphate binds to this type of di - zinc ( ii ) receptorssee figure s30 ) . [SEP]
[CLS] following this initial screening , the interaction between 2 • pv and the anions B-material was further analysed by uv - vis spectroscopy B-technique , which confirmed the ability of receptor B-material 2 to bind etidronate and alendronate with high selectivity over all other anions B-material investigatedexcept pyrophosphate which also shows some binding at high concentrations ( figure 1b ) . [SEP]
[CLS] quantitative determination of the binding affinity between receptor B-material 2 and the bisphosphonate drugs was assessed by uv - vis and 31 p nmr titrations ( figure 2 ) . [SEP]
[CLS] the uv - vis titration was performed by adding increasing amounts of either etidronate or alendronate to a solution of 2 • pv ( 50 µm ) . [SEP]
[CLS] the intensity of the uv - vis bands centred at 455 nm increases while the band centred around 650 nm decreases upon addition of the bisphosphonate anion B-material ( figure 2b ) . [SEP]
[CLS] from this data , the association constants for 2 with etidronate ( ka = 2 . 42±0 . 15 x 10 5 m - 1 ) and alendronate ( 2 . 25±0 . 16 x 10 5 m - 1 ) were calculated . [SEP]
[CLS] the interaction between 2 and the two drugs was also studied by p nmr spectroscopy B-technique in d2o at room temperature ( figure 2d ) . [SEP]
[CLS] upon addition of alendronate to a solution of 2 , a resonance at 21 . 9 ppm ( assigned to coordinated alendronate ) appeared and increased with subsequent additions of the anion B-material until ca . results were obtained for etidronate , figure s31 ) . [SEP]
[CLS] the uv - vis and 31 p nmr spectroscopic studies indicate that 2 binds to etidronate and alendronate in a 1 : 1 stoichiometry . [SEP]
[CLS] to confirm this , the method of continuous variation ( i . e . job ' s plots ) was applied ; as can be seen in figure 2c and s30c , these studies clearly show the presence of a 1 : 1 binding stoichiometry . [SEP]
[CLS] spectra of both these complexes in meod showed two doublets for the phosphorus B-material of the etidronate and alendronate in the range of 23 . 4 - 24 . 8and 21 . 9 - 22 . 9 ppm , respectively . [SEP]
[CLS] it is interesting to note that there are differences in the 31 p nmr spectra of 3 and 4 when recorded in d2o ( see figure 2d ) and meod ( see figures s10 and s12 ) . [SEP]
[CLS] in the former a single resonance is observed , while in meod two distinct doublets are observed , which is consistent with the two different phosphorous environments ( coupling to each other ) . [SEP]
[CLS] the formulation and purity of these host - guest complexes were confirmed by mass spectrometry and elemental analyses ( see experimental details ) . [SEP]
[CLS] single - crystal x - ray diffraction analysis . [SEP]
[CLS] the molecular structures of compounds 3 and 4 [SEP]
[CLS] were determined by single - crystal x - ray crystallography ( figures 3 and 4 functionalization of gold B-nanoparticle nanoparticles I-nanoparticle with receptors . [SEP]
[CLS] the next aim of this work was to explore the attachment of receptor B-material 2 ( loaded with bisphosphonates ) onto gold B-nanoparticle nanoparticles I-nanoparticle ( aunp B-nanoparticle ) to yield a potential drug delivery system . [SEP]
[CLS] as has been previously reported , dithiocarbamates are efficient groups for the attachment of small molecules onto gold B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] thus , we functionalized the backbone of receptor B-material 2 with a dithiocarbamate to yield 5 as shown in scheme 2 . [SEP]
[CLS] 2 : synthetic route for the preparation of dithiocarbamate - modified di - zinc ( ii ) receptor B-material and the corresponding complexes with etidronate and alendronate . [SEP]
[CLS] subsequently , the modified receptor B-material 5 was loaded with either etidronate or alendronate to yield compounds 6 and 7 respectively . [SEP]
[CLS] due to the limited solubility B-property of 6 and 7 , it was not possible to obtain 1 h and c nmr spectra of good enough quality to assign all the resonances . [SEP]
[CLS] however , the 31 p nmr spectra in meod of these complexes showed the expected doublets for coordinated etidronate ( δ = 22 . 1 - 24 . 9 ppm ) and alendronate ( δ = 22 . 1 - 22 . 9 ppm ) . [SEP]
[CLS] in addition , esi ( + ) - ms showed the molecular ion B-material peaks for both the host - guest complexes : [ m + h ] + and [ m - bf4 ] + at 1079 . 70 and 1120 . 12 a . m . u . for 6 and 7 respectively . [SEP]
[CLS] these complexes were then attached to aunps B-nanoparticle which were also functionalised with thiolated oligonucleotides labelled with fluorescein ( fam - dna - sh ) to give the system greater biocompatibility B-property as well as a fluorescent B-property tag to help determining the dna loading on the aunp B-nanoparticle ( see below ) . [SEP]
[CLS] it is well established that passivation of aunps B-nanoparticle with a dense layer of oligonucleotides ( often referred to as ' spherical nucleic B-material acids I-material ' ) provides nanoconjugates with good solubility B-property profile , low cytotoxicity B-property and cell B-material permeability . [SEP]
[CLS] therefore , the following systems were prepared and studied ( see figure 5 ) : aunps B-nanoparticle without receptor B-material 2 or drug ( aunp B-nanoparticle - dnaas a control ) , with receptor B-material 2 but no drug ( aunp B-nanoparticle - dna - 5 ) , and with drug - loaded receptor B-material complexes 6 or 7 ( aunp B-nanoparticle - dna - 6 and aunp B-nanoparticle - dna - 7 ) . [SEP]
[CLS] the aunps B-nanoparticle were synthesized following previously reported procedures . [SEP]
[CLS] briefly , they were grown through the reduction of haucl4 with sodium B-material citrate and tannic acid to yield quasispherical , narrowly - dispersed aunps B-nanoparticle with an average core B-material diameter of 10 . 59 ± 1 . 8 nm . [SEP]
[CLS] the characterization of the aunps B-nanoparticle was carried out through uv - vis spectroscopy B-technique , dynamic B-technique light I-technique scattering I-technique ( dls ) , and transmission B-technique electron I-technique microscopy I-technique ( tem ) ( figure s32 and table s1 ) . [SEP]
[CLS] the changes observed in the hydrodynamic diameter ( dh ) and zeta B-property potential I-property ( ζ ) of the nps B-nanoparticle after functionalization ( see table s3 ) suggest that all three methods yield aunps B-nanoparticle passivated with both the oligonucleotides and receptor - drug conjugate 6 . [SEP]
[CLS] however , a higher oligonucleotide loadingwhich , in turn , increases the stability and biocompatibility B-property of the systemswas observed by adding the fam - dna - sh and receptor - drug complex together i . e . second method described above . [SEP]
[CLS] the oligonucleotide loading was estimated by treating the corresponding dna - passivated aunps B-nanoparticle with a large excess of mercaptoethanol following reported protocols . [SEP]
[CLS] incubation B-technique with mercaptoethanol displaces the fluorescently B-property - labelled oligonucleotides from the aunp B-nanoparticle surface , allowing the number of oligonucleotides attached per aunp B-nanoparticle to be calculated . [SEP]
[CLS] having established the best synthetic method , samples of aunp B-nanoparticle - dna - 5 and aunp B-nanoparticle - dna - 7 [SEP]
[CLS] ( in addition to aunp B-nanoparticle - dna - 6 as described above ) were prepared and fully characterised by s3 in the esi ) . [SEP]
[CLS] the largest difference is observed for aunp B-nanoparticle - dna - 7 ( with ζ = - 25 . 4 ± 1 . 6 cf . - 81 . 2 ± 0 . 9 for aunp B-nanoparticle - dna ) which can be attributed to alendronate ' s positively charged alkyl ammonium group . [SEP]
[CLS] additionally , there were changes in the uv - vis absorbance of the nanoparticles B-nanoparticle before and after functionalisation , which reflects the changes of optical properties of aunps B-nanoparticle caused by the coating B-material process ( figure 6a ) . [SEP]
[CLS] the tem images showed that the spherical morphology of the aunps B-nanoparticle is preserved after the formation of the aunp B-nanoparticle - dna - 6 and aunp B-nanoparticle - dna - 7 ( figure 6c and 6d ) . subsequently , the number of fam - dna molecules attached to the aunps B-nanoparticle was determined by fluorescence B-technique spectroscopy I-technique ( as described above using mercaptoethanol ) while the number of attached host - guest complexes was indirectly determined by quantifying the amount of zn via icp - ms ( see table 1 ) . [SEP]
[CLS] these measurements indicate that there are 69 and 79 molecules of etidronate and alendronate per aunp B-nanoparticle in aunp B-nanoparticle - dna - 6 and aunp B-nanoparticle - dna - 7 respectively . [SEP]
[CLS] interestingly , in the aunp B-nanoparticle - dna - 5 , aunp B-nanoparticle - dna - 6 and aunp B-nanoparticle - dna - 7 samples , a higher oligonucleotide loading was observed in comparison with aunp B-nanoparticle - dna sample . [SEP]
[CLS] this effect could be attributed to the stabilization of the negative charge of oligonucleotides strands by the presence of the complexes . [SEP]
[CLS] in summary , we have shown that di - zinc ( ii ) complex 2 binds bisphosphonates with high affinity and good selectivity over many other physiologically - relevant anions B-material . [SEP]
[CLS] the x - ray crystal structures of this receptor B-material bound to alendronate and etidronate show for the first time the exact binding mode of these oxoanions to a di - metallic receptor B-material . [SEP]
[CLS] we also show that by modifying the backbone of the receptor B-material , it is possible to load the drug - loaded receptor B-material onto aunps B-nanoparticle that have also been passivated with oligonucleotides to make them water B-property - soluble B-property and biocompatible B-property . [SEP]
[CLS] these new nanoconjugates are likely to be good vehicles B-material for the delivery of these drugs across the cell B-material membrane . [SEP]
[CLS] future work will explore this possibility . [SEP]
[CLS] general information . [SEP]
[CLS] 1 h nmr , c nmr and 31 p nmr spectra were recorded on either a bruker avance 400 mhz ultrashield nmr spectrometer or a bruker avance 500 mhz nmr spectrometer . [SEP]
[CLS] electrospray ionisation B-property mass spectra were obtained on a bruker daltonics esquire 3000 spectrometer . [SEP]
[CLS] the x - ray crystal structures where measured using an od xcalibur px ultra diffractometer ( 1 . 54184 a ) . [SEP]
[CLS] uv - vis spectra were collected with a perkin elmer uv - vis lambda 25 spectrophotometer . [SEP]
[CLS] fluorescence B-property measurements were made on a cary eclipse fluorescence B-property spectrophotometer . [SEP]
[CLS] hydrodynamic diameters and zeta B-property potentials I-property were measured on a malvern zetasizer nano zs instrument . [SEP]
[CLS] nanoparticles B-nanoparticle images were taken using a jeol jem - 2100 plus transmission electron microscope . [SEP]
[CLS] all reagents were purchased from commercial suppliers and used without further purification . [SEP]
[CLS] etidronate and alendronate were purchased as etidronate disodium hydrate and alendronate sodium B-material trihydrate , respectively . [SEP]
[CLS] the organic precursor d was prepared following a previously reported synthetic procedure ( scheme s1 ) . [SEP]
[CLS] for numbering of the 1 h nmr assignments shown below , please see the corresponding spectra in the esi . [SEP]
[CLS] to a solution of d ( 0 . 815 g , 1 . 45 mmol ) in methanol ( 20 ml ) benzaldehyde ( 0 . 177 ml , 1 . 74 mmol ) was added dropwise . [SEP]
[CLS] the reaction mixture was stirred for 2 h at room temperature . [SEP]
[CLS] nabh4 ( 110g , 2 . 90 mmol ) was then added to the reaction mixture and after stirring it for 4 h , the solvent was removed under vacuum and the excess nabh4 neutralised with saturated nh4cl aqueous solution ( 60 ml ) . [SEP]
[CLS] the product was extracted with dichloromethane and dried over na2so4 powder . [SEP]
[CLS] after filtration , the solvent was reduced under vacuum to yield a brown oil which was purified by silica column chromatography B-technique with chcl3 / meoh / et3n ( 94 : 5 : 1 ) as eluent to afford ligand 1 as a yellow oil . [SEP]
[CLS] yield : 87 % ( 0 . 819 g , 1 . 26 mmol ) . [SEP]
[CLS] s s1 - s4 for 1 h and c nmr spectra ; s5 - s8 for 2d nmr spectra ) . [SEP]
[CLS] in the c nmr [SEP]
[CLS] 1 : colorimetric response of 2 • pv to different anions B-material in 10 mm hepes solution at ph 7 . 4 at room temperature . [SEP]
[CLS] ( a ) with 1 eq of anions B-material . [SEP]
[CLS] ( b ) uv - vis spectra of 50 µm 2 • pv in 10 mm hepes solution at ph = 7 . 4 and 1 eq of anions B-material ( c ) uv - vis spectra of 50 µm 2 • pv in 10 mm hepes solution at ph = 7 . 4 and 1 eq . of etidronate , alendronate and 10 equivalents of common inorganic B-material anions I-material . [SEP]
[CLS] 2 : ( a ) indicator B-property displacement assay between 2 • pv and alendronate ; ( b ) uv spectra of 50 µm 2 • pv titrated with increasing amounts of 1 . 25 mm alendronate in 10 mm hepes solution , ph = 7 . 4 . [SEP]
[CLS] ( c ) job´s plot of 50 µm 2 • pv and alendronate in 10 mm hepes solution at ph = 7 . 4 . [SEP]
[CLS] ( d ) 31 p nmr ( 400 mhz , d2o ) spectra showed the titration of 2 with alendronate . [SEP]
[CLS] 1 association between the receptor B-material and bisphosphonates . [SEP]
[CLS] suitable crystals of compounds 3 and 4 were grown from acetone and methanol / toluene solutions respectively . [SEP]
[CLS] in both of these structures , the four negative charges of the bisphosphonate groups are delocalized between the four oxygen B-material atoms I-material coordinated to the zinc ( ii ) centres and the two remaining uncoordinated oxygen B-material atoms I-material . [SEP]
[CLS] this effect is evidenced in the relatively narrow distribution of the p - o distances , from 1 . 510 ( 2 ) a to 1 . 546 ( 3 ) a and 1 . 518 ( 3 ) a to 1 . 543 ( 3 ) a for 3 and 4 , respectively . [SEP]
[CLS] these values are intermediate B-property to the standard bond lengths reported for p - o ( 1 . 50 a ) and p = o ( 1 . 63 a ) . [SEP]
[CLS] the average values for the n - zn , odrug - zn and oligand - zn bond distances are 2 . 210 a , 2 . 063 a , 2 . 100 a for 3 , and 2 . 212 a , 2 . 059 a , 2 . 096 a for 4 . [SEP]
[CLS] in both structures the coordination geometry around the zinc ( ii ) centres corresponds to a distorted octahedron . [SEP]
[CLS] in 4 , the amine B-material group I-material of the coordinated alendronate is protonated giving an overall positive charge to the complex . [SEP]
[CLS] 3 . the crystal structure of 3 ( 50 % probability ellipsoids ) showing the interaction between the di - zinc ( ii ) receptor B-material and etidronate . [SEP]
[CLS] 4 . the structure of the cation B-material in the structure of 4 ( 50 % probability ellipsoids ) showing the interaction between the di - zinc ( ii ) receptor B-material and alendronate . [SEP]
[CLS] 5 : schematic representation of nanoconjugates aunp B-nanoparticle - dna - 6 and aunp B-nanoparticle - dna - 7 . [SEP]
[CLS] three different methods were studied to passivate the aunps B-nanoparticle with thiolated fluoresceinlabelled oligonucleotides and the receptor - drug 6 : ( i ) by first preparing aunp B-nanoparticle - dna and then adding 6 to the nanoparticles B-nanoparticle ; ( ii ) by adding fam - dna - sh and 6 at the same time to the unfunctionalized aunps B-nanoparticle ; ( iii ) by first adding 6 to the aunps B-nanoparticle and then the fam - dna - sh . [SEP]
[CLS] vis and fluorescence B-technique spectroscopy I-technique , dls , tem and inductively coupled plasma mass spectrometry ( icp - ms ) [SEP]
[CLS] the coating B-material of aunps B-nanoparticle with fam - dna - sh to yield aunp B-nanoparticle - dna induces a change dh from 21 . 4 ± 0 . 2 nm to 45 . 7 ± 0 . 4 nm , and a change ζ from - 34 . 8 ± 1 . 2 mv to - 81 . 2 ± 0 . 9 mv . [SEP]
[CLS] addition of the complexes ( 6 and 7 ) makes ζ less negative which is consistent with less dna being present on the surface of the aunps B-nanoparticle ( see table [SEP]
[CLS] 6 : ( a ) uv - vis spectra of aunps B-nanoparticle , aunp B-nanoparticle - dna , aunp B-nanoparticle - dna - 5 , aunp B-nanoparticle - dna - 6 and aunp B-nanoparticle - dna - 7 ; ( b ) size distribution of unfunctionalised aunps B-nanoparticle ; and tem of images of ( c ) aunp B-nanoparticle - dna - 6 and ( d ) aunp B-nanoparticle - dna - 7 . [SEP]
[CLS] in a 50 ml three - necked round - bottom flask under vigorous stirring [SEP]
[CLS] when the temperature reached 70 °c , haucl4 ( 0 . 41 ml , 10 mm ) was injected , resulting in a color change from colorless to pink . [SEP]
[CLS] stirring at 70 °c for a further 10 minutes generated a solution of seed of the solution and replacing with sodium B-material citrate ( 9 . 1 ml , 2 . 2 mm ) . [SEP]
[CLS] when the temperature reached 70 °c again , two injections of haucl4 ( 0 . 2 ml , 10 mm ) on a time interval of 10 minutes were done . [SEP]
[CLS] this growing process ( sample dilution followed by the injection of haucl4 ) was followed by uv - vis spectroscopy B-technique and was repeated until the nanoparticles B-nanoparticle reached the desired size . [SEP]
[CLS] functionalization of gold B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] au nps B-nanoparticle were functionalized with fam - dna - sh and the corresponding complexes ( i . e . 5 , 6 or 7 ) based on general methodologies previously described in the literature . fam - tt - ggg - tta - ggg - tta - ggg - tta - ggg - tt - ( ch2 ) 6 - sh ( fam - dna - sh ) oligonucleotide was selected with a thiol B-material modifier on the 3 ' end and a fluorescein ( as an optical label ) amidite group on the 5 ' end . [SEP]
[CLS] before use , tris ( 2 - carboxyethyl ) phosphine ) ( tcep ) was added to fam - dna - sh in a 100x excess and the mixture was shaken for 1 hr at room temperature . [SEP]
[CLS] final concentrations of tcep and fam - dna - sh were 5 mm and 50 μm respectively . [SEP]
[CLS] 1 wt % tween 20 ( 68 . 7 μl ) was added to the aunps B-nanoparticle ( 12 . 1 nm , 6 . 87 ml ) and the mixture was stirred for 1 min . [SEP]
[CLS] fam - dna - sh ( 50 μm ) and the corresponding bimetallic zinc B-material complex ( 500 μm ) was added at a volume that allowed for 90 and 350 oligonucleotide strands and complex per aunp B-nanoparticle . [SEP]
[CLS] nacl ( 5 m ) was then added until its final concentration in the mixture reached 800 mm . the solution was incubated B-technique for 1 h at 25 °c with gentle agitation and then centrifuged at 30 , 000 rpm for 15 min at 15 °c . [SEP]
[CLS] the supernatant was removed and the nps B-nanoparticle were redispersed in milli - q water B-material . [SEP]
[CLS] this centrifugation process was repeated at least 3 times to ensure thorough washing of the aunps B-nanoparticle . [SEP]
[CLS] wr2 ( all ) = 0 . 1796 , 8585 independent observed absorption - corrected reflections [ | fo | > 4σ ( | fo | ) , completeness to θfull ( 67 . 7° ) = 98 . 4 % ] , 757 parameters . [SEP]
[CLS] ccdc 1984671 . [SEP]
[CLS] see esi for further discussion about this x - ray crystal structure . [SEP]
[CLS] samples [SEP]
[CLS] all samples were freshly prepared in 10 mm hepes buffer ( ph 7 . 1 ) prior to measurements . [SEP]
[CLS] all measurements were repeated 3 times . [SEP]
[CLS] for optical tests , 1 ml of 2 • pv ( 50 μm ) was mixed with 5 μl ( 1 eq . ) or 50 μl ( 10 eq . ) of the corresponding anion B-material ( 10 mm ) in 10 mm hepes solution at ph 7 . 4 at room temperature . [SEP]
[CLS] the mixture was stirred gently and then left to stand for 10 min . [SEP]
[CLS] to obtain the uv - vis spectra , 700 µl 2 • pv were added to a uv - vis cuvette and mixed with one or ten molar equivalent of each anion B-material under study in a buffer solution ( 10 mm hepes at ph = 7 . 4 ) at room temperature . [SEP]
[CLS] aunps B-nanoparticle of different sizes were synthesized following protocols reported from puntes et al . [SEP]
[CLS] sodium B-material citrate ( 25 ml , 2 . 2 mm ) , tannic acid ( 17 µl , 2 . 5 mm ) and potassium B-material carbonate B-material ( 170 µl , 150 mm ) was heated [SEP]
[CLS] the field of structural dna nanotechnology applies the programmability of watson - crick base pairing to the construction of custom nanostructures that are prescribed by the sequence information encoded in dna molecules . [SEP]
[CLS] precisely defined geometries , highly programmable molecular interactions , and outstanding biocompatibility B-property make dna nanostructures a new category of nanocarriers for drug delivery . [SEP]
[CLS] over the past decade , the potential of using dna nanocarrier - based formulation for cancer therapy has been extensively explored with the successful implementation of various therapeutic strategies , both in vitro and in vivo . [SEP]
[CLS] moreover , dna nanocarriers can be encoded with complex instructions via sequence design , enabling therapeutic functions to be executed in a programmed , automatic manner . [SEP]
[CLS] in this review , we summarize recent advances and discuss the challenges and opportunities in designer dna nanostructure - enabled therapeutics . [SEP]
[CLS] nanotechnology is an interdisciplinary research field focusing on the manipulation and organization of matter at the nanometer scale with the aim of achieving novel functions . [SEP]
[CLS] in particular , nanomedicine , a branch of nanotechnology , takes advantage of the unique properties of nanomaterials B-material for diagnostics and therapeutics . [SEP]
[CLS] it has been proven that including nanocarriers in therapeutic formulations can improve effectiveness and minimize adverse effects of conventional drugs . [SEP]
[CLS] by 2016 , over 50 nanomedicines had been approved by the u . s . food and drug administration ( fda ) , with many more undergoing clinical trials . [SEP]
[CLS] a majority of fda - approved nanomedicines are based on micellar , liposomal B-nanoparticle , polymeric , and inorganic B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] they are more or less limited by the heterogeneity in size and shape , chemical or physical instability , and potential cytotoxicity B-property . [SEP]
[CLS] research on molecular self - assembly has suggested the prospects of using a programmable biopolymeric B-material material B-material , dna , to enrich the choices of nanocarriers . [SEP]
[CLS] although at an early stage of development , the use of dna nanocarriers to facilitate drug delivery has demonstrated profound potential ( figure 1 ) . [SEP]
[CLS] dna is the most widely used molecular building block for nanofabrication via self - assembly . [SEP]
[CLS] besides its biocompatible B-property nature , dna possesses several uniquely advantageous characteristics , such as a well - defined geometry , predictable and programmable complementarity , abundant choices of sequences , thermostability , affordable synthesis chemistry , and readily accessible chemical and enzymatic tools for modification . [SEP]
[CLS] the history of using dna as a nanoscale building material B-material can be traced back to 1982 when ned seeman proposed the concept that ' ' migrationally immobile junctions ' ' could be assembled from rationally designed sequences and further joined together to form two - dimensional ( 2d ) or three - dimensional ( 3d ) networks through complementary single - stranded overhangs called sticky ends ( figure 2a ) . [SEP]
[CLS] the early work in structural [SEP]
[CLS] the bigger picture [SEP]
[CLS] the development of nanomedicine is closely related to the evolution of nanocarriers that endow therapeutic agents with improved efficacy , safety , and targeting specificity . [SEP]
[CLS] a grand challenge in nanomedicine is the ability to design suitable nanocarriers that can precisely transport therapeutic agents across biological barriers B-property in the complex environment of living organisms . [SEP]
[CLS] the programmable and biocompatible B-property nature of dna has enabled the rational design of tailored dna nanocarriers for targeted drug delivery , which has greatly contributed to the remarkable progress in cancer therapy . [SEP]
[CLS] additionally , dna nanotechnology - enabled therapeutic systems have demonstrated profound potential in the development of smart therapeutic robotic systems and the treatment of renal diseases . [SEP]
[CLS] in this review , we discuss the roles of dna nanostructures in different therapeutic strategies and highlight model systems that hold promise for clinical translation in the long term . [SEP]
[CLS] random dna tilings with programmable disorder a structural foundation of dna nanotechnology therapeutic applications of dna nanotechnology b ned seeman proposed the concept that " migrationally immobile junctions " could be assembled from dna oligonucleotides with rationally designed sequences and further linked into networks assembly of an immobile holliday junction from four synthetic dna oligonucleotides folate - mediated cellular uptake of dna nanotubes B-nanoparticle delivery of dna tetrahedra into mammalian cells B-material without the aid of transfection reagents gigadalton 3d polyhedra 3d molecular canvas assembled from 10 , 000 unique ssts 51 sustained delivery of dox and cpg motifs in vivo by dna hydrogels dna nanotechnology focused on exploring design principles and assembly methods for building complex structures ( figure 1a ) . [SEP]
[CLS] after more than 30 years of development , the structural complexity of dna architectures has reached an unprecedented level . [SEP]
[CLS] research on the fundamental aspects of dna - based self - assembly has facilitated the rational design of custom dna nanostructures that can be tailored for specific purposes . [SEP]
[CLS] in the past decade , numerous attempts have been made by dna nanoscientists to use the dna - nanotechnology - based platform for therapeutic applications ( figure 1b ) . [SEP]
[CLS] dna nanostructures are primarily used as carriers to deliver therapeutic agents , including anticancer B-property drugs , antisense oligonucleotides ( asos ) , photosensitizers B-property , nanoparticles B-nanoparticle , and proteins B-material , to pathogenic sites . moreover , other functional modules can be feasibly integrated into dna - based nanocarriers , allowing for real - time tracking , precise targeting , conditional activation , and controlled release . [SEP]
[CLS] the versatility of dna nanostructures enables the implementation of different therapeutic strategies , including chemotherapy , gene therapy , phototherapy , and immunotherapy , both in vitro and in vivo . [SEP]
[CLS] additionally , the dna - nanotechnology - based therapeutic platform provides new solutions to multidrug resistance ( mdr ) , tumor B-material vascularization , and acute kidney injury . [SEP]
[CLS] the successful proof - of - concept applications in therapeutics have inspired ever - increasing efforts for ' ' bench - to - bedside ' ' translation . [SEP]
[CLS] in this review , we first introduce the fundamental design strategies and principles that have transformed the field of structural dna nanotechnology . [SEP]
[CLS] criteria for developing dna - based nanocarriers are then discussed in terms of stability , cellular uptake , targeting specificity , and economical manufacturing . [SEP]
[CLS] in addition , recent advances in the therapeutic applications of dna nanostructures are categorized and summarized based on their therapeutic strategies , including chemotherapy , gene therapy , phototherapy , immunotherapy , combination therapy , and therapeutic dna nanorobots . [SEP]
[CLS] as a case study , we discuss the potential use of dna nanostructures for the treatment of kidney diseases . [SEP]
[CLS] at last , we further discuss the challenges and potential future directions for the therapeutic applications of structural dna nanotechnology . [SEP]
[CLS] structural dna nanotechnology exploits the hybridization of complementary dna sequences to create structures via the self - assembly of dna building blocks . [SEP]
[CLS] a core B-material aim of structural dna nanotechnology is to use dna nanostructures for the organization of other molecules and materials with nanometer precision in order to engineer novel functions . [SEP]
[CLS] to this end , structural dna nanotechnology initially focused on the exploration of structural motifs that could be rationally designed , which laid the foundation for the rapid evolution of the field since 2000s . [SEP]
[CLS] in this section , we provide a brief overview regarding the fundamental design strategies and principles of structural dna nanotechnology . [SEP]
[CLS] comprehensive reviews can be found elsewhere in the literature . [SEP]
[CLS] design strategies seeman ' s original concept of ' ' migrationally immobile junctions ' ' was validated soon after it was proposed through the assembly of an immobile holliday junction from four synthetic dna oligonucleotides with rationally designed sequences and prescribed complementarity . [SEP]
[CLS] based on branched junctions , various covalently closed , discrete 3d objects , such as a cube and tetrahedra , were constructed ( figure 2b ) . [SEP]
[CLS] later on , modular dna tiles were proposed as nanoscale building blocks to construct extended , higher - order structures , including discrete nanocages B-nanoparticle , nanotubes B-nanoparticle , 27 2d arrays , and macroscopic crystals , via sticky end interactions ( figure 2c ) . [SEP]
[CLS] the groundbreaking technique of scaffolded dna origami was developed by folding a long single - stranded dna ( called a scaffold ) , typically derived from m13mp18 bacteriophage , into target shapes with the help of hundreds of auxiliary ' ' staple strands ' ' ( figure 2d ) . [SEP]
[CLS] as an extension of this concept , 3d origami structures were designed by packing dna helices into various 3d lattices . [SEP]
[CLS] via targeted insertions and deletions of base pairs , as well as curved - surface rendering , dna origami with complex curvatures can be reliably created . [SEP]
[CLS] in the dna origami design , each staple strand is uniquely addressable , which means it has a unique sequence and occupies a unique position that can be addressed as a ' ' pixel ' ' on the design , making dna origami a versatile platform to organize other molecules with nanometer precision by placing them on these ' ' pixels . [SEP]
[CLS] ' ' new strategies have further simplified the design of complex dna nanostructures . [SEP]
[CLS] single - stranded tile ( sst ) is the simplest form that a tile can take , as it is only composed of concatenated sticky ends . [SEP]
[CLS] complex structures , including 2d patterns , discrete 3d objects , and crystals , can be constructed by sculpting or packing the molecular canvas formed by a large number of unique ssts ( figure 2e ) . [SEP]
[CLS] methods for designing wireframe dna origami with either single - helix or reproduced with permission from seeman . copyright 1982 elsevier . [SEP]
[CLS] ( b ) discrete nanoobjects can be assembled from oligonucleotides with rationally designed sequences and complementarities . [SEP]
[CLS] a ~ 7 nm dna cube is the earliest polyhedral object constructed from dna . [SEP]
[CLS] ( c ) dna tile - based self - assembly is a feasible approach to create higher - order structures from modular building blocks . [SEP]
[CLS] for example , a 4 - by - 4 dna tile can be assembled from several oligonucleotides and further linked into 2d lattices through complementary sticky ends . [SEP]
[CLS] ( d ) dna origami is the folding of a long , single - stranded dna ( termed ' ' scaffold ' ' ) into 2d or 3d shapes with the help of hundreds of short dna oligonucleotides ( termed ' ' staples ' ' ) . [SEP]
[CLS] ( top right ) reproduced with permission from rothemund . [SEP]
[CLS] copyright 2006 springer nature . [SEP]
[CLS] ( bottom right ) reproduced with permission from castro et al . copyright 2011 springer nature . [SEP]
[CLS] ( e ) single - stranded tiles ( ssts ) can be programmed to assemble into 2d or 3d shapes without the guidance of a scaffold . [SEP]
[CLS] ssts interact with each other through complementary domains , in a way analogous to ' ' lego ' ' bricks . [SEP]
[CLS] ( top right ) reproduced with permission from wei et al . copyright 2012 springer nature . [SEP]
[CLS] ( bottom right ) reproduced with permission from ke et al . copyright 2012 aaas . [SEP]
[CLS] ( f ) single - stranded dna origami is the unimolecular folding of a long single - stranded dna into nanostructures through paranemic cohesions . [SEP]
[CLS] reproduced with permission from han et al . copyright 2017 aaas . [SEP]
[CLS] review double - helix edges offer new possibilities for developing dna frameworks . the design of single - stranded dna origami further enables a unimolecular folding of a single - stranded dna of over several kilobases into discrete structures ( figure 2f ) . [SEP]
[CLS] the size and complexity of dna assemblies are further advanced by a series of scaling - up strategies . [SEP]
[CLS] 2d random tilings , 3d polyhedra , dynamic devices , and gigadalton - scale objects can be built with high quality by assembling individual dna origami into extended structures . [SEP]
[CLS] through a ' ' fractal assembly ' ' strategy , micron - scale , fully addressable arrays that displayed user - defined patterns were made from the hierarchical , multistage assembly of up to 64 unique components . [SEP]
[CLS] in the sst system , the molecular canvas assembled from ssts was further expanded to up to 30 , 000 unique components . [SEP]
[CLS] feasible methods for fabricating micronsized dna assemblies from a large number of unique building units will enable the development of molecular devices with sizes comparable with cellular organelles and provide the platform for various applications including biomimetics and therapeutics . [SEP]
[CLS] in addition , dna - based nanomaterials B-material such as hydrogels and noncanonical nanoflowers have also been applied to biomedical applications and are discussed in this review . [SEP]
[CLS] spherical nucleic B-material acids I-material ( snas ) , including dna - functionalized inorganic B-nanoparticle nanoparticles I-nanoparticle , polymersomes , micelles B-material , and liposomes B-nanoparticle , are distinct from the abovementioned dna nanostructures , as they are the covalent coating B-material of a core B-material material B-material with a layer of highly oriented oligonucleotides . [SEP]
[CLS] these groups of dna - functionalized materials and their derivatives have been extensively researched and comprehensively reviewed elsewhere in the literature and are not covered in this review . [SEP]
[CLS] no matter how complicated the dna structure is , the physiochemical principle it follows is the maximization of base - pairing events ( i . e . , reducing the thermodynamic free energy of the whole self - assembly system ) . [SEP]
[CLS] second , complex , higher - order structures can be produced from modular , branched building blocks . [SEP]
[CLS] moreover , given the noncovalent , reversible nature of dna hybridization , choosing proper conditions could guide the self - assembly pathway to favor the rapid production of desired nanostructures with high yield . [SEP]
[CLS] finally , dynamic properties such as reconfiguration , unidirectional motion , and reversible assembly can be integrated into structures via an enzyme - free strand displacement mechanism . [SEP]
[CLS] overall , structural dna nanotechnology offers tremendous design space for nanofabrication , setting the groundwork for diverse applications . [SEP]
[CLS] an ideal drug - delivery system improves drug efficacy and safety by fine - tuning its retention and specificity in vivo . [SEP]
[CLS] as a crucial component , the carrier for drug delivery should fulfill the following criteria : ( 1 ) it should be chemically inert and biologically stable in physiological environments ; ( 2 ) be nontoxic , nonimmunogenic , and compatible with living organisms ; ( 3 ) must have a controllable retention time and a clear clearance pathway ; ( 4 ) should function specifically at the pathogenic site and have minimal effect on healthy tissues ; and ( 5 ) be scalable and cost efficient . [SEP]
[CLS] synthetic dna nanostructures do not inherently meet all the criteria , but they can be modified to better fulfill them . [SEP]
[CLS] moreover , the programmability of dna - based platform is not found in any other systems , making it one of the most promising candidates for therapeutics . [SEP]
[CLS] in this section , we discuss prior works on quantifying and improving characteristics of dna nanostructures with respect to the abovementioned criteria . [SEP]
[CLS] stability under physiological conditions dna nanostructures are usually prepared in buffers containing approximately 10 mm mg 2 + or 1 . 0 m na + , since cations B-material can reduce charge repulsion between closely packed dna helices , keeping structures from denaturation . [SEP]
[CLS] well - formed dna origami can withstand repeated freezing and thawing cycles in buffer containing sufficient mg 2 + and cryoprotectants , facilitating long - term cryopreservation . [SEP]
[CLS] however , most dna nanostructures are susceptible to low cation B-material concentration and nuclease digestion in physiological environments . [SEP]
[CLS] the tolerance of dna nanostructures to cation B-material depletion and nuclease digestion is design dependent . [SEP]
[CLS] in general , less compact structures can better withstand lower cation B-material concentrations , while more compact structures are more resistant to nuclease digestion . [SEP]
[CLS] for example , in contrast to a 24 - helix bundle with a denser packing of dna helices , a 6 - helix bundle can be assembled in buffers containing less cations B-material and remain intact under low - mg 2 + conditions . [SEP]
[CLS] wireframe origami can even be folded in mg 2 + - free buffers commonly used in biomedical research , such as phosphate - buffered saline ( pbs ) . [SEP]
[CLS] compared with duplex dna or double - crossover tiles , paranemic crossover ( px ) tiles with a higher number of crossovers showed enhanced nuclease resistance . [SEP]
[CLS] this effect can be attributed to local steric hindrance and global conformational strain and is in agreement with real - time visualization and computational modeling of dna origami digestion by nucleases . [SEP]
[CLS] taken together , these examples suggest the possibility of tuning the stability of dna nanostructures by rational design . [SEP]
[CLS] to maintain structural integrity under physiological conditions , it is necessary to protect dna nanostructures against cation B-material depletion and nuclease digestion with proper modifications ( figure 3a ) . [SEP]
[CLS] synthetic oligonucleotides with unnatural backbone , base , or left - handed chirality are suitable building units for assembling discrete or periodic nanostructures with improved serum stability . [SEP]
[CLS] however , their usage in stabilizing complex dna nanostructures is usually hindered by the high synthesis cost . [SEP]
[CLS] chemical or enzymatic ligation of nick points and photocross - linking of thymine bases 69 make individual components topologically interlocked for a better resistance against cation B-material depletion and nuclease digestion . [SEP]
[CLS] decorating or coating B-material dna nanostructures with hexaethylene glycol ( heg ) , lipid B-material bilayer I-material , dendritic oligonucleotides , peptoids , peptides B-material , albumins , and cationic B-material block co - polymers B-material based on oligolysine - polyethylene glycol ( peg ) , poly ( 2 - ( dimethylamino ) ethyl methacrylate ) , chitosan B-material , and linear polyethylenimine ( lpei ) helps shield them from nuclease digestion , greatly extending their half - life in cell B-material - culture media ( typically supplemented with 10 % fetal bovine serum ) or in vivo . [SEP]
[CLS] unlike dna nanostructures , dna - based nanoparticles B-nanoparticle ( e . g . , nanoflowers ) acquire nuclease resistance via a dense dna packaging . [SEP]
[CLS] notably , single - stranded dna origami has extraordinary thermostability , as it is analogous to a fully ligated dna structure . [SEP]
[CLS] its potential use as a drug carrier remains to be explored . [SEP]
[CLS] despite the charge repulsion between dna nanostructures and the cellular membrane , various dna nanostructures can be readily internalized into mammalian cells B-material via endocytosis B-event . [SEP]
[CLS] cell B-material type and the properties of dna nanostructures such as size , shape , and surface chemistry are the major factors that determine the route of endocytosis B-event . [SEP]
[CLS] general trends derived from other materials can hardly be adapted to dnabased systems due to the distinct surface chemistry . [SEP]
[CLS] dna nanostructures might be internalized into mammalian cells B-material through multiple pathways , given that their sizes range from a few nanometers to the submicron scale ( figure 3b ) . [SEP]
[CLS] a selection of recent studies focusing on the pathways of cellular uptake is discussed here . [SEP]
[CLS] comprehensive reviews on the cellular delivery of dna nanostructures can be found elsewhere in the literature . [SEP]
[CLS] a dna tetrahedron was one of the earliest structures proven to enter mammalian cells B-material without the aid of transfection reagents . [SEP]
[CLS] internalized tetrahedra were found unmodified dna nanostructures are usually susceptible to cation B-material depletion and enzyme digestion due to the charge repulsion between negatively charged dna backbones and the existence of nick points between individual strands . [SEP]
[CLS] ligation of nick points , cross - linking , and surface coating B-material are widely used strategies for protecting dna nanostructures from denaturation or degradation under physiological conditions . [SEP]
[CLS] ( b ) illustration depicting the cellular uptake pathways of dna nanostructures . [SEP]
[CLS] a dna tetrahedron ( 7 nm ) and a dna origami rod ( 127 nm in length ) are both internalized into hela cells B-material via caveolaemediated endocytosis B-event . [SEP]
[CLS] a dna nanoribbon with a high aspect ratio takes the clathrin - mediated endocytic pathway into h460 cells B-material . [SEP]
[CLS] the disulfide - modified dna nanospheres B-nanoparticle are directly internalized into the cytosol of hela cells B-material through a nonendocytic pathway . [SEP]
[CLS] ( c ) a variety of targeting modules , including aptamer , antibody B-material , small - molecule ligand , peptide B-material , and protein B-material , have been integrated onto dna - based carriers , resulting in improved targeting specificity and cellular uptake efficiency . [SEP]
[CLS] localized in the cytoplasm and also substantially intact . [SEP]
[CLS] as further elucidated by single - particle tracking , caveolin - dependent endocytosis B-event , followed by microtubuledependent transportation to the lysosomes , was identified as the primary uptake and intracellular trafficking mechanism in hela cells B-material . [SEP]
[CLS] the underlying physical mechanism was obtained through a coarse - grained modeling of the tetrahedron - cell B-material membrane interaction . [SEP]
[CLS] the modeling results suggested that tetrahedra predominantly attacked the spots of slight or no charge repulsion on the semifluidic membrane with their corners . [SEP]
[CLS] dna origami can be folded into arbitrary shapes , facilitating a systematic evaluation of the effect of size and shape on endocytosis B-event . [SEP]
[CLS] screening the shape - dependent uptake in three cell B-material lines representing endothelial , epithelial , and immune cells B-material uncovered a general trend wherein larger origami with more compact helical arrangement could be more efficiently internalized than smaller , less compact ones . [SEP]
[CLS] a similar trend was observed in lung cancer cell B-material lines : larger origami were more preferred than smaller ones ; compact , rod - shaped origami were more efficiently internalized than branched , tetrahedron - shaped ones . [SEP]
[CLS] the detailed cell B-material - internalization process of the large rod was visualized in the latter study , which was mediated by scavenger receptors and was likely to follow a caveolin - dependent pathway to late endosomes and lysosomes . [SEP]
[CLS] this result is consistent with the co - localization of tubular origami and lysosomes in nih 3t3 cells B-material observed by super - resolution fluorescence B-technique microscopy I-technique . [SEP]
[CLS] dna assemblies can also be internalized via clathrin - mediated or nonendocytic pathways [SEP]
[CLS] however , not many different shapes have been tested for their internalization pathways . [SEP]
[CLS] moreover , cellular uptake is usually quantified by fluorescence - based techniques , which can be affected by a number of factors , including degradation of dna structures , cellular uptake of fluorophores , and local environmental changes . [SEP]
[CLS] given the fact that cell B-material types differ vastly regarding their uptake efficiency , there are no comprehensive rules to predict the entry , biodistribution , and fate of dna nanostructures . [SEP]
[CLS] further research is necessary to unravel the complex mechanisms of cellular uptake and to seek non - endo / lysosomal pathways to circumvent the acidic environment in the lysosome for the efficient delivery of therapeutic agents . [SEP]
[CLS] studies on the in vivo distribution of dna nanostructures in healthy mice suggest the preferential accumulation in liver or kidney after intravenous injection into the blood circulatory system . [SEP]
[CLS] then , dna nanostructures are degraded or excreted within 24 h . [SEP]
[CLS] encapsulating dna nanostructures with pegylated lipid B-material bilayers I-material can help retain over 80 % of the administered dose throughout the mice as visualized 2 h after the initial injection . [SEP]
[CLS] topical medication is another potential solution to circumvent rapid clearance in systemic administration . [SEP]
[CLS] transdermal drug delivery is especially suitable for skin cancer treatment and has been applied to the mouse melanoma model with initial success . [SEP]
[CLS] dna nanostructures interact with cells B-material primarily through receptors displayed on the cellular membrane . [SEP]
[CLS] specific targeting and enhanced cellular uptake can be achieved by tagging dna nanostructures with ligands that can be recognized by the receptors ( figure 3c ) . [SEP]
[CLS] for example , folate modification enabled the specific internalization of dna nanotubes B-nanoparticle into kb cells B-material . [SEP]
[CLS] enhanced cellular uptake of a rectangular dna origami by hek293 cells B-material was facilitated by virus - capsid protein B-material coating B-material . [SEP]
[CLS] in addition , dna aptamers that are selected via cell - selex ( systematic evolution of ligands by exponential enrichment ) are widely used as targeting modules , as they can be incorporated into most dna nanocarriers . furthermore , the cell B-material entry and transport pathways of dna nanocarriers can be readily modulated by varying the targeting molecules . [SEP]
[CLS] the endocytic pathway usually determines the destination of intracellular transport . [SEP]
[CLS] most dna nanostructures and their cargo are ultimately transported to lysosomes for degradation . [SEP]
[CLS] thus , endolysosomal escape is necessary so that dna nanostructures can circumvent the acidic interior of lysosomes . [SEP]
[CLS] dna nanostructures can be modified with a secondary transport signal in order to be re - directed to target other organelles . [SEP]
[CLS] for example , signaling peptides B-material were anchored onto a dna tetrahedron to facilitate nucleus targeting . [SEP]
[CLS] modifying dna nanostructures to take nonendocytic or endosomal escape pathways is a key strategy for targeting specific organelles which remains to be explored . [SEP]
[CLS] high synthesis cost and long reaction time are two obstacles that hinder the rapid production of complex dna nanostructures in large quantities . [SEP]
[CLS] biotechnological mass - production methods based on enzymatic amplification or bacteriophage replication have facilitated large - scale synthesis of replicable dna nanostructures , and single - stranded dna that can be used as the scaffolds or staples for assembling dna origami . [SEP]
[CLS] remarkably , by including selfexcising dnazyme sequences in the single - stranded precursor dna produced by bacteriophages , target dna can be produced without the need for costly restriction endonucleases . [SEP]
[CLS] the production cost of folded origami is estimated to be $ 25 per milligram for a liter - scale lab setup and even lower for a pilot - scale setup . [SEP]
[CLS] as a highly cooperative process , the folding of a dna origami can be completed at a constant temperature within minutes . [SEP]
[CLS] taking advantage of this characteristic , rapid and scalable self - assembly of dna origami can be carried out with widely available lab equipments . [SEP]
[CLS] moreover , scalable methods for scaffold purification by selective polymer B-material catch and release and for origami purification by rate - zonal centrifugation and peg - induced precipitation further reduced the cost of preparing pure dna nanostructures . [SEP]
[CLS] methods for economical and scalable synthesis , fabrication , and purification will benefit applications that require dna nanostructures in large quantities and high purities , including therapeutics . [SEP]
[CLS] with a better understanding of their behaviors under physiological conditions and interactions with cells B-material , dna nanostructures have been extensively explored as a multifunctional platform to deliver therapeutic agents for the implementation of various in vitro and in vivo cancer - therapeutic strategies . [SEP]
[CLS] in this section , we summarize these strategies and highlight those successful examples demonstrated in vivo . [SEP]
[CLS] as the most widely used strategy for cancer treatment , chemotherapy is conducted by delivering cytotoxic B-property therapeutic agents to their functional sites . [SEP]
[CLS] dna - based drug - delivery systems use dna nanostructures or dna - based nanoparticles B-nanoparticle as carriers to improve the biodistribution , specificity , and efficacy of conventional drugs . [SEP]
[CLS] the safety concerns on using dna nanostructures can be precluded with appropriate design of dna sequences ( e . g . , obviating the use of functional dna modules like as1411 , a 26 - base guanine - rich dna aptamer with potential apoptotic induction activity ) . [SEP]
[CLS] to date , many dna - based carriers have been proven to have little cytotoxicity B-property and are primarily used as the hub to integrate drug loading , targeting , and release modules together . [SEP]
[CLS] doxorubicin B-material ( dox ) is widely used as a model drug as it can be easily loaded onto double - stranded dna B-event by I-event intercalation I-event and released by disrupting the dna duplex . [SEP]
[CLS] daunorubicin is another intercalating drug that has been delivered in a similar way . [SEP]
[CLS] other common model drugs , such as floxuridine ( fu ) , paclitaxel B-material ( ptx ) and camptothecin ( cpt ) , are usually loaded onto the dna carriers by conjugation or encapsulation . [SEP]
[CLS] some dna nanostructures , such as tetrahedra and origami , have the intrinsic ability to transport drug molecules into cancerous cells B-material without the aid of transfection reagents . [SEP]
[CLS] the loading B-property efficiency I-property and release kinetics of intercalating drugs can be modulated by designing dna origami carriers with different degrees of global twist ( figure 4a ) . [SEP]
[CLS] notably , dna origami can enhance the cellular uptake and distribution of dox and daunorubicin in dox - resistant breast adenocarcinoma ( mcf - 7 ) cells B-material and daunorubicin - resistant leukemia ( hl - 60 / adr ) cells B-material , respectively , resulting in a higher cytotoxicity B-property than free drug at equal concentrations . [SEP]
[CLS] targeted chemotherapy was facilitated by targeting molecule - functionalized dna nanocarriers ranging from aptamer - drug conjugates , nanoflowers , to dna nanostructures ( figure 4b ) . [SEP]
[CLS] moreover , drug release can be triggered by the degradation of carriers into nontoxic molecular components in response to a series of molecular stimuli or cancer hallmarks . [SEP]
[CLS] successful demonstration of improved specificity and efficacy in vitro lays the groundwork for transferring dna - based drug delivery systems toward in vivo implementation . [SEP]
[CLS] the efficacy of several dna - based chemotherapeutic systems has been proven in mouse models . [SEP]
[CLS] an aptamer - tethered dna duplex chain ( termed ' ' nanotrain ' ' ) was self - assembled via hybridization chain reaction ( hcr ) , which specifically delivered dox to human t cell acute lymphocytic leukemia ( cem ) cells and slowed down tumor B-material growth in a mouse xenograft model . [SEP]
[CLS] due to a wide distribution in size , drugs delivered by this type of carriers are not homogeneous ; i . e . , each carrier dox can be easily loaded onto dna nanostructures by intercalating between base pairs . [SEP]
[CLS] twisted dna origami nanotubes B-nanoparticle with 12 bp / turn showed a higher loading capacity and a more sustained release , compared to straight ones with 10 . 5 bp / turn . [SEP]
[CLS] reproduced with permission from zhao et al . copyright 2012 american chemical society . ( b ) muc1 aptamer - modified dna icosahedra for targeted delivery of dox into mcf - 7 cells B-material . [SEP]
[CLS] reproduced with permission from chang et al . copyright 2011 american chemical society . [SEP]
[CLS] ( c ) dna origami for dox delivery in vivo . [SEP]
[CLS] dox - loaded dna origami showed passive tumor B-material accumulation and antitumor efficacy without observable systemic toxicity B-property . reproduced with permission from zhang et al . copyright 2014 american chemical society . ( d ) dna nanostructures for the transdermal delivery of dox . [SEP]
[CLS] the 17 - nm dna tetrahedra enabled the dox to reach ~ 400 mm beneath the surface of mouse skin . reproduced with permission from wiraja et al . copyright 2019 springer nature . [SEP]
[CLS] can deliver a different amount of drug molecules . [SEP]
[CLS] moreover , carriers of difference sizes may follow multiple endocytic pathways and result in potential side effects . [SEP]
[CLS] the drug - loading ratio can be accurately controlled by covalently integrating drug molecules into dna oligonucleotides that further self - assemble into shape - defined nanostructures . [SEP]
[CLS] fu and cpt were incorporated into dna strands composing dna nanocages B-nanoparticle , showing an improved efficacy in inhibiting the growth of hela and hct166 tumors B-material in vivo , respectively . [SEP]
[CLS] using dna origami as drug - delivery vehicles B-material was demonstrated in vivo by delivering dox - loaded triangular dna origami to nude mice bearing human orthotopic breast ( mda - mb - 231 ) tumors B-material ( figure 4c ) . [SEP]
[CLS] the triangular dna origami passively accumulated in the tumor B-material region due to the abnormal permeability of tumor B-material vessels and the lack of effective lymphatic drainage , an effect called enhanced permeability and retention ( epr ) . [SEP]
[CLS] besides conventional drug - delivery strategy , which relies on the degradation of carriers and the passive release of the cargo in the endocytic pathway , various triggered - release mechanisms have been implemented with dna - based carriers in vivo . [SEP]
[CLS] a telomeraseresponsive dna icosahedron nanostructure was designed to encapsulate and deliver platinum B-material nanodrugs into cisplatin B-material - resistant human gastric carcinomas ( bgc823 / ddp ) cells B-material for in vivo tumor B-material inhibition . [SEP]
[CLS] atp - responsive nanogels , h 2 o 2 - degradable dna nanoflowers , and self - catabolic dnazyme nanosponges were programmed to release dox in response to atp , h 2 o 2 , or an acidic microenvironment in order to inhibit human breast adenocarcinoma ( mda - mb - 231 ) , human colon cancer ( hct - 166 ) , and subcutaneous hela tumors B-material , respectively . [SEP]
[CLS] most in vivo chemotherapeutic systems were conducted via systemic administration ( e . g . , intravenous injection ) . [SEP]
[CLS] report on the transdermal delivery of dox with dna nanostructures provides new insights into topical , noninvasive drug - delivery methods ( figure 4d ) . [SEP]
[CLS] the 17 - nm dna tetrahedron can deliver dox to 400 mm from the surface of mouse skin , inhibiting melanoma tumor B-material growth with comparable efficacy to intratumoral injection and microneedle application of dox . [SEP]
[CLS] overall , dna nanotechnology provides abundant structural and functional modules for improving the safety , specificity , and efficacy of chemotherapy . [SEP]
[CLS] combining chemotherapy with other therapeutic strategies could further enhance its therapeutic potency . [SEP]
[CLS] combination therapy will be discussed later in this section . [SEP]
[CLS] gene therapy can be achieved via the delivery of healthy genes or gene regulators into abnormal cells B-material for disease prevention or treatment . [SEP]
[CLS] dna nanotechnology facilitates gene therapy via a nonviral delivery of gene regulators that include antisense dna ( asd ) , dnazyme , and small interfering rna ( sirna ) for post - transcriptional gene silencing . [SEP]
[CLS] to prevent the translation of undesirable proteins B-material , asd , dnazyme , and sirna mediate the degradation of target messenger rna ( mrna ) via different mechanisms : asd induces the hydrolysis of targeted mrna by rnase h , dnazyme recognizes and cleaves mrna at a specific site , and sirna induces mrna degradation by forming the rna - induced silencing complex ( risc ) . [SEP]
[CLS] poor cellular uptake of these nucleic B-material - I-material acid I-material gene regulators is a major issue that limits efficient gene silencing . [SEP]
[CLS] dna nanotechnology provides various types of carriers , which can be easily loaded with nucleic B-material - I-material acid I-material gene regulators and internalized via the endocytic pathway . [SEP]
[CLS] as compared with asd alone , integrating asd onto a dna tetrahedron doubled its uptake and gene silencing effect ( figure 5a ) . [SEP]
[CLS] to increase the amount of cargos that can reach the cytoplasm via the endocytic pathway , carriers with a higher loading capacity are preferred for asd delivery . [SEP]
[CLS] for example , multifunctional nanogels , which integrate asd , dnazyme , aptamer , and glutathione ( gsh ) - responsive disulfide B-property linkage I-property , can downregulate genes that are associated with the proliferation of human lung adenocarcinoma epithelial ( a549 ) cells B-material in a targeted and stimulus - responsive manner . [SEP]
[CLS] rna interference ( rnai ) has become a ubiquitous tool in biological research and a new frontier for therapeutics due to its structural simplicity , stealth administration , and universality . [SEP]
[CLS] sirnas can be delivered by various dna nanostructures and downregulate proteins B-material such as survivin and bcl2 , which are involved in apoptosis B-event inhibition of skov - 3 and dms53 cells B-material , respectively . [SEP]
[CLS] ideally , if the nucleic B-material acid I-material cargo can be directly delivered into the cytosol , the dose requirement could be greatly reduced . [SEP]
[CLS] covalently modifying sirna or asd with a disulfide group at its terminus or packaging asd with a guanidinium - disulfide monomer B-material can facilitate a transmembrane delivery to the cytoplasm within 30 min after administration . [SEP]
[CLS] these strategies have the potential to be implemented into dna nanostructures for ultrafast cytosolic delivery of cargos . [SEP]
[CLS] most of the in vivo gene therapy experiments are based on rnai . [SEP]
[CLS] one of the biggest challenges for the implementation of gene therapy in vivo is maintaining the structural integrity of the therapeutic nucleic B-material acids I-material during delivery . [SEP]
[CLS] dna tetrahedra were initially used as a scaffold to organize sirna and folate with well - defined location , density , and spatial orientation for targeted gene silencing of firefly - luciferase - expressing kb xenografts ( figure 5b ) . [SEP]
[CLS] the assembled carrier circulated 4 times longer in the blood than the sirna alone and resulted in a 60 % reduction in the bioluminescent B-property intensity without a detectable immune response . [SEP]
[CLS] microsponges and nanogels are also capable of sirna delivery for the effective knockdown of firefly luciferase in t22 - luc tumor - bearing mice and polo - like kinase 1 ( plk1 ) expression in mda - mb - 231 tumor B-material - bearing mice , respectively . [SEP]
[CLS] in these particles , dna mainly serves as an rna transcription B-event template ( b ) folate - modified dna tetrahedra were used for the targeted delivery of sirna to kb xenograft tumors B-material in vivo . [SEP]
[CLS] the spatial organization of folate on the dna tetrahedra plays an important role in determining the efficiency of gene silencing . [SEP]
[CLS] reproduced with permission from lee et al . copyright 2012 springer nature . [SEP]
[CLS] ( c ) sirna delivery by sst carriers for bcl2 depletion in vivo . [SEP]
[CLS] efficient uptake of a small rectangular structure led to an effective inhibition of dms53 tumor B-material growth in vivo . [SEP]
[CLS] reproduced with permission from rahman et al . copyright 2017 wiley - vch . [SEP]
[CLS] ( d ) crispr - cas9 delivery by using pei - coated dna nanoclews for genome editing . [SEP]
[CLS] egfp depletion was observed in ~ 25 % of u2os . egfp cells B-material after intratumoral injection . [SEP]
[CLS] rca , rolling circle amplification . [SEP]
[CLS] reproduced with permission from sun et al . copyright 2012 wiley - vch . [SEP]
[CLS] or sirna anchoring point [SEP]
[CLS] in the first report of using sst nanocarriers for therapeutics , a set of rectangular and tubular sst nanostructures were screened to deliver sirna for the depletion of an antiapoptotic protein B-material , bcl2 ( figure 5c ) . [SEP]
[CLS] efficient uptake of the smallest rectangular structure led to a 90 % knockdown of the bcl2 protein B-material in vitro and an effective inhibition of dms53 tumor B-material growth in vivo . [SEP]
[CLS] an intriguing example of genome editing was demonstrated by the intratumoral injection of cas9 / sgrna - loaded dna nanoclews for targeted enhanced green fluorescent B-property protein B-material ( egfp ) disruption ( figure 5d ) . [SEP]
[CLS] the successful delivery of crispr - cas9 by dna nanocarriers suggested new opportunities for integrating molecular - biology techniques with the dna nanotechnology platform . [SEP]
[CLS] smart carriers with specific targeting and triggered release functions are in high demand for gene therapy and combination therapy . [SEP]
[CLS] photodynamic therapy ( pdt ) and photothermal therapy ( ptt ) are the two major forms of phototherapies that have been advanced by dna nanotechnology . [SEP]
[CLS] dna carriers improve the solubility B-property , specificity , and cellular uptake of photosensitizers B-property and photothermal materials , and promote reactive oxygen B-material species ( ros ) or heat generation upon irradiation to damage malignant cells B-material . [SEP]
[CLS] compared with chemotherapy , phototherapy is less likely to cause drug resistance , long - term toxicity B-property , and side effects . [SEP]
[CLS] aptamer - photosensitizer B-property complexes such as aptamer - chlorin B-material e6 ( ce6 ) conjugates and porphyrin photosensitizer B-property ( tmpyp4 ) - loaded aptamers were the earliest constructs for targeted pdt . [SEP]
[CLS] aptamers confine photosensitizers B-property to malignant cells B-material and minimize the indiscriminate damage of ros on healthy cells B-material . [SEP]
[CLS] the pdt effect can be locally amplified with the use of a catalytic dna circuit , where the catalystsequence - bearing aptamer activates and catalyzes the revelation of ce6 on the cell B-material surface . [SEP]
[CLS] moreover , different aptamers can be multiplexed in a single circuit for logic - based cell B-material recognition and pdt . [SEP]
[CLS] for example , a 3 - arm dna ' ' nanoclaw ' ' carrying an sgc8c aptamer , a tc01 aptamer , and a ce6 photosensitizer B-property on each arm could operate based on the and logic gate and activate ce6 only when both aptamers recognized targeted surface markers on the same cell B-material ( figure 6a ) . [SEP]
[CLS] dna origami can improve the solubility B-property and photostability of bmepc , a carbazolederived photosensitizer B-property , and enhance radical production in mcf - 7 cells B-material ( figure 6b ) . [SEP]
[CLS] photothermal materials are usually bulkier and less programmable than photosensitizers B-property ; therefore , among all types of dna nanostructures , dna origami is usually used as the carrier for ptt . [SEP]
[CLS] compared with chemo - and gene therapy , there are not as many applications of dna nanotechnology - based phototherapy in vivo . [SEP]
[CLS] dna nanosponges were loaded with sgc8c aptamer , catalase , and tmpyp4 photosensitizer B-property to achieve effective pdt under hypoxic conditions ( figure 6c ) . [SEP]
[CLS] the sensitization of pdt could be enhanced by including the antisense sequence - targeting hypoxia - inducible factor 1a ( hif - 1a ) , a major transcription B-event factor associated with tumor B-material hypoxia . [SEP]
[CLS] the passive tumor B-material - targeting property of dna origami has been used to assist the delivery of gold B-material nanorods B-nanoparticle ( aunrs ) to the tumor B-material site for a more efficient elevation of local temperatures upon near - infrared ( nir ) irradiation and for better ptt efficacy than aunr itself ( figure 6d ) . [SEP]
[CLS] integrating imaging agents into these therapeutic systems enabled a real - time visualization of their biodistribution and tumor B-material uptake via fluorescence B-property or optoacoustic imaging . [SEP]
[CLS] in summary , dna nanotechnology primarily enhances the specificity and efficacy of phototherapy . [SEP]
[CLS] combining phototherapy with other therapeutic strategies could result in a more synergistic effect and light - controlled drug release . [SEP]
[CLS] unmethylated cytosine - phosphate - guanine ( cpg ) oligonucleotides with certain sequence motifs can be recognized by toll - like receptor B-material 9 ( tlr9 ) and stimulate cytokine release and an immune response . [SEP]
[CLS] tlr9 is expressed in antigen - presenting cells B-material ( apcs ) and is generally located in the endosome . [SEP]
[CLS] since dna nanostructures are usually internalized into apcs via the endocytic pathway , the delivery of synthetic cpg oligonucleotides by dna nanostructures is a convenient method for immune stimulation and immunotherapy . [SEP]
[CLS] cellular delivery of well - studied synthetic cpg motifs has been achieved with a variety of dna nanocarriers , including y - shaped tiles , dendrimers B-nanoparticle , hydrogels , dna tetrahedra , and dna origami tubes ( figure 7a ) . [SEP]
[CLS] as indicated by enzymelinked immunosorbent assay ( elisa ) , compared with cpg motif itself , a significantly higher level of cytokines ( e . g . , tumor B-material necrosis factor ( tnf ) - a , interleukin ( il ) - 6 , and il - 12 ) can be induced by the cpg motif delivered by dna nanocarriers . [SEP]
[CLS] the cytokine levels were positively correlated with the number of cpg motifs displayed on a single dna tetrahedron . [SEP]
[CLS] the activation of immune cells B-material induced by dna origami tubes carrying 62 cpg sequences was also indicated by a greater amount of the early activation marker cd69 on dendritic and b cells B-material . [SEP]
[CLS] a synthetic vaccine was rationally designed by precisely assembling cpg adjuvants and a model antigen , streptavidin ( stv ) , onto dna tetrahedra ( figure 7a ) . [SEP]
[CLS] the vaccine mimicked a natural viral particle , inducing an antibody B-material response against stv and maintaining a higher anti - stv igg level than that of free cpg and stv for the photosensitizer B-property ( green triangle ) is activated only when both targeting domains are attached to the surface biomarkers B-property on cancer cells B-material . reproduced with permission from you et al . copyright 2014 american chemical society . [SEP]
[CLS] ( b ) dna origami for photosensitizer B-property delivery into tumor B-material cells B-material . [SEP]
[CLS] the model photosensitizer B-property , bmepc , can be loaded onto dna origami by intercalation . [SEP]
[CLS] dna origami reduces the photobleaching of bmepc and enhances its radical production . [SEP]
[CLS] reproduced with permission from zhuang et al . copyright 2016 american chemical society . ( c ) dna nanosponges for enhanced pdt . [SEP]
[CLS] co - delivery of tmpyp4 and catalase by dna nanosponges can relieve hypoxia - associated resistance and enhance the efficacy of tmpyp4 . [SEP]
[CLS] reproduced with permission from pan et al . copyright 2019 wiley - vch . [SEP]
[CLS] ( d ) aunr delivery by dna origami . [SEP]
[CLS] optoacoustic imaging and photothermal therapy are simultaneously achieved in vivo . [SEP]
[CLS] reproduced with permission from du et al . copyright 2016 wiley - vch . [SEP]
[CLS] over 70 days . [SEP]
[CLS] to prevent postsurgical tumor B-material relapse , an anti - pd - 1 antibody B-material was encapsulated in a cpg - encoded nano - cocoon and locally injected into the resection bed . [SEP]
[CLS] a local proinflammatory environment triggered the sustained release of cpg oligonucleotides and anti - pd - 1 antibodies B-material , inducing an antitumor effect that was effective for both remaining and metastatic tumors B-material . [SEP]
[CLS] overall , the use of dna nanotechnology for immunotherapy mainly focuses on cpg delivery . [SEP]
[CLS] recently , ( c ) a prototype dna nanorobot for triggered cargo exposure based on and logic . [SEP]
[CLS] the sequestered antibody B-material fragments are exposed for cell B-material signaling regulation when both aptamerbased locks are attached to the corresponding cell B-material surface markers . [SEP]
[CLS] reproduced with permission from douglas et al . copyright 2012 aaas . [SEP]
[CLS] ( d ) a nucleolin - responsive dna nanorobot for tumor B-material inhibition in vivo . [SEP]
[CLS] the blood B-event coagulation I-event protease , thrombin , is exposed in response to nucleolin and induces intravascular thrombosis to inhibit tumor B-material growth . reproduced with permission from li et al . copyright 2018 springer nature . [SEP]
[CLS] ( e ) dna origami with preferred renal uptake and aki alleviation effect . [SEP]
[CLS] compared with dna origami with other geometries , rectangular dna origami showed an outstanding ros - scavenging effect , which was further used for aki treatment . [SEP]
[CLS] reproduced with permission from jiang et al . copyright 2018 springer nature . [SEP]
[CLS] aptamer - equipping strategies for immune - cell B-material engineering started to emerge , which hold great potential in adoptive immunotherapy . [SEP]
[CLS] therapeutic agents kill malignant cells B-material with chemical toxicity B-property , biological interference , or physical damage . [SEP]
[CLS] however , intrinsic limitations and adverse effects could still cause a simple therapeutic strategy to fail . [SEP]
[CLS] for example , tumor B-material cells B-material could potentially develop resistance to chemotherapy . [SEP]
[CLS] phototherapy has limited tissue - penetration depth and limited efficacy on metastatic cancer . [SEP]
[CLS] gene therapy is usually short lived and might incite an immune response . [SEP]
[CLS] to address these issues , dna nanotechnology provides a multifunctional platform that integrates therapeutic strategies into a single formulation to promote a synergistic effect of different strategies ( figure 7b ) . [SEP]
[CLS] moreover , the dose and ratio of therapeutic agents can be finely tuned on the dna platform , minimizing dose - dependent toxicity B-property and other side effects without compromising efficacy . [SEP]
[CLS] combination therapy has been proven to be effective against mdr cancer cells B-material . [SEP]
[CLS] for example , dox and the gene encoding tumor B-material suppressor p53 were delivered into mdr cancer ( mcf - 7r ) cells B-material by a triangular dna origami to inhibit tumor B-material growth without eliciting apparent systemic toxicity B-property or immune response . [SEP]
[CLS] dox , aunr , and muc1 aptamer have been combined on a triangular dna origami for targeted photo - chemo therapy in vitro . [SEP]
[CLS] the photothermal effects of aunr upon nir irradiation inhibited the expression of glycoprotein ( p - gp ) , a typical drug efflux pump involved in drug resistance , and enhanced the efficacy of dox on dox - resistant mcf - 7 / adr cells B-material . [SEP]
[CLS] another widely used formulation is the co - delivery of chemotherapy drug along with gene regulators targeting p - gp . [SEP]
[CLS] the cytotoxic B-property effect of dox on mdr tumors B-material can be effectively enhanced by various p - gp inhibitors , including asd nanoparticles B-nanoparticle and small - hairpin rna ( shrna ) transcription B-event templates [SEP]
[CLS] combination therapy explores the complementary and synergistic effects among a combination of therapeutic strategies , providing potential solutions for tumor B-material types that are difficult to treat with a single therapy . [SEP]
[CLS] therapeutic dna nanorobots therapeutic dna nanorobots are dna devices that can automatically perform therapeutic tasks , including target recognition , drug delivery , and triggered release , following pre - encoded rules . [SEP]
[CLS] for example , a prototype dna nanorobot was programmed to open and expose its payloads in response to a combination of ' ' key ' ' antigens that were present on the surface of a cell B-material ( figure 7c ) . [SEP]
[CLS] by varying its payloads , the nanorobot could carry out user - defined tasks such as cell B-material discrimination , growth arrest of aggressive natural killer leukemia ( nkl ) cells B-material , and t cell activation . [SEP]
[CLS] recently , therapeutic dna nanorobot was successfully applied to cancer therapy in vivo ( figure 7d ) . [SEP]
[CLS] rather than being internalized into tumor B-material cells B-material , the nanorobot was activated in response to nucleolin in the tumor B-material vessel and subsequently exposed thrombins that were initially sequestered to trigger intravascular thrombosis at the tumor B-material site and induced tumor necrosis . [SEP]
[CLS] no significant changes in the blood B-event coagulation I-event parameters , cytokine levels , or cytotoxicity B-property were observed when testing the nanorobot in healthy mice and bama miniature pigs , suggesting that their use in the normal tissues of large animals would be considered safe . [SEP]
[CLS] the kidney is an important organ that filters blood , removes wastes and toxins , maintains the balance of body fluids and electrolytes , and helps control blood pressure in animal bodies . [SEP]
[CLS] kidney dysfunction can lead to serious or even life - threatening health issues . [SEP]
[CLS] several recent studies that highlight the potential of applying dna nanotechnology to the treatment of kidney diseases ( or nephropathy ) are discussed here . [SEP]
[CLS] there are various biological interfaces in living bodies , which form the barriers B-property for drug delivery and affect the organ distribution of nanoparticles B-nanoparticle . [SEP]
[CLS] given the extraordinary programmability of dna nanostructures , one may envision that their abilities to pass through biological barriers B-property and target specific organs ( e . g . , brain , liver , kidney , etc . ) can be improved by simply tuning their physical properties . [SEP]
[CLS] in general , nanoparticles B-nanoparticle ranging from 30 to 150 nm are not easily cleared by kidney filtration , while those with a small hydrodynamic size ( 5 - 7 nm ) or with high aspect ratios and small diameters can be cleared as they fall below the filtration threshold of kidney . [SEP]
[CLS] taking advantage of this effect , dna nanostructures can be designed to have distinct renal uptake and retention profiles . [SEP]
[CLS] for example , the renal clearance of cu 64 - labeled small dna tetrahedra ( 7 nm ) can be monitored for the evaluation of kidney functions via positron B-technique emission I-technique tomography I-technique ( pet ) imaging . [SEP]
[CLS] in another study , pet imaging of the biodistribution of cu 64 - labeled dna origami nanostructures revealed that intact structures preferentially accumulate in kidneys within 12 h and showed whole - body clearance after 24 h postinjection . [SEP]
[CLS] in contrast , the scaffold strand and partially folded dna origami nanostructures were primarily sequestered in the liver and cleared by the mononuclear phagocyte system . [SEP]
[CLS] thus , structural compactness and intact folding are two factors that reduce hepatic sequestration and improve kidney accumulation of dna nanostructures . [SEP]
[CLS] treating aki with dna nanostructures kidney diseases can be generally categorized into acute kidney injury ( aki ) , chronic kidney disease ( ckd ) , and kidney - related diseases ( e . g . , kidney tumor B-material ) . [SEP]
[CLS] a rectangular dna origami was exploited to alleviate rhabdomyolysis - induced aki and protect renal cells B-material from nephrotoxic agents ( figure 7e ) . [SEP]
[CLS] its therapeutic effect was evaluated by dynamic pet imaging I-technique with ga - edta , blood tests , and kidney - tissue staining , which was similar to antioxidant B-property n - acetylcysteine , a clinical drug for contrast - induced aki prevention . [SEP]
[CLS] current research suggests the preferential renal uptake and the possibility of tuning the retention of dna nanostructures in kidneys . [SEP]
[CLS] therefore , it is possible to apply dna - based therapeutic formulations that have been proven effective in vivo to the treatments of other kidney diseases . [SEP]
[CLS] the key is to find target molecules that are involved in the onset of kidney diseases . [SEP]
[CLS] in addition to ros , potential targets include noncoding rnas , and proteins B-material such as kidney injury molecule - 1 ( kim - 1 ) , clusterin and transforming growth factor - b ( tgf - b ) [SEP]
[CLS] based on these targets , it is possible to design proper kidney - targeted dna drug delivery systems for the treatment of other kidney diseases . [SEP]
[CLS] in the past decade , remarkable progress has been made in structural dna nanotechnology , which facilitates the use of designer dna nanostructures for therapeutic applications . [SEP]
[CLS] complex higher - order dna assemblies can be rationally and automatically designed and efficiently scaled - up . [SEP]
[CLS] chemical methods are developed to enhance the stability of dna nanostructures in physiological environments . [SEP]
[CLS] synthesis and purification costs of dna origami structures are expected to be greatly reduced by the recently reported mass - production and purification protocols . [SEP]
[CLS] the modularity and programmability of the dna platform allow various mechanisms of target recognition and triggered release to be implemented in vivo . [SEP]
[CLS] several challenges still remain to be addressed before using dna nanocarriers in clinical practice . [SEP]
[CLS] firstly , the structural heterogeneity of dna nanocarriers may cause side effects . [SEP]
[CLS] there is evidence that partially formed and intact dna nanostructures have different preferences for organ accumulation . [SEP]
[CLS] it can be inferred that the organ - targeting ability of a dna nanocarrier changes during circulation due to its gradual denaturation . aberrant accumulation not only increases the burdens of healthy organs but also reduces the effective concentration of the drug at pathogenic sites . [SEP]
[CLS] moreover , precise dosing is challenging to achieve , especially for intercalating drugs , whose spectrum properties and loading and release profiles are affected by a number of factors . [SEP]
[CLS] in this respect , a gold B-material standard is needed to enable cross - comparison between studies . [SEP]
[CLS] drug - loading capacity can be better controlled by covalently conjugating drug molecules onto dna strands that compose a nanocarrier . [SEP]
[CLS] biotechnological methods for the economical synthesis of drug - conjugated dna rely on the enzymes that can catalyze the polymerization of nucleotide - containing unnatural base analogs . [SEP]
[CLS] although there are in vitro strategies available , in vivo production is largely elusive . [SEP]
[CLS] secondly , changes in the surface chemistry of dna nanocarriers may affect their biodistribution . [SEP]
[CLS] for example , surface coating B-material via electrostatic interaction is likely to change the circulating time , organ distribution , and toxicity B-property of a dna nanostructure . [SEP]
[CLS] the resultant effects need to be characterized in vivo . [SEP]
[CLS] protein B-material - corona I-material formation is another factor that could drastically alter the surface chemistry of dna nanostructures in vivo . [SEP]
[CLS] serum proteins B-material can adhere to dna nanostructures in both sequence - dependent and nonspecific manners , and further induce aggregation or promote clearance . [SEP]
[CLS] to solve this problem , chemical modification and coating B-material could effectively change the surface chemistry of dna nanostructures and prevent protein B-material - corona I-material formation by reducing the affinity of serum proteins B-material . [SEP]
[CLS] alternatively , the sequence of dna nanostructures can be rationally designed to alter the profile of protein B-material adsorption and facilitate organ targeting . [SEP]
[CLS] moreover , the endocytosis B-event mechanism of dna nanostructures is not comprehensively understood . [SEP]
[CLS] although dna nanotechnology has the unique advantage of designing arbitrary shapes with nanometer precision , the types of dna nanostructures that have been tested for endocytosis B-event are relatively limited . [SEP]
[CLS] comprehensive mechanistic studies that take structure characteristics , surface chemistry , and cell B-material type into account are needed to predict the endocytosis B-event and cellular fate of a specific construct . [SEP]
[CLS] getting trapped and degraded in the lysosome is usually the fate of internalized dna nanostructures . [SEP]
[CLS] therefore , simple and robust strategies to facilitate endo / lysosomal escape , organelle targeting , and nuclear entry are in demand . [SEP]
[CLS] exploring nonendocytic pathway for dna nanostructures to circumvent the acidic environment in the lysosome will greatly enhance the efficacy of therapeutic agents that function in the cytosol and nucleus . [SEP]
[CLS] given the complex environments in vivo , the pharmacokinetics and pharmacodynamics of dna nanostructures are complicated by many factors . [SEP]
[CLS] tracing the absorption , distribution , metabolism , and excretion of a dna carrier and its cargo , as well as their effects on living organisms , are the key to evaluate dosing , efficacy , and adverse effects . [SEP]
[CLS] rapid clearance is a major obstacle that limits the efficacy of dna - based drug - delivery systems at the current stage of research . [SEP]
[CLS] a higher cost and toxicity B-property will be associated with the repetitive administration of drugs at a higher dose to prolong the retention time . [SEP]
[CLS] finally , the biosafety of dna - based therapeutic systems should be evaluated in the long term . [SEP]
[CLS] for example , the contamination of immunostimulatory endotoxin , which is involved in the preparation and amplification of dna from gram - negative bacteria , must be evaluated and reduced to the safe level . [SEP]
[CLS] there is no indication of chronic toxicity B-property induced by dna nanostructures so far , which can be largely attributed to the rapid clearance of dna nanostructures from the body . [SEP]
[CLS] extending the retention of dna nanostructure might bring new safety concerns . [SEP]
[CLS] for example , given the complexity and randomness of dna sequences in a nanostructure , extraneous dna could randomly interfere with cellular rna and induce other potential chronic toxicity B-property . [SEP]
[CLS] moreover , the immune response has not been well characterized for dna nanostructures in vivo . [SEP]
[CLS] although many studies report that cpg - free dna nanostructures have minimal immunogenicity B-property , immunostimulatory activity via tlr9 - independent pathway is not negligible . [SEP]
[CLS] principles to design dna carriers that are compatible with the immune system of higher organisms are in need . [SEP]
[CLS] recent advances in dna nanotechnology - based therapeutics have brought up new opportunities for future research . [SEP]
[CLS] first , to obtain a general and comprehensive understanding of the endocytosis B-event mechanism and the pharmacokinetics and pharmacodynamics of dna nanostructures , it is necessary to summarize individual therapeutic examples into networks in order to better predict the interaction between dna nanostructures and living organisms . [SEP]
[CLS] big data analytics and machine learning could help with the exploration of the vast design space for this purpose . [SEP]
[CLS] second , more diverse drug - administration routes are in demand . [SEP]
[CLS] the successful implementation of transdermal dox delivery suggests the possibilities of developing other noninvasive drug - delivery routes such as intranasal administration . [SEP]
[CLS] topical administration via mucosa is expected to yield rapid drug absorption and reduced risk of systemic side effects , and may serve as a supplementary of radiation therapy for nasopharyngeal carcinoma and other diseases . [SEP]
[CLS] third , the ability to pattern diverse molecules with nanometer precision would facilitate the rational design of antivirals B-property or vaccines . [SEP]
[CLS] for example , a practical strategy to engineer antivirals B-property is to block and deactivate viruses with patterned ligands that are templated by a dna scaffold . [SEP]
[CLS] another example is the effective b cell receptor B-material activation in vitro by sitespecifically displaying immunogens B-property on a dna template . [SEP]
[CLS] implementation of these two systems in vivo remains to be explored . [SEP]
[CLS] fourth , dna nanostructure has the potential to be used as an immunosuppressant to regulate the excessive release of cytokines . [SEP]
[CLS] this is helpful for alleviating cytokine storm , which is a catastrophic immune problem that may cause permanent organ damage or even death . [SEP]
[CLS] at last , replicable single - stranded dna origami structures are promising candidates for therapeutics as they have suitable size , addressability , and exceptional thermostability . [SEP]
[CLS] dna nanotechnology - based therapeutics will certainly benefit from the development of new drugs , therapeutic strategies , and formulations . [SEP]
[CLS] solutions to intractable solid tumors B-material and vascularized metastases B-event will be promising to pursue . [SEP]
[CLS] as a rapidly evolving research direction , dna nanotechnology - based therapeutics would serve as a smart , custom platform for clinical therapeutics . [SEP]
[CLS] 1 . a timeline of the field and the emerging therapeutic applications of dna nanotechnology ( a ) a timeline of the field of structural dna nanotechnology since 1982 . [SEP]
[CLS] ( b ) a timeline of the emerging therapeutic applications of dna nanotechnology since 2008 . [SEP]
[CLS] representative discoveries and achievements are listed . [SEP]
[CLS] sst , single - stranded tile ; dox , doxorubicin B-material ; ros , reactive oxygen B-material species . [SEP]
[CLS] structural dna nanotechnology ( a ) ned ' s original concept : forming a 2d lattice from immobile junctions through complementary sticky ends . [SEP]
[CLS] reproduced with permission from seeman . [SEP]
[CLS] copyright 1982 elsevier . [SEP]
[CLS] ( b ) discrete nanoobjects can be assembled from oligonucleotides with rationally designed sequences and complementarities . [SEP]
[CLS] a ~ 7 nm dna cube is the earliest polyhedral object constructed from dna . [SEP]
[CLS] ( c ) dna tile - based self - assembly is a feasible approach to create higher - order structures from modular building blocks . [SEP]
[CLS] for example , a 4 - by - 4 dna tile can be assembled from several oligonucleotides and further linked into 2d lattices through complementary sticky ends . [SEP]
[CLS] ( d ) dna origami is the folding of a long , single - stranded dna ( termed ' ' scaffold ' ' ) into 2d or 3d shapes with the help of hundreds of short dna oligonucleotides ( termed ' ' staples ' ' ) . [SEP]
[CLS] ( top right ) reproduced with permission from rothemund . [SEP]
[CLS] copyright 2006 springer nature . [SEP]
[CLS] ( bottom right ) reproduced with permission from castro et al . copyright 2011 springer nature . [SEP]
[CLS] ( e ) single - stranded tiles ( ssts ) can be programmed to assemble into 2d or 3d shapes without the guidance of a scaffold . [SEP]
[CLS] ssts interact with each other through complementary domains , in a way analogous to ' ' lego ' ' bricks . [SEP]
[CLS] ( top right ) reproduced with permission from wei et al . copyright 2012 springer nature . [SEP]
[CLS] ( bottom right ) reproduced with permission from ke et al . copyright 2012 aaas . [SEP]
[CLS] ( f ) single - stranded dna origami is the unimolecular folding of a long single - stranded dna into nanostructures through paranemic cohesions . [SEP]
[CLS] reproduced with permission from han et al . copyright 2017 aaas . [SEP]
[CLS] 1156 - 1179 , may 13 , 2021 1159 [SEP]
[CLS] criteria for developing dna - based nanocarriers ( a ) common strategies to stabilize dna nanostructures under physiological conditions . [SEP]
[CLS] unmodified dna nanostructures are usually susceptible to cation B-material depletion and enzyme digestion due to the charge repulsion between negatively charged dna backbones and the existence of nick points between individual strands . [SEP]
[CLS] ligation of nick points , cross - linking , and surface coating B-material are widely used strategies for protecting dna nanostructures from denaturation or degradation under physiological conditions . [SEP]
[CLS] ( b ) illustration depicting the cellular uptake pathways of dna nanostructures . [SEP]
[CLS] a dna tetrahedron ( 7 nm ) and a dna origami rod ( 127 nm in length ) are both internalized into hela cells B-material via caveolaemediated endocytosis B-event . [SEP]
[CLS] a dna nanoribbon with a high aspect ratio takes the clathrin - mediated endocytic pathway into h460 cells B-material . [SEP]
[CLS] the disulfide - modified dna nanospheres B-nanoparticle are directly internalized into the cytosol of hela cells B-material through a nonendocytic pathway . [SEP]
[CLS] ( c ) a variety of targeting modules , including aptamer , antibody B-material , small - molecule ligand , peptide B-material , and protein B-material , have been integrated onto dna - based carriers , resulting in improved targeting specificity and cellular uptake efficiency . [SEP]
[CLS] 4 . dna nanotechnology for chemotherapy ( a ) tuning the loading capacity and releasing rate of dox by varying the global twist of dna origami nanotubes B-nanoparticle . [SEP]
[CLS] dox can be easily loaded onto dna nanostructures by intercalating between base pairs . [SEP]
[CLS] twisted dna origami nanotubes B-nanoparticle with 12 bp / turn showed a higher loading capacity and a more sustained release , compared to straight ones with 10 . 5 bp / turn . [SEP]
[CLS] reproduced with permission from zhao et al . copyright 2012 american chemical society . [SEP]
[CLS] ( b ) muc1 aptamer - modified dna icosahedra for targeted delivery of dox into mcf - 7 cells B-material . [SEP]
[CLS] reproduced with permission from chang et al . copyright 2011 american chemical society . [SEP]
[CLS] ( c ) dna origami for dox delivery in vivo . [SEP]
[CLS] dox - loaded dna origami showed passive tumor B-material accumulation and antitumor efficacy without observable systemic toxicity B-property . [SEP]
[CLS] reproduced with permission from zhang et al . copyright 2014 american chemical society . [SEP]
[CLS] ( d ) dna nanostructures for the transdermal delivery of dox . [SEP]
[CLS] the 17 - nm dna tetrahedra enabled the dox to reach ~ 400 mm beneath the surface of mouse skin . [SEP]
[CLS] reproduced with permission from wiraja et al . copyright 2019 springer nature . [SEP]
[CLS] dna nanotechnology for gene therapy ( a ) nonviral delivery of aso and dnazyme by a dna tetrahedron for post - transcriptional gene silencing . [SEP]
[CLS] dna nanocarriers enhance the cellular uptake of aso and dnazyme . [SEP]
[CLS] aso and dnazyme inhibit downstream protein B-material production by degrading the corresponding mrna . ( b ) folate - modified dna tetrahedra were used for the targeted delivery of sirna to kb xenograft tumors B-material in vivo . [SEP]
[CLS] the spatial organization of folate on the dna tetrahedra plays an important role in determining the efficiency of gene silencing . [SEP]
[CLS] reproduced with permission from lee et al . copyright 2012 springer nature . [SEP]
[CLS] ( c ) sirna delivery by sst carriers for bcl2 depletion in vivo . [SEP]
[CLS] efficient uptake of a small rectangular structure led to an effective inhibition of dms53 tumor B-material growth in vivo . [SEP]
[CLS] reproduced with permission from rahman et al . copyright 2017 wiley - vch . [SEP]
[CLS] ( d ) crispr - cas9 delivery by using pei - coated dna nanoclews for genome editing . [SEP]
[CLS] egfp depletion was observed in ~ 25 % of u2os . egfp cells B-material after intratumoral injection . [SEP]
[CLS] rca , rolling circle amplification . [SEP]
[CLS] reproduced with permission from sun et al . copyright 2012 wiley - vch . [SEP]
[CLS] 6 . dna nanotechnology for phototherapy ( a ) a logic - based dna nanoclaw for cancer targeting and pdt . [SEP]
[CLS] the photosensitizer B-property ( green triangle ) is activated only when both targeting domains are attached to the surface biomarkers B-property on cancer cells B-material . [SEP]
[CLS] reproduced with permission from you et al . copyright 2014 american chemical society . [SEP]
[CLS] ( b ) dna origami for photosensitizer B-property delivery into tumor B-material cells B-material . [SEP]
[CLS] the model photosensitizer B-property , bmepc , can be loaded onto dna origami by intercalation . [SEP]
[CLS] dna origami reduces the photobleaching of bmepc and enhances its radical production . [SEP]
[CLS] reproduced with permission from zhuang et al . copyright 2016 american chemical society . [SEP]
[CLS] ( c ) dna nanosponges for enhanced pdt . [SEP]
[CLS] co - delivery of tmpyp4 and catalase by dna nanosponges can relieve hypoxia - associated resistance and enhance the efficacy of tmpyp4 . [SEP]
[CLS] reproduced with permission from pan et al . copyright 2019 wiley - vch . [SEP]
[CLS] ( d ) aunr delivery by dna origami . [SEP]
[CLS] optoacoustic imaging and photothermal therapy are simultaneously achieved in vivo . [SEP]
[CLS] reproduced with permission from du et al . copyright 2016 wiley - vch . [SEP]
[CLS] other promising therapeutic strategies facilitated by dna nanotechnology ( a ) two strategies of using dna nanostructures for immunotherapy . [SEP]
[CLS] dna nanostructures enhance the cellular uptake of immunostimulatory cpg sequences and induce a high - level secretion of various pro - inflammatory I-property cytokines . [SEP]
[CLS] co - delivery of cpg adjuvants and a model antigen by dna tetrahedra induces strong and long - lasting antibody B-material responses against the model antigen . [SEP]
[CLS] ( top left ) reproduced with permission from li et al . copyright 2011 american chemical society . [SEP]
[CLS] ( bottom left ) reproduced with permission from schuller et al . copyright 2011 american chemical society . [SEP]
[CLS] ( right ) reproduced with permission from liu et al . copyright 2012 american chemical society . [SEP]
[CLS] ( b ) three common strategies for combination therapy . [SEP]
[CLS] combination therapy can be achieved by the co - delivery of two synergistic therapeutic agents by the same dna carrier . [SEP]
[CLS] examples of combination therapy include photo - chemo therapy , chemo - gene therapy , and photothermaldynamic therapy . [SEP]
[CLS] ( c ) a prototype dna nanorobot for triggered cargo exposure based on and logic . [SEP]
[CLS] the sequestered antibody B-material fragments are exposed for cell B-material signaling regulation when both aptamerbased locks are attached to the corresponding cell B-material surface markers . [SEP]
[CLS] reproduced with permission from douglas et al . copyright 2012 aaas . [SEP]
[CLS] ( d ) a nucleolin - responsive dna nanorobot for tumor B-material inhibition in vivo . [SEP]
[CLS] the blood B-event coagulation I-event protease , thrombin , is exposed in response to nucleolin and induces intravascular thrombosis to inhibit tumor B-material growth . [SEP]
[CLS] reproduced with permission from li et al . copyright 2018 springer nature . [SEP]
[CLS] ( e ) dna origami with preferred renal uptake and aki alleviation effect . [SEP]
[CLS] compared with dna origami with other geometries , rectangular dna origami showed an outstanding ros - scavenging effect , which was further used for aki treatment . [SEP]
[CLS] reproduced with permission from jiang et al . copyright 2018 springer nature . [SEP]
[CLS] single - base changes lead to important biological and biomedical implications ; however , the discrimination of single - base changes from normal dna always remains a grand challenge . [SEP]
[CLS] herein we developed a dna recognition and amplification system based on artificial branched dna , namely , target - triggered polymerization ( ttp ) , to realize enzyme - free and fast discrimination of single - base changes . [SEP]
[CLS] branched dna as monomers B-material rapidly polymerized into dna nanospheres B-nanoparticle only with the trigger of specific dna . [SEP]
[CLS] our ttp system worked reliably over a wide range of conditions . [SEP]
[CLS] remarkably , our ttp system was capable of discriminating base - changing dna from normal dna , including distinguishing 1 - 4 nucleotide changes and positions of single base , which was attributed to the significant amplification of small differences in hybridization thermodynamics and kinetics . [SEP]
[CLS] we further proposed a theoretical method for calculating the hybridization probability of nucleic B-material acids I-material , which performed highly consistent with experimental results . [SEP]
[CLS] nucleic B-material acids I-material are genetic biopolymers B-material that are composed of nucleotides , which are essential to all known forms of life . [SEP]
[CLS] alteration of nucleotides between nucleic B-material acid I-material sequences ( single - base substitution ) , as the most common form of nucleic B-material acid I-material variations , is often associated with important biological and biomedical implications . [SEP]
[CLS] for instance , nucleotide changes in the coding region of genome affect the sequences of amino B-material acid I-material and protein B-material function , leading to the occurrence of genetic disease . [SEP]
[CLS] nucleotide changes in the pathogen genome cause antibiotic resistance . [SEP]
[CLS] in addition , tumor - associated base changes have significant impacts on tumor B-material growth , transformations , and metastasis B-event , which can serve as noninvasive biomarkers B-property of cancers . [SEP]
[CLS] it is therefore vitally important to develop effective strategies for the discrimination of single - base changes . [SEP]
[CLS] single - base changes have only one nucleotide difference with normal counterparts ; it is extremely challenging to discriminate single - base - changing dna from normal dna because of the small energetic differences in hybridization reactions . [SEP]
[CLS] dna nanotechnology and the corresponding dna nanostructures with recognition capability and programmability provide strategies for target recognition and signal amplification to increase the small differences ( [SEP]
[CLS] the difficulty of design and use , it is necessary to develop an enzyme - free , simply designed , low - cost , and easily manipulated method for the discrimination of single - base changes . [SEP]
[CLS] herein , we constructed a branched dna - based polymerization system , namely , ' ' target - triggered polymerization ( ttp ) . ' ' [SEP]
[CLS] in ttp system , branched dna was designed to polymerize into dna nanospheres B-nanoparticle only under the trigger of specific dna , realizing target recognition and efficient signal amplification simultaneously . [SEP]
[CLS] our ttp system can be accomplished rapidly without the involvement of any enzymes . [SEP]
[CLS] in addition , the ttp system possessed robust performance in a wide range of conditions . [SEP]
[CLS] our enzyme - free , rapid , and robust ttp system enabled high specificity and sensitivity . [SEP]
[CLS] remarkably , our ttp system was capable of discriminating base - changing dna from normal dna , including different number of nucleotide changes and the positions of single - base change . [SEP]
[CLS] furthermore , thermodynamics and kinetics analyses showed that the ttp system apparently amplified the small differences of base - changing dna with normal dna in an identical hybridization reaction . [SEP]
[CLS] taking advantage of the ttp system , we developed a theoretical model for analyzing the hybridization probability of nucleic B-material acids I-material , which performed highly consistent with experimental results , making our ttp system a powerful tool for exploring the probability of nucleic B-material acid I-material hybridization . [SEP]
[CLS] enzyme - free and fast ttp system for simultaneous target recognition and signal amplification x - shaped branched dna ( x - dna ) was designed as ttp monomers B-material , which consisted of four corresponding single - stranded dna ( ssdna ) . [SEP]
[CLS] through rational sequence design , ssdna was composed of intermolecular binding region ( marked by black in scheme 1a ) for the construction of x - dna and sticky ends ( marked by red ) for recognition with specific target dna ( transfer dna ) . [SEP]
[CLS] four ssdna ( ssdna 1 , ssdna 2 , ssdna 3 , and ssdna 4 ) self - assembled to from x - dna with optimized annealing procedure . [SEP]
[CLS] in ttp system , two types of x - dna ( x 1 and x 2 ) were designed as monomers B-material to further form dna nanospheres B-nanoparticle . [SEP]
[CLS] x 1 and x 2 had the same binding core B-material and different sticky ends , which were complementary with specific t - dna . [SEP]
[CLS] through this design , t - dna functioned both as initiators to trigger the polymerization of x - dna and as linkers to hybridize with x 1 and x 2 , consequently forming dna nanospheres B-nanoparticle ( scheme 1b ) . [SEP]
[CLS] it was notable that the formation of polymeric dna nanospheres B-nanoparticle only occurred in the presence of t - dna . [SEP]
[CLS] in addition , the ttp system did not require any enzymes , reducing the operational complexity and potentially facilitating future applications such as point - of - care testing ( poct ) . [SEP]
[CLS] specifically , compared with other enzyme - free amplification methods , such as hcr and strand displacement reaction , the ttp system avoided the steps of toehold migration and hairpin structure de - conformation , which therefore enabled the formation of dna nanospheres B-nanoparticle in a very short period of time . [SEP]
[CLS] in other words , simultaneous recognition and amplification was achieved in a one - step reaction and in an enzyme - free and rapid manner . [SEP]
[CLS] we first constructed x - dna by mixing four corresponding ssdna at equimolar concentrations and with further annealing procedure . [SEP]
[CLS] the successful synthesis of x - dna was characterized and confirmed by gel B-technique electrophoresis I-technique ( figure s1a ) . [SEP]
[CLS] the addition of t - dna into the mixture of x 1 and x 2 initiated the polymerization of dna nanospheres B-nanoparticle . [SEP]
[CLS] polymeric dna nanospheres B-nanoparticle with higher molecular weights were observed in agarose B-technique gel I-technique electrophoresis I-technique ( figure s1b ) . [SEP]
[CLS] the bands in gel hole indicated the successful synthesis of dna nanospheres B-nanoparticle . [SEP]
[CLS] a long - tail smear in the lanes of x 1 + x 2 + t - dna existed , which corresponded to the dna nanospheres B-nanoparticle with broad size distribution . [SEP]
[CLS] in contrast , in the absence of t - dna or in the presence of background dna ( b - dna ) , no band of dna nanospheres B-nanoparticle was observed ( in the lanes x 1 + x 2 + pbs and x 1 + x 2 + b - dna in figure s1b ) , indicating that polymerization did not occur . [SEP]
[CLS] these results confirmed that t - dna specifically triggered the polymerization of x - dna into dna nanospheres B-nanoparticle . [SEP]
[CLS] the synthesized dna nanospheres B-nanoparticle were also verified by atomic B-technique force I-technique microscopy I-technique imaging ( figure s1c ) . [SEP]
[CLS] in addition , we explored the formation of dna nanospheres B-nanoparticle over time . [SEP]
[CLS] after the addition of t - dna in the mixture of x 1 and x 2 for 2 min , the band in the gel hole with strong fluorescence B-property intensity indicated that the formation of dna nanospheres B-nanoparticle started within 2 min ( figure s2 ) . [SEP]
[CLS] also , the fluorescence B-property intensity of the smear became relatively lighter over time , suggesting that the dynamic assembly of dna nanospheres B-nanoparticle continuously proceeded from 2 to 60 min . [SEP]
[CLS] for real - time monitoring of the ttp - based process , we took advantage of the quartz crystal microbalance ( qcm ) as a signal output device . [SEP]
[CLS] qcm has the ability to measure extremely small mass changes at the nanogram level by monitoring in real - time the frequency changes of quartz crystal resonator . [SEP]
[CLS] qcm , therefore , has been developed as a label - free biosensor for the sensitive analysis of biomolecules and kinetic evaluation . [SEP]
[CLS] the ttp - based system included two key stages : molecular recognition for t - dna and signal amplification by ttp ( figure s3a ) . [SEP]
[CLS] to efficiently recognize t - dna , we designed capture dna ( c - dna ) probes for complementary pairing with t - dna , which contained a thiol B-material group for immobilizing on the chip surface by au - s bond and a poly ( a ) tail for increasing the distance between the probes and chip surface and reducing the ' ' size dilemma ' ' . [SEP]
[CLS] in addition , the chip with c - dna attached was blocked by 6 - mercapto - 1 - hexanol ( mch ) to avoid non - specific adsorption . [SEP]
[CLS] in ttp - based system , the immobilized c - dna probes specifically hybridized with partial sequences of t - dna , generating a corresponding molecular recognition signal . [SEP]
[CLS] afterward , the remaining single - strand region of t - dna was exposed for subsequent signal amplification . [SEP]
[CLS] we first studied effective one - step signal amplification by the ttp system . [SEP]
[CLS] we synthesized dna nanospheres B-nanoparticle with x 1 in the periphery at a certain proportion of t - dna and x - dna , to allow their effective hybridization with t - dna ( figure s3b ) . [SEP]
[CLS] the frequency signals decreased slightly and then further decreased significantly after introducing t - dna and dna nanospheres B-nanoparticle with x 1 on the periphery , respectively ( figure s3c ) . [SEP]
[CLS] interestingly , we observed that the values of frequency changes ( df ) and rates ( ddf / dt ) both changed in the process of signal amplification relative to target recognition , owing to the introduction of dna nanospheres B-nanoparticle with high molecular weight . [SEP]
[CLS] in contrast , the frequency signal remained unchanged when applying b - dna and the following mixture of x 1 and x 2 ( figure s3c ) , confirming the high specificity of the ttp system . [SEP]
[CLS] we further developed a layer - by - layer ttp system to regulate the self - assembly of dna nanospheres B-nanoparticle that was in situ growing on qcm chip , achieving step - by - step signal amplification . [SEP]
[CLS] in detail , after the incubation B-technique of c - dna and mch , the chip was capable of t - dna - specific recognition and capture . [SEP]
[CLS] after the addition of t - dna , x 1 was introduced due to the complementary pairing between exposed sequences of t - dna and sticky end of x 1 , followed by t - dna and x 2 . [SEP]
[CLS] by the sequential injection of t - dna and x - dna as a cycle , layer - by - layer self - assembled dna nanospheres B-nanoparticle were in situ assembled on chip surface after several cycles , and consequently , ttp systems realized effective step - by - step signal amplification . [SEP]
[CLS] finally , the frequency signals were used as real - time outputs to monitor the polymerization of dna nanospheres B-nanoparticle , where df and ddf / dt reflected hybridization thermodynamic and kinetic behaviors , respectively . [SEP]
[CLS] in addition , the chip was regenerated after treatment with denaturant for the next round of testing , demonstrating the reusability and cost - effectiveness ( figure s4 ) . [SEP]
[CLS] to determine the optimal concentration ratio of t - dna to x - dna , we set three groups of experiments . [SEP]
[CLS] we defined the differences in df values between final equilibrium states and baseline as df . [SEP]
[CLS] the df values decreased twice after the introduction of t - dna and x - dna , which were recorded as df 1 and df 2 , respectively . [SEP]
[CLS] also , the values of df 2 / df 1 reflected the hybridization capacity of x - dna relative to t - dna . [SEP]
[CLS] by comparing the values of df 2 / df 1 , the concentration ratio of t - dna : x - dna at 2 : 1 had the highest hybridization capacity , which was used for subsequent experiments ( figure s5 ) . [SEP]
[CLS] after four cycles in layer - by - layer ttp system , df and dissipation change ( dd ) were clearly distinguished , demonstrating excellent capability of target recognition and signal amplification ( figure 1a ) . [SEP]
[CLS] during the first cycle , the frequency response showed a slowly decreasing trend ( from 0 hz to a8 hz ) and then a dramatic decrease ( from a8 to a31 hz ) , owing to the effective hybridization between t - dna and x 1 . [SEP]
[CLS] next , the accumulated introduction of t - dna and x - dna contributed to a multistep drop ( from a31 to a46 hz ) . [SEP]
[CLS] although the decrease after the first cycle was relatively slight due to space resistance and electrostatic repulsion , our layer - by - layer ttp system significantly improved the sensitivity of biosensor . [SEP]
[CLS] meanwhile , the corresponding mass changes were calculated by sauerbrey equation applied in aqueous solutions df = a f 3 = 2 0 ffiffiffiffiffiffiffiffiffiffiffiffiffi nr l h l pr q m q s = a cdm where f 0 is the resonance frequency of the bare resonator ; n is the number of the overtone ; r l and h l are the density and viscosity B-property of liquid , respectively ; r q and h q are the density and shear modulus of quartz , respectively ; and c is a proportionality constant according to the intrinsic parameters of the qcm device . [SEP]
[CLS] as a result there was a linear relationship between the change of mass ( dm ) and df when the flexible conformation of branched dna was ignored . [SEP]
[CLS] a gradient of mass change curve indicated the growth of dna nanospheres B-nanoparticle triggered by t - dna ( figure 1b ) . [SEP]
[CLS] we further examined the sensitivity of layer - by - layer ttp system . [SEP]
[CLS] the frequency signals were monitored in the range of t - dna concentrations from 2 pm to 0 . 2 mm , presenting a gradual increasing trend . [SEP]
[CLS] - df and the logarithmic values of t - dna concentrations showed linear relationships of y = 5 . 696x - 25 . 195 ( r 2 = 0 . 9286 ) and y = 8 . 511x - 37 . 456 ( r 2 = 0 . 9608 ) in the range of 20 pm to 20 nm , corresponding to one - cycle and threecycle amplifications , respectively ( figure s6 ) . [SEP]
[CLS] compared with the one - cycle curve , the slope of the df curve corresponding to three cycles was improved by nearly 1 . 5 times , which contributes to the effective signal amplification . [SEP]
[CLS] there existed two abnormal values : the point at 0 . 2 mm was mainly due to the complex nanostructures formed among individual dna nanospheres B-nanoparticle , resulting in the enhanced frequency signal ; the point at 2 pm was mainly due to the high background signal . [SEP]
[CLS] as a result , the limit of detection was determined as 20 pm . [SEP]
[CLS] the aforementioned results demonstrated that the ttp system worked well in laboratory - based conditions . [SEP]
[CLS] we further tested the robustness and stability of the ttp system in realistic conditions , including different temperature , ph , water B-material quality , and simulated biological fluids ( figure 2 ) . [SEP]
[CLS] we first verified that the formation of dna nanospheres B-nanoparticle was not affected in the temperature range of 4 c - 45 c by gel B-technique electrophoresis I-technique ( figure s7a ) . [SEP]
[CLS] we further tested the temperature stability ( 15 c - 45 c ) of the ttp system on qcm device . [SEP]
[CLS] the relative df showed only slight fluctuations within the temperature range of 15 c - 40 c , indicating the temperature stability of the ttp system ( figure s7b and 2a ) . [SEP]
[CLS] the signal became weaker at 45 c because of the instability of au - s bonds at a higher temperature . [SEP]
[CLS] a gradient of ph from 3 . 0 to 13 . 0 was tested . [SEP]
[CLS] the formed dna nanospheres B-nanoparticle maintained integrity in both weak acidic and weak basic buffers except for ph 3 . 0 ( figure s8a ) , which was mainly due to dna degradation induced by protonation of adenine ( a ) , guanine ( g ) , and cytosine ( c ) under strong acid condition . [SEP]
[CLS] the frequency signals were detected distinctly in solutions with different ph from 5 . 0 to 11 . 0 ( figure s8b ) . [SEP]
[CLS] in spite of the slight decrease in acidic buffers of ph 5 . 0 or in basic buffers of ph 11 . 0 , the relative df was sufficiently distinguishable for t - dna detection ( figure 2b ) . [SEP]
[CLS] we further tested the robustness of dna nanospheres B-nanoparticle in various water B-material qualities . [SEP]
[CLS] dna products formed in different environments showed obvious bands in gel holes ( figure s9 ) , which indicated that the formation of dna nanospheres B-nanoparticle was not affected by the water B-material quality . [SEP]
[CLS] the robust performance of ttp systems was also tested by qcm experiments . [SEP]
[CLS] the obtained relative df results clearly demonstrated that the ttp system performed reliably in a relatively wide range of non - laboratory water B-material environments , such as pure , river , tap , and rain water B-material ( figure 2c ) . [SEP]
[CLS] finally , to evaluate the clinical feasibility and future potentiality of ttp system , four simulated biological fluids as medium were used to assess the reliability of the ttp system , including simulated sweat , simulated urine , simulated saliva , and fetal bovine serum . [SEP]
[CLS] it was confirmed by both gel B-technique electrophoresis I-technique and frequency curves on qcm that dna nanospheres B-nanoparticle were extremely stable in various simulated biological fluids ( figure s10 ) . [SEP]
[CLS] the relative df of simulated biological fluids had slight fluctuations in the range of g20 % ( figure 2d ) . [SEP]
[CLS] according to these results , the ttp system demonstrated excellent robustness and reliability for t - dna detection in relatively harsh conditions . [SEP]
[CLS] in the aforementioned experiments , we observed that different t - dna concentrations simultaneously influenced df and ddf / dt , which corresponded to hybridization thermodynamics and kinetics , respectively . [SEP]
[CLS] we studied the thermodynamics and kinetics of dna hybridization at the biosensing interface ( figure 3 ) , by taking the first cycle of ttp system in figure s6a as an example . [SEP]
[CLS] in terms of hybrid thermodynamics , the horizontals of frequency signal curves after the addition of t - dna and x 1 ( marked by blue and red , respectively ) were set as the equilibrium states of two hybridization reactions , which reflected the termination of hybridization thermodynamics ( figure 3a ) . [SEP]
[CLS] as df was linear with dm , - df iatadna or df iax1 after the introduction of t - dna or x 1 represented the corresponding hybridization yields , where i was the concentration of t - dna . [SEP]
[CLS] the - df gradually improved linearly within a t - dna concentration range of 20 pm to 20 nm ( figure 3b ) . [SEP]
[CLS] the addition of t - dna and x 1 corresponded to the linear curves of y = 1 . 378x - 5 . 788 ( r 2 = 0 . 9558 ) and y = 4 . 318x - 19 . 407 ( r 2 = 0 . 9115 ) , respectively . [SEP]
[CLS] to clearly understand the hybridization thermodynamics , we defined r i as the hybridization capacity of x 1 to t - dna at t - dna concentrations of i . [SEP]
[CLS] showed that r i had a slight fluctuation around the value of 2 , which indicated that the hybridization capacity remained almost unchanged within a range of t - dna concentrations . [SEP]
[CLS] according to the molecular weight and concentration ratio ( 2 : 1 ) of t - dna and x 1 , we calculated the theoretical ratio of hybridization yield ðr t þ to be approximately 3 . 5 . [SEP]
[CLS] on the basis of experimental and theoretical ratio of hybridization yield , we obtained hybridization efficiency ( u i ) at different t - dna concentrations ( table s2 ) , which reached an average level of 60 % at the biosensing interface . [SEP]
[CLS] we analyzed the influencing factors for the hybridization efficiency between t - dna and x - dna in the following aspects : ( 1 ) the topologies of dna on the chip were uncontrollable , such as the influence of non - specific adsorption ; ( 2 ) the presence of spatial resistance restricted the effective binding of x - dna and t - dna ; ( 3 ) the high electron cloud density on chip surface caused electrostatic repulsion to hinder the binding of x - dna ; and ( 4 ) the x - dna was washed away before binding at a certain flow rate . [SEP]
[CLS] to sum up , hybridization efficiency could be further improved by overcoming these limitations . [SEP]
[CLS] in terms of hybridization kinetics , ddf / dt curves were drawn to display the instantaneous dna hybridization rates at different t - dna concentrations ( van der , where two peaks ( labeled by blue and red ) appeared when the introduction of t - dna and x - dna , respectively ( figure 3d ) . [SEP]
[CLS] we observed that the instantaneous hybridization rates reached a maximum shortly at the beginning of hybridization , and the polymerization lasted for a few minutes , reflecting the rapid performance of ttp system on the biosensing interface . [SEP]
[CLS] to describe the kinetic characteristics of dna hybridization , we plotted the instantaneous hybridization rates - ddf iatadna / dt and - ddf iax1 / dt corresponding to t - dna and x 1 as a function of t - dna concentrations , where i represented the concentration of t - dna ( figure 3e ) . [SEP]
[CLS] the - ddf / dt curves varied linearly within the t - dna concentration range ( 2310 a11 - 2310 a8 m ) . [SEP]
[CLS] the fitted curves were y = 0 . 1029x - 0 . 3270 ( r 2 = 0 . 9261 ) and y = 1 . 467x - 6 . 747 ( r 2 = 0 . 9076 ) , corresponding to the addition of t - dna and x 1 . [SEP]
[CLS] the slope of - ddf iax1 / dt curve was increased by 14 times to that of the - ddf iatadna / dt curve , illustrating the significantly enhanced hybridization kinetics by the ttp system owing to the introduction of x - dna with higher molecular weight . [SEP]
[CLS] we then defined ε i as the ratio of instantaneous hybridization rates to explore the concentration dependence of hybridization kinetics . [SEP]
[CLS] in figure 3f , the ε i increased first with the increase of t - dna concentration in the range of 2310 a12 to 1310 a7 m , reaching as high as 10 , and began to decrease when the t - dna concentration exceeded 10 nm , revealing an optimal kinetic concentration of 10 nm . because [SEP]
[CLS] hybridization kinetics were attributed to both mass transfer and effective collision , the ratio of hybridization rates was limited to effective collision at lower concentrations and to mass transfer at higher concentrations . [SEP]
[CLS] when t - dna was at a lower concentration , the amount of t - dna used for x 1 conjugation was limited , so as to generate a lower collision efficiency . [SEP]
[CLS] in contrast , when t - dna was at a relatively higher concentration , t - dna with higher density caused space crowding at biosensing interface , further obstructing effective mass diffusion and transfer . [SEP]
[CLS] as a result , both the limited t - dna number and the crowded space restricted the dna hybridization , leading to the lower hybridization kinetics . [SEP]
[CLS] from the above analysis , we observed that hybridization thermodynamics and kinetics exhibited different behaviors as a function of t - dna concentration . [SEP]
[CLS] hybridization thermodynamics presented concentration independence , whereas hybridization kinetics depended on the t - dna concentration owing to mass transfer and collision probability , proving guidance for the study of nucleic B-material acid I-material hybridization in interface chemistry . [SEP]
[CLS] to investigate the selectivity and specificity of the ttp system , a series of dna with non - complementary sequences ( m - dna ) were introduced including single - base and multi - base changes relative to normal dna ( n - dna ) ( table s3 ) . [SEP]
[CLS] n - dna functioned as linkers for binding two types of x - dna to generate stable dna nanospheres B-nanoparticle . [SEP]
[CLS] however , metastable dna nanospheres B-nanoparticle were produced when substituted bases existed in a sequence ( asterisk indicated substituted bases ) . [SEP]
[CLS] as the number of substituted bases increased , dna nanospheres B-nanoparticle became more unstable owing to the occurrence of bulges or mismatched bubbles , which reflected in the gradually decreased df signals ( figure 4c ) . [SEP]
[CLS] there were clearly distinguishable signals of m - dna with one to four altered bases compared with n - dna . [SEP]
[CLS] once the number of altered bases reached 4 ( including 4 , 6 , 8 and 10 altered bases ) , the frequency signals remained almost unchanged . [SEP]
[CLS] the phenomenon of sharp reduction was obviously observed from the curve of relative df ( figure 4d ) , suggesting four altered bases as the critical value of frequency signals . [SEP]
[CLS] as a result of the low binding energy , hybridization between m - dna and branched dna was difficult , showing unchanged frequency signals . [SEP]
[CLS] in addition , the intermolecular tension caused by mismatched bases also affected the base pairing . [SEP]
[CLS] interestingly , when only n - dna or m - dna was introduced , there were insignificant differences in the df signal of non - complementary sequences with altered bases of 1 , 2 , and 3 ; however , significant differences were observed after the introduction of x - dna ( figure 4d ) . [SEP]
[CLS] these results indicated that the introduction of x - dna achieved effective signal amplification and that the ttp system had a capability for the discrimination of base - changing dna from normal dna . [SEP]
[CLS] furthermore , we studied the ability of the ttp system to discriminate single - base changes at different sites in a stretch of dna . [SEP]
[CLS] to ensure that the frequency changes were only related to the locations of single - base changes , the single base was set with the same change from c to g at different locations , including m1 - m ( tg16 , middle ) , m1 - t ( tg30 , terminal ) , and m1 - i ( tg11 , internal ) . [SEP]
[CLS] these mismatched sequences with singlebase changes at different positions were clearly distinguishable from each other according to frequency signals , in which df ranked as m1 - m > m1 - t > m1 - i ( figure 4f ) . [SEP]
[CLS] the small differences produced by n - dna or single - base m - dna were amplified by the addition of x - dna , indicating that ttp - based signal amplification obviously distinguished the positions of single - base changes . [SEP]
[CLS] when altered base appeared at the location of 16 or 30 , the mismatch occurred only at the terminal of hybridization chains , which did not affect the pairing of surrounding bases . [SEP]
[CLS] however , for the altered base at the location of 11 , intermolecular mismatch caused incomplete pairing of adjacent bases due to the inherent molecular tension , even though adjacent base pairs were fully complementary ( figure 4g ) . [SEP]
[CLS] in addition , unexpected dna secondary structures also influenced the hybridization binding affinity . [SEP]
[CLS] for example , the gibbs free energy ( dg ) of m1 - i was about a4 kcal / mol when forming stable hairpin secondary structure , whereas m1 - m and m1 - t were only a0 . 44 kcal / mol ( calculated by www . nupack . org ) ( santalucia and hicks , 2004 ) . [SEP]
[CLS] as a result , the relative df of m1 - i was much smaller than that of m1 - m and m1 - t ( figure 4g ) . [SEP]
[CLS] the relative df of m - dna reflected the hybridization thermodynamics , which had a close relationship with binding energy of dna nanospheres B-nanoparticle . [SEP]
[CLS] we therefore systematically analyzed the theoretical binding energy of non - complementary sequences , including multi - base changes and single - base changes at different sites . [SEP]
[CLS] to predict the hybridization situation of target dna ( including n - dna and m - dna ) more effectively and reduce the complexity of algorithm , we reasonably simplified the dna nanospheres B-nanoparticle into a theoretical model of hybrid - dna nanostructures , as demonstrated in figure 4a . [SEP]
[CLS] we obtained the theoretical dg of hybrid - dna nanostructures ( dg h ) under the corresponding experimental conditions ( figure 4b , calculated by www . nupack . org ; . [SEP]
[CLS] the curve of relative dg h varied with the number of altered bases ( figure s11 ) , whose s - type trend was similar to the experimental results by and large . [SEP]
[CLS] however , the appearance of several abnormal points with 4 and 6 altered bases indicated that the probability of the existence of hybrid - dna was not only related to theoretical dg h . [SEP]
[CLS] we therefore introduced hybridization probability ( s ) of single - base changes ( s s ) and multi - base changes ( s m ) , which was affected by the combination of theoretical relative dg h and corresponding equilibrium yields ( q s and q m , described as q ) : s = dg h 3 q where q represented a fraction of equilibrium concentrations ( calculated by www . nupack . org ) of the expected structures to total concentrations . [SEP]
[CLS] q = equilibrium concentrations total concentrations [SEP]
[CLS] in figure 4b , q m values of m4 - dna and m6 - dna ( 0 . 314 % and 0 . 00034 % , respectively ) were dramatically lower than those of the other sequences ( m1 - dna , 99 . 3 % ; m2 - dna , 60 . 7 % ; m3 - dna , 47 . 2 % ) , indicating that the content of hybrid - dna constructed from m4 - dna and m6 - dna in solution was extremely low , which was another source of the abnormal points . [SEP]
[CLS] as a result , dg h and q m synergistically affected the probability of the existence of hybrid - dna in solution . [SEP]
[CLS] the resulting curve of s m had great agreement with the experimental results ( figure 4e ) . [SEP]
[CLS] in terms of m - dna with single - base changes at different sites , a similar phenomenon emerged . [SEP]
[CLS] the theoretical dg h was essentially consistent with the experimental frequency signals ( figure s12 ) except for the abnormal point of m1 - i . the lower q s of m1 - i ( 72 . 2 % ) relative to m1 - m and m1 - t was another primary cause of lower frequency signal . [SEP]
[CLS] as a result , the s s values of m - dna with single - base changes were in agreement with experimental values ( figure 4h ) , indicating the reliability of the theoretical model . [SEP]
[CLS] taking advantage of the established model , we predicted the hybridization probability of single - nucleotide change in long - strand dna sequences ( table s4 ) . [SEP]
[CLS] with the increase of strand length , dna sequences with single - base change tended to more occurrence of hybridization . [SEP]
[CLS] the hybridization probability reached 99 % when an altered base appeared in a stretch of 270 - bp sequences , approaching the length limit of discrimination . [SEP]
[CLS] in general , the abovementioned results showed that ttp system exhibited superior performances on the discrimination of altered dna , including the number of altered bases ( 1 - 4 nt ) and the positions of single - nucleotide changes . [SEP]
[CLS] moreover , ttp - based hybridization model effectively analyzed the hybridization probability of expected dna nanostructures , which was influenced by both binding energy and equilibrium yield . [SEP]
[CLS] to explore the fundamental reason why base - changing dna was effectively discriminated by the ttp system , we focused on the hybridization thermodynamics and kinetics in the first cycle of m - dna and n - dna in the ttp system ( figure 5a ) . [SEP]
[CLS] first , we studied the hybridization thermodynamics of m - dna . [SEP]
[CLS] as shown in figure 5b , the - df values after the addition of m - dna or n - dna and x 1 ( marked by blue and red , respectively ) reflected the corresponding hybridization yield , owing to the linearity between df and dm . [SEP]
[CLS] we observed a dramatic decline on the - df values of x 1 at the point of 4 - base change ; however , no dramatic change was observed on the - df values of m - dna or n - dna . [SEP]
[CLS] the phenomena indicated that the introduction of x 1 significantly increased the small differences of hybridization thermodynamics between m - dna and n - dna . [SEP]
[CLS] according to the theoretical hybridization yield ( r t = 3 . 5 ) calculated above , hybridization efficiencies ( u i ) of m - dna and n - dna were approximate 85 % . [SEP]
[CLS] second , we analyzed the hybridization kinetics by plotting the ddf / dt curves of m - dna and n - dna with time and the - ddf / dt values with the number of altered bases ( figures 5c [SEP]
[CLS] the ratio of the instantaneous hybridization rates of x 1 and n - dna or m - dna increased to approximately 5 times at altered bases less than 4 , demonstrating that the ttp system amplified the small differences of hybridization kinetics performance between n - dna and m - dna in an identical reaction . [SEP]
[CLS] the above analysis indicated that the differences of hybridization kinetics and thermodynamic properties are the fundamental reasons for m - dna discrimination in the ttp system . [SEP]
[CLS] single - base changes are associated with various biological and biomedical implications , such as genetic diseases , antibiotic resistance , and cancers ; the distinction of single - base changes was , therefore , important for revealing biological mechanisms and exploring the methods for disease treatment . [SEP]
[CLS] current methods for distinguishing single - base changes mainly include pcr , sequencing technology , and hybridization . [SEP]
[CLS] the traditional pcr and sequencing technology are mostly applied for distinguishing single - base changes ; however , these methods have some intrinsic disadvantages , such as time consumption , high cost , complicated operation , low specificity , and so on . [SEP]
[CLS] as a result , a large number of hybridization - based methods are developed for detecting base mutations , including protein - assistant hybridization , thermal denaturation - based hybridization , and direct hybridization . [SEP]
[CLS] protein - assistant hybridization needs to maintain enzyme B-property activity I-property during manipulating , and thermal denaturation - based hybridization needs the participation of precise temperature - control device , which undoubtedly increase the cost and difficulty of use . [SEP]
[CLS] relatively speaking , direct hybridization is the easiest way to operate . [SEP]
[CLS] our ttp system depended on the direct hybridization of nucleic B-material acid I-material , and there is no involvement of any enzyme and temperature control device , which made the operation very simple and low cost . [SEP]
[CLS] the key to direct hybridization - based methods is the hybridization design . [SEP]
[CLS] the common strategies are to design the constrained probes or to modify chemical molecules on hybridization probes . [SEP]
[CLS] relative to some constrained probes ( e . g . , hairpin probes and strand displacement probes ) and modified molecule ( e . g . , molecule beacons , artificial nucleic B-material acid I-material ) probes , ssdna as probes in our ttp system is the easiest and cheapest form . [SEP]
[CLS] moreover , the ssdna probes and x - dna amplifiers avoided the steps of toehold migration and structure de - conformation , enabling the rapid performance of the ttp system . [SEP]
[CLS] in addition , the use of qcm biosensor provided convenience for detecting singlebase changes with high specificity and selectivity , and for studying hybridization kinetics and thermodynamics of nucleic B-material acid I-material . [SEP]
[CLS] as a whole , compared with the serious drawbacks in current methods , such as complicated operation , time - consumption , high cost , and low specificity , our ttp system possessed multiple unprecedented advantages including portable design , rapid manner , robust performance , enzyme - free amplification , label - free signal readout , and labor - free usage , fully satisfying the requirements of point - of - care testing . [SEP]
[CLS] in conclusion , we proposed a branched dna - based ttp strategy for the enzyme - free and fast discrimination of base - changing dna from normal dna and developed a theoretical method for analyzing the hybridization probability of expected dna structures . [SEP]
[CLS] in ttp system , target dna triggered the self - assembled polymerization of two species of x - dna to construct dna nanospheres B-nanoparticle , realizing rapid target recognition and signal amplification for picomolar - level dna detection . [SEP]
[CLS] the formation of dna nanospheres B-nanoparticle only occurred in the presence of target dna , without the involvement of any enzymes , ensuring the specificity and simplicity of the system . [SEP]
[CLS] in addition , the ttp system exhibited excellent robustness and stability in a wide range of realistic conditions , including different ph and temperature , as well as a series of water B-material sources and simulated biological fluids . [SEP]
[CLS] furthermore , we analyzed the hybridization thermodynamics and kinetics of the ttp system at different t - dna concentrations : the hybridization kinetics depended on the t - dna concentrations , whereas thermodynamics remains almost constant , providing insight for interface chemistry . [SEP]
[CLS] significantly , the ttp system had a powerful capability of discriminating not only the number of multi - base changes but also the positions of single - base changes . [SEP]
[CLS] we proposed a theoretical model involving the synergistic effect of binding energy and equilibrium yields , based on which the hybridization probability of single - base and multi - base - changing dna was analyzed and had a quite good correlation with the experimental results . [SEP]
[CLS] we envision that our ttp system will be a powerful tool for the enzyme - free and fast discrimination of base - changing dna in poct and the theoretical analysis of nucleic B-material acid I-material hybridization . [SEP]
[CLS] we implemented a new system for the discrimination of single - nucleotide changes . [SEP]
[CLS] although our ttp system exhibited excellent robustness and stability in a wide range of realistic conditions , as well as a series s3 . [SEP]
[CLS] dna sequences of non - complementary dna with different number or positions of mismatched bases ( related to figure 4 ) . [SEP]
[CLS] real - time monitoring of layer - by - layer ttp system for step - by - step signal amplification ( a ) frequency and dissipation changes of the self - assembled dna nanospheres B-nanoparticle in the ttp system . [SEP]
[CLS] stepped frequency and dissipation change signals clearly showed the feature of layer - by - layer ttp system to form dna nanospheres B-nanoparticle via four cycles . [SEP]
[CLS] in the first cycle , a dramatic change reflected the efficient hybridization , and the accumulated introduction of t - dna and x - dna obviously increased the signals . [SEP]
[CLS] ( b ) corresponding dm of the self - assembled dna nanospheres B-nanoparticle in the ttp system calculated by sauerbrey equation . [SEP]
[CLS] the ttp system exhibited excellent robustness and reliability in relatively harsh environments ( a ) the relative df signals demonstrated the robustness of the ttp system under different temperatures ( n = 3 , mean g sd ) . [SEP]
[CLS] ( b ) the relative df signals demonstrated the robustness of the ttp system under different ph ( n = 3 , mean g sd ) . [SEP]
[CLS] ( c ) the relative df signals demonstrated the robustness of the ttp system under different water B-material environments ( n = 3 , mean g sd ) . [SEP]
[CLS] ( d ) the relative df signals demonstrated the robustness of the ttp system under different simulated biological fluids ( n = 3 , mean g sd ) . [SEP]
[CLS] ttp system showed enhanced hybridization thermodynamic and kinetic performance ( a ) the df curves changing with time . [SEP]
[CLS] ( b ) the - df curves after the introduction of t - dna ( marked with blue ) and x 1 ( marked with red ) changing with t - dna concentrations . [SEP]
[CLS] ( c ) r i reflecting dna hybridization thermodynamics , which remained constant within a concentration range of 2310 a7 to 2310 a12 m . [SEP]
[CLS] ( d ) the ddf / dt curves changing with time , reflecting instantaneous hybridization rates of t - dna with different concentrations . [SEP]
[CLS] ( e ) the - ddf / dt curves after the introduction of t - dna ( marked with blue ) and x 1 ( marked with red ) changing with t - dna concentrations . [SEP]
[CLS] ( f ) ε i increasing first and then decreasing within a concentration range of 2310 a7 to 2310 a12 m , which was limited by probe density and steric hindrance . [SEP]
[CLS] ttp system achieved the discrimination of multi - base changes and single - base changes at different locations from normal dna ( a ) the model of hybrid - dna based on the hybridization process of n - dna or m - dna ( non - complementary dna with altered bases ) with two types of x - dna . [SEP]
[CLS] ( b ) the theoretical values of binding energy and equilibrium yields . [SEP]
[CLS] ( c ) the frequency curves of base - changing dna with different number of altered bases . [SEP]
[CLS] ( d ) the relative df of base - changing dna with different number of altered bases ( n = 3 , mean g sd ) . [SEP]
[CLS] ( e ) the theoretical s m of base - changing dna with different number of altered bases . [SEP]
[CLS] the experimental results were in good agreement with the theoretical analysis . [SEP]
[CLS] ( f ) the frequency curves of base - changing dna with single - base changes at different positions . [SEP]
[CLS] ( g ) the relative df of base - changing dna with single - base changes at different positions ( n = 3 , mean g sd ) . [SEP]
[CLS] ( h ) the theoretical s s of base - changing dna with single - base changes at different positions . [SEP]
[CLS] the experimental results were good agreement with the theoretical analysis . [SEP]
[CLS] 5 . improved thermodynamic and kinetic performance by ttp system for the discrimination of m - dna and n - dna ( a ) the frequency curves of m - dna and n - dna changing with time . [SEP]
[CLS] ( b ) the - df values after the introduction of t - dna ( marked with blue ) and x 1 ( marked with red ) changing with the number of altered bases , reflecting hybridization efficiency with different number altered bases . [SEP]
[CLS] ( c ) the ddf / dt curves changing with time , reflecting instantaneous hybridization rates of m - dna with different number of altered bases . [SEP]
[CLS] ( d ) the - ddf / dt values varying with the number of altered bases , reflecting the hybridization kinetics properties of m - dna in ttp system . [SEP]
[CLS] s2 . [SEP]
[CLS] effect of reaction time on formation of dna nanospheres B-nanoparticle in ttp system [SEP]
[CLS] s10 . the simulated body fluids stability of ttp system ( related to figure 2 ) . [SEP]
[CLS] s11 . [SEP]
[CLS] the theoretical relative δg h curves of hybrid - dna with different [SEP]
[CLS] the altered base sites were marked in red . [SEP]
[CLS] the hybridization efficiency at different dna concentrations in ttp system ( related to figure3 ) . [SEP]
[CLS] dna - functionalized gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) are popular hybrid materials with applications in directed assembly , biosensor development , and drug delivery . [SEP]
[CLS] this system is particularly interesting due to the versatile optical and catalytic properties of aunps B-nanoparticle combined with the molecular recognition and programmable properties of dna . [SEP]
[CLS] instead of emphasizing applications , this review focuses on the interfaces including adsorption of thiol B-material and dna bases , colloidal stability of aunps B-nanoparticle , and related methods for preparing this conjugate . [SEP]
[CLS] the effects of salt B-material , ph , proteins B-material , and dna base composition are discussed for controlling the conformation , density , and stability of dna . [SEP]
[CLS] hybridization of dna on aunps B-nanoparticle , dna - directed growing of materials , and cellular uptake of this conjugate are discussed as examples to highlight the importance of the interfaces . [SEP]
[CLS] our understanding of these biointerfaces is still far from complete , and a few future research opportunities for engineering hybrid materials are described . [SEP]
[CLS] aside from the structural roles , certain dna sequences also carry chemical functions . [SEP]
[CLS] dna aptamers are popular ligands for molecular recognition . [SEP]
[CLS] aptamers are often around 20 - 50 nucleotides long , and aptamers for various small molecules , metal B-material ions B-material , and proteins B-material have been reported . [SEP]
[CLS] dna sequences with catalytic activities are called dnazymes , which often require metal B-material ions B-material for catalysis and are useful for metal B-material detection . [SEP]
[CLS] aptamers and dnazymes are collectively called functional dna , referring to their chemical functions . [SEP]
[CLS] compared with proteins B-material , dna has [SEP]
[CLS] conjugation of biomolecules on nanomaterials B-material has resulted in numerous hybrid materials . [SEP]
[CLS] the molecular - recognition , catalytic , and structure - directing functions of biopolymers B-material are coupled to nanomaterials B-material . [SEP]
[CLS] nanomaterials B-material in turn offer optical , electrochemical , catalytic , and magnetic B-property properties useful for sensing , diagnostics , and drug loading . among the diverse bio - nano combinations , dna - functionalized gold B-nanoparticle nanoparticles I-nanoparticle ( dna - aunps B-nanoparticle ) have played a key role in the field , and its legacy continues to date . [SEP]
[CLS] for applications , dna - aunps B-nanoparticle have been used for directed assembly of nanomaterials B-material to form random structures , colloidal crystals , and molecule - like nanoparticle B-nanoparticle oligomers . [SEP]
[CLS] in addition , various biosensors with colorimetric , fluorescence B-property , light - scattering , raman , and electrochemical detection were designed for nucleic B-material acids I-material , metal B-material ions B-material , small molecules , proteins B-material , and cells B-material . [SEP]
[CLS] finally , these conjugates have interesting applications in drug delivery . [SEP]
[CLS] many reviews have covered dna - aunps B-nanoparticle in various aspects including preparing the conjugates , sensing , directed materials assembly , and drug delivery . [SEP]
[CLS] the focus of the current review is on the biointerfaces . [SEP]
[CLS] biointerfaces often refer to the interfaces between biomaterials and biological fluids , cells B-material , or tissues . [SEP]
[CLS] in this article , we use biointerfaces to describe the dna - aunp B-nanoparticle interface and also interfacing dna - aunps B-nanoparticle with other species such as ions B-material , proteins B-material , and cells B-material . [SEP]
[CLS] sometimes , we just use ' ' interfaces ' ' in short . [SEP]
[CLS] the surface science of gold B-material has been well developed , and can be easily controlled by the self - assembled monolayer technology . [SEP]
[CLS] the interfaces between dna and aunps B-nanoparticle are critical for enabling and even driving their applications . [SEP]
[CLS] a fundamental reason is the strong interactions between dna bases and gold B-material surface . [SEP]
[CLS] our goal here is not a comprehensive review but to [SEP]
[CLS] with around 1 , 000 papers published yearly , dnafunctionalized gold B-nanoparticle nanoparticles I-nanoparticle are a useful model system for demonstrating many bionano concepts , exploring fundamental colloidal and materials science problems , and showing various practical applications in sensing , directed assembly of nanoparticles B-nanoparticle , and targeted drug and gene delivery . [SEP]
[CLS] the optical , electric , and catalytic properties of gold B-material are nicely combined with the molecular recognition , catalytic , and structure - directing properties of dna . [SEP]
[CLS] the rich interfacial interactions between dna and gold B-material , and between dna and solution components , have enabled and affected many of their properties . [SEP]
[CLS] by understanding the interfaces , more controllable , reproducible , and efficient hybrid materials can be designed and new applications envisioned to continue the legacy of this hybrid . [SEP]
[CLS] highlight representative examples to illustrate the role of interfaces . [SEP]
[CLS] we focus on the fundamental aspects of the field with the hope of obtaining a solid understanding for future research and applications . [SEP]
[CLS] with excellent plasmonic properties , aunps B-nanoparticle have extremely high extinction coefficients , allowing visual observation at low - nanomolar and even picomolar concentrations , while organic dyes need to be at micromolar concentrations to achieve the same extinction ( figure 1a ) . [SEP]
[CLS] in addition , the color of aunps B-nanoparticle can change from red to blue as the particles approach each other , allowing visual colorimetric detection ( figure 1b ) . [SEP]
[CLS] large aunps B-nanoparticle ( e . g . , > 40 nm ) strongly scatter light , while the scattering property is also significantly affected by the interparticle distance . [SEP]
[CLS] against a dark background , light scattering is also highly sensitive ( figure 1c ) . [SEP]
[CLS] aunps B-nanoparticle are strong fluorescence B-property quenchers I-property , with close to 100 % quenching efficiency for fluorophores from blue to far - red emission ( figure 1d ) . [SEP]
[CLS] adsorption of molecules may also affect the localized surface plasmon resonance ( lspr ) properties . [SEP]
[CLS] aunps B-nanoparticle can strongly enhance raman signals ( figure 1e ) , and are good conductors of electrochemical sensors . [SEP]
[CLS] small aunps B-nanoparticle act as catalysts B-property for many redox reactions ( figure 1f ) . [SEP]
[CLS] finally , aunps B-nanoparticle have high surface energy and can strongly adsorb thiol B-material , halides , and organic molecules including all the nucleotides , which in turn can alter the surface property of aunps B-nanoparticle . [SEP]
[CLS] clean gold B-material surfaces are very hydrophilic B-property but are easily contaminated in air , leading to contact angles greater than 90 . [SEP]
[CLS] aunps B-nanoparticle are dispersed in water B-material , which can protect them from airborne contamination , thus retaining a consistent surface property for a long time . [SEP]
[CLS] the most commonly used aunps B-nanoparticle for dna attachment are prepared by reducing haucl 4 with citrate , where some of the remaining citrate molecules are adsorbed on the surface ( figure 1a , the 13 - nm aunp B-nanoparticle surface ) . [SEP]
[CLS] raman signals from the au - cl bond can also be detected due to the chloride B-material used in the gold B-material source . [SEP]
[CLS] both ligands afford some negative charges for weak electrostatic stabilization , while adding salt B-material can result in aggregation of aunps B-nanoparticle . [SEP]
[CLS] some common buffers such as hepes can also strongly adsorb on aunps B-nanoparticle and affect its colloidal properties . [SEP]
[CLS] a key challenge is to retain the function of dna on gold B-material surfaces while still keeping the colloidal stability of the aunps B-nanoparticle . [SEP]
[CLS] since the invention of chemical synthesis of dna , dna oligonucleotides have become a very important biopolymer B-material in materials science . [SEP]
[CLS] during synthesis , dna can be modified at almost any position with a diverse range of functional groups . [SEP]
[CLS] the persistence length of a duplex dna is about 50 nm , corresponding to 150 base pairs , which is readily achievable by synthesis . [SEP]
[CLS] adding a base pair increases the length by 0 . 34 nm , and thus dna can be controlled with sub - nanometer precision . [SEP]
[CLS] dna is highly programmable , and many impressive nanostructures have been prepared by dna . [SEP]
[CLS] dna and functional dnamuch higher stability and is easier to modify . [SEP]
[CLS] with both structural and functional properties , dna is an ideal biopolymer B-material for materials science . [SEP]
[CLS] biointerfaces : adsorption via terminal thiol B-material and nucleobases within the context of aunps B-nanoparticle , a terminal thiol - modified dna is popular for its conjugation . [SEP]
[CLS] dna is highly negatively charged due to its phosphate backbone ( figure 2a ) , and aunps B-nanoparticle with a high density of dna layer have excellent colloidal stability by both electrostatic and steric stabilization from the dna . [SEP]
[CLS] thiolated dna has been used to functionalize au surfaces for a long time due to the high au - s affinity ( over 200 kj / mol ) , although dna desorption upon long - term storage was observed . [SEP]
[CLS] interestingly , the nature of the au - s bond is still under investigation [SEP]
[CLS] the interactions between the dna bases and aunps B-nanoparticle introduce complications . [SEP]
[CLS] all four dna bases can adsorb on aunps B-nanoparticle via the keto and imino groups ( figure 2b ) . [SEP]
[CLS] various theoretic and experimental studies indicate that adenine ( a ) has the highest affinity , thymine ( t ) has the lowest affinity , while the position of cytosine ( c ) and guanine ( g ) varies from different measurements . [SEP]
[CLS] the strength of adsorption is quite high ( e . g . , greater than 100 kj / mol for each base in vacuum ) . [SEP]
[CLS] the negatively charged phosphate backbone does not bind to the au surface , while a phosphorothioate replacing one of the nonbridging oxygen B-material atoms I-material with sulfur B-material has an affinity even stronger than the dna bases . [SEP]
[CLS] since citrate - aunps B-nanoparticle and dna are both negatively charged , their interactions are strongly affected by ionic strength , ph , and the local environment . [SEP]
[CLS] first , salt B-material is needed to screen their long - range charge repulsion to allow dna to approach aunps B-nanoparticle for adsorption . [SEP]
[CLS] adsorption of a nonthiolated dna is promoted when nacl is higher than 30 mm ( figure 2c ) , and different salts B-material have different abilities for charge screening . [SEP]
[CLS] among the group 1a metals B-material , cs + is more effective than li + in promoting the aggregation of citrate - capped aunps B-nanoparticle ( figure 2d ) . [SEP]
[CLS] this was attributed to the ability of cs + to dehydrate more easily , making it more effective in binding to the surface of aunps B-nanoparticle to decrease its negative surface potential . [SEP]
[CLS] the initial rate of dna adsorption was also faster in the presence of cs + . [SEP]
[CLS] salt B-material is also needed to achieve a high density of dna by screening charge repulsion among the dna strands on aunps B-nanoparticle . [SEP]
[CLS] interestingly , li + enabled a higher dna density than cs + , and this was attributed to the stronger binding B-event of I-event dna I-event by li + . [SEP]
[CLS] therefore , cations B-material are not simple point charges ; they can affect the dna - aunp B-nanoparticle interfaces in various ways . [SEP]
[CLS] the discussion of salt B-material is often focused on cations B-material for charge screening . [SEP]
[CLS] in fact , anions B-material are also important , mainly through their adsorption on aunps B-nanoparticle as they compete with dna adsorption . [SEP]
[CLS] for halides , their adsorption affinity on au increases with increasing halide size . [SEP]
[CLS] when the concentration of anions B-material is very high ( e . g . , reach molar concentration ) , another effect may come into play by perturbing the water B-material structure , and this is characterized by the hofmeister series . [SEP]
[CLS] discussion on this front has been limited within the context of citrate - capped aunps B-nanoparticle , since they are easily aggregated with even at moderate salt B-material concentrations ( e . g . , below 50 mm ) . [SEP]
[CLS] the solution ph is also critical for the dna - aunp B-nanoparticle system . [SEP]
[CLS] the dna bases are noncharged at neutral ph , and their ( de ) protonation takes place at around three ph units or more away from neutrality . [SEP]
[CLS] when ph is lower than 5 or higher than 9 , the dna duplex starts to destabilize . [SEP]
[CLS] adenine and cytosine can be protonated at around ph 3 , and they may neutralize the negative charges on the phosphate backbone . [SEP]
[CLS] lowering the solution ph also facilitates the formation of certain secondary structured dnas ( e . g . , i - motif , [SEP]
[CLS] parallel [SEP]
[CLS] poly ( a ) duplex , and dna triplex [SEP]
[CLS] citrate - capped aunps B-nanoparticle are quite stable between ph 5 and 9 , although citrate can be partially protonated at low ph . [SEP]
[CLS] despite the strong thiol B-material - gold B-material interaction , attachment of dna to aunps B-nanoparticle is complicated by two issues : protecting the colloidal stability of the aunps B-nanoparticle , and retaining the function of attached dna . [SEP]
[CLS] a simple mixing of dna with aunps B-nanoparticle results in adsorption of only a few dna strands with uncontrolled conformation . [SEP]
[CLS] with a high negative charge density , these adsorbed strands repel incoming dna . [SEP]
[CLS] at this moment , the adsorbed dna cannot hybridize to the cdna due to the strong interactions between dna bases and the aunp B-nanoparticle surface ( figure 3a ) . [SEP]
[CLS] by adding salt B-material to screen charge repulsion and pushing more dna strands to be adsorbed , the initially adsorbed dna bases may be displaced by the thiol B-material group of incoming dna . [SEP]
[CLS] once a high - density dna shell B-material is formed , the conjugate is highly stable and the upright dna strands are functional . [SEP]
[CLS] on a bulk planar gold B-material surface , even 1 m salt B-material can be added all at once to promote dna adsorption . [SEP]
[CLS] however , for aunps B-nanoparticle , a high concentration of salt B-material cannot be added in one shot or the aunps B-nanoparticle would aggregate . [SEP]
[CLS] the mirkin group invented the salt - aging method , whereby nacl was added at small increments ( around 10 - 50 mm each time ) . [SEP]
[CLS] after each salt B-material addition , a few more dna strands can be adsorbed , which in turn allow higher stability to withstand the addition of more salt B-material ( figure 3b ) . [SEP]
[CLS] the whole process takes more than a day to complete . [SEP]
[CLS] to retain the colloidal stability of aunps B-nanoparticle during faster salt B-material aging , surfactants B-property such as sds can be added ( figure 3c ) . [SEP]
[CLS] the resulting conjugates were given the name of spherical nucleic B-material acids I-material ( snas ) . [SEP]
[CLS] with the high curvature of small aunps B-nanoparticle , dna can be more densely packed than that on a planar gold B-material surface ( up to $ 4 - fold higher ) . [SEP]
[CLS] this very high density of highly negatively charged dna has produced many interesting properties , such as sharp melting transitions and higher affinity for its cdna . [SEP]
[CLS] at the same time , compared with free linear dna , snas have higher stability against degradation by nuclease , which was attributed to a high local ionic strength . [SEP]
[CLS] the concept of snas has been expanded beyond gold B-material cores B-material to include hollow snas , liposomal B-nanoparticle snas , and molecular snas . [SEP]
[CLS] in 2012 , we discovered that dna adsorption was significantly accelerated by lowering ph . [SEP]
[CLS] a high density of dna was attached in 3 min at ph 3 , and later it was found that poly ( a ) - containing dna is particularly attractive for this method due to formation of parallel duplex ( figure 3d ) . [SEP]
[CLS] sedighi and krull reported a facile method using positively charged magnetic B-property particles . [SEP]
[CLS] the positive surface generated a high local concentration of dna and aunps B-nanoparticle , achieving fast adsorption ( 1 , 000 - fold enhancement ) . [SEP]
[CLS] inserting an oligoethylene glycol spacer B-material between the thiol B-material group and dna also allowed rapid and quantitative attachment of the dna . [SEP]
[CLS] recently , we found that dna can be efficiently attached by simply freezing the dna and aunp B-nanoparticle mixtures , followed by thawing , with no other reagents needed ( figure 3e ) . [SEP]
[CLS] upon freezing , dna , aunps B-nanoparticle , and salt B-material were all concentrated into the micropockets between ice crystals . [SEP]
[CLS] in addition , dna oligonucleotides were aligned and stretched to expose the ends . [SEP]
[CLS] the basis of adding acids and freezing are still salt B-material related , but the stretched conformation of dna also played a critical role , allowing significantly increased kinetics of dna adsorption and retaining the upright conformation of dna at the same time . [SEP]
[CLS] to obtain aunps B-nanoparticle with a discrete number of dna strands , they were mixed at a low dna - to - aunp B-nanoparticle ratio , resulting in a poisson distribution of dna density . [SEP]
[CLS] a gel B-technique electrophoresis I-technique experiment was then performed to separate the conjugates . [SEP]
[CLS] another way to prepare dna - aunps B-nanoparticle with controlled density is using a nonthiolated diblock dna with a poly ( a ) block as an anchor . [SEP]
[CLS] the high affinity between adenine and gold B-material makes it useful for this purpose . [SEP]
[CLS] it was first realized on aunps B-nanoparticle by fan ' s group . [SEP]
[CLS] by using unmodified dna , this represents a paradigm - shifting example for making low - cost biosensing elements . [SEP]
[CLS] taking advantage of the high affinity of poly ( a ) , the density of dna is inversely proportional to the length of the poly ( a ) block ( figure 3f ) , and even monovalent dna - aunps B-nanoparticle can be achieved with this method . [SEP]
[CLS] aside from attaching simple dna oligonucleotides , structured dna such as tetrahedrons have also been immobilized on gold B-material surfaces . [SEP]
[CLS] such more rigid structures serve as a spacer B-material to maximally retain the function of biomolecules attached to it . [SEP]
[CLS] while attaching dna to aunps B-nanoparticle is well established , many other surfaces such as metal B-material oxides I-material , mos 2 , and some carbon - based materials could be difficult for covalent dna attachment . [SEP]
[CLS] interestingly , dna - aunps B-nanoparticle can strongly adsorb on all these materials to indirectly attach dna . [SEP]
[CLS] with a dense dna layer , each dna facing the material B-material can interact strongly to rival covalent B-property interactions I-property taking advantage of the polyvalent binding effect , while the dna strands away from the material B-material are still functional . [SEP]
[CLS] the ability to control the relative position of nanoparticles B-nanoparticle with sub - nanometer precision has been long desired by materials scientists . [SEP]
[CLS] dna - directed assembly can enable these applications , and aunps B-nanoparticle are always among the first materials tested for establishing these methods . [SEP]
[CLS] in this section , we outline a few important types of dna - aunp B-nanoparticle assemblies , emphasizing biointerfacial understanding . [SEP]
[CLS] in figure 4a , a and b are aunps B-nanoparticle respectively functionalized with two types of dna . [SEP]
[CLS] specific dna hybridization allows directed assembly of these aunps B-nanoparticle , accompanied with a red to blue color change . [SEP]
[CLS] the aunps B-nanoparticle , however , were randomly organized without a long - range order ( figure 4b ) . [SEP]
[CLS] it took a decade before researchers figured out a way to foster crystalline structures . [SEP]
[CLS] the flexibility of dna is critical , and the dna bonds between aunps B-nanoparticle cannot be too stable . [SEP]
[CLS] otherwise , the assembled aunps B-nanoparticle are trapped kinetically . [SEP]
[CLS] 4c shows an initial strategy by decreasing the hybridization region to 4 - 7 bp to achieve crystal - like aunp B-nanoparticle assemblies . [SEP]
[CLS] those individual weak but collectively strong short duplexes made it possible for the assembled structures to overcome the kinetically trapped states . [SEP]
[CLS] further incorporating flexible spacers B-material before the hybridization region could improve the crystal quality ( fig - ures 4d and 4e ) . [SEP]
[CLS] samples can be annealed at slightly lower than the melting temperature ( t m ) to maximize the collective interactions between sticky ends . [SEP]
[CLS] since then , by tuning the size of aunps B-nanoparticle and length of dna , numerous amounts of such colloidal crystals have been prepared . [SEP]
[CLS] some representative examples with transmission B-technique electron I-technique microscopy I-technique ( tem ) micrographs and the lattice structures are shown in figure 4f . [SEP]
[CLS] the major driving force in dna - directed crystallization is dna base pairing . [SEP]
[CLS] due to a high local dna density , snas behave dramatically differently from free linear counterparts . [SEP]
[CLS] for example , a sharp melting transition ( within a few degrees ) of dnalinked aunps B-nanoparticle is a hallmark of such materials , while free dna takes over 20 to complete melting . [SEP]
[CLS] this reflects cooperative behaviors of snas . [SEP]
[CLS] the distance between aunps B-nanoparticle also affects the stability of the assembly . [SEP]
[CLS] t m increased about 10 c after inserting an a 30 spacer B-material in the thiolated dna ( hs - dna ) , which was attributed to decreased interparticle repulsion . [SEP]
[CLS] salt B-material is important for stabilizing both dna duplexes , but the aunp B-nanoparticle assembly showed a higher t m at each salt B-material concentration . [SEP]
[CLS] overall , a high dna density on aunps B-nanoparticle resulted in a collective behavior of linked dna and stabilized the duplex . [SEP]
[CLS] snas are highly negatively charged due to the dense dna shell B-material . [SEP]
[CLS] electrostatic repulsion is expected when two snas approach each other . [SEP]
[CLS] the polymeric nature of dna also provides steric hindrance and osmotic repulsion in sna crystallization process . [SEP]
[CLS] therefore , salt B-material plays an important role in the sna crystallization . [SEP]
[CLS] to understand repulsion during sna crystallization , seo et al . investigated the gap distance ( distance between adjacent snas in crystals ) as a function of ionic strength in two systems : dna - driven and depletion - forced crystallization . [SEP]
[CLS] they found that the gap distance was a function of ionic strength , similar in both systems . [SEP]
[CLS] they concluded that the steric repulsion is the dominant repulsion force during sna crystallization . [SEP]
[CLS] more recently , they showed that a very high salt B-material concentration ( 1 m nacl ) even provided an additional repulsive force , rather than simple charge screening , resulting in larger - sized crystals . [SEP]
[CLS] modulation of dna - aunp B-nanoparticle crystals after preparing dna - aunp B-nanoparticle crystals , efforts have also been made to further modify them . [SEP]
[CLS] for example , to stabilize dna - linked crystals , mirkin and coworkers proposed a silica encapsulation method by forming a silica B-material shell I-material on preformed dna - aunp B-nanoparticle crystals ( figure 5a ) . [SEP]
[CLS] the solid superlattice exhibited higher stability toward variations in temperature , solvent , ionic strength , and pressure . [SEP]
[CLS] recently , ag + was introduced to stabilize dna - aunp B-nanoparticle crystals . [SEP]
[CLS] + was found to increase the thermal stability of duplex dna by insertion into multiple base pairs . [SEP]
[CLS] after ag metalation , the volume of the crystal contracted by 25 % . [SEP]
[CLS] the formed ag crystal showed excellent stability from ph 5 to ph 11 in various organic solvents and temperatures . [SEP]
[CLS] such high stability was attributed to the chemical bonding between ag + and dna bases . [SEP]
[CLS] superstructures of dna - aunps B-nanoparticle without linker dna assembly of dna - aunps B-nanoparticle without linker dna is also possible . [SEP]
[CLS] luo ' s group obtained monolayered dna - aunp B-nanoparticle sheets with hexagonal packing by evaporating dna - aunps B-nanoparticle in confined microholes . [SEP]
[CLS] different from base - pairing - driven crystallization , the structure was directed by nonspecific interactions between the dna layers during evaporation . [SEP]
[CLS] the interparticle spacing can be well controlled by tuning the dna length . [SEP]
[CLS] further kinetic study by small - angle x - ray scattering ( saxs ) revealed that the dna - aunps B-nanoparticle formed crystals only after a certain period of drying , suggesting a concentration threshold was needed . [SEP]
[CLS] tan et al . examined crystallization of dna ( poly - t ) - modified aunps B-nanoparticle at the air - water B-material interface using saxs and found that salt B-material concentration was critical . [SEP]
[CLS] the charge repulsion between dna - aunps B-nanoparticle prevented ordered structures when nacl was lower than 0 . 3 m , while a long - range order structure formed between 1 . 2 m and 2 . 4 m nacl . [SEP]
[CLS] interestingly the authors found that base - pairing - induced crystallization only occurred in a narrow salt B-material range ( around 0 . 3 m ) . [SEP]
[CLS] such a ' ' salting - out ' ' phenomenon of dna - aunps B-nanoparticle without linker dnas was also reported by kewalramani et al . [SEP]
[CLS] they proposed a three - stage model from gas - like to face - centered cubic crystal and then to glass - like with increasing ionic strength . [SEP]
[CLS] another direction of dna - directed assembly is to form molecule - type structures with each ' ' atom B-material ' ' being a aunp B-nanoparticle . [SEP]
[CLS] to achieve this , each aunp B-nanoparticle is often functionalized with just one or a few dna strands , which define the ' ' valency ' ' of the aunp B-nanoparticle . [SEP]
[CLS] liu and liedl reviewed dna - assembled plasmonic materials . [SEP]
[CLS] here , we emphasize the surface chemistry of tuning inter - aunp B-nanoparticle interactions . [SEP]
[CLS] alivisatos and coworkers demonstrated that single - stranded dna ( ssdna ) - linked au - ag dimers exhibited a strong color change under a dark - field microscope . [SEP]
[CLS] since ssdna is more flexible than double - stranded dna ( dsdna ) , the interparticle distance can be tuned by adjusting the ionic strength . [SEP]
[CLS] deng ' s group developed a ' ' ag + soldering ' ' strategy to stabilize the formed aunp B-nanoparticle oligomers and enhance the plasmon coupling efficiency between aunps B-nanoparticle ( figures 5b - 5d ) . [SEP]
[CLS] bis ( p - sulfonatophenyl ) phenylphosphine ( bspp ) - capped aunps B-nanoparticle modified with monovalent dna were used to form dimers , and the excess bspp on the surface served as a binding ligand for ag + ( figure 5b ) . [SEP]
[CLS] the cryo - tem images showed that the interparticle distance dropped from $ 60 nm ( figure 5c ) to $ 2 nm ( figure 5d ) after adding ag + . [SEP]
[CLS] such shortened distance also resulted in a broadened plasmon peak , which was attributed to the strong longitudinal coupling . [SEP]
[CLS] the deng group further prepared aunp B-nanoparticle dimers with strong plasmon coupling directly from bspp - capped aunps B-nanoparticle and ag + . [SEP]
[CLS] different from previous works using aunps B-nanoparticle with preloaded dnas , the investigators found that mechanically shortened fish sperm dnas could stabilize partially aggregated aunps B-nanoparticle . [SEP]
[CLS] with sub - nanometer gaps , the dimers served as an ideal substrate for surface - enhanced raman B-technique spectroscopy I-technique applications . [SEP]
[CLS] these dimers were further thermally sintered to form conductive junctions ( cj ) between aunps B-nanoparticle . [SEP]
[CLS] for 13 - nm aunps B-nanoparticle , with increased temperature , the cj distance increased from $ 7 nm to 17 nm . [SEP]
[CLS] by tuning the particle size and sintering temperature , au structures with charge - transfer plasmon over an 800 - nm range were achieved . [SEP]
[CLS] chan and coworkers demonstrated that cationic B-material polymers B-material can stabilize assembled dna - aunp B-nanoparticle superstructures by condensing the dna layers via electrostatic interactions . [SEP]
[CLS] a core - satellite superstructure was assembled based on dna - directed assembly and the conjugate was dispersed in pbs containing 0 . 01 % ( w / v ) tween 20 ( pbst ) ( figure 5e ) . [SEP]
[CLS] after adding the cationic B-material polyelectrolyte ( i . e . , poly ( allylamine ) ) , the morphology of the assembled superstructures did not change , which was also observed in silica coating B-material . [SEP]
[CLS] however , the overall size of the structure decreased from 87 to 62 nm in diameter , and a compact shell B-material was observed from the tem ( figures 5f - 5h ) , which was attributed to the condensation of dna . [SEP]
[CLS] with a more compact structure , enhanced plasmon coupling was achieved as indicated by the drastic color change after polymer B-material coating B-material ( figure 5i ) . [SEP]
[CLS] after coating B-material with the polyelectrolyte , the structure also exhibited higher stability in biological medium against endogenous nuclease - induced degradation . [SEP]
[CLS] taking advantage of the interactions between dna and metal B-material ions B-material , metal B-material oxides I-material , and metal B-nanoparticle nanoparticles I-nanoparticle , dna has been extensively used for templated growth of nanomaterials B-material . [SEP]
[CLS] some early examples relied on ag + binding to duplex dna followed by adding a reducing B-property agent I-property . [SEP]
[CLS] nam ' s group used hs - dna - modified aunps B-nanoparticle as seeds to grow au @ ag and au @ au core - shell structures ( figure 6a ) . [SEP]
[CLS] they designed a diblock dna sequence with an a 10 - peg 18 block close to the thiol B-material . [SEP]
[CLS] by optimizing the spacer B-material sequence and the amount of silver B-material - staining reagent , they were able to control the thickness of the ag shell B-material without affecting the recognition function of the other dna block . [SEP]
[CLS] by controlling the nacl concentration in the growth process , asymmetric silver B-material shells B-material were achieved . [SEP]
[CLS] control experiments showed that the dna shell B-material was particularly important , and citrate - capped aunps B-nanoparticle only form branched structures or nanoshells B-nanoparticle without gaps . [SEP]
[CLS] later , the same investigators found that the dna sequence affected the morphology of the final products . [SEP]
[CLS] while hs - poly - a - and hs - poly - c - modified aunps B-nanoparticle resulted in uniform nanogaps of $ 1 nm , hs - poly - g - and hs - poly - t - capped aunps B-nanoparticle gave nanohole - like gaps with aggregates and popcorn - like au shells B-material , respectively ( figure 6b ) . [SEP]
[CLS] using saxs , tem , and uv - visible ( uv - vis ) spectra , shen et al . examined the growth process in detail and proposed a five - stage reaction model . [SEP]
[CLS] instead of using hs - dna as capping ligands , the lu group reported nonthiolated dna to direct the growth of various metal B-nanoparticle nanoparticles I-nanoparticle on aunp B-nanoparticle seeds . [SEP]
[CLS] in an initial report , flower - like nanostructures were obtained with a 30 or c 30 , but spherical particles were formed with t 30 . [SEP]
[CLS] a 30 and c 30 adsorb on aunps B-nanoparticle more strongly compared with t 30 , suggesting that dna adsorption could direct heterogeneous growth . [SEP]
[CLS] later , a detailed study of dna - mediated growth on au nanoprisms was carried out to elucidate the role of dna sequence ( figures 6c and 6d ) . [SEP]
[CLS] in the presence of t 30 , the au seeds transformed from prisms to nonagons , hexagons , and sixpoint stars . [SEP]
[CLS] interestingly , the final products in the presence of other sequences were actually the intermediates B-property of that observed with t 30 . [SEP]
[CLS] a two - stage model was then proposed . [SEP]
[CLS] in the first stage , interaction of dna with au controls the deposition of precursors and particle growth . [SEP]
[CLS] for example , at the same dna concentration , high - affinity dnas ( poly ( a ) or poly ( c ) ) retarded the particle growth , and kinetically trapped the formed particles . [SEP]
[CLS] in the second stage , the thickness of growth was mainly controlled by the remaining precursors in the reaction solution . [SEP]
[CLS] biointerfaces in sensing aunps B-nanoparticle possess many useful signaling mechanisms ( figure 1 ) , and dna can take full advantage of the physical and chemical properties of aunps B-nanoparticle for sensing applications . [SEP]
[CLS] the related analytical biosensors need careful consideration of the surface chemistry of aunps B-nanoparticle and its interaction with dna and targets . [SEP]
[CLS] early examples demonstrated target dna - induced assembly of dna - aunps B-nanoparticle into large aggregates , resulting in a red to blue or purple color change ( figure 7a ) . [SEP]
[CLS] such aggregates possess sharp melting transitions , allowing highly selective discrimination of dna with single mismatches ( figure 7b ) . [SEP]
[CLS] the sensitivity of the assays can be enhanced by using larger particles with even higher extinction coefficients , and a 50 pm detection limit can be detected with 50 - nm aunps B-nanoparticle ( $ 1 nm detection limit with 13 - nm aunps B-nanoparticle ) . [SEP]
[CLS] due to a high local dna density , collective hybridization of dna - aunps B-nanoparticle can generate very stable assemblies . [SEP]
[CLS] with such strong interactions , even single terminal base stacking can support interparticle assembly . [SEP]
[CLS] maeda ' s group found that dna - aunps B-nanoparticle hybridized with their cdna to form aggregates at a high salt B-material concentration ( 0 . 5 m ) , but if the cdna had a terminal mismatch , the aunps B-nanoparticle remained dispersed . [SEP]
[CLS] a detailed force measurement revealed that fully hybridized dna on aunps B-nanoparticle exhibited a strong attractive force within a few nanometers , whereas dna - aunps B-nanoparticle with terminal mismatch did not show such an attraction . [SEP]
[CLS] in dna - aunp B-nanoparticle conjugates , the density and thickness of the dna layer can affect its colloidal stability . [SEP]
[CLS] when the adenosine aptamers were attached , in the absence of the target the aptamer adopted a random conformation , and the conjugate aggregated with more than 5 mm mgcl 2 ( figures 7c and 7d ) . [SEP]
[CLS] however , the conjugate remained well dispersed , even with 30 mm mgcl 2 in the presence of adenosine ( figure 7d ) . [SEP]
[CLS] it seems that the structured aptamer - target complex generated more steric repulsion to prevent aunp B-nanoparticle aggregation . it could also be that the folded aptamer disallowed interparticle dna base - pairing interactions . [SEP]
[CLS] without a thiol B-material label , dna can still adsorb on aunps B-nanoparticle via nucleobase - au interactions . [SEP]
[CLS] typically , ssdna and nonstructured dnas adsorb on aunps B-nanoparticle stronger and faster than dsdna or dna with secondary structures ( e . g . , aptamer - target complexes ) . [SEP]
[CLS] in other words , the surface of aunps B-nanoparticle is able to discriminate the folding state of probe dna . [SEP]
[CLS] this difference can be easily visualized by adding salt B-material to induce aggregation of nonprotected aunps B-nanoparticle ( figures 7e and 7f ) . [SEP]
[CLS] since the aunps B-nanoparticle do not have a dense dna shell B-material , they might aggregate because of other reasons , leading to false results . [SEP]
[CLS] for example , most of the sensing work did not consider potential interactions between the analyte and aunp B-nanoparticle surface . [SEP]
[CLS] we recently found that as ( iii ) can strongly adsorb on aunps B-nanoparticle and inhibit dna adsorption , which could cause artifacts in aptamer - based arsenic B-material detection . [SEP]
[CLS] taking advantage of the strong fluorescence B-property - quenching properties of aunps B-nanoparticle , the concept of nano - flare was proposed by mirkin and coworkers in 2007 ( figure 8a ) . [SEP]
[CLS] in their initial design , aunps B-nanoparticle ( 13 nm ) were densely functionalized with an hs - dna , which was then hybridized with a fluorophore - labeled reporter dna resulting in quenched fluorescence B-property . [SEP]
[CLS] target rna can hybridize with the probe dna and release the reporter dna with an increased fluorescence B-property signal . [SEP]
[CLS] this design was named ' ' nano - flare ' ' and has been used to detect and regulate mrna inside cells B-material ( figures 8b and 8c ) . [SEP]
[CLS] compared with the hybridization of free dna in solution , the hybridization on aunps B-nanoparticle behaved thermodynamically differently . [SEP]
[CLS] considering dna - aunps B-nanoparticle as a whole ( i . e . , snas ) , its hybridization with cdna was significantly enhanced compared with that of the free dna , with this enhancement being driven by enthalpy rather than entropy . [SEP]
[CLS] fluorescent B-property melting experiments showed that the dh of sna ( a91 . 3 g 5 . 5 kcal / mol ) was significantly more than that of linear dna ( a40 . 6 g 2 . 4 kcal / mol ) , while the entropy cost for the sna was much higher . overall , the free energy decrease upon hybridization to cdna for sna ( a15 . 5 g 0 . 2 kcal / mol ) was lower than that of a linear dna ( a13 . 4 g 0 . 1 kcal / mol ) , yielding a two - order - of - magnitude higher binding constant for sna . [SEP]
[CLS] this experiment only considered hybridization of one equivalent of cdna ( sna treated as a single entity ) . [SEP]
[CLS] further isothermal titration calorimetry experiments gave more insights into the collective behavior of the dna on sna . [SEP]
[CLS] the linear dna exhibited an expected sigmoidal 1 : 1 binding isotherm . [SEP]
[CLS] however , the sna showed a double sigmoidal binding isotherm , and the ratio was determined to be 4 and 15 , respectively . [SEP]
[CLS] by inserting a longer spacer B-material dna in the sna design , the hybridization enthalpy was close to that of the free linear dna . [SEP]
[CLS] confining dna on aunps B-nanoparticle also affects the efficiency of dna hybridization on sna . [SEP]
[CLS] sna without a spacer B-material ( i . e . , thiolated 12 - mer dna ) can only have 4 % of dna hybridized , which was 10 - fold lower than that of sna with an a 20 spacer B-material . [SEP]
[CLS] while the initial enthalpy - driven hybridization to cdna was very strong , further hybridization became progressively more difficult . [SEP]
[CLS] the melting curves showed that the dsdna on sna became less stable with increasing density . [SEP]
[CLS] the preloaded cdnas on sna generated higher enthalpy penalty for subsequent incoming cdna . [SEP]
[CLS] the hybridization kinetics were also investigated . [SEP]
[CLS] the observed rate of association ( k obs ) of linear dna in solution was 58 % of that of sna . [SEP]
[CLS] aside from simple dna oligonucleotides , functional dna such as dnazymes were also immobilized on aunps B-nanoparticle ( figure 8d ) . [SEP]
[CLS] for example , the lu group prepared uranylspecific dnazyme ( 39e ) - modified aunps B-nanoparticle ( 13 nm ) , and the fluorescently B-property labeled substrate was hybridized to the enzyme strand forming the dnazyme complex on the aunps B-nanoparticle . [SEP]
[CLS] with this nanoprobe B-nanoparticle , these authors were able to image the uranyl ion B-material ( uo 2 2 + ) in a hela cell B-material ( figures 8e and 8f ) . [SEP]
[CLS] this strategy was further modified by introducing caged dnazymes to avoid background cleavage for detection of na + in cells B-material . [SEP]
[CLS] dynamic dna networks were also designed on aunps B-nanoparticle . [SEP]
[CLS] a three - dimensional ( 3d ) dna walker was designed in which aunps B-nanoparticle were densely functionalized with short fluorophore - labeled substrates and a few long walkers ( figure 8g ) . [SEP]
[CLS] the dna walker can hybridize with the substrate and induce substrate cleavage by a nicking endonuclease , indicated by the rapid fluorescence B-property increase ( figure 8h ) . [SEP]
[CLS] in the absence of endonuclease , the fluorescence B-property increase was much slower ( figure 8i ) . [SEP]
[CLS] engineering the surface chemistry is critical for rapid dna walking . [SEP]
[CLS] li and coworkers found that threshold values exist for dna walker density with specific lengths . [SEP]
[CLS] increasing the number of walkers from 0 to 20 per nanoparticle B-nanoparticle accelerated the reaction , while a further increase to 60 decreased the signal . [SEP]
[CLS] the authors proposed that a high density of walkers would interfere with one another due to the interstrand charge repulsion and increased steric hindrance . [SEP]
[CLS] the investigators were able to detect dna targets down to 5 pm using this method . [SEP]
[CLS] sensing based on catalytic activity of aunps B-nanoparticle aunps B-nanoparticle are excellent catalysts B-property with a diverse range of catalytic activities . [SEP]
[CLS] some early applications include the use of aunps B-nanoparticle as seeds to grow large silver B-material particles for enhanced colorimetric sensing . [SEP]
[CLS] even pcr - like sensitivity was achieved with multiple stages of signal amplification . [SEP]
[CLS] using aunps B-nanoparticle to mimic enzyme - like activities has also been extensively reported , such as glucose oxidase ( gox ) and peroxidase . [SEP]
[CLS] dna adsorption may modulate such activities , allowing sensing applications . [SEP]
[CLS] for example , adsorption of ssdna and dsdna on aunps B-nanoparticle inhibited its goxlike activity by 4 - fold and 25 % , respectively . [SEP]
[CLS] since h 2 o 2 is one of the products of glucose oxidation , less h 2 o 2 was produced in the presence of ssdna , which was detected by a colorimetric assay . [SEP]
[CLS] adsorption of ssdna on the aunps B-nanoparticle would block such growth , and adding cdna forming dsdna can further recover the reaction . [SEP]
[CLS] the generated h 2 o 2 can catalyze the further growth of aunps B-nanoparticle with haucl 4 . [SEP]
[CLS] interestingly , the growth was self - limited , which could be inhibited by the adsorption of another oxidation product , gluconic acid , or due to the decreased ph . [SEP]
[CLS] dna adsorption on aunps B-nanoparticle can also alter its peroxidase - like activity . [SEP]
[CLS] based on an inhibition effect , bansal and coworkers detected kanamycin , an antibiotic compound , with high sensitivity ( limit of detection = 1 . 49 nm ) . [SEP]
[CLS] however , hizir et al . found that dna enhanced the peroxidase - like activity of aunps B-nanoparticle for a positive charged substrate , 3 , 3 0 , 5 , 5 0 - tetramethylbenzidine . [SEP]
[CLS] therefore , dna can exert a diverse range of effects , including surface blocking and electrostatic attraction of substrate , to modulate the activity of aunps B-nanoparticle . [SEP]
[CLS] biointerfaces in biomedical applications dna - aunps B-nanoparticle have been widely used in biomedical applications , including drug delivery , regulation of gene expression , and live cell B-material imaging . [SEP]
[CLS] the blood and intracellular environments are very complex . [SEP]
[CLS] since the aunps B-nanoparticle are fully shielded by the dna shell B-material , this shell B-material is the main interface within biological fluids . [SEP]
[CLS] despite being highly negatively charged , the dna - aunp B-nanoparticle conjugates are readily internalized by cells B-material . [SEP]
[CLS] the efficiency of uptake was found to correlate with the density of dna on aunps B-nanoparticle , which was attributed to the adsorption of scavenger proteins B-material leading to cellular uptake . [SEP]
[CLS] interaction of dna - aunps B-nanoparticle with serum exposure of dna - aunps B-nanoparticle to cell B-material culture media resulted in an immediate adsorption of proteins B-material , and such a protein B-material corona I-material may then alter the cellular uptake . [SEP]
[CLS] the formation of a protein B-material corona I-material was indicated by the increased hydrodynamic size and smaller negative surface charge of dna - aunps B-nanoparticle . [SEP]
[CLS] further fluorescence - based assays confirmed that each aunp B-nanoparticle ( 13 nm with 80 dnas ) can adsorb ca . 23 proteins B-material on its surface , and the number of bound proteins B-material positively correlates with the dna density . [SEP]
[CLS] the adsorption of proteins B-material on sna is sequence dependent ( figure 9a ) . [SEP]
[CLS] g - rich dna immobilized on aunps B-nanoparticle showed enhanced ability to form g - quadruplex , and the conjugates are more likely to bind with serum proteins B-material . [SEP]
[CLS] the larger hydrodynamic - sized sna with g - rich dna indicated that more proteins B-material adsorbed on the dna layer ( figure 9b ) . [SEP]
[CLS] the bicinchoninic acid ( bca ) assay also showed that poly - g - aunps B-nanoparticle adsorbed more proteins B-material ( $ 40 ) in comparison with poly - t - aunps B-nanoparticle ( figure 9c ) . [SEP]
[CLS] in addition , more types of proteins B-material were identified on the corona of poly - g - aunps B-nanoparticle , indicated by the page gel ( figure 9d ) . [SEP]
[CLS] mass spectrometric analysis showed that proteins B-material of the immune system preferentially bind to poly - g - aunps B-nanoparticle . [SEP]
[CLS] western blotting analysis was further used to analyze the adsorption of five representative proteins B-material : apolipoprotein b100 , complement factor h , transferrin , complement c3b , and serum albumin . [SEP]
[CLS] three of them , apolipoprotein b100 , complement factor h , and complement c3b , prefer poly - g - aunps B-nanoparticle . [SEP]
[CLS] interestingly , such sequence - dependent protein B-material preference only exists in the sna architecture but not in the linear form of the dna . [SEP]
[CLS] enzyme - induced dna degradation serum components ( e . g . , nucleases , biothiols ) may decrease the dna stability via displacement and / or degradation , which ultimately affects the functionality of dna - aunps B-nanoparticle inside cells B-material . [SEP]
[CLS] the integrity of dna - aunps B-nanoparticle in the presence of various nucleases has been studied . [SEP]
[CLS] the stability of dna - aunps B-nanoparticle hybridized with its cdna has a longer lifetime ( $ 100 min ) in the presence of dnase i compared with its free counterpart ( $ 23 min ) . [SEP]
[CLS] mechanistic studies showed that dnase i binds to dna - aunps B-nanoparticle stronger than to the free dna , but its cleavage of the dsdna on dna - aunps B-nanoparticle was slower . [SEP]
[CLS] the highly charged surface and high local salt B-material concentration were believed to be the major reasons for the increased stability . [SEP]
[CLS] this was also confirmed by a dna density - dependent study , in which lower dna density gave rise to shorter lifetime . [SEP]
[CLS] interestingly , an rna - dna duplex in dna - aunps B-nanoparticle degraded 2 . 5 - fold faster by ribonuclease h ( rnase h ) compared with the free duplex . [SEP]
[CLS] rnase h degrades the rna strand of the rna - dna duplex . [SEP]
[CLS] compared with dnase i , rnase h was more salt B-material tolerant , still showing activity in high kcl concentrations . [SEP]
[CLS] similar to dnase i , rnase h can bind more strongly to the rna - dna duplex on aunps B-nanoparticle than to the free duplex . [SEP]
[CLS] therefore , with retained activity on the dna - aunp B-nanoparticle surface , the high local substrate concentration and enhanced binding affinity actually promoted the rna cleavage . [SEP]
[CLS] the stability of dna - aunps B-nanoparticle in biological fluids was systematically studied by chan and coworkers , and possible pathways of degradation were investigated . [SEP]
[CLS] dna - aunps B-nanoparticle were incubated B-technique with mouse serum and the dna remaining on aunps B-nanoparticle was then analyzed . [SEP]
[CLS] first , physiological concentration of endonucleases ( e . g . , dnase i ) did not have a pronounced effect on dna - aunps B-nanoparticle , and a 500 - fold higher concentration was needed to induce obvious degradation . [SEP]
[CLS] second , these authors found that 3 0 exonuclease - induced degradation was the major pathway . [SEP]
[CLS] third , although dna may be displaced by small molecules ( e . g . , biothiols ) and proteins B-material , they pointed out most biothiols are bound to proteins B-material , and the free cysteine B-material ( 20 mm ) and glutathione ( 1 mm ) did not cause much dna desorption . [SEP]
[CLS] they proposed that proteins B-material were the major factor for dna desorption . [SEP]
[CLS] the dna desorption problem might be solved by modifying dna with multiple thiols B-material , using stronger se - au chemistry , or coating B-material aunps B-nanoparticle with a shell B-material of platinum B-material . [SEP]
[CLS] 144 [SEP]
[CLS] in 2006 , the mirkin group reported the high - efficiency cellular uptake of snas into many cell B-material lines without transfection agents . [SEP]
[CLS] the dna density is important for cellular uptake . [SEP]
[CLS] a critical dna - to - aunp B-nanoparticle ratio of $ 60 ( for 13 aunps B-nanoparticle ) is required to achieve maximal uptake . [SEP]
[CLS] zhang and coworkers recently created molecular snas with a precisely controlled number of dna strands on each polymer B-material core B-material , and found that the efficiency of cellular uptake was linearly dependent on dna density . [SEP]
[CLS] the adsorption of protein B-material was thought to enhance the cellular uptake , but adsorption of bsa or transferrin actually inhibited the cellular uptake . [SEP]
[CLS] to probe the uptake pathway of sna , patel et al . examined the effect of inhibitors on different cellular uptake mechanisms . [SEP]
[CLS] after screening several inhibitors , poly i and fucoidan were found to inhibit more than 60 % of sna uptake . [SEP]
[CLS] poly i and fucoidan are known to bind to scavenger receptors . [SEP]
[CLS] it was suggested that a high density of dna on sna surfaces behaves like polynucleotides B-material with a complex structure , which binds with scavenger receptors . [SEP]
[CLS] more specifically , the sna was internalized into cells B-material by lipid - raft - mediated rather than clathrin - mediated endocytosis B-event . [SEP]
[CLS] enzyme - linked immunosorbent assays were performed to determine the specific receptor B-material of the scavenger receptor B-material family , and the class a of sr ( sr - a ) has a high affinity with sna , with an apparent dissociation constant of $ 5 nm . this affinity is at least 10 - fold higher than that of free linear dna , highlighting the importance of multivalent interactions . [SEP]
[CLS] as described above , the dna sequence on snas affects the composition of the protein B-material corona I-material , which further affects the uptake by macrophage cells B-material . [SEP]
[CLS] while protein B-material corona I-material can form on both poly - t and poly - g snas , the latter showed much higher uptake , which was attributed to the specific adsorption of opsonin proteins B-material on the poly - g sna . [SEP]
[CLS] blocking the complement receptor B-material greatly inhibited the uptake of serum - treated poly - g sna . [SEP]
[CLS] combined with the protein B-material corona I-material analysis , they concluded that poly - g sna facilitated its internalization into macrophage cells B-material by accumulating more specific protein B-material components , which supports the binding with cell B-material - I-material surface I-material receptors I-material . [SEP]
[CLS] since 1996 , this field has been through 24 years of rapidly growing research . [SEP]
[CLS] nowadays we are seeing about 1 , 000 papers on dna - functionalized gold B-nanoparticle nanoparticles I-nanoparticle each year . [SEP]
[CLS] so far , most of the materials - related work is probably driven by curiosity , rational design , and observations . [SEP]
[CLS] it has gradually become clear that understanding the dna - aunp B-nanoparticle interfaces is critical for realizing the full potential of this conjugate . [SEP]
[CLS] all four dna bases have affinities with the aunps B-nanoparticle , and for dna to be functional , such dna base adsorption needs to be avoided . [SEP]
[CLS] for certain applications , such adsorption could also be useful . [SEP]
[CLS] we believe that dna - aunps B-nanoparticle will continue to serve as a model system for learning about bio - nano interfaces . [SEP]
[CLS] by tightly packing a high density of dna , the colloidal stability problem of the aunps B-nanoparticle can be solved . [SEP]
[CLS] at the same time , many useful properties such as tighter binding of cdna and sharp melting transitions have been observed . [SEP]
[CLS] applications in many fields were demonstrated , and we have presented examples for directed materials assembly , biosensors , and drug delivery . [SEP]
[CLS] in each case , we described important surface and interface features . [SEP]
[CLS] looking forward , there are many interesting research opportunities for this material B-material . [SEP]
[CLS] the first enabling aspect is to prepare dna - aunp B-nanoparticle conjugates . [SEP]
[CLS] so far , simple conjugation with a high density of thiolated dna is no longer a difficult task . [SEP]
[CLS] custom - designed dna - aunp B-nanoparticle conjugates are also commercially available . [SEP]
[CLS] however , preparing dna - aunp B-nanoparticle conjugates with a defined number of dna strands and functionalization of different regions of aunps B-nanoparticle with different dna sequences remains challenging . [SEP]
[CLS] it is desirable to develop simple methods to achieve these goals . [SEP]
[CLS] success in this front can enable asymmetrically assembled materials and multiplexed biosensors . [SEP]
[CLS] to achieve this goal , precise engineering of the interface is key . [SEP]
[CLS] for more complicated assembly , more purification steps are required , and the yield of the final product could be very low . [SEP]
[CLS] with this limitation , most of the work on this side is only for proof of concept . [SEP]
[CLS] on the fundamental colloid and interface science level , the role of various cations B-material , anions B-material , and important small molecules needs to be carefully studied . [SEP]
[CLS] cations B-material often act on dna , while anions B-material can compete with dna for the aunp B-nanoparticle surface . [SEP]
[CLS] small molecules may play complex roles depending on their chemical structures and charge . [SEP]
[CLS] these studies are fundamentally interesting and practically important when the materials are interfaced with practical and complex samples . [SEP]
[CLS] for the development of robust biosensors , long - term stability of the conjugate and its reproducible hybridization are very important . [SEP]
[CLS] studies have shown that the attached dna can slowly dissociate and degrade in buffer . [SEP]
[CLS] developing methods to build highly active and stable interfaces is a long - standing challenge that needs to be addressed to move the technologies from the lab to the market . [SEP]
[CLS] while enhanced stability against nucleases compared with the free dna has been shown , the difference has been quite moderate in most cases . [SEP]
[CLS] a recent report showed that nuclease degradation of dna nanostructures caused significant false positives inside cells B-material . [SEP]
[CLS] surface modification could be considered to further enhance the intracellular and plasma stability of the conjugates . [SEP]
[CLS] finally , while the formation of protein B-material corona I-material on dna - aunps B-nanoparticle has been well recognized , its effect on the target recognition of the conjugates ( e . g . , by using aptamers for targeting purposes ) needs to be systematically studied , and strategies to overcome related problems are also needed [SEP]
[CLS] attempts have been made to commercialize dna - functionalized aunps B-nanoparticle , and the most recent effort is probably by exicure to commercialize the sna technology for therapeutic applications . [SEP]
[CLS] with further understanding of biointerfaces and the intrinsic property of the conjugate , the chance of commercial success will be higher . [SEP]
[CLS] in turn , the field will benefit from commercial successes . [SEP]
[CLS] 1 . optical properties of aunps B-nanoparticle ( a ) large aunps B-nanoparticle have high extinction coefficients , ε , much higher than organic dyes . [SEP]
[CLS] ( b ) the color of aunps B-nanoparticle changes from red to blue upon aggregation . [SEP]
[CLS] ( c ) aunps B-nanoparticle can also strongly scatter light , and the scattered color is dependent on the aggregation state of aunps B-nanoparticle . [SEP]
[CLS] ( d ) aunps B-nanoparticle can strongly quench fluorescence B-property , and adsorption can also be detected by lspr . [SEP]
[CLS] ( e ) aunps B-nanoparticle can form hot spots for surface - enhanced raman B-technique spectroscopy I-technique ( sers ) . [SEP]
[CLS] ( f ) aunps B-nanoparticle can catalyze the growth of a silver B-material shell B-material and also act as oxidase or peroxidase mimics . [SEP]
[CLS] interfacing dna with aunps B-nanoparticle ( a ) a dna oligomer with four bases with a backbone phosphorothioate ( ps ) and a terminal disulfide modification . [SEP]
[CLS] the purple circles indicate the possible binding sites with aunps B-nanoparticle . [SEP]
[CLS] ( b ) binding modes and the ranking of adsorption affinity of thiol B-material , ps , and the four bases adsorbed on gold B-material surface with the structures of the bases and ps shown . [SEP]
[CLS] ( c ) kinetics of fluorescence B-property quenching by aunps B-nanoparticle as a function of nacl , indicating the dna adsorption . [SEP]
[CLS] ( d ) effect of cations B-material on aggregation of citrate - capped aunps B-nanoparticle , cs + being stronger than li + for inducing aggregation . [SEP]
[CLS] reprinted with permission from : ( c ) zhang et al . , 41 copyright 2012 , acs ; and ( d ) liu et al . , 42 copyright 2014 , acs . [SEP]
[CLS] methods for conjugating dna on aunps B-nanoparticle ( a ) a low density of dna tends to wrap around the au surface , and the dna cannot hybridize with the cdna . [SEP]
[CLS] ( b ) the typical salt - aging process involving initial dna adsorption , and gradual increase of nacl concentration . [SEP]
[CLS] ( c ) attaching dna in the presence of stabilizing ligands ( e . g . , surfactants B-property ) to improve the colloidal stability of aunps B-nanoparticle . [SEP]
[CLS] ( d ) low - ph loading requiring the dna containing a poly ( a ) block to assemble into a parallel duplex . [SEP]
[CLS] ( e ) the freezing method does not require additional reagents . [SEP]
[CLS] ( f ) anchoring poly ( a ) containing nonthiolated dna on aunps B-nanoparticle with controlled density ; a longer poly ( a ) block yields a lower dna density . [SEP]
[CLS] 4 . dna - directed assembly of aunps B-nanoparticle ( a and b ) scheme ( a ) and tem image ( b ) showing assembly of dna - aunps B-nanoparticle without crystallization . [SEP]
[CLS] aunps B-nanoparticle were modified with two different dnas , and a partial duplex linker . [SEP]
[CLS] ( c ) scheme of aunps B-nanoparticle modified with partially hybridized dna with a four - base overhang for hybridization . [SEP]
[CLS] ( d and e ) saxs pattern of dna - aunp B-nanoparticle single - crystalline domains ( d ) and the integrated data ( e ) . [SEP]
[CLS] with this design , dna - aunps B-nanoparticle formed crystallized structures ( fcc ) . [SEP]
[CLS] ( f ) different crystal structures formed by dna - aunps B-nanoparticle . [SEP]
[CLS] reproduced with permission from : ( b ) mirkin et al . , 5 copyright 1996 , springer nature ; ( d and e ) park et al . , 6 copyright 2008 , springer nature ; and ( f ) macfarlane et al . , 79 copyright 2013 , wiley - vch verlag gmbh & co . kgaa , weinheim . [SEP]
[CLS] modification B-event of dna I-event - aunp B-nanoparticle assemblies ( a ) solidifying dna - aunp B-nanoparticle crystals by a silica layer . [SEP]
[CLS] ( b - d ) scheme ( b ) showing that ag + can shorten and stabilize dna - linked aunp B-nanoparticle dimers by interacting with the surface bspp ligands . [SEP]
[CLS] the tem micrographs of the dimers before ( c ) and after ( d ) adding ag + are shown . [SEP]
[CLS] ( e - i ) enhancing the surface plasmon coupling by cationic B-material polymers B-material . [SEP]
[CLS] ( e ) a scheme showing the assembled core - satellite superstructure . [SEP]
[CLS] tem micrographs of the assembly in ( f ) pbst buffer , ( g ) with mgcl 2 , and ( h ) with a cationic B-material polyelectrolyte ( pe ) are shown . [SEP]
[CLS] ( i ) tem micrographs and photographs showing the addition of pe to the satellite - to - core B-material with increasing stoichiometry ( from 3 : 1 to 30 : 1 ) . [SEP]
[CLS] reprinted with permission from : ( a ) auyeung et al . , 84 copyright 2012 , wiley - vch verlag gmbh & co . kgaa , weinheim ; ( b - d ) wang et al . , 86 copyright 2015 , wiley - vch verlag gmbh & co . kgaa , weinheim ; and ( e - i ) chou et al . , 87 copyright 2016 , acs . [SEP]
[CLS] 6 . dna - directed growth of aunps B-nanoparticle ( a and b ) schemes ( a ) and tem micrographs ( b ) showing the growth of au shell B-material on aunps B-nanoparticle functionalized with a dense hs - dna layer of different sequences . [SEP]
[CLS] ( c and d ) schemes ( c ) and scanning electron B-technique microscopy I-technique micrographs ( d ) showing the growth on au nanoprism seeds in the presence of various dna sequences at different time points . [SEP]
[CLS] reprinted with permission from : ( a and b ) oh et al . , 99 copyright 2014 , acs ; and ( c and d ) tan et al . , 100 copyright 2015 , acs . [SEP]
[CLS] typical designs of colorimetric assays using dna - aunps B-nanoparticle ( a ) aunps B-nanoparticle are densely functionalized with hs - dna , and the target dna assembles the aunps B-nanoparticle to large aggregates with a color change . [SEP]
[CLS] ( b ) melting curves of dna without aunps B-nanoparticle ( red ) and with aunps B-nanoparticle ( black ) by monitoring the absorbance at 260 nm . [SEP]
[CLS] insets showing the derivative curve of each set . [SEP]
[CLS] ( c ) aunps B-nanoparticle are functionalized with sh - aptamer dna . [SEP]
[CLS] the target - aptamer complex can better protect the core B-material particles from salt - induced aggregation . [SEP]
[CLS] ( d ) uv - vis absorbance spectra of au - aptamer ( black ) and au - aptamer with mgcl 2 ( blue ) , and au - aptamer - target with mgcl 2 . [SEP]
[CLS] insets show images of ( 1 ) au - aptamer - target , ( 2 ) au - aptamer , ( 3 ) au - aptamer - nontarget , and ( 4 ) au - nonaptamer - target . [SEP]
[CLS] all solutions contained mgcl 2 ( 30 mm ) . [SEP]
[CLS] ( e ) nonthiolated can adsorb on aunps B-nanoparticle in the absence of targets and prevent salt - induced aggregation . [SEP]
[CLS] ( f ) uv - vis absorbance of aunps B-nanoparticle ( diamonds ) with ssdna ( circles ) , with cdna ( triangles ) , and with duplex ( squares ) . [SEP]
[CLS] inset shows the color changes of aunps B-nanoparticle - ssdna in presence of different amounts of target dna . [SEP]
[CLS] the ratio of target - to - probe dna was 0 , 0 . 2 , 0 . 4 , 0 . 6 , and 1 ( from left to right ) [SEP]
[CLS] reprinted with permission from : ( b ) elghanian et al . , 105 copyright 1997 , american association for the advancement of science ; ( d ) zhao et al . , 107 copyright 2008 , acs ; and ( f ) li et al . , 108 copyright 2004 , national academy of sciences . [SEP]
[CLS] 8 . typical designs of fluorescent B-property assays using dna - aunps B-nanoparticle ( a - c ) scheme ( a ) showing nano - flare for mrna detection . [SEP]
[CLS] a reporter dna is hybridized with dna - aunps B-nanoparticle and displaced by the target dna to produce fluorescence B-property . [SEP]
[CLS] confocal fluorescent B-property micrographs of hela cells B-material incubated B-technique with survivin nano - flare ( b ) and control nano - flare ( c ) . [SEP]
[CLS] ( d - f ) scheme ( d ) showing dnazyme - aunps B-nanoparticle for metal B-material ion B-material detection . [SEP]
[CLS] the substrate was modified with a fluorophore and optionally also a quencher . [SEP]
[CLS] confocal fluorescent B-property microscopic images of hela cells B-material treated with ( e ) or without ( f ) uranyl ions B-material and dnazyme - aunp B-nanoparticle probes . [SEP]
[CLS] the dnazyme signal is in the red channel and the nuclei are stained blue . [SEP]
[CLS] ( g - i ) 3d dna walkers ( g ) . [SEP]
[CLS] target dna unblocks the walker dna ( in blue ) , allowing its hybridization with the fluorophore - labeled substrates , which are subsequently cleaved by a nuclease . [SEP]
[CLS] a walk can induce multiple cleavage events for signal amplification . [SEP]
[CLS] kinetics of fluorescence B-property signal from dna walker ( h ) and control ( only substrate - modified aunps B-nanoparticle ) ( i ) are shown . [SEP]
[CLS] reprinted with permission from : ( b and c ) seferos et al . , 113 copyright 2009 , acs ; ( e and f ) wu et al . , 115 copyright 2013 , acs ; and ( h and i ) yang et al . , 116 copyright 2016 , acs . [SEP]
[CLS] interaction of sna with biological media scheme ( a ) showing protein B-material adsorption onto two different dna ( t - rich and g - rich ) modified aunps B-nanoparticle . [SEP]
[CLS] analysis of proteins B-material on sna after incubating B-technique in 10 % human serum for 24 h at 37 c by dynamic B-technique light I-technique scattering I-technique ( b ) , bca assay ( c ) , and page gel ( d ) . [SEP]
[CLS] reprinted with permission from chinen et al . , 138 copyright 2015 , wiley - vch verlag gmbh & co . kgaa , weinheim . [SEP]
[CLS] in a recent work published in the journal of the american chemical society , the deng group at the university of science & technology of china ( ustc ) reported the use of butanol to dehydrate gold B-nanoparticle nanoparticle I-nanoparticle / dna reaching a record high dna density of spherical nucleic B-material acids I-material ( snas ) . [SEP]
[CLS] dna - functionalized gold B-nanoparticle nanoparticles I-nanoparticle ( dna - aunps B-nanoparticle ) are critical agents in a broad range of applications from biosensing , dna - directed assembly to drug delivery . [SEP]
[CLS] when dna oligonucleotides are densely packed on aunps B-nanoparticle , such conjugates feature unique physicochemical properties such as sharp melting transitions , stronger binding B-event to I-event complementary I-event dna I-event , and highly efficient cellular uptake . [SEP]
[CLS] as such , spherical nucleic B-material acids I-material ( snas ) were used to describe these conjugates . [SEP]
[CLS] the function of snas has a lot to do with its preparation , where a thiolated dna is typically used . [SEP]
[CLS] despite the high affinity between thiol B-material and gold B-material , this conjugation reaction is complicated due to the charge repulsion between dna and aunps B-nanoparticle , as well as the colloidal stability of the aunps B-nanoparticle . [SEP]
[CLS] the classic conjugation method is called salt - aging , in which nacl is stepwise added to screen the charge repulsion and carefully avoid aunp B-nanoparticle aggregation . [SEP]
[CLS] the higher the final concentration of nacl reached , the higher dna loadings on aunps B-nanoparticle . [SEP]
[CLS] the dna strands attached at a later stage can use their thiol B-material group to displace non - specifically adsorbed dna bases from the initially attached dna , forcing the attached dna in an upright conformation . [SEP]
[CLS] to maximize dna loadings , the salt - aging process takes more than a day . [SEP]
[CLS] to shorten the aging time , surfactants B-property were used to stabilize aunps B-nanoparticle , which allowed dna loading to finish within a few hours . [SEP]
[CLS] in 2012 , our group found that at ph 3 , the reaction could be completed in minutes . [SEP]
[CLS] later , we developed a freezing method . [SEP]
[CLS] without additional reagents added , snas were prepared after freezing and thawing aunp B-nanoparticle / dna mixtures . [SEP]
[CLS] recently , deng and coworkers introduced an instant dehydration method for preparing snas and obtained conjugates with record - high dna density . [SEP]
[CLS] the dehydration - driven method has two fast solution - mixing steps ( figure 1 ) . [SEP]
[CLS] first , butanol is added to extract water B-material from the dna and aunps B-nanoparticle mixture , leading to an extremely crowded and volume - reduced dna / aunp B-nanoparticle solution . [SEP]
[CLS] it is believed that rapid thiolated dna - aunp B-nanoparticle interactions happened during this dehydration process . [SEP]
[CLS] second , concentrated snas are diluted or redispersed in an aqueous buffer for stock or use . [SEP]
[CLS] with this dehydration method , the dna ( 59 - mer ) adsorption density on each 15 nm aunp B-nanoparticle reached nearly 400 strands , which was over two to three times of that achieved by the salt - aging or freezing method based on side - byside comparisons . [SEP]
[CLS] the maximum capacity of thiol B-material groups on aunp B-nanoparticle can be estimated by assuming a typical alkythiol self - assembled monolayer structure packed on gold B-material with a footprint of 0 . 214 nm 2 . [SEP]
[CLS] on a 15 nm aunp B-nanoparticle , around 3300 alkylthiols can be adsorbed . [SEP]
[CLS] the dna density obtained from the dehy - dration method reached 12 % of this theoretical maximal capacity for alkylthiols . [SEP]
[CLS] since oligonucleotides are more bulky than simple alkyl chains , the density should be a lot lower . [SEP]
[CLS] while the absolute density may be susceptible to experimental errors , the side - by - side comparison was still impressive . [SEP]
[CLS] as such , this record - high density has broken our current understanding of snas . [SEP]
[CLS] with such a dense dna shell B-material , the snas were still functional for recognizing and hybridizing with target dna . [SEP]
[CLS] two experiments were designed to compare the performance of snas respectively prepared by the freezing and dehydration methods . [SEP]
[CLS] using a fluorescent B-property sensor based on the quenching property of aunps B-nanoparticle , greater sensitivity was observed with the sna prepared with the dehydration method . [SEP]
[CLS] in a dna - directed assembly experiment , the dehydration prepared snas also hybridized with more satellite nanoparticles B-nanoparticle . [SEP]
[CLS] this method worked for spherical aunps B-nanoparticle of different sizes and also for ctab - capped gold B-material nanorods B-nanoparticle , a challenging material B-material for dna attachment . [SEP]
[CLS] the key to this success is to use butanol to extract water B-material from dna / aunps B-nanoparticle . [SEP]
[CLS] butanol has been widely used in concentrating nucleic B-material acids I-material or extracting dyes from nucleic B-material acids I-material . [SEP]
[CLS] the deng group previously used it to concentrate silver B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] the dna density increased as the volume ratio of butanol to water B-material was increased , and peaked when the volume ratio reached 9 : 1 . [SEP]
[CLS] during dehydration , the dna , aunps B-nanoparticle and salt B-material were concentrated in the aqueous phase to accelerate the conjugation . [SEP]
[CLS] in this regard , freezing has a similar effect of removing water B-material . [SEP]
[CLS] it might be that the extent of dehydration caused by freezing is lower , resulting in a lower dna density . [SEP]
[CLS] the authors speculated that the dehydration made the dna slimmer , so that a high dna density can be achieved . [SEP]
[CLS] we believe that the conformation of dna might be important . [SEP]
[CLS] for the low ph method , it prefers to dna sequences with a poly - a spacer B-material due to the formation of the parallel poly - a structure . [SEP]
[CLS] by exploring dna in the frozen state , dna strands were found to be stretched and aligned , which also exposes the terminal thiol B-material . [SEP]
[CLS] considering the nearly irreversible nature of dna attachment in such a short reaction time , dna strands must have the correct conformation upon attachment . [SEP]
[CLS] therefore , besides dehydration , other effects of butanol on sna synthe - sis such as the conformation of dna , remain to be explored . [SEP]
[CLS] besides butanol , tris ( 2 - carboxyethyl ) phosphine hydrochloride ( tcep ) is another key reagent , which is used to reduce disulfide bonds in thiolated dna . [SEP]
[CLS] without tcep , the dna density achieved by the dehydration method was often lower . [SEP]
[CLS] adding a low concentration of nacl ( e . g . , 5 mm ) also showed to be important . [SEP]
[CLS] studying the roles of tcep , nacl and potentially other molecules in this system may provide fundamental insights into this system . [SEP]
[CLS] dna density has a lot to do with the special properties of snas , and previous methods can hardly achieve such a high density . by doubling and even tripling the dna density , new properties and applications may arise from further studies . [SEP]
[CLS] scavenger receptors clear pathogens , transport lipid B-material , and mediate polyanionic ligand uptake in macrophages , but their expression and role in the skin are poorly understood . [SEP]
[CLS] although the epidermal barrier B-property typically excludes nucleic B-material acid I-material entry , topically applied , spherically arranged oligonucleotide nanoconjugates ( spherical nucleic B-material acids I-material [ snas ] ) penetrate mouse skin , three - dimensional ( 3d ) skin equivalents , and human skin . [SEP]
[CLS] we explored the mechanism of sna uptake in normal human epidermal keratinocytes and 3d skin equivalents . [SEP]
[CLS] normal human epidermal keratinocytes and 3d raft treatment with sr - a inhibitors reduced sna uptake by > 80 % . [SEP]
[CLS] the human epidermis expresses sr - as scara3 and , to a lesser extent , marco . [SEP]
[CLS] simultaneous lentiviral knockdown of scara3 and marco reduced sna uptake in normal human epidermal keratinocytes and 3d rafts after topical application , affirming a role for sr - as in sna uptake and 3d raft penetration . [SEP]
[CLS] incubation B-technique of normal human epidermal keratinocytes at 4 o c or with sodium B-material azide prevented sna uptake , suggesting active endocytosis B-event . [SEP]
[CLS] endocytosis B-event inhibitors , immunofluorescence , immunoprecipitation , and knockdown studies localized functional sr - as to flot - 1 - containing lipid B-material rafts throughout the epidermis and cav - 1 - containing rafts only in the upper epidermis . [SEP]
[CLS] these studies suggest a central role for sr - a complexes in epidermal lipid B-material rafts in mediating the uptake of nucleic acid - laden nanoparticles B-nanoparticle . [SEP]
[CLS] spherical nucleic B-material acid I-material ( sna ) nanoconjugates have recently been introduced as promising therapeutic agents for gene regulation therapy , including for skin disorders . [SEP]
[CLS] these conjugates typically consist of short , highly anionic oligonucleotides arranged in a densely packed , spherical configuration on a nanoparticle B-nanoparticle template ( supplementary figure s1 ) . [SEP]
[CLS] this architecture facilitates cellular uptake and stabilizes the oligonucleotides with regard to nuclease - mediated degradation . [SEP]
[CLS] therefore , compared with their linear counterparts , these nanoconjugates are more stable and require neither cationic B-material transfection materials nor additional physical or chemical assistance for their cellular uptake and gene suppression , including in difficult - to - transfect normal human epidermal keratinocytes ( nheks ) . [SEP]
[CLS] snas have exhibited excellent penetration into threedimensional ( 3d ) skin equivalents ( also called organotypic raft cultures ) , mouse skin , and human abdominoplasty skin explants and have been shown to suppress targets in 3d human skin and mouse models of diabetic wounds and psoriasis . [SEP]
[CLS] in contrast , linear nucleic B-material acids I-material are unable to penetrate these tissues , presumably because of the key ability of the stratum corneum and epidermal tight junctions ( components of the epidermal barrier B-property ) to preclude the entry of nucleic B-material acids I-material and other foreign materials . [SEP]
[CLS] indeed , snas targeting tnf for psoriasis are the only known nucleic B-material acid I-material nanoconstructs to be topically delivered in human skin in phase 1 clinical trials . [SEP]
[CLS] sr - as were found to play an important role in the uptake of snas in a mouse endothelial cell B-material line . [SEP]
[CLS] sr - as are type ii transmembrane proteins B-material , originally identified by their ability to recognize and scavenge modified lipoproteins . [SEP]
[CLS] sr - as are now known to perform a wide range of functions , including pathogen clearance , lipid B-material transport , and mediating the uptake of a variety of polyanionic ligands . [SEP]
[CLS] sr - as have been extensively studied for their critical role in immune response in macrophages , but their expression , distribution patterns , and role in other cells B-material and tissues , including in human keratinocytes ( kcs ) and epidermis , are not well - understood . [SEP]
[CLS] using prototypical small interfering rna snas with a 13nm gold B-nanoparticle nanoparticle I-nanoparticle core B-material ( supplementary figure s1 ) , we have explored how snas are taken up so readily in nheks and are able to traverse the differentiated epidermis of 3d human skin equivalents ( hses ) . [SEP]
[CLS] we found that sr - as scara3 and marco are the predominant scavenger receptors expressed in the human epidermis and responsible for sna penetration and uptake after topical delivery . [SEP]
[CLS] knockdown of these scavenger receptors using short hairpin rna ( shrna ) significantly reduced sna uptake in nheks and penetration in 3d hses . [SEP]
[CLS] we also discovered a central role of flotillin , a noncaveolar lipid B-material raft - associated protein B-material , in associating with sr - as to mediate scavenger receptor - promoted sna uptake . [SEP]
[CLS] snas enter primary human kcs ( nheks ) in a dose - and time - dependent manner as demonstrated by confocal microscopy B-technique , as little as 0 . 1 nm cyanine dye cy5 - - labeled gold B-material - core B-material sna ( concentration based on gold B-material and equivalent to 4 nm nucleic B-material acid I-material concentration ) was visible within 2 hours in nheks , with progressively more uptake seen with higher concentrations of snas ( figure 1a ) . [SEP]
[CLS] similarly , the uptake of snas increased as a function of concentration . [SEP]
[CLS] indeed , quantitative analysis of gold B-material by inductively coupled plasma mass spectrometry ( icp - ms ) showed 1 , 890 ae 250 nanoparticles B-nanoparticle on average per cell B-material after 2 hours of incubation B-technique with 1 nm sna ; with 5 nm sna , this value increased to 6 , 128 ae 530 nanoparticles B-nanoparticle per cell B-material ( figure 1a , right ) . [SEP]
[CLS] uptake also depends on time . [SEP]
[CLS] confocal images of cells B-material after the addition of 0 . 25 nm snas showed particle entry as early as 120 minutes with progressively greater fluorescence B-property - labeled accumulation during 8 hours of investigation ( figure 1b ) . [SEP]
[CLS] for the 0 . 25 nm snas , icp - ms quantification showed 2 , 619 ae 390 nanoparticles B-nanoparticle per cell B-material in the nheks after 4 hours and 22 , 824 ae 1 , 930 nanoparticles B-nanoparticle per cell B-material by 8 hours . [SEP]
[CLS] we have previously reported that snas are taken up in hairless mouse skin ( about 7 cell B-material layers thick , in contrast to the 2 - 3 layers of most mouse epidermis ) and human organotypic raft cultures ( hses ) . [SEP]
[CLS] note that all these data assume that the sna architecture remains intact over the time course of the experiments , a reasonable assumption based on previous stability studies . [SEP]
[CLS] we studied whether endocytosis B-event of snas into kcs depends on sr - as , as has been suggested by the requirement for scara1 for gold - cored sna uptake in an endothelial cell B-material line . [SEP]
[CLS] fucoidan ( sigma - aldrich , st . louis , mo ) and polyinosinic acid ( sigma - aldrich ) , ligands that specifically block binding to sr - as but not sr - bs , reduced the uptake of snas by nheks by approximately 90 % , as shown by flow B-technique cytometry I-technique ( p < 0 . 001 for both , not shown ) and confocal imaging ( figure 1c ) . [SEP]
[CLS] icp - ms was used to quantify blockade of uptake by these inhibitors of sr - a function ( 87 . 7 ae 0 . 7 % by fucoidan and 90 . 4 ae 0 . 8 % by polyinosinic acid ; figure 1c ) . [SEP]
[CLS] these data strongly implicate sr - as as the main mediators for sna uptake in nheks . [SEP]
[CLS] consistent with results in monolayer - cultured nheks , sna penetration to basal kcs was blocked in a dose - dependent manner when organotypic raft cultures were pretreated for 24 hours with increasing amounts of polyinosinic acid , as visualized by confocal imaging ( figure 1d ) . [SEP]
[CLS] snas accumulated particularly well in the basal epidermis of 3d - cultured skin equivalents , suggesting the possibility that uptake is accentuated in these cells B-material . [SEP]
[CLS] scara3 is required for sna penetration in 3d human skin models [SEP]
[CLS] little is known about the composition , localization , and role of scavenger receptors in human skin . [SEP]
[CLS] five major subclasses of sr - a transmembrane proteins B-material have been described : scara1 ( also called mrs1 , two isoforms ) , marco ( or scara2 ) , scara3 ( with its two isoforms ) , scara4 , and scara5 . [SEP]
[CLS] to determine sr - a expression in kcs , rt - pcr was performed using primers specific for each sr - a and agarose B-technique gel I-technique electrophoresis I-technique . [SEP]
[CLS] marco , scara3 ( isoforms i and ii ) , and scara5 were expressed , but scara1 ( isoforms i and ii ) and scara5 were undetectable ( figure 2a ) . [SEP]
[CLS] under standard nhek culture conditions ( 0 . 07 mm calcium B-material ion B-material ) , rt - qpcr showed scara3 isoform ii to be the predominant sr - a transcript B-event , comprising 85 . 5 ae 6 . 5 % of the total sr - a mrna , whereas scara3 isoform i , marco , and scara5 comprised 13 . 3 ae 2 . 1 % , 0 . 90 ae 0 . 01 % , and 0 . 2 ae 0 . 01 % , respectively ( figure 2b ) . [SEP]
[CLS] in nheks differentiated by increasing the calcium B-material concentration to 1 . 2 mm , expression of marco and scara5 increased to 8 . 7 ae 2 . 1 % and 2 . 3 ae 0 . 3 % of total sr - a mrna , respectively , whereas scara3 isoform ii expression decreased but continued to predominate ( 76 . 9 ae 8 . 5 % of the total expressed sr - a mrna ) ( figure 2b ) . [SEP]
[CLS] immunofluorescence evaluation of human abdominoplasty skin confirmed the predominance of scara3 and marco expression . [SEP]
[CLS] although scara3 was noted throughout the epidermis , it was preferentially expressed in basal kcs , whereas marco was primarily expressed in the differentiated skin layers , consistent with our rt - qpcr results from two - dimensional kcs ( figure 2c ) . [SEP]
[CLS] in addition , sna uptake decreased as calcium B-material concentration increased in nheks , showing an inverse relationship between kc differentiation and sna uptake ( figure 2d ) . [SEP]
[CLS] to test the relative roles of scara3 , given its predominance overall , and marco , given its increased expression in the more differentiated superficial epidermis , we generated lentiviral shrnas directed against scara3 and marco as well as a lentiviral control vector ( supplementary figures s2 and s3a and b ) . [SEP]
[CLS] we measured the effect of knockdown of specific sr - a classes as well as serial addition of both scara3 and marco shrnas on sna uptake in stably transduced monolayer ( undifferentiated ) nheks . [SEP]
[CLS] as shown by immunofluorescence ( figure 2e ) and quantified by flow B-technique cytometry I-technique ( figure 2f ) , sna uptake was reduced by 48 . 9 ae 3 . 2 % with scara3 knockdown , 21 . 4 ae 5 . 0 % with marco knockdown , and 57 . 2 ae 2 . 0 % with the knockdown of both scara3 and marco in monolayer , undifferentiated nheks . [SEP]
[CLS] to test the relative role of these sr - a classes in epidermal [SEP]
[CLS] penetration [SEP]
[CLS] , we used the shrna - treated nheks with specific sr - a knockdown and the lentiviral vector as a control to generate 3d - cultured hses . [SEP]
[CLS] transduction of scara3 shrna alone or serial addition of both scara3 and marco shrnas markedly reduced the uptake of topically applied 100 nm snas ( figure 2g ) , resembling the reduction in uptake by polyinosinic acid treatment . [SEP]
[CLS] 3d skin equivalents comprising marco shrna - treated nheks alone showed increased basal kc uptake and no reduction in overall uptake compared with control untreated and vector - treated cultures . [SEP]
[CLS] culture medium from each 3d skin equivalent was collected and centrifuged to further test the inhibition of sna penetration ; the visible red color of the accumulated gold B-material particles was absent in the scara3 shrna - transduced cultures and was slightly present in the cultures serially transduced with scara3 and marco shrnas , in contrast to that in the vector - and marco shrna - transduced 3d cultures ( supplementary figure s4 ) . [SEP]
[CLS] to confirm the importance of scara3 in epidermal sna uptake , we tracked the ability of snas to reach basal cell B-material layers by electron B-technique microscopy I-technique . [SEP]
[CLS] by 24 hours after topical application to 3d skin equivalents , snas were visualized easily in basal kcs , either trapped inside the endosomal vesicles or freely released in the cytoplasm ( figure 3a ) . [SEP]
[CLS] consistent with the predominant localization of scara3 in the 3d epidermis , snas were largely detected in basal cells B-material . [SEP]
[CLS] in 3d cultures with scara3 knockdown , the number of gold B-nanoparticle nanoparticles I-nanoparticle in basal kcs was reduced by 86 % compared with that in lentiviral vector controls on the basis of manual counts by two blinded investigators ( mean counts 74 ae 8 sna per basal cell B-material in vector - transduced 3d rafts versus 10 ae 2 sna per basal cell B-material in 3d rafts with scara3 knockdown , p < 0 . 001 with n ¼ 50 basal cells B-material counted for each condition ) ( figure 3b ) . [SEP]
[CLS] to determine whether uptake requires active endocytosis B-event , an energy - dependent process , nheks were preincubated at 4 o c or in the presence of sodium B-material azide ( figure 4a ) . [SEP]
[CLS] pretreatment of nheks at 4 o c for 1 hour , followed by a 1 - hour incubation B-technique with 0 . 25 nm snas at 4 o c reduced the uptake of snas by 68 . 9 ae 3 . 2 % in comparison with incubation B-technique at 37 o c . [SEP]
[CLS] pretreatment of nheks with sodium B-material azide , which blocks adenosine triphosphate synthesis needed for endocytosis B-event , also decreased the uptake of 0 . 25 nm sna by 72 . 8 ae 2 . 8 % , as determined by icp - ms quantification ( figure 4a ) . [SEP]
[CLS] these studies confirmed that sna uptake requires endocytosis B-event . [SEP]
[CLS] to evaluate whether this endocytosis B-event of snas occurs through clathrin - mediated endocytosis B-event , cav - and / or lipid raft - mediated endocytosis B-event , or macropinocytosis , the three major endocytotic mechanisms for cellular uptake , we treated cells B-material with inhibitors of endocytotic pathways . [SEP]
[CLS] initially , we established concentrations of inhibitors that caused minimal to no toxicity B-property in nheks to determine test concentrations ( supplementary figure s5 ) . [SEP]
[CLS] sna uptake did not appear to require clathrin - mediated endocytosis B-event based on lack of change in sna uptake with potassium B-material depletion using transferrin as a positive control ( supplementary figure s6a and b ) or macropinocytosis given the failure of 100 mm wortmannin to block uptake with dextran B-material as a positive control ( supplementary figure s6c and d ) . [SEP]
[CLS] we next explored the contribution of lipid rafts by treating with ( i ) 2 . 5 mg / ml methyl - b - cyclodextrin , which depletes cholesterol and dissociates lipid B-material rafts , and ( ii ) 100 mm genistein , a tyrosine B-material kinase inhibitor , which blocks lipid raft - mediated endocytosis B-event . [SEP]
[CLS] methyl - b - cyclodextrin and genistein reduced sna uptake by 54 . 3 ae 2 . 1 % and 68 . 9 ae 4 . 3 % , respectively , as determined by icp - ms quantification , and by 56 . 6 ae 5 . 2 % and 66 . 3 ae 2 . 1 % , respectively , as assessed by flow B-technique cytometry I-technique ( all p < 0 . 001 ) ( figure 4b ) . [SEP]
[CLS] lactosylceramide , which requires lipid B-material rafts for endocytosis B-event , served as a positive control for these studies ( figure 4c ) . [SEP]
[CLS] these studies indicated the involvement of lipid rafts in sna uptake . [SEP]
[CLS] uptake in lipid B-material rafts often occurs through a cav - 1mediated process . [SEP]
[CLS] however , the knockdown of cav1 ( encoding cav - 1 ) through lentiviral shrna ( supplementary figures s2 and s7a ) in monolayer ( low calcium B-material ion B-material ) nheks did not block sna uptake ( figure 5a ) . [SEP]
[CLS] flot - 1 and flot - 2 are lipid raft - associated , highly homologous proteins B-material that form a specific domain in the noncaveolar region of lipid B-material rafts . [SEP]
[CLS] this flotillin domain is thought to function in several cellular activities , including signaling , endocytosis B-event , and interaction with the cytoskeleton . [SEP]
[CLS] indeed , we found that lentiviral shrnamediated knockdown of flot1 ( encoding flot - 1 ) ( supplementary figures s2 and s7b ) , which prevents the formation of the flotillin domain , reduced sna uptake in undifferentiated nheks ( 0 . 07 mm calcium B-material ion B-material ) by 70 % ( 81 , 526 ae 5 , 044 - 23 , 364 ae 1 , 269 gold B-material particles per cell B-material ) , whereas serial transduction of both cav1 and flot1 shrnas did not further suppress sna uptake ( figure 5a ) . [SEP]
[CLS] in differentiated nheks ( 1 . 2 mm calcium B-material ion B-material ) , however , cav1 knockdown reduced sna uptake by 50 % ( from 51 , 235 ae 2 , 222 gold B-material particles per cell B-material to 25 , 325 ae 701 gold B-material particles per cell B-material ) compared with vector - transduced nheks , whereas the transduction of flot1 shrna alone or serial transduction of both flot1 and cav1 shrna reduced sna uptake by 78 % ( from 51 , 235 ae 2 , 222 gold B-material particles per cell B-material to 11 , 223 ae 338 gold particles per cell B-material ) or 77 % ( from 51 , 235 ae 2 , 222 gold B-material particles per cell B-material to 11 , 752 ae 781 gold particles per cell ) , respectively ( figure 5a ) . [SEP]
[CLS] furthermore , flot - 1 coimmunoprecipitated ( as shown by western blotting ) and colocalized ( as shown by total internal reflection fluorescence B-technique microscopy I-technique ) with both scara3 and marco in undifferentiated ( pearson ' s correlation coefficient , r ¼ 0 . 713 ae 0 . 018 and 0 . 753 ae 0 . 020 , respectively ) and differentiated ( r ¼ 0 . 758 ae 0 . 019 and 0 . 764 ae 0 . 010 , respectively ) nheks ( figure 5b and c ) . [SEP]
[CLS] cav1 was only able to coimmunoprecipitate and colocalize in differentiated nheks with scara3 ( r ¼ 0 . 684 ae 0 . 021 ) and , to a lesser extent , marco ( r ¼ 0 . 502 ae 0 . 032 ) ( figure 5b and c ) . [SEP]
[CLS] marco coimmunoprecipitated with scara3 , and scara3 coimmunoprecipitated with marco , suggesting that both sr - as are in the same complex regardless of nhek differentiation status ( figure 5b ) . [SEP]
[CLS] in 3d skin equivalents , flot - 1 strongly colocalized with scara3 in both basal ( r ¼ 0 . 526 ae 0 . 012 ) and differentiated ( r ¼ 0 . 514 ae 0 . 012 ) layers of 3d equivalents . [SEP]
[CLS] flot - 1 colocalized with marco , although to a lesser extent ( r ¼ 0 . 436 ae 0 . 016 ) , in the differentiated layers of the 3d skin ( figure 5d ) . [SEP]
[CLS] the pattern of association of flot - 1 with scara3 , however , differed from that with marco in the differentiated cell B-material layers , with strong colocalization at the cellular membrane of scara3 and flot - 1 but localization of marco and flot - 1 in the cytoplasm ( figure 5d ) . [SEP]
[CLS] marco weakly colocalized with flot - 1 in the basal cell B-material layer ( r ¼ 0 . 310 ae 0 . 026 ) of 3d skin ( figure 5d ) . [SEP]
[CLS] cav - 1 , in contrast , weakly colocalized with scara3 in the basal and differentiated layers ( r ¼ 0 . 206 ae 0 . 021 and 0 . 264 ae 0 . 018 , respectively ) but colocalized with marco in the differentiated layers of the 3d skin equivalents ( r ¼ 0 . 468 ae 0 . 015 ) and weakly in the basal layer ( r ¼ 0 . 198 ae 0 . 030 ) ( figure 5d ) . [SEP]
[CLS] these data begin to define the localization and role of scavenger receptors in the skin and suggest that scara3 is the principal sr - a involved in sna uptake in the skin , primarily in complex with flot - 1 and , to a lesser extent , with cav - 1 in differentiated layers , enabling uptake throughout the epidermis . [SEP]
[CLS] scavenger receptors have been extensively studied in macrophages , mainly for their immune responses to pathogen clearance , and in the formation of foam cells B-material . [SEP]
[CLS] however , little is known about their function in the epidermis . [SEP]
[CLS] sr - b1 is known to mediate oxidized low - density lipoprotein endocytosis B-event through a noncaveolar lipid B-material raft pathway and has previously been described in nheks . [SEP]
[CLS] we have now found that sr - a subclasses , predominantly scara3 , are expressed in nheks and human skin . [SEP]
[CLS] expression of scara3 , similar to that of sr - b1 , is reduced in differentiated nheks . [SEP]
[CLS] however , the second most prevalent sr - a , marco , is most strongly expressed in differentiated epidermis . [SEP]
[CLS] human epidermis functions as an important barrier B-property that prevents transepidermal water B-material loss and entry of foreign molecules , including pathogens . [SEP]
[CLS] this epidermal barrier B-property also limits the transcutaneous delivery of nucleic B-material acids I-material and proteins B-material above 300 - 500 da . [SEP]
[CLS] snas are unusual with regard to their ability to traverse the epidermal barrier B-property without adjunctive chemical or physical agents . [SEP]
[CLS] using specific sr - a subclass inhibitors and gene knockdown , we have now shown that uptake into kcs and penetration into 3d hses of the snas primarily require the function of scara3 . [SEP]
[CLS] in the c166 mouse endothelial cell B-material line , sr - a msr1 ( also called scara1 ) was shown to be the receptor B-material for sna uptake , suggesting cell B-material - type specificity . [SEP]
[CLS] mechanistic studies in intact human skin will be important for further delineating the role of scavenger receptors in skin traversal in vivo . [SEP]
[CLS] the mechanism for uptake through sr - as is not wellunderstood . [SEP]
[CLS] sna uptake is greatly reduced by methyl - bcyclodextrin - induced depletion of cholesterol and genistein , both inhibitors of uptake through lipid B-material rafts . [SEP]
[CLS] in undifferentiated nheks , scara3 and marco both colocalize with flot - 1 in the flotillin domain of lipid B-material rafts and not in the caveolar domain . [SEP]
[CLS] however , in differentiated nheks , scara3 and marco show more colocalization with cav - 1 , suggesting the ability to utilize both flotillin and caveolar domains in the upper epidermis for cellular uptake . [SEP]
[CLS] sna uptake through sr - a msr1 or scara1 in the c166 mouse endothelial cell B-material line was shown to require cav - 1 , suggesting the localization of uptake to caveolar lipid B-material rafts domains , but the potential role of flotillins in c166 cells B-material was not explored . [SEP]
[CLS] we have now shown that cav - 1 associates with sr - as and reduces sna uptake only in differentiated nheks , consistent with its preferential membrane - based localization in the upper epidermis . [SEP]
[CLS] in contrast , flot - 1 , a marker of noncaveolar lipid B-material rafts , associates with sr - as in both undifferentiated and differentiated states , colocalizing with scara3 , and is essential for sna uptake in both differentiated and undifferentiated nheks . [SEP]
[CLS] the accentuation of uptake in basal epidermal cells B-material may further reflect a role for a flotillinescara3 complex in the uptake of snas and other highly anionic oligonucleotides in epidermal cells B-material . [SEP]
[CLS] scavenger receptors interact with toll - like receptors in macrophages and dendritic cells B-material . [SEP]
[CLS] in bone marrow - derived dendritic cells B-material , sr - a1 can interact with toll - like receptor B-material 4 to promote the phagocytosis B-event of e . coli , whereas sr - a1 and toll - like receptor B-material 2 cooperate in the phagocytosis B-event of staphylococcus aureus . [SEP]
[CLS] in macrophages , marco can partner with toll - like receptor B-material 2 and cd14 in the recognition of mycobacterium tuberculosis glycolipid trehalose 6 , 6 0 - dymycolate and is required for the optimal production of proinflammatory cytokines in response to this bacterial product . [SEP]
[CLS] whereas scavenger receptors have largely been unexplored in the epidermis , demonstrated a role for marco in the epithelial adsorption and infection of human herpes simplex virus type i and vaccinia viruses . [SEP]
[CLS] unlike msr1 or scara1 and marco , which have been studied for decades , scara3 has only recently been discovered . [SEP]
[CLS] scara3 was originally recognized to be a cellular stress response gene that could protect cells B-material by scavenging ros , inducing cell B-event death I-event , and suppressing tumor B-material growth and metastasis B-event . [SEP]
[CLS] studies with myeloma and ovarian carcinoma cells B-material suggested that scara3 could function as an antioxidant B-property and its expression was upregulated during disease progression . [SEP]
[CLS] we have now shown a central role in facilitating the penetration and uptake of the highly anionic , oligonucleotide - laden snas in the skin . [SEP]
[CLS] taken together , our observations suggest that sr - as , and particularly scara3 , likely play a role in the uptake of these spherical B-nanoparticle gold I-nanoparticle nanoparticle I-nanoparticle constructs and thus their ability to effect sequence - specific target knockdown in the skin . [SEP]
[CLS] further efforts to untangle the complex interplay between physical skin penetration and receptor - mediated cellular uptake are underway . [SEP]
[CLS] see the supplementary materials and methods for details . [SEP]
[CLS] nheks were isolated from neonatal foreskin and cultured in supplemented kcs serum - free medium ( cascade biologics , portland , or ) with 0 . 07 mm calcium B-material chloride B-material unless otherwise indicated . [SEP]
[CLS] hses were generated by the northwestern university skin biology and diseases resource - based center ( chicago , il ) and were harvested on day 12 when fully differentiated . [SEP]
[CLS] the accrual of foreskins ( discarded ) for cultured cells B-material was approved by the northwestern university institutional review board and patient consent was not required . [SEP]
[CLS] lentiviral shrnas for human cav1 , flot1 , scara3 , and marco were prepared in the northwestern university skin biology and diseases resource - based center . [SEP]
[CLS] transduction was performed with polybrene ( 8 mg / ml ) ( thermo fisher scientific , waltham , ma ) on primary nheks on day 7 or 8 , and stably transduced nheks were selected in complete serum - free medium with 0 . 07 mm calcium B-material chloride B-material ( cascade biologics , portland , or ) and 5 mg / ml puromycin ( cascade biologics ) . [SEP]
[CLS] lentiviral vectors were transduced as a negative control , and serial addition of scara3 and marco shrnas or flot1 and cav1 shrnas were used to knockdown both concurrently . [SEP]
[CLS] snas were composed of 13 - nm gold B-nanoparticle nanoparticles I-nanoparticle functionalized with duplex rna nonsense oligonucleotides that were cyanine dye cy5 labeled at the 3 0 end and functionalized at the 5 0 terminus with a thiol B-material functional B-material group I-material . [SEP]
[CLS] sequences of the cyanine dye cy5 nonsense and complementary nonsense sequences are shown in supplementary table s1 and were synthesized as previously described . [SEP]
[CLS] cell B-material samples were fixed with 4 % paraformaldehyde ( thermo fisher scientific ) for 10 minutes and permeabilized with 0 . 1 % triton - x ( thermo fisher scientific ) for 10 minutes . [SEP]
[CLS] 3d skin ( from the skin biology and diseases resourcebased center ) or abdominoplasty skin samples ( obtained through the skin biology and diseases resource - based center after northwestern university institutional review board approval that required no patient consent ) were embedded in optimal cutting temperature and cut into 4 - mm sections . [SEP]
[CLS] all samples were incubated B-technique with anti - marco ( santa cruz biotechnology , dallas , tx ) , anti - scara3 ( novus biologicals , littleton , co ) , anti - flot - 1 ( santa cruz biotechnology ) , and / or anti - cav - 1 ( santa cruz biotechnology ) overnight at 4 o c , and after treatment with secondary fluorescencelabeled antibodies B-material , the samples were counter - stained with dapicontaining medium . [SEP]
[CLS] cell B-material lysates were prepared in radioimmunoprecipitation assay buffer ( sigma - aldrich ) , precleared , and incubated B-technique with mouse anti - human scara3 or marco antibody B-material ( or anti - igg antibody B-material as a control ) overnight ( sigma - aldrich ) at 4 o c . [SEP]
[CLS] the lysate - antibody B-material mixture was pulled down using protein a / g magnetic B-property beads mixture ( new england biolabs , ipswich , ma ) . [SEP]
[CLS] proteins B-material were eluted and western blots were performed . [SEP]
[CLS] unless otherwise mentioned , reported values represent mean ae sd from three independent experiments performed at least in triplicate . [SEP]
[CLS] significance was determined across two independent groups using a student ' s t - test . [SEP]
[CLS] when comparing multiple groups within a single variable ( e . g . , dose ) , one - way anova with tukey ' s posthoc adjustment was used . [SEP]
[CLS] multiple variable interactions were tested using a two - way anova with a tukey ' s posthoc adjustment ( e . g . , figures 2b , 5b , and 5d ) . [SEP]
[CLS] p < 0 . 05 was considered significant . [SEP]
[CLS] graphpad prism ( version 8 . 2 . 0 ) ( graphpad software inc , san diego , ca ) was used to analyze data . [SEP]
[CLS] a total of 13 ae 1 nm citrate - capped gold B-material particles were prepared through the reduction of gold ( iii ) salt B-material ( sigma - aldrich , st . louis , mo ) in a citric buffer . [SEP]
[CLS] to sterilize the particles of all nucleases , 0 . 01 % diethyl pyrocarbonate ( sigma - aldrich ) was added to the nanoparticles B-nanoparticle for 1 hour , and the solution was subsequently autoclaved . [SEP]
[CLS] on cooling , 0 . 2 % surfactant B-property ( tween 20 ) ( sigma - aldrich ) was added to the particles , and the solution was vigorously mixed for 5 minutes . [SEP]
[CLS] next , 150 mm sodium B-material chloride I-material ( sigma - aldrich ) was added to the gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) . [SEP]
[CLS] all stock solutions were made using nuclease - free water B-material ( integrated dna technologies , coralville , ia ) . [SEP]
[CLS] the thiolated small interfering rna ( sirna ) duplex was added to the solution of aunps B-nanoparticle and shaken on a benchtop shaker for four hours . [SEP]
[CLS] after 4 hours , the final sodium B-material chloride I-material concentration in the aunpseoligonucleotide solution was brought to 350 mm and incubated B-technique overnight at room temperature ( rt ) . [SEP]
[CLS] a total of 10 mm thiol - conjugated polyethylene glycol 2 , 000 ( sigma - aldrich ) , was added to the particle solution for 3 hours at rt to complete the spherical nucleic B-material acid I-material ( sna ) functionalization process . [SEP]
[CLS] unbound sirna and thiol - conjugated polyethylene glycol from the snas were removed using centrifugation . [SEP]
[CLS] uv - visible spectroscopy B-technique measurements were performed to determine sna concentration on the basis of the absorbance of aunps B-nanoparticle ( cary 5000 , agilent technologies , santa clara , ca ) . [SEP]
[CLS] the measured absorbance was related to the concentration of gold B-material particles using beer ' s law ( a ¼ εbc ) , where ε ¼ 2 . 77 a 10 8 . [SEP]
[CLS] the size distribution and polydispersity of the snas were determined using dynamic B-technique light I-technique scattering I-technique . [SEP]
[CLS] a 5 nm aliquot of sna was used to make dynamic B-technique light I-technique scattering I-technique measurements ( nanozs , malvern , united kingdom ) . [SEP]
[CLS] to study the energy dependency of uptake , normal human epidermal keratinocytes ( nheks ) were treated either at 4 c or at 37 c , and to block adenosine triphosphate synthesis , nheks were treated at 37 c with or without 3 . 0 mg / ml sodium B-material azide ( sigma - aldrich ) beginning at 1 hour after treatment with cyanine dye cy5 snas . [SEP]
[CLS] to block sr - as , cells B-material were preincubated with 50 mg / ml fucoidan ( sigma - aldrich ) or polyinosinic acid ( sigma - aldrich ) for 1 hour . [SEP]
[CLS] to block clathrin - mediated endocytosis B-event , cells B-material were pretreated with either potassium B-material - depleted medium ( 140 mm sodium B-material chloride I-material , 1 mm calcium B-material chloride B-material , 1 mm magnesium B-material chloride B-material , 5 . 5 mm d - glucose , 20 mm 4 - [ 2 - hydroxyethyl ] - 1piperazineethanesulfonic acid ) ( sigma - aldrich ) or 20 mm potassium B-material chloride B-material ( sigma - aldrich ) supplemented for 1 hour . [SEP]
[CLS] to block macropinocytosis , cells B-material were pretreated with or without 100 mm wortmannin ( sigma - aldrich ) for 1 hour . [SEP]
[CLS] to block caveolar - and / or lipid raft - mediated endocytosis B-event , cells B-material were pretreated with 2 . 5 mg / ml methyl - b - cyclodextrin ( sigma - aldrich ) or 100 mm genistein ( sigma - aldrich ) for 1 hour . [SEP]
[CLS] after each condition , nheks were treated with either 0 . 25 nm ( for confocal fluorescence B-technique microscopy I-technique ) or 0 . 5nm sirna sna ( for flow B-technique cytometry I-technique or inductively coupled plasma mass spectrometry quantification ) for another 2 hours . [SEP]
[CLS] stably transduced human cav1 and flot1 knockdown in nheks lentiviral short hairpin ( shrna ) for human cav1 was prepared in the dna / rna delivery core B-material at the northwestern university ( chicago , il ) skin biology and diseases resourcebased center . [SEP]
[CLS] transduction was performed with polybrene ( 8 mg / ml ) ( sigma - aldrich ) on primary nheks on day 7 or 8 , and nheks stably transduced with cav1 shrna , flot1 shrna , or both concurrently were selected in complete serum - free medium ( cascade biologics , portland , or ) with 0 . 07 mm calcium B-material chloride B-material ( cascade biologics ) and 5 mg / ml puromycin ( cascade biologics ) . [SEP]
[CLS] stably transduced sr - a - deficient nheks and threedimensional skin equivalents inoculated competent e . coli strains with shrna - expressing plko . 1 - puro vectors were purchased from sigma - aldrich . [SEP]
[CLS] the strains were incubated B-technique at 30 c in ampicillincontaining lysogeny broth for 24 hours before extraction of plasmids using the qiagen plasmid plus maxi kit ( qiagen , valencia , ca ) . [SEP]
[CLS] lentiviral shrna was prepared in the dna / rna delivery core B-material at the northwestern university skin biology and diseases resource - based center . [SEP]
[CLS] transduction was performed with polybrene ( 8 mg / ml ) on primary nheks on day 7 or 8 , and nheks stably transduced with marco and / or scara3 shrnas were selected in a complete serumfree medium with 0 . 07 mm calcium B-material chloride B-material and 5 mg / ml puromycin . [SEP]
[CLS] human skin equivalents with marco and / or scara3 knockdown were generated from stably transduced nheks with suppression of marco and / or scara3 expression as appropriate for the knockdown . [SEP]
[CLS] knockdown was confirmed by western blotting . [SEP]
[CLS] lentiviral vectors were transduced into cells B-material as a negative control . [SEP]
[CLS] marco and scara3 shrnas were added serially when both were transduced . [SEP]
[CLS] uptake of sna in nheks , three - dimensional rafts using confocal microscopy B-technique nheks were cultured on glass coverslips in 12 - well plates to 50 - 60 % confluence . [SEP]
[CLS] after pretreatment with endocytosis B-event inhibitors and incubation B-technique with cy5 - sirna snas , cells B-material were washed with pbs , fixed for 10 minutes in 4 % paraformaldehyde ( thermo fisher scientific ) , stained with 8 mm dapi ( thermo fisher scientific ) , for 10 minutes at rt , and mounted onto slides using prolong gold B-material antifade mounting medium ( life technologies , grand island , ny ) . [SEP]
[CLS] three - dimensional organotypic rafts were cultured on transwell inserts ( corning , corning , ny ) before topical application of sna . [SEP]
[CLS] cy5 - sirna snas were mixed with aquaphor ( beiersdorf , wilton , ct ) ( 1 : 1 v / v ) , and the mixture was topically applied for designated time points before harvesting . [SEP]
[CLS] the three - dimensional rafts with sna were fixed in 4 % paraformaldehyde and embedded in an optimal cutting temperature medium . [SEP]
[CLS] sections ( 4 mm ) were cut , stained with 8 mm dapi for 10 minutes at rt , and mounted onto slides in a mounting medium . [SEP]
[CLS] samples were visualized using the nikon a1r confocal fluorescence B-technique microscopy I-technique ( nikon instruments , melville , ny ) ( northwestern university center for advanced microscopy B-technique and / or nikon imaging center , chicago , il ) . [SEP]
[CLS] total cell B-material lysates were extracted in a boiling buffer containing 10 mm tris ( ph 7 . 4 ) , 1 % sds , and 1 mm sodium B-material orthovanadate ( sigma - aldrich ) . [SEP]
[CLS] the solution was centrifuged at 12 , 000 r . p . m . for 10 minutes at rt to remove debris , and protein B-material concentrations were determined using the bca protein B-material assay kit ( thermo fisher scientific ) . [SEP]
[CLS] equal amounts of proteins B-material were subjected to 10 % sds - page , transferred to a nitrocellulose membrane ( bio - rad , hercules , ca ) , and blocked for 1 hour in tris - buffered saline , ph 7 . 5 , containing 0 . 5 % tween 20 and 5 % milk ( santa cruz biotechnology , dallas , tx ) . [SEP]
[CLS] membranes were then incubated B-technique overnight at 4 o c with an antibody B-material recognizing marco , flot - 1 , cav - 1 ( santa cruz biotechnology ) , and scara3 ( novus biologicals , littleton , co ) for 24 hours . [SEP]
[CLS] the membranes were then incubated B-technique with horseradish peroxidase - conjugated secondary antibody B-material ( jackson immunoresearch laboratories , west grove , pa ) for 1 hour , and the bound antibodies B-material were detected using chemiluminescence B-property ( pierce chemical , rockford , il ) . [SEP]
[CLS] images were digitized , and band densities were measured using national institutes of health image software . [SEP]
[CLS] electron B-technique microscopy I-technique imaging and semiquantitative analysis sna - treated human skin equivalents with or without scara3 knockdown were fixed with 2 % paraformaldehyde , and 2 . 5 % glutaraldehyde ( thermo fisher scientific ) in pbs solution and postfixed with osmium tetroxide ( sigma - aldrich ) and uranyl acetate ( sigma - aldrich ) before a graded series of ethanol dehydration . [SEP]
[CLS] samples were further dehydrated utilizing propylene oxide B-material . [SEP]
[CLS] finally , the skin equivalents were infiltrated with spurr ' s epoxy resin ( sigma - aldrich ) and embedded in blocks . [SEP]
[CLS] the resin was cured at 60 c for 48 hours . [SEP]
[CLS] the embedded blocks of skin were trimmed in cross - section with a block face of approximately 0 . 7 mm a 0 . 5 mm and sectioned at a thickness of 200 nm in a leica uc7 ultramicrotome ( leica microsystems , wetzlar , germany ) . [SEP]
[CLS] sections were collected on slotted grids with a formvar - carbon B-material membrane ( vwr , radnor , pa ) . [SEP]
[CLS] the grids were analyzed unstained in a hitachi hd - 2300 scanning B-technique transmission I-technique electron I-technique microscopy I-technique running a schottky feg at 200 kv with a high angular annular dark field detector ( hitachi , tokyo , japan ) so that the aunps B-nanoparticle were easier to find . [SEP]
[CLS] images were collected with gatan digital micrograph ( gatan , pleasanton , ca ) , and contrast was later inverted with software to simulate transmission B-technique electron I-technique microscopy I-technique bright field imaging . [SEP]
[CLS] semiquantitation of sna uptake was conducted by two blinded reviewers counting the overall gold B-material snas from a total of 50 basal keratinocytes from each human skin equivalents with or without scara3 knockdown with pooling of data . [SEP]
[CLS] total rna was extracted with the rneasy mini kit ( qiagen ) . [SEP]
[CLS] one microgram of rna per sample was reverse transcribed with qscript cdna synthesis kit ( vwr ) according to the manufacturer ' s instructions . [SEP]
[CLS] subsequent pcr reactions were performed using 2 ml of undiluted cdna in a 20 ml reaction mix ( vwr ) or , for qpcr , 5 ml of 1 : 20 diluted cdna in a 20 ml reaction using sybr green master mix ( thermo fisher scientific ) . [SEP]
[CLS] a t100 thermal cycler ( bio - rad ) was used for pcr amplification , whereas a lightcycler 96 system ( roche , basel , switzerland ) was used for qpcr . [SEP]
[CLS] primer sequences used to amplify different scara subclasses were described previously . [SEP]
[CLS] after pcr amplification of cdna , products were analyzed by 2 % agarose gel ( sigma - aldrich ) electrophoresis B-technique . [SEP]
[CLS] inductively coupled plasma mass spectrometry nheks were cultured in 12 - well plates to 60 - 70 % confluence , treated , and harvested as for flow B-technique cytometry I-technique , but the pellet was digested with 0 . 3 ml of 3 % hydrogen B-material chloride I-material ( thermo fisher scientific ) in concentrated nitric acid at rt overnight . [SEP]
[CLS] after adding 5 ml of 5 parts per million indium B-material ( internal standard ) and 5 ml of matrix solution ( 2 % hydrogen B-material chloride I-material and 2 % nitric acid ) , the gold B-material 197 content of the resultant solution was measured by an x series ii inductively coupled plasma mass spectrometry ( thermo fisher scientific ) after subtracting the background gold B-material content of untreated cells B-material . [SEP]
[CLS] nheks for flow B-technique cytometry I-technique were grown in six - well plates to 60 - 70 % confluence , pretreated with inhibitors and then cyanine 5 snas , washed , and trypsinized , and the centrifuged pellet was suspended as single cells B-material in 4 % paraformaldehyde . [SEP]
[CLS] fluorescence B-property was quantified as the number of cells B-material that emitted above the level of control cell B-material autofluorescence using a bd lsrii flow cytometer ( bd biosciences , san jose , ca ) ( northwestern university immunobiology center flow B-technique cytometry I-technique ) . [SEP]
[CLS] cell B-material debris was excluded on the basis of the forward and side scatter characteristics of the cell B-material populations . [SEP]
[CLS] cell B-material lysates were prepared in radioimmunoprecipitation assay buffer ( sigma - aldrich ) then precleared with a 20 ml mixture of protein B-material a and protein g magnetic B-property beads ( new england biolabs , ipswich , ma ) . [SEP]
[CLS] precleared cell B-material lysates ( 400 mg total protein B-material concentration ) were incubated B-technique with mouse anti - human scara3 or marco antibody B-material ( or anti - igg antibody B-material as a control ) ( sigma - aldrich ) overnight at 4 o c . [SEP]
[CLS] a 40 ml mixture of protein B-material a and protein B-material g was added to the lysateantibody solution for 4 hours at 4 o c . [SEP]
[CLS] the beads were then washed three times with 500 ml of radioimmunoprecipitation assay buffer and collected using a magnetic B-property rack . [SEP]
[CLS] elution buffer ( 20 ml ) was added to the beads and boiled at 95 o c for 10 minutes . [SEP]
[CLS] the beads were then removed , and the solution containing immunoprecipitated proteins B-material was resolved by western blots . [SEP]
[CLS] cell B-material samples were fixed with 4 % paraformaldehyde for 10 minutes and permeabilized with 0 . 1 % triton - x ( thermo fisher scientific ) for 10 minutes . [SEP]
[CLS] three - dimensional skin was embedded in optimal cutting temperature , and 4 mm sections were washed and permeabilized by 4 % paraformaldehyde for 15 minutes . [SEP]
[CLS] all samples were then blocked with 5 % goat serum and 1 % bsa in 0 . 1 % tween 20 in pbs and incubated B-technique with anti - marco ( santa cruz q song et al . [SEP]
[CLS] epidermal sr - as and nanoparticle B-nanoparticle uptake [SEP]
[CLS] sna dose - , time - , and scavenger receptor - - dependent uptake . [SEP]
[CLS] ( a - - c ) nheks were pretreated for ( a ) 2 hours with 0 . 1 - 5 nm cyanine dye cy5 - labeled sirna snas , ( b ) 30 min to 8 hours with 0 . 25 nm cy5labeled snas , or ( c ) 1 hour with sr - a inhibitors fucoidan or polyi ( 50 mg / ml each ) before incubation B-technique with 1 nm sirna sna . [SEP]
[CLS] cells B-material were then visualized by confocal microscopy B-technique . [SEP]
[CLS] graphs show the uptake measured by icp - ms . [SEP]
[CLS] ( d ) organotypic rafts were pretreated with 50 mg / ml polyi for 24 hours before topically applying 100 nm sirna sna . [SEP]
[CLS] sna penetration decreased in polyi - treated rafts compared with that in nontreated rafts . [SEP]
[CLS] red indicates cy5 - labeled sirna sna ; blue indicates dapistained nuclei . [SEP]
[CLS] bars ¼ 25 mm ( a - - c ) and 50 mm ( d . [SEP]
[CLS] error bars indicate sd ; n ¼ 3 . [SEP]
[CLS] * p < 0 . 05 , * * p < 0 . 001 , * * * p < 0 . 0001 . [SEP]
[CLS] aunp B-nanoparticle , gold B-nanoparticle nanoparticle I-nanoparticle ; icp - ms , inductively coupled plasma mass spectrometry ; min , minute ; nhek , normal human epidermal keratinocyte ; polyi , polyinosinic acid ; sna , spherical nucleic B-material acid I-material ; sirna , small interfering rna . [SEP]
[CLS] impact of lentiviral shrna knockdown of marco and scara3 on sirna sna uptake and penetration . [SEP]
[CLS] ( a ) rt - pcr products were resolved by agarose B-technique gel I-technique electrophoresis I-technique to detect mrna expression of sr - a isoforms in nheks . [SEP]
[CLS] ( b ) expression of scara3 , marco , and scara5 in undifferentiated and differentiated nheks . [SEP]
[CLS] ( c ) scara3 and marco expression in a 3d raft and human intact skin by immunofluorescence . [SEP]
[CLS] ( d ) sna uptake was measured by icp - ms in undifferentiated and differentiated nheks . [SEP]
[CLS] ( e , f ) reduced cyanine dye cy5 sna uptake after transduction of marco shrna , scara3 shrna , or both added serially ( combo ) was shown by ( e ) immunofluorescence staining and ( f ) flow B-technique cytometry I-technique to quantify fluorescent B-property uptake . [SEP]
[CLS] ( g ) penetration of 100 nm sna in scara3 shrna - transduced or both marco and scara3 shrna - transduced ( added serially ) ( combo ) human 3d rafts was reduced ( vs . control or untreated vector shrna and marco shrna transduced ) . [SEP]
[CLS] bars ¼ 50 mm in c and g and 25 mm in f . [SEP]
[CLS] error bars indicate sd , n ¼ 3 . [SEP]
[CLS] * * p < 0 . 001 , * * * p < 0 . 0001 . [SEP]
[CLS] 3d , three - dimensional ; aunp B-nanoparticle , gold B-nanoparticle nanoparticle I-nanoparticle ; bp , base pair ; ca 2þ , calcium B-material ion B-material ; combo , combination ; hse , human skin equivalent ; icp - ms , inductively coupled plasma mass spectrometry ; nhek , normal human epidermal keratinocyte ; shrna , short hairpin rna ; sirna , small interfering rna ; sna , spherical nucleic B-material acid I-material . [SEP]
[CLS] scanning B-technique transmission I-technique electron I-technique microscopy I-technique showing sna uptake in serial sections of 3d - cultured skin equivalents with and without scara3 knockdown . [SEP]
[CLS] ( a ) snas were clearly identified in basal keratinocytes of vector shrna - transduced 3d rafts , either trapped inside the endosomal vesicles or freely released in the cytoplasm . [SEP]
[CLS] no particles were found in these thin sections between intracellular spaces . [SEP]
[CLS] ( b ) significantly lower numbers of sna were found in the basal keratinocytes of scara3 shrna - transduced 3d rafts . [SEP]
[CLS] arrows point to clusters of snas . [SEP]
[CLS] bars ¼ 500 mm in the top row , 5 mm in middle and bottom rows , and 100 mm in the stitched images ( vertical arrangement to left ) . [SEP]
[CLS] 3d , three - dimensional ; sb , stratum basale ; sc , stratum corneum ; sg , stratum granulosum ; shrna , short hairpin rna ; sna , spherical nucleic B-material acid I-material ; ss , stratum spinosum . [SEP]
[CLS] endocytosis B-event of snas requiring sr - as occurs in lipid rafts . [SEP]
[CLS] ( a ) nheks were pretreated for 1 hour at 37 o c , 4 o c , or 37 o c with 3 . 0 mg / ml nan B-technique 3 before exposure to cyanine dye cy5 - labeled snas to confirm endocytotic uptake . [SEP]
[CLS] ( b ) nheks were pretreated with mbcd to reduce cholesterol or with genistein to suppress lipid B-material raft endocytosis B-event . [SEP]
[CLS] ( c ) laccer uptake was tested in lieu of sna because laccer is known to require lipid B-material rafts for endocytosis B-event . [SEP]
[CLS] icp - ms was used to measure sna uptake ( graph in a , left graph in b ) . [SEP]
[CLS] flow B-technique cytometry I-technique was used to quantify sna ( right graph in b ) and laccer ( graph in c ) uptake . [SEP]
[CLS] bars ¼ 25 mm . [SEP]
[CLS] red indicates cy5 - labeled sirna sna , and blue indicates dapi - stained nuclei . [SEP]
[CLS] error bars indicate sd ; n ¼ 3 . [SEP]
[CLS] * p < 0 . 0001 . [SEP]
[CLS] aunp B-nanoparticle , gold B-nanoparticle nanoparticle I-nanoparticle ; icp - ms , inductively coupled plasma mass spectrometry ; laccer , lactosylceramide ; mbcd , methyl - b - cyclodextrin ; nan B-technique 3 , sodium B-material azide ; nhek , normal human epidermal keratinocyte ; sirna , small interfering rna ; sna , spherical nucleic B-material acid I-material . [SEP]
[CLS] featured article : mirkin ca , letsinger rl , mucic rc , storhoff jj . [SEP]
[CLS] a dna - based method for rationally assembling nanoparticles B-nanoparticle into macroscopic materials . [SEP]
[CLS] nature 1996 ; 382 : 607 - 9 . [SEP]
[CLS] 2 spherical nucleic B-material acids I-material , or snas , were first introduced in our 1996 paper in nature featured here . [SEP]
[CLS] the original nanoconstructs were composed of gold B-nanoparticle nanoparticles I-nanoparticle modified with a layer of dna at a density high enough to force the strands upright , forming a shell B-material that adopted the spherical shape of the nanoparticle B-nanoparticle template . [SEP]
[CLS] since then , many other core B-material and nucleic B-material acid I-material compositions have been synthesized and explored . [SEP]
[CLS] snas were born from an idea i had after attending a seminar on colloidal crystallization . [SEP]
[CLS] researchers were exploring ways of engineering such crystals on the basis of electrostatic and simple small - molecule interactions between particles . [SEP]
[CLS] i imagined a new type of chemistry in which the interactions between nanoparticles B-nanoparticle could be programed using sequence - specific dna interactions - a system in which the particles could be viewed as atoms B-material and the dna as programmable bonds between them , offering an almost limitless number of possibilities for controlling the assembly of particles into hierarchical materials , crystals in particular . [SEP]
[CLS] to test this concept , i asked then - northwestern graduate students bobby mucic and james storhoff to seek out bob letsinger , the pioneer of oligonucleotide synthesis , and learn how to make strands of dna that could be chemically immobilized on gold B-material . [SEP]
[CLS] soon after , bobby and james synthesized two sets of gold B-material particles , each approximately 13 nm in diameter , with dna strands linked to their surfaces via thiol B-material adsorption on gold B-material ; they observed a reversible red - to - blue color change when these particles were mixed with the appropriate complementary linker , which we now know indicates the assembly of these particles via programmable hybridization . [SEP]
[CLS] these nanostructures have since ushered in whole new areas of materials and clinical chemistry . [SEP]
[CLS] initial discoveries involving the color change associated with sna assembly led to my group ' s development of simple biosensing schemes based mainly upon visualization or ultraviolet - visible spectroscopy B-technique ; but as we learned more , we devised ones with increased complexity and sophistication . [SEP]
[CLS] for example , we found that snas displayed sharp , highly cooperative melting transitions and enhanced binding properties ( compared to the same sequence of linear nucleic B-material acids I-material ) . [SEP]
[CLS] not long after we synthesized the first sna , we introduced the sna - based scanometric and biobarcode assays in 2000 and 2003 papers in science , respectively . [SEP]
[CLS] these papers and subsequent related ones showed the power of utilizing well - characterized nanomaterials B-material as probes for the detection of biomolecules at analytical sensitivities rivaling and , in some cases , even surpassing pcr and elisa . [SEP]
[CLS] taking what we had learned from a decade of using snas in extracellular biological environments , we then envisioned how snas could be used for monitoring and ultimately regulating what goes on inside living cells B-material . [SEP]
[CLS] both snas and cell B-material walls are highly negatively charged , yet we made the surprising observation that snas naturally entered many cell B-material types in high quantities without the use of positively charged transfection agents . [SEP]
[CLS] we tracked their mode of entry and determined that it was based upon scavenger receptor B-material and caveolin - mediated endocytosis B-event . [SEP]
[CLS] this understanding made the development of snas as highly potent , nontoxic , and nonimmunogenic classes of intracellular gene regulatory ( antisense ( 4 ) and small interfering rna ( 5 ) ) and immunomodulatory materials ( 6 ) possible . [SEP]
[CLS] by 2012 , it had become clear that snas constituted a new form of matter ( 1 ) - spherical forms of nucleic B-material acids I-material with no known natural equivalent and with properties vastly different from those of their linear counterparts that would make them exceedingly useful in clinical medicine . [SEP]
[CLS] many different classes of snas , defined by core B-material and nucleic B-material acid I-material shell B-material composition , now exist and are the basis for commercial clinical products and systems that are improving and , in certain cases , saving the lives of patients . [SEP]
[CLS] the fda - cleared verigene tm system , now a part of luminex ' s product portfolio , represents the first commercialized molecular medical diagnostic system in the modern era of nanotechnology , and it is used in over half of the nation ' s top hospitals with capabilities in rapid point - of - care ultrasensitive nucleic B-material acid I-material analysis . [SEP]
[CLS] snabased [UNK] , which have been commercialized by aurasense with merck millipore , can be used intracellularly to identify and quantify genetic content at the level of a single live cell B-material . [SEP]
[CLS] the [UNK] therapeutic platform for gene regulation and immunomodulation B-property is being commercialized by exicure via several key partnerships . [SEP]
[CLS] snas are useful therapeutic modalities because they can be delivered to almost any tissue , including topically to the skin and systemically to the brain . [SEP]
[CLS] promising results from first - of - their - kind human clinical trials have pointed to the suitability of a sna - based lead compound , ast - 005 , for the topical treatment of psoriasis and proved snas to be a class of nucleic B-material acids I-material with privileged access to tissues and organ systems that linear nucleic B-material acids I-material normally do not enter , major milestones in nucleic B-material acid I-material drug development . [SEP]
[CLS] in addition to being heavily used within the field of medicine , snas illustrate a very important point - structure matters . [SEP]
[CLS] in this case , when arguably one of the most important classes of molecules chemists have learned to synthesize and mass produce - nucleic B-material acids I-material - is restructured on the nanoscale , a new material B-material with previously unobserved and enabling properties in biology and medicine emerges . [SEP]
[CLS] multiferroic composites of ferromagnetic and ferroelectric phases are of importance for studies on mechanical strain mediated coupling between the magnetic B-property and electric subsystems . [SEP]
[CLS] this work is on dna - assisted self - assembly of superstructures of such composites with nanometer periodicity . [SEP]
[CLS] the synthesis involved oligomeric dna - functionalized ferroelectric and ferromagnetic nanoparticles I-nanoparticle , 600 nm batio 3 ( bto ) and 200 nm nife 2 o 4 ( nfo ) , respectively . [SEP]
[CLS] mixing bto and nfo particles , possessing complementary dna sequences , resulted in the formation of ordered core - shell heteronanocomposites held together by dna hybridization . [SEP]
[CLS] the composites were imaged by scanning electron B-technique microscopy I-technique and scanning microwave microscopy B-technique . [SEP]
[CLS] the presence of heteroassemblies along with core - shell architecture is clearly observed . [SEP]
[CLS] the reversible nature of the dna hybridization allows for restructuring the composites into mm - long linear chains and 2d - arrays in the presence of a static magnetic B-property field and ring - like structures in a rotating - magnetic B-property field . [SEP]
[CLS] strong magneto - electric ( me ) coupling in as - assembled composites is evident from static magnetic B-property field h induced polarization and low - frequency magnetoelectric voltage coefficient measurements . [SEP]
[CLS] upon annealing the nanocomposites at high temperatures , evidence for the formation of bulk composites with excellent cross - coupling between the electric and magnetic B-property subsystems is obtained by h - induced polarization and lowfrequency me voltage coefficient . [SEP]
[CLS] the me coupling strength in the self - assembled composites is measured to be much stronger than in bulk composites with randomly distributed nfo and bto prepared by direct mixing and sintering . [SEP]
[CLS] manipulation of materials at the nanoscale has recently become an area of great interest to the materials science community . [SEP]
[CLS] the generation of one - and two - dimensional nanostructures provides materials with interesting magnetic B-property , optical , and electronic properties . [SEP]
[CLS] creating anisotropy from isotropic materials presents challenges that still need to be overcome , however . [SEP]
[CLS] assembly of composites using nanoparticles B-nanoparticle tethered with dna has recently been explored . [SEP]
[CLS] methods of covalently attaching single stranded dna ( ssdna ) to nanoparticle B-nanoparticle surfaces have included gold B-material - thiolate interaction , michael addition , disulfide bond formation , phosphate - amino linkage B-property via carbodiimide activation , amide B-material bond formation , schiff - base formation , glutaraldehyde - assisted cross - linking , and cross - linking using compounds containing sulfhydryl and amino - ( nhydroxysuccinimide ) groups [SEP]
[CLS] employment of dna to assemble nanoparticle B-nanoparticle via hybridization into superlattices has been an example of particle - to - materials fabrication . [SEP]
[CLS] the cu ( i ) - catalyzed azide - alkyne cycloaddition ( cuaac ) " click " reaction has also been employed to achieve attachment of dna to nanoparticles B-nanoparticle . [SEP]
[CLS] this report is on the self - assembly of multiferroic nanocomposites using dna - dna hybridization . [SEP]
[CLS] multiferroic composites consisting of ferromagnetic and ferroelectric phases are of interests for studies on coupling between magnetic B-property and electric subsystems that is mediated by mechanical forces . [SEP]
[CLS] the magneto - electric ( me ) interaction in such composites is a product property , i . e . , mechanical strain in the ferromagnetic phase due to magnetostriction in an applied magnetic B-property field resulting in charge generation due to piezoelectric effect in the ferroelectric phase . [SEP]
[CLS] strong me coupling was reported in several bulk and thick / thin film composites with ferromagnetic / ferrimagnetic metals B-material , alloys or oxides B-material and ferroelectrics such as barium titanate ( bto ) , lead zirconate titanate ( pzt ) , and lead magnesium B-material niobate - lead titanate ( pmn - pt ) . [SEP]
[CLS] the strain mediated me interactions in nanocomposites is expected to be very strong due to a surface - to - volume ratio that is a factor of 10 3 to 10 6 higher than for bulk and thick / film composites . [SEP]
[CLS] studies so far on nanocomposites have primarily focused on thin films and nanopillars in which substrate clamping weakens the strength of me coupling . [SEP]
[CLS] such clamping effects are expected to be absent in nanostructures of core - shell particles , and coaxial tubes and wires . [SEP]
[CLS] in a recent study 12 - 200 nm particles of barium titanate ( bto ) and cobalt B-material ferrite were functionalized by attaching carboxylate B-material and amine B-material groups I-material , respectively , and a core - shell particulate composite was formed by covalent bonds between the two particles with the addition of a coupling agent . [SEP]
[CLS] in our recent work on chemical self - assembly we utilized the ( cuaac ) " click " reaction to synthesize heteronanocomposites by functionalizing bto and nickel B-material ferrite nife 2 o 4 ( nfo ) with complementary azide and alkyne reaction groups . [SEP]
[CLS] the assembled composite showed evidence for strong me coupling . [SEP]
[CLS] but these efforts also clearly indicated the strengths and weaknesses of pure chemical assembly . [SEP]
[CLS] the technique is very effective in creating covalent attachments between the nfo and bto nanoparticles B-nanoparticle and is not limited by the diameters for these particles that have been employed . [SEP]
[CLS] a shortcoming , however , is that the attachments are non - dynamic ( cannot be reorganized once assembled ) and create nanocomposites that are limited in size which may be due to low coupling efficiency for the click reaction . [SEP]
[CLS] here we discuss results of a new approach for assembling core - shell particulate composites based on biological tenets utilizing dna covalently attached to nanomaterials B-material to effect assembly of nanocomposites through specific base - pairing interactions between complimentary sequences . [SEP]
[CLS] in our efforts to assemble ordered heteroassemblies at the nanoscale comprised of ferromagnetic and ferroelectric nanoparticles B-nanoparticle , we have employed the sequence specificity of ssodn to achieve this goal . [SEP]
[CLS] in this work , we describe the covalent attachment of complementary ssodn to particles of 600 nm barium titanate ( bto ) and 200 nm nickel B-material ferrite ( nfo ) and the interaction of nanoparticles B-nanoparticle possessing this modification to form a core - shell structure that was inferred from sem images and scanning microwave microscopy B-technique ( smm ) phase , amplitude and capacitance images at 14 ghz . [SEP]
[CLS] this approach is of importance since the strength of bonding , specificity of interaction , and distance between nanoparticles B-nanoparticle can be controlled using custom designed dna . [SEP]
[CLS] furthermore , we show that the thermal reversibility of binding allows a reorganization of the morphology of the nanocomposites to assemble mm - size or greater structures based on nm periodicity . [SEP]
[CLS] melting the dna - dna interactions at 80 • c followed by reassembly and cooling in a static or rotating magnetic B-property field results in formation of mm - size linear chains and 100 µm diameter rings , respectively . [SEP]
[CLS] magneto - electric characterization of as - assembled particles by static field induced polarization and low - frequency me voltage coefficient reveal strong coupling between the ferromagnetic and ferroelectric phases . [SEP]
[CLS] further strengthening of me interactions is measured in samples that were annealed at high temperatures so that dna - linkers are eliminated and efficient transfer of mechanical strain occurs at the ferromagnetic - ferroelectric interface . [SEP]
[CLS] details on nanoparticles B-nanoparticle assembly , characterization by several microscopy B-technique techniques and studies on me coupling are provided in the sections that follow . [SEP]
[CLS] the composite synthesis involved the following steps . [SEP]
[CLS] ( i ) synthesis of nfo particles by coprecipitation . [SEP]
[CLS] vendor supplied bto was . [SEP]
[CLS] ( ii ) azide modification of nanopartilces . [SEP]
[CLS] ( iii ) attachment of alkyn - functionalized complementary dna to nanoparticles B-nanoparticle by click - reaction . [SEP]
[CLS] ( iv ) composite formation by dna - dna interaction . [SEP]
[CLS] batio 3 ( dia . 600 nm ) was purchased from inframat advanced materials and nife 2 o 4 ( dia . 200 nm ) nanoparticles B-nanoparticle were prepared by co - precipitation followed by sintering at 650 • c . [SEP]
[CLS] the particle size ranges were verified by sem . materials required for the synthesis [SEP]
[CLS] ii . experimentare as follows . [SEP]
[CLS] sodium B-material azide and allyl alcohol B-material ( ≥ 99 % ) were obtained from sigma - aldrich . [SEP]
[CLS] 3 - buten - 1 - ol ( 98 + % ) was purchased from gfs organic chemicals . [SEP]
[CLS] sodium B-material l - ascorbate was purchased from tci . [SEP]
[CLS] methanesulfonyl chloride B-material was purchased from alfa aesar . [SEP]
[CLS] copper ( ii ) acetate monohydrate and trimethylamine were purchased from acros . [SEP]
[CLS] dmso was obtained from mallinckrodt . [SEP]
[CLS] tris ( 3 - hydroxypropyltriazolylmethyl ) - amine B-material was prepared by a known method . [SEP]
[CLS] dichloromethane was purified using an innovative technologies , inc . solvent purification system . [SEP]
[CLS] methanol , chloroform , and isopropyl alcohol B-material ( fischer ) were used as received . [SEP]
[CLS] a milli - q water B-material purification system ( millipore corporation ) was used to generate purified water B-material . [SEP]
[CLS] oligonucleotides were purchased from keck laboratories ( yale university ) and used as received . [SEP]
[CLS] the sequences were xa 10 attatcact ( 1 ) , xa 10 agtgataat ( 2 ) , xagtgataatcgctatat ( 3 ) , and xatatagc - gattatcact ( 4 ) ( x = 5 ' - hexynyl ) . [SEP]
[CLS] azide modified nanoparticles B-nanoparticle : 1 g of each batio 3 ( dia . 600 nm ) and nife 2 o 4 ( dia . 200 nm ) nanoparticles B-nanoparticle were functionalized with a terminal azide group using a previously published method . [SEP]
[CLS] under nitrogen B-material , the nanoparticles B-nanoparticle were immersed in 3 - buten - 1 - ol ( 4 ml ) in a 50 ml round bottom flask which was stoppered . [SEP]
[CLS] the flask was sonicated while being exposed to ultraviolet ( uv ) light ( 254 nm , [UNK] mw / cm 2 ) for 17 h to graft the 3 - buten - 1 - ol molecules to the nanoparticle B-nanoparticle surface via the alkene group . [SEP]
[CLS] a small fan was used to keep the samples from heating above 40 • c . the samples were then rinsed sequentially with meoh , chcl 3 , and i proh . [SEP]
[CLS] the terminal −oh groups were converted to methanesulfonyl ( mesyl ) groups by immersing the sample in a 10 : 1 : 1 ( by volume ) ch 2 cl 2 / triethylamine at 0 • c then adding methanesulfonyl chloride B-material and allowing the mixture to sit for 1 h at 0 • c . after rinsing with ch 2 cl 2 , meoh , and i proh the mesyl groups were converted to azide groups by immersing the sample in a solution of supersaturated nan B-technique 3 in dmso for 15 h at 80 • c . [SEP]
[CLS] rinsing with deionized B-material water I-material for one minute followed by i proh resulted in azide - modified nanoparticles B-nanoparticle . [SEP]
[CLS] after washing the nanoparticles B-nanoparticle with water B-material and isopropyl alcohol B-material , they were dried under high vacuum and transferred to separate 100 ml round bottom flasks . [SEP]
[CLS] attachment of dna to nanoparticles B-nanoparticle : alkyne - functionalized dna , ( 1 µmol , keck lab , yale university ) was added to a flask containing each azide - functionalized nanoparticle B-nanoparticle followed by 1 . 5 ml deionized B-material water I-material , 25 ml dmso containing 1 . 2 mg copper ( ii ) acetate monohydrate , 10 . 6 mg tris [ ( 1 - benzyl - 1h - 1 , 2 , 3 - triazol - 4 - yl ) methyl ] amine B-material ( tbta ) , and 8 . 8 mg sodium B-material ascorbate . [SEP]
[CLS] the mixtures were stoppered and sonicated for 15 min . [SEP]
[CLS] after this , the mixtures were allowed to sit for 14 h at 25 • c . [SEP]
[CLS] the samples were washed with 100 mm pbs buffer three times then deionized B-material water I-material . [SEP]
[CLS] after drying under high vacuum , the samples were stored at −20 • c . [SEP]
[CLS] dna - functionalized particles were combined in 100 mm pbs buffer in various ratios and then washed with deionized B-material water I-material . [SEP]
[CLS] preparation of composites : 10 mg nife 2 o 4 , ( nfo , 200 nm ) functionalized with 3 and 10 mg batio 3 , ( bto , 600 nm ) functionalized with 4 were placed in a test tube and 2 ml of 100 mm nacl was added . [SEP]
[CLS] the sample was vortexed and then placed in a bath at 80 • c for 5 min . [SEP]
[CLS] the sample was sonicated for 1 min and allowed to cool to room temperature . [SEP]
[CLS] the sample was centrifuged and the supernatant was discarded . [SEP]
[CLS] the sample was washed with water B-material and placed under high vacuum for drying . [SEP]
[CLS] clusters and individual core - shell nanoparticles B-nanoparticle were analyzed with a scanning electron microscope ( model : jsm - 6510 / gs from jeol , usa ) and an agilent scanning microwave microscope ( smm ) . [SEP]
[CLS] the smm is essentially an atomic force microscope that operates in contact mode , but is interfaced to a vector network analyzer . [SEP]
[CLS] the smm probe directs microwave power at frequencies of 1 to 26 ghz onto the sample surface and measures the phase and amplitude of the scattering parameter s 11 . [SEP]
[CLS] since nfo and bto have their unique response to microwave power in terms of phase and amplitude of reflected power , it is possible to obtain well resolved images showing the structure of assembled core - shell particles . [SEP]
[CLS] due to the ability of microwaves to penetrate the assembled particles up the skin depth , smm is an ideal tool for studies on the core - shell particles . [SEP]
[CLS] another smm imaging mode of importance for this study is the capacitance image . [SEP]
[CLS] the smm tip and the particle from a metal B-material oxide - semiconductor ( mos ) capacitor yields an image of the capacitance profile over the entire particle . [SEP]
[CLS] the strength of me coupling was measured by static magnetic B-property field induced polarization by p vs e measurements under h using a ( radiant ) ferroelectric tester . [SEP]
[CLS] discs of as - assembled core - shell particles and high temperature annealed samples were used . [SEP]
[CLS] for comparison similar measurements were carried out on bulk nfo - bto samples made by mixing the nanoparticles B-nanoparticle . [SEP]
[CLS] low - frequency me voltage coefficient ( mevc ) was measured on thin discs of the composites made by sintering as - assembled composite particles . [SEP]
[CLS] we choose ferroelectric bto and ferrimagnetic nickel B-material ferrite ( nfo ) due to their robust ferroic response at room temperature and high chemical , thermal and mechanical stability . [SEP]
[CLS] nickle ferrite particles with an average size of 200 nm and bto of diameter 600 nm were used in this study . [SEP]
[CLS] nanometer sized nfo particles were prepared by co - precipitation techniques , followed by high temperature annealing . [SEP]
[CLS] vendor supplied bto particles were used . [SEP]
[CLS] 1 shows the sem images of the nfo particles and data on the room temperature magnetization B-property m versus static field h . [SEP]
[CLS] the saturation magnetization B-property of 35 emu / g is in agreement with expected value for bulk nfo . [SEP]
[CLS] a . ferromagnetic and ferroelectric nanoparticlesnanoparticle surfaces . [SEP]
[CLS] a sequence in the literature touted as leading to specific dna - dna hybridization when complementary strands were brought together was employed . [SEP]
[CLS] the complementary sequences are shown below : odn - 1 : xaaaaaaaaaaattatcact odn - 2 : xaaaaaaaaaaagtgataat x = 5 ' hexynyl 3 - buten - 1 - ol was grafted to the surfaces of 600 nm bto and 200 nm nfo using 254 nm light under anaerobic conditions . these groups were then converted to alkyl azide groups . [SEP]
[CLS] to attach the dna , we mixed the particles with the appropriate odn ( odn - 1 for bto and odn - 2 for nfo ) in distilled B-material water I-material . [SEP]
[CLS] a mixture of copper ( ii ) acetate monohydrate , tris [ ( 1 - benzyl - 1h - 1 , 2 , 3 - triazol - 4yl ) methyl ] amine B-material , and ascorbate in dmso were added to effect coupling ( scheme 1 ) . [SEP]
[CLS] the mixtures were allowed to react for 14 h and then centrifuged and washed with 100 mm saline solution and then water B-material . [SEP]
[CLS] the samples were then dried under high vacuum and stored at fig . 2 . [SEP]
[CLS] sem images of 1 : 1 mixtures of 600 nm bto - odn - 1 and 200 nm nfo - odn - 2 at various magnifications after being washed with deionized B-material water I-material . [SEP]
[CLS] −20 • c . when bto - odn - 1 was mixed with nfo - odn - 2 in saline buffer ( 100 mm ) , sonicated , and washed with water B-material to remove excess saline , clear heteronanoparticle interaction was observed with minimal self - aggregation ( figure 2 ) . [SEP]
[CLS] formation of bto core nfo shell B-material structures is clearly present . [SEP]
[CLS] the presence of these core - shell structures ( figure 3 ) was further investigated by smm . [SEP]
[CLS] 3 shows s 11 phase and amplitude images obtained at 14 ghz . [SEP]
[CLS] a well resolved bto core B-material and nfo shell B-material are seen in the images . [SEP]
[CLS] both the phase ( figure 3 ( a ) image shows a near - spherical particle with a core B-material diameter of [UNK] 400 nm and shell B-material thickness of [UNK] 300 nm that compare favorably with dimensions of 600 nm bto core B-material and 200 nm nfo shell B-material . [SEP]
[CLS] thus a distribution in the size of core B-material and shell B-material was evident for the smm data . [SEP]
[CLS] the s 11 amplitude image ( figure 3 ( b ) shows likely distortions in the particle shape that are not resolved in the phase image . [SEP]
[CLS] the data in figure 3 do show the expected core - shell structure with contrast in both the amplitude and phase of the reflected microwave power . [SEP]
[CLS] we also carried out smm capacitance c measurements for isolated core - shell particles ( figure 4 ) . [SEP]
[CLS] the capacitance c which is directly proportional to the dielectric constant of the particles can be measured for frequencies ranging from 1 to 26 ghz . [SEP]
[CLS] 4 shows the capacitance image obtained at 14 ghz . [SEP]
[CLS] a line profile showing the c variation across the core B-material and the shell B-material is also shown . [SEP]
[CLS] since c is directly proportional to the permittivity , the image is essentially a permittivity map of the particle . [SEP]
[CLS] upon tracking the profile from left to right , c begins to increase rapidly at the periphery of the particle shell B-material , shows a discontinuity at the shell - core interface and a constant c value over the core B-material . [SEP]
[CLS] a discontinuity at the core - shell interface , followed by a decrease in c in the shell B-material region and a rapid drop in c just beyond the shell B-material region on the right is also seen in the image ( figure 4 ) . [SEP]
[CLS] one could model the capacitance image data to obtain the permittivity of the core B-material and the shell B-material . [SEP]
[CLS] magnetic B-property field assisted assembly ( mfaa ) was utilized to form superstructures of the core - shell particles . [SEP]
[CLS] the assembly was carried out in a uniform field generated by an electromagnet or in the field generated by a rotating permanent magnet . [SEP]
[CLS] a uniform field is expected to magnetize B-property the core - shell particles and align them into a linear chain . [SEP]
[CLS] in a non - uniform field of a permanent magnet the particles will experience an attractive force due to field gradient and will be moved to regions of high field strength . [SEP]
[CLS] for h - assisted assembly a solution was prepared by mixing 20 mg of the particles with 1 ml of deionized B-material water I-material and was ultrasonicated for uniform mixing . [SEP]
[CLS] the assembly procedure involved heating the solution to 80 • c for dehybridization . [SEP]
[CLS] this was followed by slowly cooling the solution on a glass slide under a uniform field of 2 koe produced by an electromagnet or a rotating bar magnet that generated a field gradient of 50 oe / mm across the width of the substrate so that dna - assisted assembly and field directed assembly could occur simultaneously . [SEP]
[CLS] 5 shows the sem micrographs of the samples obtained by mfaa . [SEP]
[CLS] 5 ( a ) shows 2d images of isolated core - shell structures with bto core B-material and nfo shell B-material in different contrast . [SEP]
[CLS] the figure also shows high resolution images of millimeter long linear chains formed under uniform h . a ring - like assembly obtained under the rotation magnetic B-property field is also shown in the figure . [SEP]
[CLS] magneto - electric characterization of the composites usually involve the sample response to ( i ) an applied magnetic B-property field ( termed direct - me effect ) and ( ii ) an applied electric field ( converse aip advances 6 , 045202 ( 2016 ) [SEP]
[CLS] in this study we investigated the direct - me effect by two different techniques : static magnetic B-property field h induced polarization p in the sample and low - frequency me voltage coefficient . [SEP]
[CLS] measurements were first carried out on pressed pellets of as - assembled core - shell composites . [SEP]
[CLS] following this , the disks were annealed at high temperatures so that the samples are free of dna and other coupling agents and the me measurements were carried out on them . [SEP]
[CLS] for comparison we also measured the me interaction strengths in bulk composites prepared by mixing equal amount ( by weight ) of nfo and bto nanoparticles B-nanoparticle to form composites with random distribution of the ferroic phases . [SEP]
[CLS] the sem images in figure 6 for samples annealed at 1200 c clearly show figure 7 shows results on the nature of direct magneto - electric ( dme ) coupling studied by static magnetic B-property field h induced polarization through p vs e measurements in a disk of dna assembled sample . [SEP]
[CLS] data on both as - pressed and high temperature annealed samples are shown . [SEP]
[CLS] the samples were subjected to a uniform in - plane magnetic B-property field . [SEP]
[CLS] a decrease in p due to h is measured in both cases . [SEP]
[CLS] the fractional change in the remnant polarization is plotted as a function of h . the ratio [UNK] r / p r ( h = 0 ) = [ p r ( h ) - p ( h = 0 ) ] / p r ( h = 0 ) versus h measured from p vs . e for h = 0 - 7 koe are shown . [SEP]
[CLS] as h is increased from 0 to 7 koe both samples show an increase in the magnitude of [UNK] r with [UNK] r / p r [UNK] 7 % at h = 7 koe for as - pressed sample and [UNK] % for sintered sample . [SEP]
[CLS] when h is decreased back to zero , a hysteresis is seen in [UNK] r / p r vs h with the magnitude of [UNK] r / p r increasing with decreasing h and reaching a value of [UNK] % for as - assembled and 20 % for sintered sample . [SEP]
[CLS] thus the results in fig . 7 provide clear evidence for magnetostriction induced change in polarization for the composite . [SEP]
[CLS] annealing the sample results in an enhancement of the strength of me coupling by 50 % . [SEP]
[CLS] results on similar measurements on a bulk composite made by mixing equal amount of bto and nfo nanoparticles B-nanoparticle are shown in figure 8 for comparison . [SEP]
[CLS] this particular case corresponds to random mixing of the ferromagnetic and ferroelectric particles , without the nm - periodicity in the case of dna - assembled samples . [SEP]
[CLS] application of h results in a reduction in the polarization . [SEP]
[CLS] in figure 8 one observes identical characteristics in [UNK] r / p r vs h for both unsintered and sintered samples as in fig . 7 for dna - assembled samples . [SEP]
[CLS] but the overall fractional change in p r is a factor of 2 to 3 smaller than for the dna - assembled composites [SEP]
[CLS] thus the most important inferences from the data in figures 7 and 8 of mechanical deformation at the ferromagnetic - ferroelectric interface and strengthening of me interactions . [SEP]
[CLS] we also performed low frequency me voltage coefficient ( mevc ) measurements on sintered discs of dna - assembled sample and composites made by mixing nfo - bto . [SEP]
[CLS] the samples were first poled in e = 1 kv / cm at room temperature and then subjected to a dc bias magnetic B-property field h and an ac field of h ac = 1 oe at 30 hz . [SEP]
[CLS] the ac and dc magnetic B-property fields were parallel to each other and applied either parallel or perpendicular to the disc plane . [SEP]
[CLS] the me voltage induced across the disk thickness was measured as a function of h and field orientations . [SEP]
[CLS] 9 shows mevc vs h data for dna - assembled sample and for mixed nfo - bto samples and for magnetic B-property fields parallel or perpendicular to sample plane . [SEP]
[CLS] consider first the data for dna - assembled samples . [SEP]
[CLS] results in figure 9 ( a ) are for h and h ac parallel to the disc plane . [SEP]
[CLS] the mevc shows a zero - bias ( h = 0 ) value of 20 mv / cm oe and is indicative of a built - in magnetic B-property field arising from dipole - dipole interactions between nfo particles in the core - shell structures . [SEP]
[CLS] with increase in h , mevc decreases and the coupling vanishes at h = 2 koe due to saturation of magnetostriction resulting in zero value for the piezomagnetic coupling q . [SEP]
[CLS] a hysteresis is also seen in mevc vs h data . [SEP]
[CLS] when h is reversed the mevc becomes negative ( a 180 0 phase difference with h ac ) . [SEP]
[CLS] when the magnetic B-property fields are applied perpendicular to the disc plane , mevc vs h data in figure 9 ( b ) show similar features as for in - plane fields . [SEP]
[CLS] but the zero - bias mevc is much smaller than for in - plane fields and the peak in mevc occurs at a much higher h - value compared to in - plane magnetic B-property field due to demagnetizing field . [SEP]
[CLS] the maximum mevc , however , is the same for both in - plane and out - of - plane field orientations . [SEP]
[CLS] similar mevc vs h measurements were done on sintered bulk samples with random distribution of nfo and bto and the results are shown in figure 9 for both in - plane and out - of - plane magnetic B-property field orientations . [SEP]
[CLS] the features in figure 9 ( c ) and 9 ( d ) are essentially identical to the dna - assembled case . [SEP]
[CLS] but the overall strength of me coupling is weaker by a factor 2 compared to the dna - assembled sample . [SEP]
[CLS] now we compare the me coupling in dna - assembled samples with results for similar multiferroic nanocomposites . [SEP]
[CLS] past efforts on ferrite - ferroelectric core B-nanoparticle - I-nanoparticle shell I-nanoparticle nanoparticles B-nanoparticle involved synthesis by chemical self - assembly and me characterization by h - induced polarization , magnetodielectric effect ( change in the permittivity in h ) and me voltage coefficient measurements . [SEP]
[CLS] a change in polarization by 3 % and mevc of 2 to 8 mv / cm oe were reported in these systems . [SEP]
[CLS] the h - induced polarization in figure 8 and mevc in figures 9 for dna - assembled core - shell particulate composites are the highest reported for any ferrite - ferroelectric particulate nanocomposite and the mevc is much higher than low - frequency me coefficient of 1 v / cm oe for nickel B-material ferrite - pzt core - shell nanofibers B-nanoparticle . [SEP]
[CLS] we present here results of the first studies on the assembly of multi - ferroic nanocomposites using dna hybridization . [SEP]
[CLS] the availability of custom - designed dna allows for control over the strength and specificity of interaction between nanoparticles B-nanoparticle . [SEP]
[CLS] the advantage of this method for assembling nanoparticle B-nanoparticle composites is that one can disassemble the composites and reassemble them under various conditions . [SEP]
[CLS] core B-material ( bto 600 nm ) - shell B-material ( nfo 200 nm ) assemblies were studied by sem and smm . [SEP]
[CLS] images of the phase and amplitude of reflected power and capacitance at 14 ghz clearly show a well - defined core - shell structure for the assembled nanoparticles B-nanoparticle . [SEP]
[CLS] melting the dna - dna interactions at 80 • c followed by reassembly and cooling in a static or rotating magnetic B-property field results in formation of mm - size linear chains and 100 µm diameter rings , respectively . [SEP]
[CLS] magneto - electric characterization by static field induced polarization and low - frequency me voltage coefficient reveal strong cross - coupling between the ferroic phases . [SEP]
[CLS] further strengthening of me interactions is measured in samples that were annealed at high temperatures so that dna - linkers are eliminated and efficient transfer of mechanical strain occurs at the ferromagnetic - ferroelectric interface . [SEP]
[CLS] fig [SEP]
[CLS] . 1 . sem images for ( a ) nfo particles with an average size of 200 nm and ( b ) 600 nm bto particles . [SEP]
[CLS] ( c ) roomtemperature m vs h for nfo particles and ( d ) p vs e for a sintered disk of bto particles [SEP]
[CLS] scheme 1 . scheme to assemble dna - linked heteronanocomposites composed of bto - nfo [SEP]
[CLS] fig . 3 . scanning microwave microscopy B-technique images showing well resolved core B-material and shell B-material for an isolated assembled particle . [SEP]
[CLS] the images obtained at 14 ghz show ( a ) phase and ( b ) amplitude of reflected microwave power from the core - shell particle . [SEP]
[CLS] fig . 4 . capacitance ( c ) image at 14 ghz and the line profile of c across the middle of the core - shell particle . [SEP]
[CLS] fig . 5 . ( a , b ) sem images showing a 2d superstructure of 600 nm bto and 200 nm nfo core - shell particles . [SEP]
[CLS] ( c , d ) images of core - shell particle chains / wires obtained in a uniform h - field and ( e ) a ring of core - shell particles assembled in a rotating magnetic B-property field . [SEP]
[CLS] fig . 7 . h - induced polarization in dna - assembled samples . [SEP]
[CLS] data in ( a ) and ( b ) are for a disk of as - assembled composite . ( c ) and ( d ) are for samples annealed at 1200 c . [SEP]
[CLS] fig . 8 . similar results as in figure6for a sample of bulk composite made by mixing equal amount of nfo and bto . [SEP]
[CLS] fig . 9 . low - frequency me voltage coefficient ( mevc ) versus bias field h data for high temperature annealed samples of dna - assembled and sample with random distribution of nfo and bto . [SEP]
[CLS] new neutralizing agents against sars - cov - 2 and the associated mutant strains are urgently needed for the treatment and prophylaxis of covid - 19 . [SEP]
[CLS] herein , we develop a spherical cocktail neutralizing aptamer - gold nanoparticle B-nanoparticle ( snap ) to synergistically block the interaction of sars - cov - 2 receptor B-event - binding domain ( rbd ) and angiotensin - converting enzyme - 2 ( ace2 ) . [SEP]
[CLS] taking advantage of the simultaneous recognition of multi - homologous and multi - heterogenous neutralizing aptamers and dimensionally matched nano - scaffolds , the snap exhibits increased affinity to the rbd with a dissociation constant value of 5 . 46 pm and potent neutralization against authentic sars - cov - 2 with a half - maximal inhibitory concentration of 142 . 80 am . additional benefits include the multi - epitope blocking capability of the aptamer cocktail and the steric hindrance of the nano - scaffold , which further covers the ace2 binding interfaces and affects the conformational transition of the spike protein B-material . [SEP]
[CLS] as a result , the snap strategy exhibits broad neutralizing activity , almost completely blocking the infection of n501y and d614g mutant strains . [SEP]
[CLS] overall , the snap strategy provides a new direction for development of anti - virus infection mechanisms , both to fight the covid - 19 pandemic and serve as a powerful technical reserve for future unknown pandemics . [SEP]
[CLS] the global pandemic of sars - cov - 2 and the associated covid - 19 disease has emphasized the importance of developing new therapeutics against novel viruses . [SEP]
[CLS] the entrance of sars - cov - 2 into host cells B-material is mediated by the rbd of trimeric - spike ( s - rbd ) binding to ace2 expressed on the host cells B-material . [SEP]
[CLS] at present , some neutralizing antibodies B-material against sars - cov - 2 are being developed to block viral entry by binding to the ace2 interaction interfaces on the s - rbd . [SEP]
[CLS] however , as an rna virus , sars - cov - 2 has a high mutation rate , leading to the emergence of many mutant strains such as d614g and n501y , which may escape the therapy of the existing neutralizing antibodies B-material . [SEP]
[CLS] moreover , most neutralizing antibodies B-material against sars - cov - 2 are derived from animal immunizations and convalescent serum , both of which involve complex and time - consuming procedures , and may not result in sufficient immunity . [SEP]
[CLS] therefore , alternative novel strategies to rapidly produce potent neutralizing biologics that are broadly effective against sars - cov - 2 and mutants , as well as future related coronaviruses , are urgently needed . [SEP]
[CLS] in vitro selection for neutralizing aptamers against sars - cov - 2 can be performed without infected individuals , and only proteins B-material are needed as targets . [SEP]
[CLS] compared to neutralizing antibodies B-material ( ~ 150 kda ) , the smaller size of neutralizing aptamers ( ~ 15 kda ) allows them to penetrate tissues that may not be accessible to classical antibodies B-material . [SEP]
[CLS] in addition , neutralizing aptamers can be easily and rapidly produced by chemical synthesis without batch - to - batch differences . [SEP]
[CLS] however , as monomers B-material , neutralizing aptamers cannot ensure simultaneous binding of the three rbd s on the trimeric - spike , resulting in unsatisfactory therapeutic application . [SEP]
[CLS] to form a simultaneous interaction , we engineered spherical cocktail neutralizing aptamer - gold B-nanoparticle nanoparticle I-nanoparticle ( snap ) conjugates to synergistically block the trimeric - spike thus inhibiting sars - cov - 2 infection . [SEP]
[CLS] snap strategy offers several unique advantages . [SEP]
[CLS] first , homoand hetero - neutralizing aptamers can be easily anchored with high density and homogeneous orientation on the same gold B-nanoparticle - I-nanoparticle nanoparticle I-nanoparticle , affording improved binding affinity and simultaneous blocking of different rbds on the trimeric - spike . [SEP]
[CLS] second , the particle scaffold can provide a dimensionally matched interaction between spherical neutralizing aptamers ( [UNK] nm ) and the trimeric - spike ( [UNK] nm ) . [SEP]
[CLS] the resultant spatially organized interaction effectively adds a " hat " to the trimeric - spike ' s apex of s - rbd . [SEP]
[CLS] the nanoscale " hat " not only largely covers the ace2 binding interfaces on the s - rbd , but also affects the conformational transition of the trimeric - spike , which is another crucial step associated with infection and pathogenicity , as well as the interaction of s - rbd and ace2 . [SEP]
[CLS] therefore , snap provides appropriate steric hindrance against both viral recognition and fusogenic conformational formation , resulting in excellent neutralization potency . [SEP]
[CLS] finally , gold B-nanoparticle nanoparticles I-nanoparticle provide aptamers with a reduced the propensity toward nuclease degradation compared to the linear aptamer counterparts . [SEP]
[CLS] therefore , multi - homologous and multiheterogenous aptamers on the snap conjugate are expected to target not only distinct epitopes of rbd monomers B-material within a trimeric - spike , but also different rbd s on the trimeric - spike , thus inhibiting the infection of sars - cov - 2 with ideal potency . [SEP]
[CLS] the mechanism of ( a ) sars - cov - 2 infection by interaction of trimericspike and ace2 . [SEP]
[CLS] the three rbd in the trimeric - spike ( grey ) are depicted in violet , green , and pink , respectively . [SEP]
[CLS] ace2 is depicted in light pink . [SEP]
[CLS] ( b ) the mechanism of sars - cov - 2 neutralization by snap . [SEP]
[CLS] snap is depicted in gold B-material , and the three neutralizing aptamers depicted in blue , green , and pink , respectively . [SEP]
[CLS] since sars - cov - 2 is known to mutate , to avoid the increased risk of mutation - induced neutralization escape , it is crucial to apply a potent series of neutralizing aptamers as a cocktail for therapy . [SEP]
[CLS] to form multiheterogenous binding , three aptamers ( cov2 - 1c , cov2 - 4c , and cov2 - 6c3 ) , each targeting sars - cov - 2 , were chosen for snap formation . [SEP]
[CLS] the results of cross - competition experiments ( figure 1a ) and the reported molecular dynamics simulations ( figure 1b ) suggest that these three aptamers have very little interference with each other in rbd recognition and interact with three non - overlapping epitopes on rbd . [SEP]
[CLS] we then investigated the neutralization efficiency by determining the binding tendency of ace2 with rbd with or without pre - blocking by the individual aptamers or the aptamer cocktail . [SEP]
[CLS] consistent with the predicted results , the aptamer cocktail has the highest rbd neutralization efficiency compared to the any of the single aptamers ( figure 1c ) . [SEP]
[CLS] the rbd neutralization efficiency of aptamer cocktail is 10 . 3 times , 2 . 7 times , and 1 . 4 times that of cov2 - 1c , cov2 - 4c , and cov2 - 6c3 aptamers , respectively . [SEP]
[CLS] it is worth noting that although cov2 - 6c3 aptamer plays the leading role of inhibition , cov2 - 1c ( dissociation constant ( k d ) value of 5 . 8 nm ) and cov2 - 4c ( k d of 19 . 9 nm ) aptamers have better rbd binding affinities than that of cov2 - 6c3 aptamer ( k d of 44 . 8 nm ) [SEP]
[CLS] thus , addition of cov2 - 1c and cov2 - 4c aptamers not only reduces the escape of mutant strains , but also improves the recognition dynamics of snap . [SEP]
[CLS] to improve binding affinity and neutralization potency , we utilized 5 nm gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) as the scaffold to assemble the aptamer cocktail and form snap conjugates , with the goal of blocking multiple rbds on the same spike simultaneously . [SEP]
[CLS] aunps B-nanoparticle were functionalized with sh - labeled cocktail aptamers by the freeze - thaw method . [SEP]
[CLS] as shown in the results of transmission B-technique electron I-technique microscopy I-technique ( fig 2a and b ) and dynamic B-technique light I-technique scattering I-technique ( fig 2c and d ) , the snap conjugate has a slightly larger diameter and a smaller polydispersity index value than those of bare aunps B-nanoparticle , suggesting the successful modification and good monodispersibility of snap conjugates . [SEP]
[CLS] the aptamer number on each aunp B-nanoparticle was evaluated to be ca . 340 , guaranteeing multivalent binding for better neutralization . [SEP]
[CLS] the k d value of snap conjugate against rbd protein B-material was found to be 5 . 46 pm , about 1000 - 8000 - fold higher affinity than the standalone aptamers [SEP]
[CLS] moreover , snap also recognizes the sars - cov - 2 spike protein B-material ( figure s2 ) , but no to the rbd or spike protein B-material of other viruses ( figure s3 ) , indicating the ideal universality and selectivity of snap . [SEP]
[CLS] in addition to multivalent interaction , the dense oligonucleotide shell B-material of the snap conjugate can deactivate the enzymes in close proximity to the aptamers due to the high local sodium B-material ion B-material concentration . [SEP]
[CLS] therefore , snap can evade nucleases , resulting in a decreased rate of hydrolysis compared to the single aptamers ( figure 2g and h ) , thereby improving the stability in vivo . [SEP]
[CLS] to confirm the neutralization of virus infection by snap inhibition of s - rbd and ace2 interaction , we utilized a pseudotyped virus , which was packed with the sars - cov - 2 trimetric - spike and contained the plasmid consisting of a gfp and a luciferase reporter . [SEP]
[CLS] as shown in fig 3a , snaps protected more than 96 % of the cells B-material from pseudotyped sars - cov - 2 infection , displaying much higher potency than the respective aptamer cocktail ( 64 . 52 % ) . [SEP]
[CLS] this success is attributed to the simultaneous interaction of snap with multiple epitopes on multiple rbds within the trimeric - spike using homo - and hetero - aptamers , thus more fully occluding the ace2 binding interfaces via the aunp B-nanoparticle scaffold . [SEP]
[CLS] in addition , the neutralizing efficiency of snap is 1 . 3 times that of cov2 - 6c3 - aunp B-nanoparticle , indicating that the cov2 - 1c and cov2 - 4c aptamers are also useful in the overall binding to rbd and inhibition of the virus infection , possibly due to the higher binding affinities of cov2 - 1c and cov2 - 4c aptamers [SEP]
[CLS] it is also worth noting that even for the cov2 - 1c aptamer with a weak inhibition ability ( 39 . 22 % ) , the inhibition effect was obviously improved after incorporation of cov2 - 1c - aunp B-nanoparticle with the 5 nm gold B-material - scaffold ( 50 . 83 % ) . [SEP]
[CLS] the inhibition ability of snap ( 96 . 16 % ) is also greater than that of a commercial neutralizing antibody B-material ( 77 . 52 % , research resource identifiers number : ab _ 2857935 ) at the same concentration . [SEP]
[CLS] and snap conjugates exhibited excellent inhibitory activity against pseudotyped sars - cov - 2 virus with a half - maximal inhibitory concentration ( ic 50 ) of 207 . 70 fm ( figure 3c ) , which is about four orders of magnitude smaller than cov2 - 6c3 aptamer . [SEP]
[CLS] moreover , as reported in the literature of 5 nm gold B-nanoparticle nanoparticles I-nanoparticle , snap showed no obvious cytotoxicity B-property ( figure s4 ) and immunogenicity B-property ( figure s5 and s6 ) . [SEP]
[CLS] cryo - electron microscopy B-technique imaging was carried out to validate the interaction between snap and the pseudovirus . [SEP]
[CLS] snap acts as a nanoscale " hat " to cover the spike of the pseudovirus , resulting in a slight deformation of the spike protein B-material ( figure 3b , figure s7 ) . [SEP]
[CLS] overall , based on neutralization by the aptamer cocktail and the steric effect of the gold B-material scaffold , which hinders the interaction of trimeric - spike - ace2 and the conformational transition of the trimeric - spike , the snap strategy is highly likely to enable synergistic - blocking of the trimeric - spike for potent inhibition of sars - cov - 2 infection . [SEP]
[CLS] recently , two new sars - cov - 2 variants with n501y mutation have shown growth rates and effective reproductive numbers in the united kingdom ( 501y . v1 ) and south africa ( 501y . v2 ) , respectively . [SEP]
[CLS] several studies have shown that the two mutated strains have higher transmissibility and severity than that of the original version , are more infectious , and are less susceptible to some existing vaccines or treatment regimens . [SEP]
[CLS] therefore , there is an urgent need to develop new strategies that can respond to different mutant strains . [SEP]
[CLS] in addition to the original version of sars - cov - 2 , the snap conjugate ' s binding performance targeting s - rbd and its neutralization ability against pseudotyped virus strain with n501y mutation were investigated . [SEP]
[CLS] as shown in figure 4a , snap retains its binding capability targeting s - rbd with n501y mutation . [SEP]
[CLS] therefore , snap is expected to be useful for neutralization of the mutated virus strains . [SEP]
[CLS] as expected , snap conjugates block almost 100 % of n501y mutated pseudotyped viruses from infecting cells B-material ( figure 4b and c ) . [SEP]
[CLS] in addition , the binding affinity and neutralization of snap against the pseudotyped virus with d614g mutation were also evaluated ( figure 4d ) . [SEP]
[CLS] snap conjugates block more than 96 % of d614g mutated pseudotyped viruses from infecting cells B-material ( figure 4f ) , with a neutralization ic 50 value of 1 . 24 pm ( figure 4e ) . [SEP]
[CLS] however , the aptamer cocktail almost loses its neutralizing ability , probably because the binding modes of the mutated spike - ace2 complex have changed . [SEP]
[CLS] therefore , although the aptamers themselves can bind to the mutated proteins B-material , the binding sites of aptamers on the mutated spike may no longer be the amino B-material acids I-material involved in the binding of mutated spike ( d614g ) and ace2 . [SEP]
[CLS] these results suggest that the ideal neutralization capacity of snap for different mutated virus strains may be due to the multi - site recognition of cocktailed aptamers and the synergistic - blocking mechanism . [SEP]
[CLS] since the inhibition mechanism of the snap strategy involves both the conventional neutralization strategy of competing ace2 binding sites on rbd by the aptamers themselves , and the steric resistance effect of the nano - scaffold , in theory the snap strategy still can inhibit virus infection to a certain extent even if the interaction mode of the mutated strain and ace2 have changed . [SEP]
[CLS] to further demonstrate the potential of snap for the treatment of covid - 19 , the inhibitory activity of snap using authentic sars - cov - 2 with d614g mutation ( genbank : mt835143 . 1 ) was assessed . [SEP]
[CLS] the ic 50 value of the authentic sars - cov - 2 neutralization was determined by quantitative polymerase chain reaction of the sars - cov - 2 genome in cellular rna . [SEP]
[CLS] snap conjugates exhibited excellent inhibitory activity against authentic sars - cov - 2 virus with a half - maximal inhibitory concentration ( ic 50 ) of 142 . 8 am ( figure 5a ) , 10000 - to 10000 - fold lower than that of the reported neutralizing antibodies B-material . [SEP]
[CLS] moreover , the results of immunofluorescent staining by the antibody B-material against sars - cov - 2 nucleocapsid protein B-material further validated the neutralization effect of snap strategy against authentic sars - cov - 2 virus . [SEP]
[CLS] as shown in figure 5b , snap protected about 93 % of host cells B-material from sars - cov - 2 infection . [SEP]
[CLS] therefore , the neutralization effects against both the pseudovirus and authentic sars - cov - 2 virus confirm that snap conjugates can inhibit viral entry into the host cell B-material , suggesting that the snap strategy has great potential for covid - 19 prophylactics and therapeutics . [SEP]
[CLS] in conclusion , we have developed a synergistical - blocking strategy against trimeric - spike to potently inhibit sars - cov - 2 infection using a spherical neutralizing aptamer with several key advantages . [SEP]
[CLS] first , homoand hetero - neutralizing aptamers themselves can simultaneously block distinct epitopes on rbd which are involved in the interaction with ace2 . [SEP]
[CLS] second , the 5 nm gold B-material scaffold matches the size of the trimeric - spike to ensure that the neutralizing aptamers enable multi - homologous and multiheterogenous recognition and thus promote simultaneous binding to the three rbds on the trimeric - spike , but also provides steric hindrance to block the interaction of rbd - ace2 . [SEP]
[CLS] based on the synergistical - blocking mechanism , snap strategy is vastly capable of neutralizing pseudotyped and authentic sars - cov - 2 , and even the n501y or d614g mutant strains . [SEP]
[CLS] moreover , the u . s . food and drug administration has approved several aunp B-nanoparticle - based therapeutic strategies because of the non - toxic and nonimmunogenic nature of aunps B-nanoparticle . [SEP]
[CLS] based on the previous complete biosafety studies on aunp B-nanoparticle , the clinical application of snap strategy for sars - cov - 2 therapy may be accelerated . [SEP]
[CLS] therefore , the snap strategy holds great potential for sars - cov - 2 prevention and treatment , as well as for in - depth research and development of new anti - virus infection mechanisms , which could greatly assist in preventing future unknown pandemics . [SEP]
[CLS] ( a ) the results of cross - competition experiments between any two of cov2 - 1c , cov2 - 4c and cov2 - 6c3 aptamers . [SEP]
[CLS] one fluoresces labeled aptamers were mixed two unlabeled aptamers and rbd - beads were incubated B-technique , washed and analyzed by flow B-technique cytometry I-technique . [SEP]
[CLS] the percentage of the cross - competition was calculated by the equation ( the fluorescence B-property signal of the fluorescent B-property aptamer with the other two non - labeled aptamers - background signal ) / the fluorescence B-property signal of the fluorescent B-property aptamer without the other two non - labeled aptamers - background signal ) . [SEP]
[CLS] ( b ) molecular dynamics simulated binding modes of cov2 - 1c , cov2 - 4c and cov2 - 6c3 aptamers against sars - cov - 2 rbd ( in grey ) . [SEP]
[CLS] the ace2 binding amino B-material acids I-material on rbd are red . [SEP]
[CLS] ( c ) rbd neutralization efficiency of candidate sequences and aptamer cocktail . [SEP]
[CLS] the percentage of rbd neutralization in the presence of cov2 - 1c , cov2 - 4c and cov2 - 6c3 was compared to rbd binding without aptamers , calculating by the equation ( 1 - ( the fluorescence B-property signal of the sample with the aptamer incubation - background signal ) / ( the fluorescence B-property signal of the sample without the aptamer incubation - background [SEP]
[CLS] 2 . transmission B-technique electron I-technique microscopy I-technique images of the aunps B-nanoparticle ( a ) and snaps ( b ) . [SEP]
[CLS] ( a ) inhibition of sars - cov - 2 pseudovirus infection of ace2 - expressing 293t cells B-material by 340 nm aptamers and 1nm snap . [SEP]
[CLS] ( b ) the neutralization of pseudotyped sars - cov - 2 virus by snap assessed through ic50 . [SEP]
[CLS] ( c ) the cryo - electron B-technique microscopy B-technique image ( left ) and schematic diagram ( right ) of interaction between snap conjugate and sars - cov - 2 pseudovirus . [SEP]
[CLS] the concentration of virus is about 100 times the concentration of snap . [SEP]
[CLS] the scale bar is 50 nm . [SEP]
[CLS] binding affinity of snap against s - rbd with n501y mutation ( a ) and spike with d614g mutation ( d ) . [SEP]
[CLS] the neutralization of pseudotyped sars - cov - 2 virus with n501y mutation ( b ) and pseudotyped sars - cov - 2 virus with d614g mutation ( e ) by snap assessed through ic50 . [SEP]
[CLS] images of snap and the antibody B-material neutralization of sars - cov - 2 pseudovirus with n501y mutation ( c ) and d614g mutation ( f ) infection of ace2 - expressing 293t cells B-material . [SEP]
[CLS] ( a ) the neutralization of authentic sars - cov - 2 virus by snap assessed through ic50 . [SEP]
[CLS] ( b ) images of snap neutralization of authentic sars - cov - 2 infection of vero e6 cells B-material . [SEP]
[CLS] the cells B-material were fixed and stained using an anti - sars - cov - 2 nucleocapsid antibody B-material for the virus and hoechst dye for the cell B-material nucleus . [SEP]
[CLS] using self - assembly , nanoscale materials can be fabricated from the bottom up . [SEP]
[CLS] opals and inverse opals are examples of self - assembled nanomaterials B-material made from crystallizing colloidal particles . [SEP]
[CLS] as self - assembly requires a high level of control , it is challenging to use building blocks with anisotropic geometry to form complex opals , which limits the realizable structures . [SEP]
[CLS] typically , spherical colloids are employed as building blocks , leading to symmetric , isotropic superstructures . [SEP]
[CLS] however , a significantly richer palette of directionally dependent properties are expected if less symmetric , anisotropic structures can be created , especially originating from the assembly of regular , spherical particles . [SEP]
[CLS] here we show a simple method to introduce anisotropy into inverse opals by subjecting them to a post - assembly thermal treatment that results in directional shrinkage of the silica matrix caused by condensation of partially hydrated sol - gel silica structures . [SEP]
[CLS] in this way , we can tailor the shape of the pores , and the anisotropy of the final inverse opal preserves the order and uniformity of the self - assembled structure , while completely avoiding the need to synthesize complex oval - shaped particles and crystallize them into such target geometries . [SEP]
[CLS] detailed x - ray photoelectron spectroscopy B-technique ( xps ) and infrared ( ir ) spectroscopy B-technique studies clearly identify increasing degrees of sol - gel condensation in confinement as a mechanism for the structure change . [SEP]
[CLS] a computer simulation of structure changes resulting from the condensation - induced shrinkage further confirmed this mechanism . [SEP]
[CLS] as an example of property changes induced by the introduction of anisotropy , we characterized the optical spectra of the anisotropic inverse opals and found that the optical properties can be controlled in a precise way using calcination temperature . [SEP]
[CLS] self - assembly provides a way to structure materials with nanoscale dimensions without using expensive , complicated , and time consuming top - down fabrication methods . [SEP]
[CLS] however , selfassembly requires a high level of control over the assembly conditions and the morphology and composition of individual unit elements . [SEP]
[CLS] this limits the choice of constituent building blocks that can be used and hence the variety of accessible assembly structures formed by the building blocks ' minimum - free - energy arrangements . [SEP]
[CLS] in particular , spherical colloids form highly symmetric , face - centered cubic crystals , called opals . [SEP]
[CLS] this structure can be inverted to form an inverse opal by filling in the interstitial spaces and removing the colloidal template , resulting in a continuous matrix of periodically arranged , interconnected voids with the same symmetry as the original colloidal template . [SEP]
[CLS] inverse opals with high long - range order can be made using a co - assembly process . [SEP]
[CLS] a thin film is grown by evaporative deposition of polymeric colloids in the presence of a silica sol - gel precursor that condenses in the colloidal crystal interstitial spaces . [SEP]
[CLS] the co - assembly method allows the growth of extremely highly ordered crystals with single domains stretching over macroscopic dimensions . [SEP]
[CLS] upon calcination , the polymeric colloids are completely combusted and the silica precursor fuses into a dense silicon B-material dioxide I-material scaffold , creating a hexagonally closepacked array of interconnected voids . [SEP]
[CLS] highly interconnected porosity and long - range order are two key attributes of inverse opal structures that make them valuable for a variety of applications . [SEP]
[CLS] due to their periodic nature , inverse opals are a versatile material B-material platform for controlling electromagnetic and acoustic wave propagation . [SEP]
[CLS] in particular , if their periodicity is tailored to the length scale of the wavelength of visible light , inverse opals interact strongly with visible light , allowing applications in solar cells B-material , as light emitting diodes , and as structural color - based coatings B-material . [SEP]
[CLS] in addition , inverse opals ' interconnected porosity and high surface area are essential for their use in many other contexts , including batteries and catalysts B-property , as well as sensors of liquid media , scaffolds for tissue engineering , substrates for omniphobic surfaces , and for studying diffusion processes in confinement . [SEP]
[CLS] all of these remarkable properties of inverse opals depend strongly on precise control over the structural uniformity . spherical colloids are simple to synthesize and crystallize , so they are typically used as templates . [SEP]
[CLS] however , anisotropic structures would offer a range of unique , direction - dependent properties due to the changes in the angular periodicities and directional porosity , and are therefore highly desirable , but a simple method to obtain anisotropic inverse opals is necessary in order to investigate these directional properties . [SEP]
[CLS] prior research efforts to form ordered structures with different morphologies have primarily focused on the design and crystallization of anisotropically shaped colloidal particles . [SEP]
[CLS] however , their synthesis is demanding and crystallization into uniform , defect - free structures remains a challenge . [SEP]
[CLS] successful examples include crystallization of gold B-material nanorods B-nanoparticle , assembly of anisotropic magnetic B-property particles in magnetic B-property fields , dna templating of anisotropic colloidal crystals , and more recently , assembly of anisotropic silica rods in an electric field . [SEP]
[CLS] these anisotropic structures have important directional properties and have been used for plasmonic sensing . [SEP]
[CLS] alternatively , the challenges of synthesizing and crystallizing anisotropic particles can be avoided by introducing anisotropy after the assembly of isotropic building blocks , for example by mechanical deformation of elastomeric inverse opal structures . [SEP]
[CLS] here we describe a simple approach to create anisotropy in inverse opal structures consisting of an inorganic , non - flexible metal B-material oxide I-material matrix . [SEP]
[CLS] our approach allows us to precisely tailor the anisotropy while preventing the need for complex crystallization techniques . [SEP]
[CLS] instead , we take advantage of the molecular state of the structure to introduce anisotropy . [SEP]
[CLS] silica sol - gels undergo condensation of remaining hydroxyl B-material groups I-material at high temperature , leading to volume decrease via loss of water B-material . [SEP]
[CLS] in the co - assembly process , the material B-material is deposited on a substrate that holds the sol - gel material B-material in place . [SEP]
[CLS] this surface constraint prevents shrinking in the plane of the substrate and leads to directional out - of - plane contraction of the film , as shown schematically in cross - section in scheme 1 . [SEP]
[CLS] this provides a facile method to introduce anisotropy into the system after assembly , resulting in controlled changes in the inverse opal properties . [SEP]
[CLS] we present a detailed investigation of this calcination - induced anisotropy in self - assembled inverse opals . [SEP]
[CLS] the calcination temperature controls the degree of condensation , as confirmed using x - ray photoelectron spectroscopy B-technique ( xps ) and infrared B-technique spectroscopy I-technique ( ir ) . [SEP]
[CLS] the amount of shrinkage directly relates to the degree of condensation , allowing us to precisely alter the pore shape and to tailor properties that depend strongly on the pore geometry of the structure . [SEP]
[CLS] in particular , we demonstrate how the optical properties of inverse opals change with increasing anisotropy . [SEP]
[CLS] it is important to note that the anisotropy is generated during post - assembly processing , thus circumventing the complex synthesis and assembly of anisotropic building blocks . [SEP]
[CLS] instead , a generalized and reliable assembly strategy of spherical particles is used to generate crystals with adjustable pore shape anisotropy . [SEP]
[CLS] chemicals and materials [SEP]
[CLS] inverse opals were synthesized using a process described in detail elsewhere . [SEP]
[CLS] briefly , polystyrene colloids with diameters of 230 , 330 , and 410 nm were synthesized using a surfactant - free emulsion polymerization process . [SEP]
[CLS] they were then diluted in water B-material to make 0 . 1 % solid content in 20 ml total volume . [SEP]
[CLS] 150 μl of pre - hydrolyzed teos solution ( 1 : 1 : 1 . 5 by weight of tetraethyl orthosilicate : 0 . 1 m hcl : ethanol , stirred for 1 h ) scheme 1 . [SEP]
[CLS] condensation in the silica matrix of an inverse opal causing anisotropic shrinking . [SEP]
[CLS] was added and the mixture was sonicated . [SEP]
[CLS] a silicon B-material wafer was suspended vertically in the vial . [SEP]
[CLS] the vial was then placed inside an oven for two days to allow for evaporation of the solvent , leaving behind a colloidal crystal with a silica matrix surrounding it . [SEP]
[CLS] a memmert uf110 oven set to 65°c stabilized by a pneumatic vibration - free table was used for the co - assembly process . [SEP]
[CLS] the colloidal crystal template was removed either by submersion in toluene or tetrahydrofuran overnight to dissolve the polystyrene , or by calcination to 500°c , 600°c , 800°c , 1000°c , or 1100°c using a 2°c / min heating and cooling rate and a two - hour hold , except for the case of 1100°c , which was held at the final temperature for an extended five hours . [SEP]
[CLS] a lindberg blue m box furnace from thermo scientific was used for calcination . [SEP]
[CLS] calcination temperatures below 500°c were not used to ensure complete removal of the polystyrene template . [SEP]
[CLS] deionized B-material water I-material was used from a milli - q system , ethanol was obtained from koptec , and all other reagents ( 0 . 1m hcl , styrene , tetraethylorthosilicate ) were obtained from sigma aldrich . [SEP]
[CLS] ultra plus field emission sem . [SEP]
[CLS] imagej was used for subsequent image analysis including the width : height ratio calculations , based on the pixel size from the zeiss sem software . [SEP]
[CLS] x - ray photoelectron spectroscopy B-technique ( xps ) was performed using a thermo scientific k - alpha xps system . [SEP]
[CLS] lower resolution survey scans were taken followed by higher resolution scans with 50 ms dwell time and 0 . 1 ev resolution for the individual elements , with 10 scans averaged . [SEP]
[CLS] the higher resolution scans were used for quantification . [SEP]
[CLS] attenuated total reflectance ( atr ) infrared ( ir ) spectroscopy B-technique was performed using a bruker vertex 70 ftir spectrometer equipped with a miracle attenuated total reflectance accessory . [SEP]
[CLS] for each spot , 128 scans were averaged over the range 600 - 4000 cm - 1 with 4 cm - 1 resolution . [SEP]
[CLS] for the optical measurements taken at normal incidence , a leica dmrx microscope was used with an ocean optics ussb2000 + spectrometer . [SEP]
[CLS] a > 96 % reflective mirror was used as reference . [SEP]
[CLS] measurements of reference and sample spectra were performed under identical configurations , thus the data is displayed as absolute intensity . [SEP]
[CLS] for the variable angle spectrometry studies , an ocean optics dh - 2000 uv - vis - nir light source was used to illuminate a small spot ( 3 mm ) of the sample at a given incidence angle . [SEP]
[CLS] for each angle of illumination , light was collected at half degree increments for - 75° to + 75° relative to the sample normal and spectrally analyzed using an ocean optics maya pro 2000 spectrometer . [SEP]
[CLS] a reference was taken with the detector at 180° to the light source ; however , the intensity data shown here is displayed in arbitrary units as the light source needed to be attenuated with a pinhole when acquiring the reference in order to prevent saturation of the detector . [SEP]
[CLS] no quantitative color bar is shown , as only the spectral position of the reflectance bands is relevant for the argument made here . [SEP]
[CLS] simulation . [SEP]
[CLS] a 2d finite element simulation was carried out using the commercial software abaqus . [SEP]
[CLS] a linear elastic material B-material model was used . [SEP]
[CLS] the shrinkage of the material B-material was simulated as thermal strain . [SEP]
[CLS] the displacement and rotation of the inverse opal structure proximate to the substrate were constrained , and a periodic boundary condition was applied on a unit cell B-material in the lateral direction . [SEP]
[CLS] the ratio of the pore width to the pore height was recorded as a function of the linear shrinkage . [SEP]
[CLS] anisotropic inverse opals were made using a slight modification to the synthesis procedure . [SEP]
[CLS] the co - assembly of inverse opals involves one crystal growth step , in which a substrate ( usually a silicon B-material wafer ) is submerged in a dispersion of polymeric colloids and pre - hydrolyzed tetraethyl orthosilicate ( teos ) in water B-material . [SEP]
[CLS] the water B-material is evaporated at 65°c , forming a thin polymer B-material / silica opal film on the substrate . [SEP]
[CLS] next , the sample is calcined at 500°c in order to combust the colloids and sinter the sol - gel silica , leaving behind an ordered , porous silica structure . [SEP]
[CLS] changing this final calcination step results in an altered pore structure ( figure 1 ) , giving access to different pore geometries . [SEP]
[CLS] with spherical pore symmetry arising from the shape and size of the colloids , the as - grown structure can be obtained by dissolving the colloids with toluene or tetrahydrofuran ( figure 1a , left ) . [SEP]
[CLS] by raising the calcination temperature , different degrees of anisotropy of the structure , with increasingly deformed pores , can be obtained ( figure 1a ) . [SEP]
[CLS] growing the matrix and template in a single , one - pot synthesis allows for large ( cm - scale ) domains , and this high order is retained even at high calcination temperatures ( figure s1 ) . [SEP]
[CLS] using the ratio of pore width and height to quantify the variation in the geometry , we see that the anisotropy introduced into the structure as a function of calcination temperature follows a roughly linear trend with temperature ( fig 1b ) . [SEP]
[CLS] in all cases , the same heating rate ( 2°c / min ) was used , and the final temperature was held for two hours , except for the case of 1100°c , which was held for an extended five hours . [SEP]
[CLS] chemical analysis of si - oh condensation . in order to determine what causes the observedstructural changes on a molecular level , we used xps and atr - ir spectroscopy B-technique ( figure 2 ) . [SEP]
[CLS] xps provides nanoscale surface - sensitive elemental information , whereas atr - ir gives information on the chemical environment and probes farther into the structure . [SEP]
[CLS] together , the results allow us to probe the level of condensation at different calcination temperatures . [SEP]
[CLS] xps confirms that silicon B-material and oxygen B-material are the only elements present in our silica film ( figure 2a ) . [SEP]
[CLS] further , the ratio of these two elements provides a way to monitor the condensation of the sol - gel silica : the si : o ratio increases when two residual si - oh groups crosslink to form si - o - si and release a water B-material molecule ( scheme 1 ) . [SEP]
[CLS] as shown in figure 2b , we see an increase in the si : o ratio with higher calcination temperatures , indicating that additional condensation is occurring . [SEP]
[CLS] this analysis is further confirmed with the atr - ir spectra shown in figure 2c . [SEP]
[CLS] the peak at 970 cm - 1 , characteristic of the si - oh vibration , is shown in the inset . [SEP]
[CLS] in order to better compare the peaks , the peak area at 970 cm - 1 for each temperature is normalized by the peak area of the antisymmetric si - o - si vibration at 1070 cm - 1 and shown in figure 2d . [SEP]
[CLS] the si - oh peak disappears at the higher calcination temperatures , indicating that si - oh bonds are no longer present and further confirming that condensation is occurring . [SEP]
[CLS] shrinking of inverse opal films . [SEP]
[CLS] condensation of silica sol - gels is known to cause shrinkage of the resulting silica . [SEP]
[CLS] since the silica inverse opals are confined in the lateral plane by the attachment to the silicon B-material wafer substrate , volume reduction of the solid phase upon condensation causes shrinkage only in the direction perpendicular to the substrate ( figure 3a ) . [SEP]
[CLS] to confirm that the substrate is preventing the matrix from shrinking in the lateral directions , we decoupled the inverse opal film from the substrate upon calcination using a thin layer of photoresist as a sacrificial layer deposited prior to the growth step . [SEP]
[CLS] after assembly , this inverse opal film on a photoresist layer underwent calcination as before , and the sacrificial layer disintegrated with the colloids , causing the film to separate from the substrate ( figure 3b ) . [SEP]
[CLS] while films bound to a substrate show anisotropic pores ( figure 3c ) , the pores from samples with a sacrificial layer appear isotropic ( figure 3d ) . [SEP]
[CLS] the anisotropy ratios of the pores from the bound films match those in figure 1b , but the pores in the released films are the same size in both measured dimensions ( table 1 ) . [SEP]
[CLS] comparing the 500°c sample and 1100°c samples in table 1 , the pores shrink ~ 18 % in each direction without a confining substrate , whereas the width stays the same but the height shrinks ~ 42 % for the substrate - bound sample . [SEP]
[CLS] the pores of the unbound films shrink less in the vertical direction ( 18 % vs 42 % ) , but they shrink in all directions equally , whereas all of the condensation - induced volume loss must be accommodated by the perpendicular direction on the substrate - bound films . [SEP]
[CLS] in both cases , this corresponds to ~ 40 - 45 % volume loss , similar to what is seen in silica sol - gel films . [SEP]
[CLS] the effect of the substrate is also visible in samples with cracks . [SEP]
[CLS] when cracks occasionally form in inverse opals , the top layers become less confined along the substrate . [SEP]
[CLS] the cracks can provide stress release sites in films annealed to high temperatures . [SEP]
[CLS] thus , while the bottom layers still deform perpendicular to the substrate , the top layers can now shrink more isotropically ( figure 4 ) , creating a difference in pore dimensions between the upper and lower layers . [SEP]
[CLS] to verify experimental data , the structural changes upon calcination were modeled using the commercial simulation software abaqus . [SEP]
[CLS] a linear elastic material B-material model was used in which the shrinkage was simulated as thermal strain . [SEP]
[CLS] a 2d model of the pore structure was used with periodic boundary conditions in the lateral direction . [SEP]
[CLS] thus , the matrix height was shrunk without allowing any changes in the horizontal direction , similar to the schematic shown in figure 3a . [SEP]
[CLS] the results are presented in the supplemental movie , and images from two points in the video are shown in figure 3e . [SEP]
[CLS] the ratio of pore width to the pore height was recorded as a function of the linear shrinkage . [SEP]
[CLS] the simulated shape qualitatively matches our experimental shape , indicating that the shape change is entirely due to matrix shrinkage upon continuing polycondensation . [SEP]
[CLS] the formation of the anisotropic structure is due to the shrinkage of the material B-material while constrained on the bottom , so we expect a dependence on thickness . [SEP]
[CLS] one way that this manifests itself is in edge effects . [SEP]
[CLS] the size of the region influenced by the edge is comparable with the thickness of the film . [SEP]
[CLS] in the middle part of the sample far away from the edges , the film is under homogenous deformation . [SEP]
[CLS] as long as the lateral dimension of the sample is much larger than its thickness , there is always a region in the middle part of the sample where uniform opal structures are formed . [SEP]
[CLS] since the films were prepared over macroscopic dimensions , we expect a high homogeneity with little influence of sample edges . [SEP]
[CLS] previous studies found that cracks naturally form in co - assembled inverse opals once the film exceeds a thickness of approximately 20 colloid layers . [SEP]
[CLS] as shown in figure 4 , such cracks prevent homogeneous pore shrinkage , and thus compromise the quality of the anisotropic film . [SEP]
[CLS] fortunately , large areas without cracks are possible with the co - assembly method . [SEP]
[CLS] as long as the crack formation threshold is maintained , further annealing does not induce more cracking ( figure s1 ) . [SEP]
[CLS] the simulation and experimental results were compared using the ratio of the pore width to the pore height ( table 2 ) . [SEP]
[CLS] specific values of the simulation ( 3 % , 10 % , and 22 % shrinkage ) were chosen to match the pore ratio to experimental examples ( 1 . 12 , 1 . 5 , and 2 ) . [SEP]
[CLS] thus , we were able to correlate the experimental calcination temperature with the amount of contraction that caused the geometry shown in the simulation . [SEP]
[CLS] assuming both horizontal directions are equivalent in the three - dimensional experimental case , this gives an estimate that the silica matrix shrinks 40 % in volume upon calcination at 1100°c . [SEP]
[CLS] this agrees with the one - and three - dimensional contraction values we see , as well as with previous results measuring shrinking of silica sol - gels . [SEP]
[CLS] optical properties of anisotropic inverse opals . [SEP]
[CLS] the diameter of the templating colloidal particles was chosen to be several hundred nanometers , giving the inverse opals a periodic structure on the scale of the wavelength of visible light ; thus , they form strongly colored photonic crystals . [SEP]
[CLS] the perceived color of the films depends on several factors , including the number of layers in the inverse opal , the refractive B-property index I-property contrast between pore and matrix material B-material , and , most importantly , the period of the repeating structure . [SEP]
[CLS] following bragg ' s law , the layer spacing and the peak wavelength are directly proportional . [SEP]
[CLS] the size of the colloidal building blocks and the calcination temperature ( via the amount of shrinking ) control this spacing . [SEP]
[CLS] to minimize errors due to inter - sample variability , measurements of variations in the optical properties of inverse opal films as a function of preparation conditions were performed at the same spot on a single sample . [SEP]
[CLS] the colloids were removed with solvent before measuring the reflectivity . [SEP]
[CLS] the film then underwent calcination to subsequently higher temperatures , obtaining an optical spectrum after each heating step ( figure 5a ) . [SEP]
[CLS] this experimental protocol differs slightly from the one used to obtain the samples from which previous results presented in this paper were deduced . [SEP]
[CLS] however , similar pore anisotropies develop following this sequential protocol , and they are comparable to the anisotropies described above ( figure s2 ) . [SEP]
[CLS] we used 410 nm colloids as the colloidal template to ensure localization of the reflection peak across a range within the visible spectrum . [SEP]
[CLS] the degree of anisotropy affects the periodicity and thus the wavelengths of bragg resonance of light within the inverse opal structure . [SEP]
[CLS] consequently , annealing the same inverse opal sample to subsequently higher temperatures results in an incremental blue shift of the reflection peak ( figure 5a ) , originating from the decrease in period along the perpendicular direction . [SEP]
[CLS] the shift in peak wavelength varied nearly linearly ( r 2 = 0 . 95 ) with anisotropy ( figure 5b ) , as expected from bragg ' s law . [SEP]
[CLS] the small deviation from linearity can be attributed to an expected increase in the refractive B-property index I-property from annealing , which would produce a red shift in the spectrum at higher temperatures . [SEP]
[CLS] the peak intensity decreases slightly at elevated calcination temperatures , perhaps due to additional crack formation at higher temperatures due to condensation - induced stresses in the material B-material . [SEP]
[CLS] these cracks lead to a change in periodicity from the top layer to the bottom layer of the crystal , as shown in figure 4 . [SEP]
[CLS] this leads to nonuniformity in pore geometry and thus layer thickness , resulting in peak broadening and lower reflection intensity . [SEP]
[CLS] the angular dependence of the optical spectra was also studied . [SEP]
[CLS] because the anisotropic shape of the inverse opal pores introduces a different periodicity as a function of incident angle , the angular dependence of the optical spectrum also changes when anisotropy is introduced . [SEP]
[CLS] we obtained angularly resolved reflectance spectra to further probe the change in structure ( figure 5c ) . [SEP]
[CLS] specular reflectance spectra were obtained at incident angles from - 75° to 75° in 0 . 5° increments with an angular resolution of less than 1° . [SEP]
[CLS] these optical spectra were then plotted against the light incidence angle to give the graphs shown in figure 5c , where the gray - scale encoded ( black = high , white = low ) reflection intensity is displayed as a function of light incidence angle and wavelength . [SEP]
[CLS] the spectra qualitatively agree with other spectra in the literature . [SEP]
[CLS] the undulations in the background of the spectra in figure 5c and figure 6 are due to thin film interference based on the thickness of the inverse opal film , which depends on the measurement method and is present because the light source probes a small area of uniform sample . [SEP]
[CLS] different crystal planes present in the structure cause reflections at different angles . [SEP]
[CLS] when they are not parallel to the substrate plane , they lead to dips in the angular spectra that appear as diagonal white lines ( red box in figure 5c ) . [SEP]
[CLS] the presence of these dips indicates that the other crystal planes are still ordered enough to reflect light by constructive interference , indicating that a high degree of order remains in the samples , even those annealed to 1100°c . [SEP]
[CLS] to further probe the angular dependence , we compared inverse opals with the same normal reflection peak with two different degrees of anisotropy ( figure 6 ) . [SEP]
[CLS] in one sample , 410 nm colloids were used for the template , and calcination to 1000°c created anisotropy in the structure ( figure 6a ) . [SEP]
[CLS] the periodicity of the anisotropic structure gave a normal reflection peak around 510 nm ( lateral dashed line ) . [SEP]
[CLS] a structure with similar periodicity will produce a similar wavelength of constructive interference , which can also be achieved by changing the colloid size . [SEP]
[CLS] thus , a second sample was made with 330 nm colloids as the template , and their removal with toluene left spherical pores ( figure 6b ) . [SEP]
[CLS] this sample also has a normal reflection peak at 510 nm . [SEP]
[CLS] the biggest difference in the two samples is the angle of the dip in the spectra that appears as a white diagonal line ( boxed ) . [SEP]
[CLS] in the isotropic sample , the dip makes a much lower angle than the anisotropic sample on the right . [SEP]
[CLS] this agrees with the fact that these crystal planes should be very different for the two samples . [SEP]
[CLS] while changing the size of the colloids can control the normal reflection peak ( although in a much more challenging way than simply altering the calcination temperature ) , it would not achieve the same angular dependence of the spectrum based on the different underlying geometry . [SEP]
[CLS] anisotropic colloidal crystals have generated much interest recently . [SEP]
[CLS] here , we provide a method to controllably adjust the anisotropy present in an inorganic inverse opal matrix and describe the mechanism of formation . [SEP]
[CLS] controlling the shape of the pores by varying the calcination temperature gives us control over the order and porosity of the resulting film and introduces defined anisotropic pore shapes not easily achievable by direct assembly of anisotropic particles . [SEP]
[CLS] based on xps and ir results , as well as a finite element simulation , we propose that matrix shrinkage from sol - gel condensation is causing this change in geometry . [SEP]
[CLS] the decreased height of the pores leads to a blue shift in the optical spectrum at higher calcination temperatures , as expected for the smaller inter - layer spacing . [SEP]
[CLS] a complete angular characterization of the optical spectra shows that three - dimensional order in the crystal is retained even with a high degree of anisotropy . [SEP]
[CLS] calcination temperature can control the shape and anisotropy of inverse opal pores , allowing wide ranges of anisotropy perpendicular to the surface to be generated . [SEP]
[CLS] in addition to the controlled tuning of inverse opals ' optical properties described here , we expect anisotropy to affect other properties as well , such as the mechanical stability or wetting characteristics . [SEP]
[CLS] this simple yet precise method for the control of anisotropy in three - dimensional periodic microstructures enables inverse opals to be a versatile platform for fundamental studies probing tailored confinements and structure - property relationships . [SEP]
[CLS] supporting information available . [SEP]
[CLS] videos of structural simulation ; volume loss calculations ; [SEP]
[CLS] sem cross - sections of samples after optics experiments . [SEP]
[CLS] this material B-material is available free of charge via the internet at http : / / pubs . acs . org . [SEP]
[CLS] ( a ) cross - sectional scanning electron microscope ( sem ) images of samples made from 230 , 330 , and 410 nm polymer B-material colloids as templates . [SEP]
[CLS] different samples are shown after template removal by solvent dissolution at room temperature ( left ) and by calcination at 500°c , 800°c and 1100°c ( from left to right ) . [SEP]
[CLS] all scale bars are 400 nm . [SEP]
[CLS] ( b ) quantification of the anisotropy as a function of calcination temperature . [SEP]
[CLS] the ratio of pore width ( w ) to pore height ( h ) for all samples was measured from the sem images ( ~ 25 measurements per sample ) . [SEP]
[CLS] error bars represent the standard deviation of these measurements assuming a gaussian distribution . [SEP]
[CLS] analytical spectroscopy B-technique of the inverse opals . [SEP]
[CLS] ( a ) xps spectrum of a typical inverse opal sample , with the silicon B-material region expanded in the inset . [SEP]
[CLS] ( b ) an increase in the si : o ratio with higher calcination temperatures is detected by comparing the peak areas . [SEP]
[CLS] the horizontal dotted line represents a silicon B-material oxide B-material layer as reference . [SEP]
[CLS] ( c ) atr - ir vibrational spectra of the inverse opals , with the inset showing the 1000 - 900 cm - 1 region characteristic of the si - oh stretch . [SEP]
[CLS] ( d ) graph of the area of the si - oh peak ( 970 cm - 1 ) , normalized by the area of the 1070 cm - 1 si - o - si peak . [SEP]
[CLS] 3 . the effect of the substrate on pore anisotropy during calcination . [SEP]
[CLS] ( a - b ) schematics of the calcination step in inverse opals on a substrate ( a ) , and with a sacrificial layer between the inverse opal and the substrate ( b ) . [SEP]
[CLS] ( c - d ) cross - sectional sem images of the samples attached to the substrate ( c ) , and with a sacrificial photoresist layer ( d ) , annealed to 500°c ( top ) and 1100°c ( bottom ) . [SEP]
[CLS] scale bars are 400 nm . [SEP]
[CLS] ( e ) 2d theoretical model of structure change based on uniaxial shrinkage of the matrix in the vertical direction . [SEP]
[CLS] 4 . sem image of a crack in an inverse opal made from a 230 nm colloidal template annealed to 800°c . [SEP]
[CLS] 5 . calcination - induced anisotropy effects lead to variations in the optical properties of inverse opal films . [SEP]
[CLS] ( a ) optical spectra of a single sample calcined sequentially to higher temperatures . [SEP]
[CLS] ( b ) peak positions from ( a ) plotted against calcination temperature . [SEP]
[CLS] ( c ) effect of temperature on angular reflectance spectra . [SEP]
[CLS] 6 . comparison of angular reflection of inverse opals made from ( a ) 410 nm colloidal template removed by calcination to 1000°c , and ( b ) 330 nm colloidal template removed by solvent . [SEP]
[CLS] insets are sem images of the structures , with 400 nm scale bars . [SEP]
[CLS] comparison of inverse opal pores with and without a substrate . [SEP]
[CLS] comparison of experimental and simulated anisotropic structures . [SEP]
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[CLS] integration of nucleic B-material acids I-material ( nas ) and nanoparticles B-nanoparticle ( nps B-nanoparticle ) has generated a wealth of research in the area of nanotechnology , with particular emphasis on nanostructures with unique physicochemical properties such as noble metal nanoparticles I-nanoparticle . [SEP]
[CLS] as a result , an increasing number of na - based nanotechnologies are continuously emerging , finding a diverse set of applications in biomedicine , sensing , bioelectronics , biocomputing , etc . [SEP]
[CLS] while countless efforts have been devoted to the study of chemically modified oligonucleotides ( mostly , thiolated dna ) cova - lently attached to plasmonic nanoparticles B-nanoparticle , the understanding of the mechanisms involved in the noncovalent interaction between native dna duplexes ( dsdnas ) , as well as the impact that such an interaction has on the double - helix structure , is far less explored , and reports in the literature often diverge significantly in their findings . [SEP]
[CLS] the interaction between unmodified dsdna and nanoparticles B-nanoparticle is largely determined by the physicochemical properties of the interface , with the nanoparticle B-nanoparticle surface charge playing a major role in this process . [SEP]
[CLS] in particular , cationic B-material nanoparticles B-nanoparticle with sufficiently high surface charge are capable of irreversible interactions with the negatively charged phosphate groups of nucleic B-material acids I-material , leading to dna damage . [SEP]
[CLS] partial denaturation of dna duplexes upon complexation with positively charged nanoparticles B-nanoparticle was previously revealed mainly by direct monitoring of the hyperchromic shift of the dna absorption band at 260 nm and by indirect examination of the optical ( absorbance or fluorescence B-property ) response of intercalating dyes sensitive to the structures of the bound dna ( i . e . , duplex vs single strand ) . [SEP]
[CLS] interestingly , fluorescence B-property studies as well as molecular dynamics ( md ) simulations suggested that only high concentrations of nanoparticles B-nanoparticle can bend and separate dna strands . [SEP]
[CLS] this led to the hypothesis that the partial strand separation may only occur via a cooperative action of an ensemble of interacting nanoparticles B-nanoparticle . [SEP]
[CLS] however , insertion of intercalating molecules causes itself local structural changes to the duplex , including unzipping of the double helix and lengthening of the strand . [SEP]
[CLS] additionally , the near - field interaction of the excited - state fluorophore with the surface plasmon resonances ( spr ) of metallic B-nanoparticle nanoparticles I-nanoparticle perturbs the optical properties of the dye and , thus , its fluorescence B-property emission in a way that is largely dependent on the properties of the plasmonic nanoparticles B-nanoparticle and the fluorophore , as well as the distance between the two . [SEP]
[CLS] on the other hand , uv - vis spectroscopy B-technique analysis of dna - np B-nanoparticle complexes can only be performed on soluble B-property complexes , thus restricting the case study to very small nanoparticles B-nanoparticle and / or large dna / np B-nanoparticle molar ratios when phase separation is avoided . [SEP]
[CLS] to a certain extent , this trend is mirrored in theoretical studies , where the nanoscale size of dna - np B-nanoparticle systems in most cases prevents their direct investigation through atomistic simulations . [SEP]
[CLS] thus , to the best of our knowledge , md simulations have been carried out only for very small nanoparticles B-nanoparticle , with diameters not exceeding 1 . 5 nm , so as to keep the problem computationally manageable . [SEP]
[CLS] within this fragmented picture , a plasmon - assisted spectroscopy B-technique such as surface - enhanced raman scattering ( sers ) has the potential to be used as a highly informative , ultrasensitive analytical method for the direct interrogation of nucleic B-material acid I-material / nanoparticle B-nanoparticle interactions and their impact on the structure of the genetic material B-material . [SEP]
[CLS] notably , the largest sers enhancements usually show a high degree of spatial localization , such as those that take place at the interparticle junctions ( i . e . , hot spots ) of highly interacting nanoparticles B-nanoparticle in colloidal aggregates . [SEP]
[CLS] under this scenario , sers can provide a unique tool to isolate the vibrational signature of those molecules that are trapped at the nanoparticle B-nanoparticle junctions . [SEP]
[CLS] herein , we combined optical methods ( sers and spr spectroscopies B-technique ) and theoretical simulations ( md and finite difference time domain ( fdtd ) ) to directly investigate the structural perturbation on nucleic B-material acids I-material imposed by the binding with cationic B-material silver B-nanoparticle nanoparticles I-nanoparticle ( ≈22 nm diameter ) with tuned ζ potential . [SEP]
[CLS] remarkably , the intrinsic nature of the sers effect unlocked the possibility of selectively examining the impact of nanoparticle B-nanoparticle clustering on the double - helix integrity over a wide degree of aggregation ( i . e . , dna / np B-nanoparticle ratios ) and without the need of exogenous fluorescence B-property reporters . [SEP]
[CLS] on the other hand , spr analysis and ms integrated critical complementary information to the indepth characterization of the dna / np B-nanoparticle interactions and the underlying mechanisms . [SEP]
[CLS] a large set of different duplex structures q4 with different lengths ( from short segments to long genomic chain ) , base composition , sequence and form were investigated . [SEP]
[CLS] the results show that , when the nanoparticle B-nanoparticle ζ potential is lowered below a certain threshold , the double helixes trapped at the interparticle gaps undergo major unzipping , which is largely independent of the investigated nucleic B-material acids I-material ' properties ( i . e . , the final outcome of the interaction is mostly determined by the electrostatic binding between phosphate groups and surface positive charges ) . [SEP]
[CLS] on the other hand , for colloids with fixed ζ potentials , strand separation preferably occurs in the case of small dna - mediated clusters ( i . e . , for very low dna / np B-nanoparticle ratios ) and , to a lesser extent , in the presence of a large excess of duplexes ( i . e . , for very high dna / np B-nanoparticle ratios ) . [SEP]
[CLS] in this case , however , the length of the duplex does play a relevant role in determining the pattern of the duplex structural reshaping , as the biomolecular size directly affects the features of the colloidal aggregation . [SEP]
[CLS] these results are complemented and confirmed by md - based simulations performed on model systems representing cluster aggregates of realistic size . [SEP]
[CLS] this section is organized as follows : first , the chemico - physical properties of the cationic B-material nanoparticles B-nanoparticle are discussed . [SEP]
[CLS] then , we describe the experimental scenario under which the direct sers analysis of dna takes place and , by means of computational tools , qualitatively predicts the spatial origin of the sers signal . [SEP]
[CLS] this is propaedeutic to the interpretation of the sers spectra revealing strand separation of both short and genomic dna duplexes upon interaction with silver B-material cationic B-material nanoparticles B-nanoparticle . [SEP]
[CLS] two main variables of the nanoparticle - induced unzipping process were identified ( degree of nanoparticle B-nanoparticle aggregation and nanoparticle B-nanoparticle surface charge ) and investigated separately by sers and spr spectroscopies B-technique , as well as via molecular dynamic simulations . [SEP]
[CLS] the wet - chemical synthesis of positively charged silver B-nanoparticle nanoparticles I-nanoparticle ( agsp ) was performed by reducing silver B-material ions B-material with sodium B-material borohydride in the presence of spermine tetrahydrochloride . [SEP]
[CLS] the colloidal synthesis yields nanoparticles B-nanoparticle of ≈22 nm diameter ( figure s1a , b , supporting information ) , with a localized surface plasmon resonance ( lspr ) band centered at ≈392 nm and a ζ potential of ≈ + 40 mv ( figure 1a , b ) . [SEP]
[CLS] ζ measurements provide the potential at the start of the diffuse layer which is indirectly related to the surface charge at the nanoparticle B-nanoparticle surface . [SEP]
[CLS] nanoparticle B-nanoparticle concentration ( ≈1 . 00 × 10 −9 m ) was determined by uv - vis spectroscopy B-technique using an extinction coefficient of 54 . 8 × 10 8 m −1 cm −1 at 392 nm , as obtained from the literature . [SEP]
[CLS] spermine molecules are linear quadrivalent cationic B-material polyamines that remain bound to the nanoparticle B-nanoparticle surfaces via electrostatic interaction mediated by the chloride B-material anions B-material . [SEP]
[CLS] these ligands provide an overall positive charge to the nanoparticles B-nanoparticle , and , due to their aliphatic nature , sers background of the agsp colloids shows only minimal contribution from the tetramine adlayer ( figure s1c , d , supporting information ) . [SEP]
[CLS] dilution of the agsp colloids with milli - q water B-material promotes a progressive reduction in plasmon intensity ( figure 1a ) with null or minimal perturbation of the colloidal stability ( figure s2 , supporting information ) . [SEP]
[CLS] at the same time , we register a decrease of ζ potential ( figure 1b ) consistent with the partial desorption of spermine molecules from the metallic surface upon dilution with milli - q water B-material . [SEP]
[CLS] this is further confirmed by the fact that ζ potential value remains unchanged when nanoparticles B-nanoparticle are diluted with the colloidal supernatant ( figure s3 , supporting information ) . [SEP]
[CLS] we have previously shown that short dna duplexes of 21 base pairs ( dsdna 21 ) mediate agsp nanoparticle B-nanoparticle aggregation acting as electrostatic molecular linkers . [SEP]
[CLS] under physiological conditions , double - stranded dna persistence length is of ≈150 bp , corresponding to ≈50 nm . [SEP]
[CLS] as a result , dsdna 21 behaves as a rigid rod of ≈7 nm length and 2 nm diameter , which is collected at the surface of individual quasispherical nanoparticles B-nanoparticle of around 22 nm diameter with minimal bending of the double helix . [SEP]
[CLS] molecular dynamics simulations of dsdna 21 adsorption on agsp nanoparticles B-nanoparticle previously indicated that the duplex rapidly adheres onto the metal B-material surface adopting an elongated conformation ( for low surface coverage ) . [SEP]
[CLS] consequently , interparticle gaps of ≈2 . 5 nm are generated in dsdna 21 - mediated clusters ( i . e . , the diameter of the duplex rod - like structure sandwiched between spermine molecules bound to the metallic surfaces ) . [SEP]
[CLS] it is also worth noting that , in addition to their physical size , dna molecules coordinated at the metallic surfaces screen further adsorption of unbound duplexes by electrostatic repulsion . [SEP]
[CLS] the extent of this electrostatic repulsion is larger at low salt B-material concentrations such as for the investigated agsp colloids ( ≈1 - 2 × 10 −3 m nacl resulting from chemicals employed in the nanoparticle B-nanoparticle synthesis and the pbs buffer of dna solutions ) . [SEP]
[CLS] thus , the formation of a hypothetical compact mono - q6 layer of dsdna 21 , corresponding to ≈100 molecules per particle ( based on the average np B-nanoparticle diameter and dsdna 21 footprint ) , is disfavored , and duplexes are expected , on the contrary , to be rather scattered over the nanoparticle B-nanoparticle surfaces even for high dna concentrations in the bulk . [SEP]
[CLS] direct sers analysis was performed on the colloidal suspension by focusing a 532 nm laser with a long working distance lens so as to acquire averaged bulk sers spectra with reproducible and well - defined spectral profiles . [SEP]
[CLS] under this scenario - relatively small gaps and nanoparticle B-nanoparticle radius ( i . e . , large curvature at the interparticle junctions ) , combined with an excitation wavelength that matches the red - shifted gap plasmon resonances - the sers intensity of aggregated colloidal systems is largely dominated by those few molecules located at the gap volume ( hot spot ) . [SEP]
[CLS] in support of this claim , we used an fdtd method to calculate the electromagnetic field distribution around a silver B-nanoparticle nanoparticle I-nanoparticle dimer in water B-material ( particle diameter = 22 nm ; gap distance = 2 . 5 nm , such as that generated by a dsdna 21 duplex extended over the metallic surface ) illuminated by a 532 nm laser . [SEP]
[CLS] in figure 2a , we show a horizontal and a vertical cut of the field intensity ( xy - and xz - planes , respectively ) . [SEP]
[CLS] we can appreciate that the em field is strongly confined in the gap . [SEP]
[CLS] knowing how the electric field decays , we can estimate the q7 volume of the hot spot considering the full width at half maximum ( fwhm ) of the electric intensity , | e | 2 ( figure 2b ) , in each direction . [SEP]
[CLS] approximating the hot - spot volume , v , as a cylinder whose height is given by the gap distance ( i . e . , 2 . 5 nm ) and the radius is ≈3 nm ( i . e . , fwhm ) , we can estimate v = 70 nm [SEP]
[CLS] based on the intrinsic size of the adsorbed dsdna 21 surface complex ( ≈30 - 35 nm ) and the sparse surface coverage imposed by electrostatic repulsions between duplexes , we can safely estimate that no more than one dsdna 21 molecule is located at the hot spot . [SEP]
[CLS] this means that one individual duplex placed at the gap is responsible for the vast majority of the sers signal arising from the dimer . [SEP]
[CLS] such a conclusion can be qualitatively extended to the large ensembles of dna - mediated clusters in suspension ( i . e . , the acquired sers spectra specifically inform about the fraction of duplex molecules bridging cationic B-material nanoparticles B-nanoparticle into aggregates , even for high dna concentration ) . [SEP]
[CLS] while agsp colloids immediately aggregate upon addition of a short duplex , the complexation process between long genomic calf thymus dna ( ctdna ) and nanoparticles B-nanoparticle is slower and requires a longer time to reach its final equilibrium state . [SEP]
[CLS] for this reason , sers analysis was performed on samples in the suspension after overnight incubation B-technique to reach binding equilibrium ( aggregates sitting at the bottom of the eppendorf tube were resuspended just before the optical measurement ) . [SEP]
[CLS] salt B-material concentration was maintained low ( millimolar of q8 nacl ) throughout the whole study to minimize unspecific saltinduced aggregation of the nanoparticles B-nanoparticle ( however , it is worth reminding that ion B-material concentration at the nanoparticle B-nanoparticle surfaces differs from the bulk ) . [SEP]
[CLS] 3 illustrates the sers spectra of ctdna at a fixed concentration on agsp colloids with different ζ potential and nanoparticle B-nanoparticle concentration . [SEP]
[CLS] the spectra were normalized to the phosphate stretching band at ≈1089 cm −1 , whose large insensitivity to changes in dna structure makes it an ideal candidate for an internal standard . [SEP]
[CLS] the spectral profile of ctdna undergoes a progressive reshaping upon addition to gradually diluted colloids . [SEP]
[CLS] among others , we highlight the drop in intensity of the guanine ( g ) ring deformation band at ≈620 cm −1 , the red - shifts of the ring - breathing modes of adenine ( a ) ( ≈730 cm −1 ) and cytosine ( c ) + thymine ( t ) ( 785 cm −1 , main contribution from cytosine ) , the intensity increase and large red - shift of the cytosine + ribose vibration at ≈1019 cm −1 , and the remarkable weakening of the cytosine ring stretching at ≈1247 cm −1 . [SEP]
[CLS] all these changes are fully consistent with the dna base unstacking and unpairing transition . [SEP]
[CLS] additionally , the broad composite carbonyl stretching band at ≈1651 cm −1 ( mainly ascribed to thymine ) suffers a large shift to lower frequency as also observed during thermal denaturation of b - form dna . [SEP]
[CLS] on the other hand , changes in ν ( co ) band intensity are largely affected by the increasing relevance of the sers background when the dna signal drops as a result of the nanoparticle B-nanoparticle dilution and , thus , such spectral alterations cannot be reliably associated with perturbations of the dna structure . [SEP]
[CLS] similar spectral changes are registered in the sers spectra of the short dsdna 21 duplex ( figure s4 , supporting information ) . [SEP]
[CLS] due to the particle size ( ≈22 nm diameter ) , the insertion of agsp nanoparticles B-nanoparticle into the dna grooves is hindered ; thus , we can safely discard this process as a possible driving cause of the strand separation . [SEP]
[CLS] on the contrary , minor spectral changes were observed when the colloidal supernatant was employed to similarly dilute agsp ( figure s4 , supporting information ) demonstrating the key role of the surface charge in determining the extension of the duplex unzipping . [SEP]
[CLS] it is also important [SEP]
[CLS] q9to stress that the sers profile of dna - spermine complexes , formed by the interaction of the duplex with free spermine molecules in the bulk solution , differs drastically from that of dna on agsp colloids ( figure s5 , supporting information ) , where the spermine molecules are restrained at the metal B-material surface . [SEP]
[CLS] this demonstrates that unbound spermine molecules in agsp colloids do not play a significant role in shaping the final dna sers spectra , and , thus , the spectral changes illustrated in figure 3 can be safely associated with strand separation . [SEP]
[CLS] the sers spectra shown in figure 3 were obtained by adding a fixed amount of ctdna to differently diluted agsp colloids . [SEP]
[CLS] this means that both surface charge ( correlated with the ζ potential ) and dna / np B-nanoparticle ratios , which affect the degree of nanoparticle B-nanoparticle clustering , were modified at the same time . [SEP]
[CLS] to isolate these variables and separately investigate their role in the duplex unzipping , we performed two different sers studies : in the first one , we progressively varied the dna / np B-nanoparticle ratio while keeping the colloidal ζ potential constant ; [SEP]
[CLS] in the second study , the effect of the nanoparticle B-nanoparticle surface charge on the spectral profile was monitored at fixed dna / np B-nanoparticle ratios . [SEP]
[CLS] a short 21 - mer single - stranded dna ( ssdna 21 ) was included as a probe molecule besides the long ( ctdna ) and short ( dsdna 21 ) duplexes . [SEP]
[CLS] peak positions of the narrow and intense ring breathing bands at ≈730 and 785 cm −1 , ascribed to a and c + t nucleobases , respectively , were selected as spectral markers of the state of the dna tertiary structure ( figure 3b ) . [SEP]
[CLS] in figure 4 , we plotted the peak frequencies of the spectral markers observed at different dna / np B-nanoparticle ratios ( milligrams of dna per nanomoles of nps B-nanoparticle , in logarithmic scale ) for four sets of colloids at fixed surface charges ( ζ potential is equal to ≈40 . 0 , 38 . 6 , 33 . 2 , and 26 . 8 mv , respectively ) . [SEP]
[CLS] the extension of the investigated ranges of dna / np B-nanoparticle ratios was defined by the acquisition of sufficiently intense and well - defined sers spectra . [SEP]
[CLS] at low dna concentrations , we are mainly restricted by the overall number of scattering molecules in the sample , whereas in the presence of an excess of analyte , dna saturation of the nanoparticle B-nanoparticle surfaces prevents the formation of efficient hot spots ( i . e . , small number of hot spots in the sample ) . [SEP]
[CLS] the results show that no spectral shifts were observed for ssdna 21 , whereas both short and genomic duplexes reveal notable changes in their spectral profiles ( figure s6 , supporting information ) , with the appearance of a frequency minimum at intermediate B-property dna / np B-nanoparticle ratios , corresponding to the minimal perturbation of the double - helix stability by cationic B-material nanoparticles B-nanoparticle of a specific ζ potential value . [SEP]
[CLS] overall , the position of such frequency minima moves to larger raman shifts when the nanoparticle B-nanoparticle surface charge is decreased . [SEP]
[CLS] it is also worth noting that the peak frequencies of both a and c + t ring breathing bands consistently remain far below than those of ssdna 21 , except when very dna / np B-nanoparticle ratios are investigated in colloids with lowest surface charge ( figure 4 , agsp with a ζ potential of + 26 . 8 mv ) . [SEP]
[CLS] these general observations are valid for both short and genomic dna duplexes . [SEP]
[CLS] on the contrary , for agsp colloids at a given ζ potential , we observe that minimal duplex separation ( corresponding to the most blue - shifted ring breathing bands ) always takes place at lower dna / np B-nanoparticle ratios for dsdna 21 than ctdna ( figure 4 ) . [SEP]
[CLS] besides the obvious difference in length , dsdna 21 and ctdna deviate in nucleobase sequence and composition ( gc content of ≈52 . 4 % for short and ≈41 . 9 % for genomic dnas ) . [SEP]
[CLS] to examine the role of these latter variables , we performed the identical spectral analysis described in figure 4 but for two 22 - bp duplexes either composed of cg or at nucleobases ( dscg and dsat , respectively ; see figure s7 in the supporting information ) . [SEP]
[CLS] overall , the trends of peak frequency shifts for dscg and dsat largely match those of dsdna 21 ( figure s7 , supporting information ) indicating that , rather than base sequence and composition , the strand length parameter is likely to play the major role in determining the marked offset in double - strand unzipping for dsdna 21 and ctdna at different dna / np B-nanoparticle ratios ( figure 4 ) . [SEP]
[CLS] given these results , questions arise as to which mechanisms lie behind the connection between dna / np B-nanoparticle ratio factor and the characteristic changes in the tertiary structure of dna duplexes electrostatically trapped at the interparticle junctions . [SEP]
[CLS] to gain insights on this intriguing issue , we have undertaken detailed studies on the evolution of the optical properties of nanoparticle B-nanoparticle clusters in suspension as well as performed md simulations on model dna - mediated clusters . [SEP]
[CLS] in addition to the large sers enhancements localized at the interparticle gaps , the plasmonic coupling of interacting silver B-nanoparticle nanoparticles I-nanoparticle is responsible for the reshaping of the colloidal extinction profile with the appearance of new gap plasmon resonances red - shifted with respect to the lspr . [SEP]
[CLS] however , the sers enhancement distribution and far - field spr responses from large ensembles of nanoparticle B-nanoparticle clusters have different spatial averaging properties due to the different spatial localization of the plasmon resonances . [SEP]
[CLS] thus , uv / vis / nir spectroscopy B-technique analysis of dna - mediated aggregates can provide important complementary information on the correlation between strand separation , as revealed by sers , and general geometrical features of nanoparticle B-nanoparticle clusters ( i . e . , gaps separation , size , and shape ) . [SEP]
[CLS] 5a displays the extinction spectra of agsp colloids ( [ np B-nanoparticle ] ≈ 1 . 00 × 10 −9 m ; ζ potential ≈ 40 mv ) registered 60 min ( red line ) or 1 d ( pink line ) after the addition of increasing amounts of dsdna 21 . [SEP]
[CLS] at low dna / np B-nanoparticle ratios ( 0 . 64 mg nmol −1 ) , we observe a very mild nanoparticle B-nanoparticle aggregation after 60 min , consistently with the small number of duplexes per nanoparticle B-nanoparticle that limits the extensive formation of dnamediated clusters . [SEP]
[CLS] however , the perturbation introduced in the colloidal system eventually triggers further aggregation events that yield larger features with broader and weaker plasmonic contributions . [SEP]
[CLS] in fact , after 1 d , the particles undergo an irreversible coalescence which also leads to the visible deposition of films of silver B-nanoparticle nanoparticles I-nanoparticle at the eppendorf tube surfaces . [SEP]
[CLS] differently , for higher dsdna 21 concentrations , the colloidal system rapidly aggregates into stable clusters that can be easily redispersed after sedimentation . [SEP]
[CLS] notably , when dna concentration is further increased as much as to start saturating metallic surfaces , the extent of interparticle coupling decreases as revealed by the overall blue - shift of the gap plasmon resonances for dna / np B-nanoparticle ratio of 12 . 8 mg nmol −1 as compared to 3 . 2 mg nmol −1 ( figure 5a ) . [SEP]
[CLS] the absorbance ratio at 700 and 391 nm ( i 700 / i lspr ) was selected to better visualize the effect of time and dna / np B-nanoparticle ratio on the ensemble of gap - plasmon resonances ( figure 5b ) . [SEP]
[CLS] here , we can observe that , for dna / np B-nanoparticle ratios below ≈2 mg nmol −1 ( ≈0 . 3 in the logarithmic scale ) , the duplex coverage of the silver B-material surface is not sufficient to either induce extensive nanoparticle B-nanoparticle aggregation or impart long - term colloidal stability . [SEP]
[CLS] in this regard , sers analysis was also performed on each sample at 10 min , 60 min , and 1 d , showing neither appreciable peak shifts of the spectral markers nor significant changes in absolute intensity over time . [SEP]
[CLS] this suggests that the unspecific colloidal aggregation that follows the initial dna - mediated cluster formation does not affect in a tangible way the final sers profile . [SEP]
[CLS] on the other hand , for dna / np B-nanoparticle ratios above ≈3 mg nmol −1 ( ≈0 . 5 in the logarithmic scale ) , we observe the rapid formation of stable clusters in suspension . [SEP]
[CLS] the extent of respective plasmon coupling appears to gradually decrease as the dna / np B-nanoparticle ratio is progressively raised ( up to the largest dna / np B-nanoparticle value presented in this study ) . [SEP]
[CLS] the 60 min aged mixtures were also analyzed by dynamic B-technique light I-technique scattering I-technique ( dls ) to extract the corresponding hydrodynamic size and size distribution of the dna - mediated aggregates ( figure s8a , supporting information ) . [SEP]
[CLS] these samples were selected as they provide a close picture of the colloidal system at the equilibrium while largely avoiding the unspecific colloidal aggregation observed for low dna / np B-nanoparticle ratio at longer [SEP]
[CLS] q11average hydrodynamic diameter of the aggregates shows a continuous increment with the increasing of the dna content . [SEP]
[CLS] on a purely qualitative basis , such a trend can also be captured in scanning electron B-technique microscopy I-technique ( sem ) images of spin - coated samples ( figure s8b - d , supporting information ) . [SEP]
[CLS] this result [SEP]
[CLS] q12directly entails that decrease of the i 700 / i lspr value observed for dna / np B-nanoparticle ratios larger than ≈3 mg nmol −1 cannot be ascribed to a reduction of the cluster size but to an enlargement of the average gaps within nanoparticle B-nanoparticle aggregates . [SEP]
[CLS] here , when a large number of dna molecules compete for adhesion on a few surface sites , the surface crowding and the electrostatic repulsion between neighboring duplexes hamper the necessary conformational changes required to maximize the number of contacting points with the surface . [SEP]
[CLS] thus , we can reasonably expect that , instead of homogeneously extending over the metallic surface , dna molecules bind the silver B-material surface adopting a broad set of tilted conformations , which lead to larger interparticle separations in aggregates . [SEP]
[CLS] this picture is fully consistent with previous molecular dynamic simulations that described the dna adsorption on a spermine - bound silver B-material surface as a multistep process . [SEP]
[CLS] according to this mechanism , after a fast recog - nition phase , the total adhesion of short dna strands is achieved by conformational changes triggered by electrostatic interactions with spermine molecules adsorbed on the surface . [SEP]
[CLS] this second step can be hindered by an overcrowded silver B-material surface and / or without the necessary amount of free spermine molecules , thus leading to aggregates characterized by properly adsorbed duplexes coexisting with partly bound dna molecules . [SEP]
[CLS] the evolution of the dna - driven nanoparticle B-nanoparticle clusterization was also investigated for ctdna ( figure s9 , supporting information ) . [SEP]
[CLS] the semiquantitative analysis of the extinction spectra did reveal minimal interparticle plasmonic coupling at very low or very high ctdna concentrations , but it was not possible to discern any further distinctive pattern . [SEP]
[CLS] most likely , this is the consequence of the coexistence of many additional aggregation routes . [SEP]
[CLS] opposite to what happens for the adsorption of ≈7 nm rod - like duplexes on agsp nanoparticles B-nanoparticle , in the case of long genomic dna , the silver B-material particles are the nanoscale objects that accumulate at the duplex chain , leading to the progressive compaction of the elongated coil which can also involve the wrapping of the chain around individual nanospheres B-nanoparticle . [SEP]
[CLS] such a compaction process consists of several intermediate B-property states with uneven distributions of nanoparticles B-nanoparticle along the chain . [SEP]
[CLS] furthermore , nanoparticles B-nanoparticle in suspension tend to agglomerate at the surfaces of other nanoparticles B-nanoparticle previously coordinated to the genomic strand . [SEP]
[CLS] the antithetical role played by dna of different chain lengths on the nanoparticle B-nanoparticle aggregation pattern ( i . e . , nanoparticles B-nanoparticle collect short dsdna 21 at their surfaces versus long genomic ctdna collects nanoparticles B-nanoparticle at the duplex chain ) is also well reflected in the uv - vis analysis of the colloidal supernatants upon removal of dna - mediated clusters ( figure s10 , supporting information ) . [SEP]
[CLS] monitoring of the optical absorption of dna at 260 nm reveals that a progressively higher concentration of unbound dsdna 21 is present in the supernatant for dna / np B-nanoparticle ratio above ≈3 mg nmol −1 ( ≈0 . 5 in the logarithmic scale ; see figure s10c in the supporting information ) , which precisely corresponds to the stability threshold for dsdna 21 - driven nanoparticle B-nanoparticle assemblies ( i . e . , when the metallic surfaces are largely saturated with short duplexes that act in this situation also as stabilizing ligands ) . [SEP]
[CLS] notably , unbound dsdna 21 in the supernatant are observed for agsp colloids with higher ζ potential , which indicates that duplex surface density increases with the nanoparticle B-nanoparticle surface charge . [SEP]
[CLS] on the other hand , in the case of ctdna , residual duplexes in the supernatant are only detected for the largest dna / np B-nanoparticle ratio ( figure s10d , supporting information ) together with a weak additional band at ≈398 nm ascribable to very sparse individual nanoparticles B-nanoparticle attached to the chain . [SEP]
[CLS] for all ctdna / np B-nanoparticle ratios below that value , the relatively high weight of the ctdnananoparticle complexes leads to sedimentation . [SEP]
[CLS] the illustrated sers and far - field spr data are the highly averaged responses of very complex samples containing a large number of polydispersed aggregates . [SEP]
[CLS] modeling systems of such sizes and complexity are , at present , unfeasible . [SEP]
[CLS] nonetheless , md - based simulations of model dna - mediated clusters may give a hint of the potential connection between the cluster geometrical features and the perturbation of the interacting dna at the junctions . [SEP]
[CLS] in particular , two model systems were envisioned , a dimer ( 2np ) and a trimer ( 3np ) with a compact geometry ( see figure 6 and figure s11a , supporting information ) , as the simplest prototypes of small and larger aggregates , respectively . [SEP]
[CLS] both models included a single dsdna 21 molecule adopting an extended conformation at the interface of each gap . [SEP]
[CLS] in figure 6 , we show the free energy surfaces calculated as a function of the separation between nanoparticles B-nanoparticle and the average distance between base pairs belonging to different dna strands . [SEP]
[CLS] in both 2np and 3np systems , the deepest minima corresponded to states showing the double helix of dna in the b - form . [SEP]
[CLS] specifically , in the 2np cluster model , the global minimum was found at a relatively large interparticle distance of about 29 a ( figure 6a ) . [SEP]
[CLS] conversely , in the trimer model , the most stable state was located at much narrower separations ( 24 - 25 a , figure 6b ) . [SEP]
[CLS] this finding clearly indicates a trend , whereby smaller aggregates tend to stretch or , at least , to easily interconvert between states of different contractions , while more compact clusters are highly preferred in the case of larger aggregates . [SEP]
[CLS] this effect can be explained through a dna - mediated binding cooperativity between the three nanoparticles B-nanoparticle , which is missing in the 2np model . [SEP]
[CLS] most importantly , however , is that the investigated systems show a remarkably different behavior along the reaction coordinate describing the separation between the two strands of the duplex . [SEP]
[CLS] indeed , an incipient unzipping could be observed in the 2np system , as it can be assessed by the multiple minima located in the free energy surface at strand separations greater than 5 a . [SEP]
[CLS] this behavior is only marginally found in the 3np model , most likely as a consequence of the cluster aggregate compaction that hinders the double strand unzipping . [SEP]
[CLS] taken as a whole , the outcome of md simulations suggests that the different sizes and geometrical features of dna - mediated clusters play indeed an important role in determining the structural features of the duplex at the gaps . [SEP]
[CLS] this provides a new tool for interpreting the experimental trends as illustrated in figures 4 and 5 . [SEP]
[CLS] on these bases , we propose the following . [SEP]
[CLS] minimal strand separation is fulfilled at intermediate B-property dna / np B-nanoparticle values when cluster aggregate compaction is maximized . [SEP]
[CLS] however , when dna concentration is low , molecules extend homogenously over the metallic surface , and cluster compaction is improved only by increasing the number of nanoparticles B-nanoparticle per aggregate . [SEP]
[CLS] on the other hand , for large dna / np B-nanoparticle ratios , cluster compaction diminishes as a result of the increased separation between nanoparticles B-nanoparticle imposed by reorientation of the dna molecules over the silver B-material surfaces . [SEP]
[CLS] based on the outcome of the data illustrated in figure 4 , we subsequently fixed the dna / np B-nanoparticle ratios at those values associated with minimal duplex perturbation ( ≈19 mg nmol −1 for ctdna and 3 . 2 mg nmol −1 for dsdna 21 ) while monitoring the c + t ring breathing peak position on nanoparticles B-nanoparticle at decreasing surface charge ( figure 7 ) . [SEP]
[CLS] in this case , short and genomic duplexes share the same qualitative behavior , presenting a jump in peak frequency for ζ potential values in the ≈30 - 35 mv range . [SEP]
[CLS] an identical result was observed for the a spectral marker ( figure s12 , supporting information ) . [SEP]
[CLS] the same study was further extended to a 21 - bp rna duplex ( dsrna 21 ) which , in addition to nucleobase sequence and com - position , differs from dsdna also by the helix geometry ( mainly the b - form for dsdna 21 and a - form for dsrna 21 ) and the sugar molecular structure . [SEP]
[CLS] despite the structural differences , our sers data indicate that the spectral shift dependency of dsrna 21 from the colloidal surface charge is qualitatively analog to those of short and long dna duplexes ( figure s12 , supporting information ) . [SEP]
[CLS] thus , when the cluster geometry variable , which is strictly correlated to the chain length , is removed by fixing the dna / np B-nanoparticle ratios at the minimal double - helix perturbation , the differences of spectral behavior for short and genomic duplexes are largely levelled out . [SEP]
[CLS] a possible explanation may be found in the electrostatic nature of the dna / np B-nanoparticle interaction . [SEP]
[CLS] dsdna initially adsorbs B-property onto a nanoparticle B-nanoparticle , then screens its positive charge thereby favoring the subsequent approaching of a second nanoparticle B-nanoparticle to generate the corresponding interparticle junction . [SEP]
[CLS] as previously simulated by means of md methods , the duplex binding onto the first nanoparticle B-nanoparticle occurs on the nanosecond time scale , where the flexibility of the surface spermine molecules contributes to promoting the optimum interaction with the phosphate groups aligned along the dna backbone . [SEP]
[CLS] on the other hand , the second approaching nanoparticle B-nanoparticle " sees " a duplex whose degrees of freedom are largely reduced due to the interaction with the solid phase . [SEP]
[CLS] thus , we may speculate that such a second dna - np B-nanoparticle interaction could occur to the detriment of the duplex stability , especially when the spermine surface density is decreased below a critical level ( i . e . , when the ζ potential is lowered ) . [SEP]
[CLS] under this scenario , no significant differences are expected to be revealed by the sers signal of duplexes with different lengths , since their sers spectra largely result from the contribution of chain fragments with similar size ( limited by the hot spot volume ) and carrying equally spaced phosphate groups on the dna backbone . [SEP]
[CLS] in summary , the outcomes of the sers analysis can be summarized as follows . [SEP]
[CLS] overall , the results indicate that the interaction of the cationic B-material nanoparticles B-nanoparticle with double - stranded dna leads to ( at least partial ) strand separation of dsdna population that molecularly mediates the nanoparticle B-nanoparticle clustering ( i . e . , dna molecules at the interparticle junctions ) . [SEP]
[CLS] the extent of such structural deformation is highly dependent on ( i ) the dna / np B-nanoparticle ratio and ( ii ) the nanoparticle B-nanoparticle surface charge . [SEP]
[CLS] the former parameter directly affects the aggregation profile which is , in turn , strictly related to the length of the duplex ( whereas base composition did not appear here to play a relevant role ) . [SEP]
[CLS] regardless of the chain length , it was possible to recognize a qualitatively common trend where unzipping of the double helixes is progressively maximized at high and low dna / np B-nanoparticle ratios , for a given ζ potential . [SEP]
[CLS] the potential correlation between the aggregation features of a dna - mediated cluster and the tendency of the dna molecule trapped at the interparticle gap to undergo unzipping was further investigated and confirmed by molecular dynamics in combination with additional spr analysis . [SEP]
[CLS] differently , once fixed the dna / np B-nanoparticle ratio ( i . e . , the degree of aggregation ) to a value corresponding to the minimal duplex perturbation and monitored sers spectra for different nanoparticle B-nanoparticle surface charges , we observe a sudden transition from a dominant duplex conformation to mainly separated strands below a critical ζ potential threshold . [SEP]
[CLS] this transition appears to be largely independent not only of nucleobase composition but also of the chain length and double helical geometry . [SEP]
[CLS] this extensive work provides new fundamental insights into the interaction between nucleic B-material acids I-material and nanoparticles B-nanoparticle , and how the integrity of the double - helical structure is perturbed by the cooperative binding of nano B-nanoparticle - I-nanoparticle objects I-nanoparticle . [SEP]
[CLS] materials : all reagents were of analytical grade and used as received . [SEP]
[CLS] all chemicals were obtained from sigma - aldrich except for short ssdna and ssrna oligonucleotides ( table s1 , sup - porting information ) , which were purchased from eurofins genomics . [SEP]
[CLS] cationic B-material silver B-nanoparticle nanoparticles I-nanoparticle : synthesis of positively charged colloids was carried as previously described . [SEP]
[CLS] ζ potential measurements were performed by diluting the nanoparticles B-nanoparticle with either the colloidal supernatant or milli - q water B-material . [SEP]
[CLS] the supernatant was obtained by removing the nanoparticles B-nanoparticle from colloids via centrifugation at 13 400 rpm for 40 min . [SEP]
[CLS] sers measurements : sers spectra at a fixed ctdna concentration in differently diluted agsp colloids ( figure 3 ) were obtained by adding 10 . 2 µl of ctdna 117 . 2 mg l −1 to 150 µl of colloids . [SEP]
[CLS] the data illustrated in figure 4 and figure s7 ( supporting information ) were extracted from the sers spectra of ctdna 21 and short ss - and ds - oligonucleotides ( ssdna 21 , dsdna 21 , dscg , and dsat ) for different dna / np B-nanoparticle ratios ( and fixed agsp ζ potential ) . [SEP]
[CLS] dna / np B-nanoparticle ratio ( r ) was expressed in milligrams of dna per nanomoles of np B-nanoparticle . [SEP]
[CLS] in each case , the ratios were expressed as follows : for ctdna 21 , r = 3 . 6 , 5 . 4 , 10 . 8 , 27 , 54 , 107 . 9 , and 186 mg nmol −1 ; for ssdna 21 , r = 0 . 21 , 0 . 32 , 0 . 64 , 1 . 61 , 3 . 21 , 6 . 43 , and 11 . 10 mg nmol −1 ; and finally for dsdna 21 , r = 0 . 43 , 0 . 64 , 1 . 29 , 3 . 21 , 6 . 43 , 12 . 85 , and 22 . 16 mg nmol −1 ( identical values were used for dscg and dsat analyses ) . [SEP]
[CLS] these samples were pre - q13 pared by adding different aliquots of dna solutions , obtained upon proper dilution of the stock solutions , to 150 µl of colloids . [SEP]
[CLS] the volume of such aliquots ranged between 3 and 10 µl in order to avoid significant alteration of the final nanoparticle B-nanoparticle concentration . [SEP]
[CLS] data reported in figure 7 and figure s12 ( supporting information ) were extracted from sers spectra of dna / rna in agsp at different ζ potentials and fixed dna / np B-nanoparticle ratios ( r = 19 mg nmol −1 for ctdna , 3 . 2 mg nmol −1 for dsdna 21 and dsrna 21 , and 1 . 6 mg nmol −1 for ssdna 21 ) . [SEP]
[CLS] these samples were also prepared according to the previously described procedure . [SEP]
[CLS] all samples were prepared and left to equilibrate overnight at 4 • c . [SEP]
[CLS] the samples were quickly redispersed before the sers measurements . [SEP]
[CLS] ζ potential and dls measurements : ζ potential and dls measurements were performed on a malvern nano zetasizer nano - zs ( malvern instruments inc . , uk ) equipped with a 4 mw he - ne 633 nm laser . [SEP]
[CLS] ζ potential measurements were acquired under the smoluchowski model , used for most aqueous solutions . [SEP]
[CLS] all samples were measured by triplicate at 25 • c and constant ph ( 6 . 2 ) . [SEP]
[CLS] a universal dip cell B-material ( containing the electrodes ) was inserted into the samples , previously placed in disposable ps cuvettes . [SEP]
[CLS] q14 hydrodynamic size measurements were acquired with a 173 • backscattered angle and calculated via the stokes - einstein equation . [SEP]
[CLS] in this case , all samples were diluted six times in milli - q water B-material , in order to avoid issues related to absorption and multiple scattering events from the sample . [SEP]
[CLS] as for ζ potential measurements , the samples were measured by triplicate at 25 • c , using disposable cuvettes . [SEP]
[CLS] equipment and instrument settings : a thermo scientific evolution 201 uv - visible spectrophotometer was employed to acquire uv - vis spectra . [SEP]
[CLS] ζ potential and dls measurements were performed on a malvern nano zetasizer . [SEP]
[CLS] sem images were acquired with an environmental scanning electron microscope ( jeol 6400 ) . [SEP]
[CLS] sers experiments were performed with a renishaw invia reflex confocal microscope equipped with a highresolution grating consisting of 1800 grooves mm −1 for visible wavelengths , additional bandpass filters , and a ccd camera . [SEP]
[CLS] a ) extinction spectra of agsp colloids at different concentration upon dilution with milli - q water B-material . [SEP]
[CLS] from the top curve to the bottom curve , the estimated nanoparticle B-nanoparticle concentrations are 1 . 00 × 10 −9 , 0 . 91 × 10 −9 , 0 . 79 × 10 −9 , 0 . 70 × 10 −9 , 0 . 55 × 10 −9 , 0 . 44 × 10 −9 , 0 . 34 × 10 −9 , 0 . 24 × 10 −9 , 0 . 20 × 10 −9 , 0 . 16 × 10 −9 , and 0 . 12 × 10 −9 m . [SEP]
[CLS] a representative tem image of the dried nanoparticles B-nanoparticle is also included ( the scale bar represents a length of 20 nm ) . [SEP]
[CLS] b ) ζ potential values for agsp colloids at q5 different concentrations upon dilution with milli - q water B-material . [SEP]
[CLS] 2 . electromagnetic near - field intensity calculations showing the volume size of the hot spot . [SEP]
[CLS] a ) the spatial near - field map of | e | 2 along the xy - and xz - planes ( black color represents a value of 0 while white represent a value of 100 ) . [SEP]
[CLS] b ) the values of | e | 2 along each axis are also shown , where we can appreciate that the x - cut has the gap width while the field along the other two directions has a half - width at half - height around 3 nm . [SEP]
[CLS] a ) sers spectra of ctdna ( final concentration : 7 . 5 µg ml −1 ) in agsp colloids at different nanoparticle B-nanoparticle concentrations and ζ potential . [SEP]
[CLS] b ) detail of the 710 - 820 cm −1 spectral region . [SEP]
[CLS] the spectra were normalized to the phosphate band at ≈1089 cm −1 . [SEP]
[CLS] a general vibrational band assignment is also reported . [SEP]
[CLS] [ 27 ] , [ 45 ] a , adenine ; c , cytosine ; t , thymine ; q10 g , guanine ; rib . , ribose ; po 2 − , phosphate group ; and ( c o ) , carbonyl B-material group I-material . [SEP]
[CLS] frequency shifts of adenine and cytosine ( + thymine ) ring breathing bands for sers spectra of ctdna , dsdna 21 , and ssdna 21 acquired in agsp colloids at fixed ζ potentials . [SEP]
[CLS] for each batch of colloids , the dna concentration , expressed as milligrams of analyte per nanomoles of nanoparticles B-nanoparticle ( logarithmic scale ) , was progressively increased up to values that still yield detectable signals . [SEP]
[CLS] error bars equal to two standard deviations ( n = 3 ) . [SEP]
[CLS] a ) extinction spectra of agsp colloids ( [ np B-nanoparticle ] ≈ 1 . 00 × 10 −9 m , ζ potential ≈ 40 mv ) after the addition of different amounts of analyte , corresponding to ≈0 . 64 , 3 . 2 , and 12 . 8 mg of dsdna 21 per nmol of nanoparticles B-nanoparticle . [SEP]
[CLS] the spectra were acquired 60 min and 1 d after the addition of dsdna 21 to the colloids ( red and pink curves , respectively ) . [SEP]
[CLS] b ) ratio of the extinction intensities at 700 nm over the intensity at the lspr maximum ( i 700 / i lspr ) against dna / np B-nanoparticle ratios ( mg of dsdna 21 per nmol of np B-nanoparticle , in logarithmic scale ) . [SEP]
[CLS] the intensity ratios were recorded at different times after the addition of the dsdna 21 in the as - synthesized agsp colloids . [SEP]
[CLS] aggregates sitting at the bottom of the eppendorf tubes were briefly resuspended just before the optical measurement . [SEP]
[CLS] the blue dotted lines broadly separate the lower and upper dna / np B-nanoparticle ratio ranges corresponding to the formation of unstable and stable clusters in suspension , respectively . [SEP]
[CLS] a , b ) free energy surfaces computed as a function of the interparticle distance and the average distance between complementary base pairs for 2np and 3np model systems . [SEP]
[CLS] iso - lines are plotted with a step of 1 kcal mol −1 . [SEP]
[CLS] pictorial representations of the mechanical features of the cluster aggregates , and a representative configuration showing partial dna unzipping extracted from md - based simulations , are also shown ( left and right panels , respectively ) . [SEP]
[CLS] frequency shift of cytosine ( + thymine ) ring breathing bands for sers spectra of ctdna , dsdna 21 , and ssdna 21 acquired in agsp colloids at different ζ potentials upon dilution with milli - q water B-material . [SEP]
[CLS] dna / np B-nanoparticle ratio was kept constant to 19 mg nmol −1 for ctdna , 3 . 2 mg nmol −1 for dsdna 21 , and 1 . 6 mg nmol −1 for ssdna 21 . [SEP]
[CLS] error bars equal to two standard deviations ( n = 4 ) . [SEP]
[CLS] due to their size and tailorable physicochemical properties , nanomaterials B-material are an emerging class of structures utilized in biomedical applications . [SEP]
[CLS] there are now many prominent examples of nanomaterials B-material being used to improve human health , in areas ranging from imaging and diagnostics to therapeutics and regenerative medicine . [SEP]
[CLS] an overview of these examples reveals several common areas of synergy and future challenges . [SEP]
[CLS] this nano focus discusses the current status and future potential of promising nanomaterials B-material and their translation from the laboratory to the clinic , by highlighting a handful of successful examples . [SEP]
[CLS] advances in medicine in the areas of genomics , proteomics , tissue engineering , and regenerative medicine are occurring at a rate that was previously unthinkable . [SEP]
[CLS] the development of new materials resulting from these breakthroughs , such as those that can be used to replace blood vessels , to promote tissue growth , to monitor blood glucose levels , or to improve the bioavailability B-property of drugs , has been equally rapid and diverse . [SEP]
[CLS] 1 , 2 one of the most exciting frontiers is the development of nanomaterials B-material for biomedical applications . [SEP]
[CLS] nanomaterials B-material have size - , shape - , and composition - dependent physical , chemical , optical , and electronic properties , among others , that can be designed and tuned , and they are showing great promise for the diagnosis , treatment , monitoring , and control of disease . [SEP]
[CLS] 5 - 8 a recent survey found that more than 247 nanomaterial - based medical products have been approved by the food and drug administration ( fda ) and are currently in various stages of clinical study . [SEP]
[CLS] 9 their intended uses range from the treatment of clinically unresectable cancers to the preparation of antibacterial hand gels to the regeneration of heart tissue . [SEP]
[CLS] at the same time , common themes emerge when discussing nanomaterials B-material in medicine . [SEP]
[CLS] indeed , one of the biggest issues is how to translate nanomaterials B-material from the laboratory to the clinic effectively . [SEP]
[CLS] and to discuss criteria for the selection , development , synthesis , and utilization of nanomaterials B-material . [SEP]
[CLS] in this nano focus , we highlight these discussions , which fall into three categories : nanotherapeutics , imaging and diagnostics , and tissue regeneration . [SEP]
[CLS] among the numerous nanomaterials B-material explored in therapeutic applications , those often found in clinical trials are gold B-nanoparticle nanoparticles I-nanoparticle , polymeric B-nanoparticle nanoparticles I-nanoparticle , liposomes B-nanoparticle , and carbonbased nanomaterials B-material . [SEP]
[CLS] by sharing expertise across fields , researchers can accelerate the utilization of nanomaterials B-material in addressing the challenges faced by traditional therapeutic agents . [SEP]
[CLS] since the development of gold B-material - nanoparticle - based spherical nucleic B-material acids I-material ( snas ) in 1996 , the mirkin group at northwestern university has exploited the properties of this class of nanostructures in many areas of biomedicine . spherical nucleic B-material acids I-material typically consist of a nanoparticle B-nanoparticle core B-material and a highly oriented and densely packed shell B-material of oligonucleotides . [SEP]
[CLS] the properties of snas emerge from the orientation and arrangement of oligonucleotides on the surfaces of these particles . [SEP]
[CLS] for example , snas are taken up into many different cell B-material types ( over 60 tested to date ) at high levels and rates without the need for transfection agents , have high affinities for nucleic B-material acid I-material targets ( 100 times greater than linear dna of the same sequence ) , and cross both the blood - brain barrier B-property and the epidermis to reach dificultto - target tissues in therapeutic applications . [SEP]
[CLS] success in the utilization of snas for intracellular mrna detection has led to the commercialization of nanoflare technology under the trade name smartflares ( merck millipore in partnership with aurasense , llc , skokie , illinois ) , and snas have also been applied as agents in gene regulation as therapeutics for a host of cancers , including glioblastoma multiforme ( an aggressive form of brain cancer ) , and skin disorders , among others . [SEP]
[CLS] the mirkin group has made progress in using snas as immunomodulatory agents . [SEP]
[CLS] spherical nucleic B-material acids I-material functionalized with oligonucleotides displaying toll - like receptor B-material ( tlr ) - agonist or tlr - antagonist activity were shown to be capable of either stimulating or modulating the activity of the immune system , respectively . [SEP]
[CLS] such structures do so with potencies up to several orders of magnitude higher than the conventional linear nucleic B-material acids I-material from which they are composed . [SEP]
[CLS] this discovery paves the way for the development of snas as therapeutic cancer vaccines . [SEP]
[CLS] mirkin ' s demonstration of the ability of snas to reduce tumor B-material burden and to enhance survival in vivo , in mouse models of lymphoma , proved that immunomodulatory snas can be directed to activate the response of the immune system to destroy tumors B-material in an antigen - specific manner ( figure 1 ) . [SEP]
[CLS] a fundamental challenge is the delivery of therapeutic molecules inside target cells B-material in the body . [SEP]
[CLS] nanoparticles B-nanoparticle have shown immense promise as vehicles B-material for intracellular delivery , with proof - of - principle experiments in humans being completed with small interfering rna ( sirna ) . [SEP]
[CLS] however , there are many barriers B-property to achieving safe and effective delivery systems ( figure 2 ) , and potential delivery systems must have multiple functionalities to allow in vivo delivery , making the design criteria dificult to define for nanoparticles B-nanoparticle capable of accomplishing intracellular delivery . [SEP]
[CLS] to accelerate the design and discovery process , the anderson group at the massachusetts institute of technology has pioneered combinatorial methods for nanoparticulate drug delivery . [SEP]
[CLS] combinatorial chemical methods have been developed to enable the rapid synthesis and characterization of a range of nanoformulations based on biodegradable B-property polymers B-material , lipid - like materials , and other materials . [SEP]
[CLS] these have generated new formulations with particular promise as delivery vehicles B-material for rna and other nucleic B-material acids I-material ; these formulations have the potential to be used as therapies for many diseases , including cancer [SEP]
[CLS] furthermore , shi at brigham and women ' s hospital / harvard medical school described the rational design and development of lipid - polymer B-material hybrid nanoparticle B-nanoparticle platforms to address the bottlenecks faced in the delivery of rna interference ( rnai ) therapeutics , such as sirna . [SEP]
[CLS] specifically , the clinical applications of rnai in cancer therapy are currently hindered by the challenge of achieving the effective systemic in vivo delivery of sirna to tumors B-material . [SEP]
[CLS] multiple physiological barriers B-property , such as enzymatic degradation , rapid elimination by renal excretion or the mononuclear phagocyte system , and poor cellular uptake and endosomal escape , must be overcome . [SEP]
[CLS] by utilizing hybrid nanoparticles B-nanoparticle , shi achieved sustained gene silencing , and prolonged circulation of sirna in the blood for high tumor B-material extravasation and accumulation . [SEP]
[CLS] the successful application of these rnai nanoparticles B-nanoparticle to validate the therapeutic role of prohibitin1 in non - small cell B-material lung cancer treatment indicates the significant potential of this platform for the validation of many other potential cancer targets and for the clinical development of novel cancer therapies . [SEP]
[CLS] kataoka at the university of tokyo has pioneered the synthesis and development of " polymeric micelle B-material drugs " , which have proven useful for targeting a variety of drugs to tissues and organs and to tumors B-material in particular . [SEP]
[CLS] there has been significant recent progress in the clinical development of polymeric micelles B-material loaded with a variety of cytotoxic B-property reagents . [SEP]
[CLS] notably , five different micellar formulations have already been explored in clinical trials in asia and the united states . [SEP]
[CLS] a version loaded with paclitaxel B-material is in the final stage of a phase iii clinical trial in japan for the treatment of recurrent breast cancer , and it is expected to proceed into the application for approval within a year . [SEP]
[CLS] more recently , kataoka and colleagues have been active in developing a second generation system of polymeric micelles B-material installed with ligand moieties at their peripheries . [SEP]
[CLS] particularly , crgd - conjugated polymeric micelles B-material were able to cross the blood - brain tumor B-material barrier B-property via a transcytosis mechanism , achieving high efficacy in treating intractable orthotopic glioblastoma in animal experiments . [SEP]
[CLS] antibody B-material fragments can also be conjugated to polymeric micelles B-material . [SEP]
[CLS] in this way , higher drug payloads can be achieved without antibody B-material precipitation or impaired binding compared to when drugs are directly conjugated to antibody B-material molecules using more conventional approaches . [SEP]
[CLS] in 1989 , the kabanov group at the university of north carolina at chapel hill investigated the use of polymeric micelles B-material for targeted drug delivery . [SEP]
[CLS] they discovered that pluronic block copolymers can be used in the sensitization of multi - drug - resistant ( mdr ) cancer and cancer stem cells B-material and elucidated the mechanisms responsible for these effects . [SEP]
[CLS] this research led to first - in - man polymeric micelle B-material drug candidates for the treatment of cancer ( sp1049c ) that show high efficacy against advanced esophageal cancer in phase ii trials . [SEP]
[CLS] recently , they discovered polymeric micelles B-material based on amphiphilic B-property poly ( 2 - oxazoline ) blocks with unprecedented high capacities for poorly soluble B-property , uncharged drugs ( e . g . , taxanes ) and drug combinations , enabling increasing therapeutic indices compared to current drug formulations . [SEP]
[CLS] liposomes B-nanoparticle are another popular nanomaterial B-material used in preclinical and clinical studies , but it is dificult to sustain release from these structures for more than a few days . [SEP]
[CLS] the venkatraman group at nanyang technological university has developed a subconjunctivally injected nanoliposome drug delivery system for the long - term ( 3 - 5 months ) delivery of latanoprost that can be used in glaucoma treatment , which went from concept to clinic in less than 5 years ( figure 3 ) . [SEP]
[CLS] glaucoma is a chronic progressive optic neuropathy that is characterized by optic nerve changes and visual field loss . [SEP]
[CLS] elevated intraocular pressure ( iop ) is the main modifiable risk factor . [SEP]
[CLS] the chronic instillation of daily eye drops to lower iop is the primary treatment of choice , although this regimen requires patient adherence and correct performance . [SEP]
[CLS] hence , a sustained - delivery system would be a big boon to patients with glaucoma . [SEP]
[CLS] venkatraman ' s group also explored the nanoparticle - mediated sustained delivery of sirna for preventing fibrosis following surgery . [SEP]
[CLS] this method has applications in ocular and other types of surgeries / implantations . [SEP]
[CLS] they have shown sustained efficacy of action with a sirna - incorporated nanoparticle B-nanoparticle . [SEP]
[CLS] as shown in the above two examples , the premise of their work rests on the ability of nanoparticles B-nanoparticle to sustain drug / protein B-material / sirna release . [SEP]
[CLS] the chow and ho groups at the national university of singapore and the university of california , los angeles ( ucla ) , respectively , study nanodiamonds , which are an emerging class of carbon - based nanomaterials B-material , due to their advantageous surface characteristics . [SEP]
[CLS] nanodiamond facets mediate electrostatic properties that have resulted in potent and scalable anthracycline B-material drug binding as well as marked enhancements in magnetic B-property resonance imaging ( mri ) efficiency . [SEP]
[CLS] with regard to drug delivery , nanodiamond - doxorubicin B-material compounds ( ndx ) were administered to treat drug - resistant breast and liver tumors B-material in mouse models . [SEP]
[CLS] this study demonstrated the nanodiamond - mediated improvement of drug tolerance ; lethal dosages of doxorubicin B-material delivered as ndx resulted in the smallest tumors B-material observed ( compared to saline controls and unmodified drug administration ) , and all of the treated mice survived the full duration of the study . [SEP]
[CLS] the active targeting of triple - negative breast tumors B-material in vivo using nanodiamond - epirubicin complexes functionalized with the epidermal growth B-material factor I-material receptor I-material ( egfr ) antibody B-material resulted in complete tumor B-material regression . [SEP]
[CLS] more recently , nanodiamond - anthracycline B-material agents have been used to treat hepatic cancer stem cells B-material . [SEP]
[CLS] toward the further enhancement of the potency and safety of cancer therapies , a prevalent challenge in the field of nanomedicine is the ability to move beyond monotherapies toward combinatorial cancer treatments . [SEP]
[CLS] using phenotypic instead of genotypic profiling to drive combinatorial optimization , ho and colleagues developed a powerful mechanismindependent engineering optimization platform , termed feedback system control . ii ( fsc . ii ) , to identify globally optimized nanodiamond - anthracycline B-material drug combinations rapidly . [SEP]
[CLS] fsc . ii does not require the use of feedback , and instead , it utilizes a selected set of experimental validation assays to formulate phenotypic profiles from which drug combinations can rapidly be pin - pointed . [SEP]
[CLS] mitragotri at the university of california , santa barbara , also translated nanomaterials B-material to clinical applications ( figure 4 ) . [SEP]
[CLS] specifically , he used gold B-material - coated silica B-nanoparticle nanoparticles I-nanoparticle for the treatment of acne . [SEP]
[CLS] these 150 nm , poly ( ethylene glycol ) - coated , silica - gold nanoparticles B-nanoparticle are designed to absorb near - infrared light and produce localized heating . [SEP]
[CLS] the delivery of these nanoparticles B-nanoparticle into skin is a major hurdle due to the skin ' s barrier B-property properties . [SEP]
[CLS] mitragotri and colleagues showed that these nanoparticles B-nanoparticle can be delivered deep into the skin ' s sebaceous glands using low - frequency ultrasound . [SEP]
[CLS] ultrasound induces cavitation on the surface of the skin , which produces microjets and shock waves that open transport pathways into the glands . [SEP]
[CLS] once delivered deep into the glands , the thermal activation of the nanoparticles B-nanoparticle using near - infrared light caused thermolysis and the inactivation of overactive sebaceous glands , the underlying pathology of acne . [SEP]
[CLS] this nanoparticle - based technology provides several advantages over standard treatments for acne ; for instance , systemic side effects are avoided with this treatment . [SEP]
[CLS] the stevens group at the imperial college of london and collaborators at the houston methodist research institute have recently reported engineering a platform of mesoporous silicon B-material nanoneedles for the delivery of nanoparticle B-nanoparticle and other therapeutic payloads to cells B-material and tissues ( figure 5a , b ) . [SEP]
[CLS] this technology could prove transformative in the fields of drug delivery , regenerative medicine , and biosensing . [SEP]
[CLS] the dynamics of the nanoneedle entry to the cell B-material and study of the nanoneedle - cell B-material interface have been elucidated and pave the way for highly controlled delivery of a range of nanoparticle B-nanoparticle payloads intracellularly . [SEP]
[CLS] furthermore , the nanoneedle array can simultaneously deliver both dna and sirna with high efficiency ( over 90 % ) and in vivo proved successful in upregulating blood vessel formation in muscle by delivery of the vegf - 165 gene ( figure 5c - e ) . [SEP]
[CLS] the stevens group has also developed several other notable nanomaterials - based technologies , particularly enzyme - response nanoparticle B-nanoparticle systems that have a wide range of important impacts in the field of biosensing . [SEP]
[CLS] from the above discussion of a variety of nanomaterials B-material as nanotherapeutic agents for enhancing treatment efficacy , we conclude that successful translation of nanomaterials B-material relies on the identification of a clinical problem and innovative ideas to solve it through rational design . [SEP]
[CLS] in addition , more and more versatile nanomaterials B-material are being exploited in emerging research themes , areas such as cancer vaccines and genome editing . [SEP]
[CLS] besides their use as therapeutic agents , nanomaterials B-material are also being used for imaging and diagnostics purposes . [SEP]
[CLS] the most well - known examples include silica B-nanoparticle nanoparticles I-nanoparticle , quantum B-nanoparticle dots I-nanoparticle , magnetic B-nanoparticle nanoparticles I-nanoparticle , and microbubbles . [SEP]
[CLS] these nanostructures have been used to detect small molecules ( like h 2 o 2 ) , cells B-material including circulating tumor B-material cells B-material ( ctcs ) , and tumor B-material tissues . [SEP]
[CLS] the sun group at brown university is interested in monitoring cellular h 2 o 2 , which is an important reactive oxygen B-material species generated via oxygen B-material metabolism ; it is actively involved in cell B-material signaling and cell growth [SEP]
[CLS] unfortunately , its uncontrolled overproduction can cause the detrimental oxidation of biomolecules and lead to aging , cancer , and other diseases . [SEP]
[CLS] sun recently developed dumbbell ( au - fe 3 o 4 and pdpt - fe 3 o 4 ) and core / shell ( au / mno ) magnetic B-nanoparticle nanoparticles I-nanoparticle as sensitive probes for h 2 o 2 detection . [SEP]
[CLS] dumbbell magnetic B-nanoparticle nanoparticles I-nanoparticle were prepared by the controlled nucleation and growth of fe 3 o 4 on presynthesized noble metal nanoparticles B-nanoparticle , while the core / shell au / mno nanoparticles B-nanoparticle were made by the controlled oxidation of aumn alloy nanoparticles B-nanoparticle . [SEP]
[CLS] both dumbbell and core / shell nanoparticles B-nanoparticle are active for the electrochemical reduction of h 2 o 2 with detection limits reaching as low as 5 nm . [SEP]
[CLS] highly sensitive electrochemical sensors have been used to monitor h 2 o 2 concentration levels released from living cells B-material ; tumorigenic cells B-material were found to have higher levels of h 2 o 2 than nontumorigenic ones . [SEP]
[CLS] these composite nanoparticle B-nanoparticle probes can be used in high - sensitivity cancer detection schemes and may also help to increase the efficacy of cancer therapies . [SEP]
[CLS] the chen group at the national institute of biomedical imaging and bioengineering of the national institutes of health uses nanomaterials B-material as platforms to provide imaging contrast in positron B-technique emission I-technique tomography I-technique ( pet ) . [SEP]
[CLS] in medical imaging , pet can provide a direct , highly sensitive , and quantitative readout of organ / tissue targeting efficiency and pharmacokinetics . [SEP]
[CLS] compared with radiolabeled antibodies B-material , proteins B-material , peptides B-material , and other biologically relevant molecules , radiolabeled nanoparticles B-nanoparticle represent a new frontier in molecular imaging probe design because they can combine different imaging modalities and targeting B-material ligands I-material in a single vector , synergistically improving the imaging quality . [SEP]
[CLS] however , the applications of radiolabeled nanoparticles B-nanoparticle are based on the premise that the radioisotopes B-material are stably attached to the nanomaterials B-material . [SEP]
[CLS] chen has developed general rules for selecting appropriate isotopes B-material for given types of nanoparticles B-nanoparticle as well as adjusting the labeling reaction according to specific applications . [SEP]
[CLS] the stability ( colloidal and radiochemical ) of the radiolabeled nanoparticles B-nanoparticle as well as their biological fate must be assessed ; special attention should be paid to labeling strategies as they affect the stability of radiolabeled nanoparticles B-nanoparticle and might cause discrepancies in the interpretation of pet data ( owing to the distribution of nanoparticles B-nanoparticle ) . [SEP]
[CLS] wang ' s group at the chinese academy of sciences is interested in creating nano - bio interfaces with controllable adhesion properties . [SEP]
[CLS] the cell - adhesive biointerfaces are based on the cooperative effects of multiscale structural matching and molecular recognition . [SEP]
[CLS] they explore the relationship between cell - I-event specific I-event adhesion and surface structure ( with nanowires B-nanoparticle , nanofibers B-nanoparticle , nanofractals , and soft nanotubes B-nanoparticle [SEP]
[CLS] also , they developed a series of biointerfaces with specific recognition and stimuli - responsive capture and release properties ( i . e . , temperature , 61 electric , enzymatic , and ph [SEP]
[CLS] they have made progress in the isolation of viable rare ctcs from the blood via their designed cell - adhesive biointerfaces and developed adhesion - based ctc isolation approaches as cancer diagnostics with high efficiencies ( > 97 % ) . [SEP]
[CLS] in particular , these biointerfaces with controlled cell B-event adhesion I-event are capable of capturing rare viable ctcs for early cancer detection and the monitoring of cancer therapy , single - cell gene analysis , and other purposes . [SEP]
[CLS] nanomaterials B-material for tissue engineering involve a broad spectrum of nanoscale formulations and structures developed to mimic tissue complexity and to modulate cellular function to yield therapeutic benefits . [SEP]
[CLS] the xia group at the georgia institute of technology and emory university has been developing practical nanomaterials B-material for medical applications . [SEP]
[CLS] they use electrospun nanofibers B-nanoparticle in tissue engineering . [SEP]
[CLS] electrospinning has been widely explored to process polymeric materials into nanofibers B-nanoparticle with tunable and controllable compositions , diameters , porosities , and surface properties . [SEP]
[CLS] owing to its small feature size , high porosity , and large surface area , a nonwoven mat of electrospun fibers can serve as a superb scaffold that mimics the extracellular matrix ( ecm ) , which is critical to cell B-material attachment and spreading . [SEP]
[CLS] the nanofibers B-nanoparticle themselves can also be functionalized through the encapsulation or attachment of bioactive species , such as ecm proteins B-material , enzymes , and growth factors . [SEP]
[CLS] in addition , the nanofibers B-nanoparticle can be readily assembled into a wide variety of arrays or hierarchical structures by manipulating their alignment , stacking , or folding . [SEP]
[CLS] all these attributes make electrospinning a powerful tool for generating nanostructured B-material materials I-material for a broad range of biomedical applications , including controlled release , drug delivery , and tissue engineering . [SEP]
[CLS] xia highlighted the use of aligned nanofibers B-nanoparticle to control the differentiation of embryonic stem cells B-material into different types of neural lineages and to guide the outgrowth of neurites for peripheral nerve repair . [SEP]
[CLS] he also pointed out that nanofiber B-nanoparticle scaffolds could be designed for repairing injuries to the flexor tendon and the tendon - tobone insertion site ; 67 they could also be used as wound dressings for brain surgery . [SEP]
[CLS] karp from brigham and women ' s hospital suggests that different approaches are required for solving medical problems versus solving basic science problems . [SEP]
[CLS] he asserts that one must develop design criteria relevant to solving the problem in animal models , while considering the multiple steps required to bring a technology from the laboratory to the clinic . [SEP]
[CLS] one must think through scale - up , manufacturing , regulatory issues , and patent strategy and then impose these criteria to advance toward a potential solution . [SEP]
[CLS] in particular , turning to nature for solutions can aid in the problem - solving process , recognizing that everything living has overcome challenges , and thus we are surrounded by solutions . [SEP]
[CLS] through elucidating mechanisms behind these solutions with state - of - the - art tools , he asserts that we can identify ideas to help overcome even the most challenging of problems . [SEP]
[CLS] karp has created tissue adhesives using inspiration from slugs and snails , spiny - headed worms , porcupine quills , and spider webs . [SEP]
[CLS] one of the adhesive technologies led to the formation of a company , gecko biomedical ( paris , france ) , and is on track to be tested for vascular graft applications in humans in late 2015 . [SEP]
[CLS] of note is that , in these developments , failure should be embraced as part of the problem - solving process and is likely a prerequisite to success . [SEP]
[CLS] in this work , it was important to build highly functional and multi - disciplinary teams that know what resources are available in their environment and how to access them . [SEP]
[CLS] teoh from nanyang technological university is interested in processing biomaterials in an environmentally friendly way . [SEP]
[CLS] to this end , his group has unearthed a solvent - free approach known as cryomilling , where biomaterials are processed at near cryogenic temperatures . [SEP]
[CLS] polymer B-material particle size reduction has been achieved due to the high - energy collision process , which occurs below the glass transition temperature . [SEP]
[CLS] particle sizes may be reduced to the nanoscale , where surface thermodynamics play critical roles in determining their behavior , particularly the interactions between two chemically distinct phases , as in the case of composite biomaterials . [SEP]
[CLS] notably , there is also a significant challenge in obtaining reproducible , well - distributed composites . [SEP]
[CLS] they have demonstrated that a variety of composite biomaterials that incorporate second phases , such as inorganic elements , trace elements , and even drugs , may be processed efficiently via cryomilling . [SEP]
[CLS] this technique also addresses second phase distribution issues that plague many other processing techniques , such as solvent - casting , electrospinning , and melt extrusion . [SEP]
[CLS] tremendously exciting advances in nanomaterials B-material - based medical treatments , from cancer therapies , diabetes administration , and vaccine development to molecular diagnostics , tissue repair , and regeneration , are both underway and yet to come . [SEP]
[CLS] the confluence of nanomaterials B-material and emerging biomedical science provides vast commercial opportunities . [SEP]
[CLS] while fundamental science has been fueled with numerous innovations over the past decade , as evidenced by the number of nanomaterial - associated formulations and devices in clinical development , the number of marketed products is still small , compared to traditional medications . [SEP]
[CLS] remaining challenges include effectively improving efficacy , while minimizing potential concerns , through the rational design and thorough evaluation of nanomaterials B-material . [SEP]
[CLS] in oncology nanomedicine , for example , the acceleration of the translation process relies profoundly on a thorough understanding of how nanocarriers interact with the physiological environment . [SEP]
[CLS] in addition to general evaluations based on the enhanced permeability and retention effect , biodistribution , and clearance mechanisms , more precise details should be taken into account , such as how the particles pass through the tumor B-material microenvironment and enter cells B-material to reach active sites . [SEP]
[CLS] moreover , in designing stimuli - responsive or programmable nanocarriers , a current theme in nanomedicine , closer investigations of the dynamic relationships between the phase transitions of materials and the relevant gradients in the biological environment , such as ph , redox , glucose , atp , and enzyme B-property activity I-property , should enable more precise targeting and release . [SEP]
[CLS] second , a major stumbling block in the translation of nanomaterials B-material for biomedical applications is safety concerns , especially for invasive administration . [SEP]
[CLS] on one hand , clinical use requires the careful , prolonged evaluation of the local and systemic toxicity B-property of nanomaterials B-material as well as their potential immunogenicity B-property . [SEP]
[CLS] on the other hand , there is an urgent need to invent and to tailor new materials with excellent biocompatibility B-property . [SEP]
[CLS] ideas inspired by nature , mimicking the structures and composites of natural particles , including viruses , vesicles , and cells B-material , have attracted increasing interest and brought promising outcomes . [SEP]
[CLS] third , regarding the rational design of nanomaterials B-material with specific physicochemical properties for clinical applications , it is important to set uniformity in preclinical trials . [SEP]
[CLS] variability in particle size , surface properties , and stability as well as differences in cell B-material lines , tumor B-material properties , therapeutic doses , and pharmacokinetics / pharmacodynamics analysis have prevented the systematic comparison of relevant nanomaterials B-material and have been an impediment to creating design rules for optimizing a specific formulation or device . [SEP]
[CLS] last but not least , the design , development , and ultimately commercialization of clinically used nanomaterials B-material require seamless collaboration and commitment between a broad range of research investigators , investors , and regulatory authorities . [SEP]
[CLS] key to these activities are platforms for fusing ideas to shepherd emerging technologies further along safe and effective pipelines . [SEP]
[CLS] overall , given the progress that has been made so far , we are optimistic that nanomaterialsbased clinical development will continue to be exciting , with growing numbers of innovations as well as those currently garnering fda approval entering the clinic soon . [SEP]
[CLS] we hope the ideas and concepts presented in this nano focus will be useful in the development of " ideal " nanomaterials B-material features , the expansion of design criteria , and the enlightenment of research opportunities for evolving the next generations of biomedical materials . [SEP]
[CLS] in vivo activity of immunomodulatory snas as cancer vaccines . [SEP]
[CLS] 2 . barriers B-property to successful in vivo delivery of nucleic B-material acids I-material using nonviral vectors . [SEP]
[CLS] reprinted with permission from ref 19 . [SEP]
[CLS] copyright 2014 macmillan publishers ltd . [SEP]
[CLS] 3 . subconjunctival instillation of nanocarriers incorporating latanoprost lowers eye pressure in glaucoma patients for up to 3 months . [SEP]
[CLS] reproduced from ref 37 . [SEP]
[CLS] copyright 2014 american chemical society [SEP]
[CLS] 4 . schematic for the nanoparticle - based treatment of acne . [SEP]
[CLS] silica - gold nanoparticles B-nanoparticle are delivered into sebaceous glands using low - frequency ultrasound . [SEP]
[CLS] nanoparticles B-nanoparticle are then activated using near - infrared light to induce thermolysis . [SEP]
[CLS] reprinted with permission from ref 45 . [SEP]
[CLS] copyright 2015 elsevier . [SEP]
[CLS] nanoneedle for the delivery of vegf - 165 to upregulate blood vessel formation in muscle . [SEP]
[CLS] ( a ) scanning electron microscope ( sem ) micrographs showing the morphology of porous silicon B-material nanoneedle arrays with pitches of 2 μm . [SEP]
[CLS] scale bars , 2 μm . [SEP]
[CLS] ( b ) high - resolution sem micrographs of nanoneedle tips showing the nanoneedles ' porous structure and the tunability of tip diameter from less than 100 nm to over 400 nm . [SEP]
[CLS] scale bars , 200 nm . [SEP]
[CLS] ( c ) intravital bright - field ( top ) and confocal ( bottom ) microscopy B-technique images of the vasculature of untreated ( left ) and hvegf - 165 - treated muscles with either direct injection ( center ) or nanoinjection ( right ) . [SEP]
[CLS] the fluorescence B-property signal originates from systemically injected fitc - dextran . [SEP]
[CLS] scale bars , bright - field 100 μm ; confocal 50 μm . [SEP]
[CLS] ( d , e ) quantification of the fraction of fluorescent B-property signal ( dextran B-material ) ( d ) and the number of nodes in the vasculature per mm 2 ( e ) within each field of view acquired for untreated control , intramuscular injection ( im ) and nanoinjection . [SEP]
[CLS] * p = 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . [SEP]
[CLS] error bars represent the sd of the averages of five areas taken from three animals . [SEP]
[CLS] reprinted with permission of figure and caption text from ref 48 . [SEP]
[CLS] copyright 2015 macmillan publishers ltd . [SEP]
[CLS] recently dnacoated gold B-nanoparticle nanoparticles I-nanoparticle have emerged as ideal tools for the detection of mrna in cells B-material using dye modified oligonucleotides . [SEP]
[CLS] however , the tracking of the gold B-material core B-material has been hindered by the small size of the particle core B-material . [SEP]
[CLS] in this work we utilize a home built set up and 43 nm dnacoated spherical B-nanoparticle gold I-nanoparticle nanoparticles I-nanoparticle for the simultaneous imaging of mrna detection using fluorescence B-technique microscopy I-technique and the gold B-nanoparticle nanoparticle I-nanoparticle core B-material using two photon photoluminescence B-property ( tppl ) . [SEP]
[CLS] in recent years the vast capabilities of dnacoated gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) have been explored including their use as sensors and as therapeutic agents for the targeted treatment of diseases and / or skin conditions . [SEP]
[CLS] in particular spherical aunps B-nanoparticle modified with a monolayer of synthetic oligonucleotides have been shown to accurately detect specific mrna targets in live cells B-material . [SEP]
[CLS] in this design , aunps B-nanoparticle are coated with a thiol modified sense strand that is conjugated to the aunp B-nanoparticle surface . [SEP]
[CLS] to the sense , a shorter fluorophore modified oligonucleotide is hybridized termed flare strand . [SEP]
[CLS] the sense sequence can be designed to detect a specific mrna target thus when the target mrna binds , the flare strand will be released due to competitive hybridization and a fluorescence B-property signal will be detected . [SEP]
[CLS] in the absence of the target the flare will remain bound and the fluorescence B-property signal corresponding to the dye will be quenched by the gold B-material core B-material as shown in scheme 1 [SEP]
[CLS] since their initial development , this design has not only been shown to be capable of the targeted detection of biomolecular targets such as mrna and microrna but has also been developed into a successful drug delivery agent . [SEP]
[CLS] by incorporating intercalating drugs into the dna duplex , it was demonstrated the targeted release only to cells B-material expressing a specific mrna target leading to efficient cell B-event death I-event . [SEP]
[CLS] however , the intracellular fate of dnacoated aunp B-nanoparticle probes following cellular uptake is still under investigation . [SEP]
[CLS] in order to assess nanoparticle B-nanoparticle intracellular fate , imaging of the gold B-material core B-material in conjunction with flare release would be of substantial interest . [SEP]
[CLS] apart from acting as a scaffold for dna attachment , the gold B-nanoparticle nanoparticle I-nanoparticle core B-material can also demonstrate interesting optical properties that can be used for its direct imaging within cells B-material . [SEP]
[CLS] aunps B-nanoparticle that display strong tppl are attractive as contrast B-technique agents I-technique for bioimaging as it allows for non - invasive imaging of living tissue with high penetration depths , 3d mapping capabilities and reduced photobleaching . [SEP]
[CLS] furthermore , tppl strongly increases the signal to noise ratio and is appealing due to the absence of a background signal , which is important when imaging in scattering media . [SEP]
[CLS] for prolonged live cell B-material tracking , quantum B-nanoparticle dots I-nanoparticle are usually utilized as a feasible option due to their enhanced stability . [SEP]
[CLS] however , they are composed of heavy metals B-material that are cytotoxic B-property making them unsuitable for in vivo clinical applications . [SEP]
[CLS] aunps B-nanoparticle on the other hand , are biocompatible B-property and have a large two - photon action cross - section making them ideal alternative candidates . [SEP]
[CLS] research by gao et al . suggested that tppl is shape and size dependent with branched aunps B-nanoparticle and aunrs displaying the strongest tppl . [SEP]
[CLS] aunrs as small as 8 × 40 nm can be easily detected with a high signal to noise ratio . [SEP]
[CLS] furthermore , they are easily functionalized , they are non - cytotoxic B-property and their longitudinal plasmon resonance can be tuned to the near infrared wavelengths where biological tissue exhibits relatively small extinction coefficients . [SEP]
[CLS] for example , a recent study involved the use of folate nanorods B-nanoparticle for the targeted delivery to kb cells B-material ( a tumor B-material cell line derived from oral epithelium ) , which is known to overexpress folate B-material receptors I-material . [SEP]
[CLS] via tppl imaging it was noticed that 6 h after incubation B-technique a large number of nanoparticles B-nanoparticle was observed on the outer membrane with complete internalization occurring after 17 h . [SEP]
[CLS] furthermore , when nanorods B-nanoparticle were incubated B-technique with a cell B-material line lacking these receptors little to no uptake was observed . [SEP]
[CLS] however , tppl imaging has also proven useful for 3d imaging using cancer cells B-material supported in a collagen matrix to mimic tissue . [SEP]
[CLS] durr et al . also showed that by conjugating anti - egfr antibodies B-material to aunrs of 14 nm in width and 48 nm in length , egfr overexpressing a431 skin cancer cells B-material could be imaged by tppl with characteristic bright rings being observed as well as bright spots in the cytoplasm , which they suggested that it was indicative of endosomal uptake . [SEP]
[CLS] furthermore , wang et al . successfully demonstrated the tppl imaging of nanorods B-nanoparticle in vivo . [SEP]
[CLS] by monitoring the tppl signal the flow of aunrs through mouse ear blood vessels could be tracked . [SEP]
[CLS] tppl signal was found to be 3 times higher than the background and interestingly no signal could be detected after 30 minutes indicating successful clearance from circulation . [SEP]
[CLS] on the other hand , liu et al . demonstrated how branched nanoparticles B-nanoparticle could also be imaged in vivo . [SEP]
[CLS] branched aunps B-nanoparticle have a high tppl signal mainly due to their sharp spikes that result in larger tppl action crosssection . [SEP]
[CLS] their study highlighted the ability of successfully imaging branched nanoparticles B-nanoparticle of two different sizes ( 30 and 60 nm ) after intravenous injection into mice with xenograft sarcomas . [SEP]
[CLS] however , when testing 12 nm aunps B-nanoparticle no tppl signal could be detected . [SEP]
[CLS] in contrary to this result , rane et al . found that tppl imaging of aunps B-nanoparticle ranging from 10 to 70 nm coated with an oligonucleotide shell B-material could be successfully utilized to image in 2d and 3d models of the hct116 cell B-material line , a colorectal carcinoma cell B-material line . [SEP]
[CLS] in this paper , we demonstrate the simultaneous detection of mrna using fluorescence B-property and the gold B-material core B-material by using tppl in cells B-material . [SEP]
[CLS] for these experiments , a home built setup was designed capable of performing both tppl and fluorescence B-technique microscopy I-technique . [SEP]
[CLS] sodium B-material tetrachloroaurate ( iii ) dihydrate , bis ( p - sulfonatophenyl ) phenyl phosphine dihydrate dipotassium salt B-material ( bspp ) , trisodium citrate and phosphate buffered saline ( pbs ) [SEP]
[CLS] materialswere purchased from sigma aldrich . [SEP]
[CLS] minimum essential media ( mem ) , pen / strep , l glutamine , trypsin , nystatin , fetal bovine serum ( fbs ) , and hbss for cell B-material culturing were purchased from invitrogen . [SEP]
[CLS] oligonucleotides , including their modifications , were obtained from atd bio . [SEP]
[CLS] for the synthesis of larger particles ( > 20 nm ) a kinetically controlled seeded growth strategy was followed developed by puntes et al . [SEP]
[CLS] this method allows for the synthesis of monodisperse citrate - stabilized aunps B-nanoparticle with a quasi - spherical shape and a narrow size distribution . [SEP]
[CLS] in detail , the au seed solution was firstly prepared by heating a solution of sodium B-material citrate in milli - q water B-material to boiling conditions ( 150 ml , 2 . 2 mm ) under vigorous stirring . [SEP]
[CLS] a three - necked round - bottomed flask ( 250 ml ) was used , which was fitted with a condenser to avoid solvent evaporation . [SEP]
[CLS] once boiling had commenced , a solution of naaucl 4 ( 25 mm , 1 ml ) was quickly injected . [SEP]
[CLS] after boiling for a further 10 min the color of the solution had changed from yellow to bluish grey to light pink . [SEP]
[CLS] immediately after , the temperature of the solution was cooled to 90 °c and a naaucl 4 solution ( 1 ml , 25 mm ) was injected . [SEP]
[CLS] after 30 min one more addition of a gold B-material solution ( 1 ml , 25 mm ) was performed to form the initial generation of particles ( g0 ) . [SEP]
[CLS] then , the sample was diluted by extracting 55 ml of sample and adding 53 ml of milli - q water B-material and sodium B-material citrate solution ( 2 ml , 60 mm ) . [SEP]
[CLS] this solution was used as a seed solution , which was injected with three consecutive gold B-material solution additions every 30 min at 90 °c to generate the next generation of particles ( g1 ) . [SEP]
[CLS] after synthesis was complete , citrate - capped gold B-material nanospheres B-nanoparticle were purified by two rounds of centrifugation ( 6 , 000 rpm , 20 min ) , decantation and re - dispersion in milli - q water B-material . [SEP]
[CLS] then , larger aggregates were removed by filtration ( 0 . 2 µm , vwr ) . [SEP]
[CLS] sample was stored at 4 °c prior to further functionalization [SEP]
[CLS] oligonucleotide attachment to larger spherical aunps B-nanoparticle was successfully achieved using a ph - assisted protocol . [SEP]
[CLS] this method allows for the instantaneous dna adsorption onto the gold B-material surface via the use of a ph 3 citrate buffer . [SEP]
[CLS] briefly citrate capped 43 ± 4 nm spherical aunps B-nanoparticle ( 1 . 35 ml , 10 pmol ) were mixed with a 3000 x excess of thiol terminated oligonucleotides ( 200 . 88 μl , 30 nmol ) and vigorously stirred for 1 min . [SEP]
[CLS] this was followed by the addition of a ph 3 citrate buffer ( 30 μl , 500 mm ) . [SEP]
[CLS] after 3 min , the na + ion B-material concentration was increased via the addition of a nacl solution ( 225 μl , 2 m ) to reach a final concentration of 0 . 3 m within the final solution . [SEP]
[CLS] the solution was left to stir for 1 min before three rounds of centrifugation ( 12 , 000 rpm , 15 min ) , decantation and redispersion in pbs . [SEP]
[CLS] the purified solution was stored at 4 °c prior to their use in further applications . [SEP]
[CLS] for flare hybridization , the oligonucleotide flare strands ( 150 . 2 µl , 15 nmol ) were incubated B-technique with purified spherical aunps B-nanoparticle . [SEP]
[CLS] the solution was heated to 60 °c and allowed to slowly cool to room temperature followed by purification via three rounds of centrifugation ( 16 , 400 rpm , 20 min ) and redispersion in pbs . [SEP]
[CLS] solution was stored at 4 °c . [SEP]
[CLS] human fetal lung fibroblast ( mrc 5 ) cells B-material were grown on a coverslip in mem until 90 % confluent . [SEP]
[CLS] once this confluency was reached , media was removed and exchanged with fresh media containing dnacoated aunps B-nanoparticle ( 55 µl , 2 pmol ) . [SEP]
[CLS] cells B-material were then incubated B-technique with the solution at 37 °c for 18 h . [SEP]
[CLS] media was removed from confluent cells B-material , which were washed with pbs ( 3 × , 5 min ) . [SEP]
[CLS] after washing , cells B-material were rinsed with 4 % paraformaldehyde ( pfa ) in piperazine - n , n ' - bis ( 2 - ethanesulfonic acid ) ( pipes ) and left for 1 h at 37 °c . [SEP]
[CLS] cells B-material were then washed with pbs ( 3 × , 5 min ) followed by an incubation B-technique step with 4 ' , 6 - diamidino - 2 - phenylindole ( dapi ) ( 1 / 500 ) in pbs for 15 min . [SEP]
[CLS] coverslips were then mounted with mowiol glycerol onto glass slides . [SEP]
[CLS] in order to accommodate both size and dispersity requirements , larger aunps B-nanoparticle were synthesized according to a protocol developed by the puntes group ( see section 2 . 2 ) . [SEP]
[CLS] this synthesis involves the use of spherical aunps B-nanoparticle as seeds , which are gradually grown by further additions of both the gold B-material precursor and citrate . [SEP]
[CLS] after every three additions half the sample was extracted ( termed generation n ) for further analysis whereas the other half was further grown into larger size aunps B-nanoparticle . [SEP]
[CLS] in this case aunps B-nanoparticle of generation 1 ( g1 ) were synthesized ( ~ 40 nm in size ) , analyzed and used in further applications . [SEP]
[CLS] citrate ligands were replaced with bspp to increase aunp B-nanoparticle stability and allow for concentration via centrifugation . [SEP]
[CLS] a representative tem image and size distribution analysis is presented in figure 1 . [SEP]
[CLS] particles were found to have a narrow size distribution . [SEP]
[CLS] the mean size was determined to be 43 . 5 nm with a standard deviation of 4 . 3 nm ( counting ~ 500 nps B-nanoparticle using image j software ) . [SEP]
[CLS] the increase in np B-nanoparticle size was also assessed by uv - vis spectroscopy B-technique as seen in figure 2 . [SEP]
[CLS] increasing aunp B-nanoparticle size is known to cause a red shift in the lspr band of aunps B-nanoparticle . [SEP]
[CLS] in this case the plasmon resonance was found to be 528 nm for 43 ± 4 nm bspp coated aunps B-nanoparticle . [SEP]
[CLS] this shift , which is in good agreement with previously published results , is attributed to the effect of electromagnetic retardation in larger nanoparticles B-nanoparticle as well as to contributions from multipolar terms where higher order oscillations start to take important roles . [SEP]
[CLS] however , an increase in aunp B-nanoparticle size also causes a change in the extinction spectrum . [SEP]
[CLS] this can be explained by an increase in the sphere volume as well as an increase in the relative contribution of scattering to the total extinction ( c sca / c abs ) whereas for smaller size spheres ( < 20 nm ) the total extinction is nearly all contributed by absorption . [SEP]
[CLS] these trends therefore suggest that larger nps B-nanoparticle ( > 40 nm ) could be suited for imaging applications . [SEP]
[CLS] for the functionalization of larger spherical aunps B-nanoparticle ( > 40 nm ) salt - ageing protocols have been reported . [SEP]
[CLS] nevertheless , it has been found that the oligonucleotide density decreases as the aunp B-nanoparticle size increases . [SEP]
[CLS] this is thought to be due to the surface curvature of spherical aunps B-nanoparticle , which significantly affects the loading of oligonucleotides . [SEP]
[CLS] oligonucleotide strands are found closer together thus intensifying inter - strand repulsion . [SEP]
[CLS] therefore , the salt B-material - ageing procedure has been stated to not work as efficiently for achieving a high density of oligonucleotides on the surface of larger spherical aunps B-nanoparticle . [SEP]
[CLS] in order to overcome the limitations associated with the salt - ageing process , a low ph assisted method was employed for the functionalization of larger spherical aunps B-nanoparticle ( see section 2 . 3 for experimental procedure ) . [SEP]
[CLS] this involves the use of a ph 3 citrate buffer , which can lead to efficient dna adsorption even at low salt B-material concentrations . [SEP]
[CLS] the role of the citrate buffer is to overcome the kinetic barrier B-property of dna attachment , which is more pronounced for larger aunps B-nanoparticle . [SEP]
[CLS] this occurs via the protonation of bases namely adenine and cytosine at ph 3 . [SEP]
[CLS] these protonated bases reduce the repulsion between the oligonucleotides and aunps B-nanoparticle but most importantly between oligonucleotide strands on a aunps B-nanoparticle surface . [SEP]
[CLS] in this study we employed the ph method for the surface functionalization of 43 nm spherical aunps B-nanoparticle and uv - vis spectroscopy B-technique was employed to qualitatively assess the oligonucleotide attachment to the np B-nanoparticle surface . [SEP]
[CLS] by examining the uv - vis spectra of both coated and citrate stabilized aunps B-nanoparticle , a red shift of 4 nm in the spr peak was observed ( see figure 3 a ) . [SEP]
[CLS] the absence of a second peak at a longer wavelength also indicates that no aggregation took place during the attachment procedure . [SEP]
[CLS] successful oligonucleotide coating B-material was also assessed by dispersing B-property particles in pbs and comparing it against citrate stabilized spherical aunps B-nanoparticle ( see figure 3 b ) . [SEP]
[CLS] whereas the citrate stabilized nanoparticle B-nanoparticle solution immediately changed color to dark blue indicating aunp B-nanoparticle aggregation due to weak citrate electrostatic stabilization , the oligonucleotide modified spherical aunps B-nanoparticle retained their dark red color . [SEP]
[CLS] 43 ± 4 nm aunps B-nanoparticle were coated with a shell B-material of sensing oligonucleotides comprised of a polyt sequence ( see section 2 . 3 for protocol of dna attachment ) designed to detect all mature cellular mrna via their distinct polya tail ( see table 1 for detailed oligonucleotide sequence ) . [SEP]
[CLS] sense and flare oligonucleotide sequences . [SEP]
[CLS] x : thiol B-material modifier 6 s - s ( cpg resin from glen research ) dnacoated aunps B-nanoparticle were further incubated B-technique with mrc 5 cells B-material and fixed 18 h post incubation ( see section 2 . 4 and 2 . 5 for experimental details ) in order to preserve and stabilize the cell B-material morphology and allow for continuous imaging over a prolonged period of time without the need of special imaging conditions ( temperature of 37 °c and continuous supply of co 2 ) . [SEP]
[CLS] prior to tppl imaging , the sample was imaged via confocal microscopy B-technique to ensure that a fluorescence B-property signal due to mrna sensing could be detectable . [SEP]
[CLS] a representative confocal image is shown below in as can be seen in figure 4 a fluorescence B-property signal corresponding to detection of all cellular mrna was detected ( green color ) . [SEP]
[CLS] the absence of a fluorescence B-property signal from the dye modified sense strand suggested that detection was specific and not due to degradation of surface bound oligonucleotides . [SEP]
[CLS] after verifying the suitability of the sample , this was further imaged on the setup designed for tppl and fluorescence B-technique imaging I-technique . [SEP]
[CLS] tppl and fluorescence B-property spectra were acquired on an improved home built setup first presented in figure 5 . [SEP]
[CLS] the light source for tppl was a fianium laser ( 10 ps , 20 mhz ) at a wavelength of 1060 nm whereas for fluorescence B-property measurements a laser source of 532 nm or 405 nm were used for cy3 ( release of flare ) and dapi ( nuclear counterstain to image cell B-material nucleus ) respectively . [SEP]
[CLS] the laser beam was guided through a set of mirrors and lenses to a 60 × objective lens . [SEP]
[CLS] emission signal was guided back down through the objective and finally filtered by a 430 nm long pass ( lp ) and 500 nm short pass ( sp ) filter ( dapi ) or a 550 nm lp filter ( cy3 ) or a 800 nm sp ( ttpl ) . [SEP]
[CLS] depending on the type of measurement the filter was changed accordingly . [SEP]
[CLS] for clarity a schematic diagram of the setup showing how the laser beam was guided from the laser source , to the sample and back to the detector is shown below in figure 5 . [SEP]
[CLS] the setup was used to further image tppl and fluorescence B-property corresponding to both cy3 and dapi sequentially by using the appropriate laser source and adding the correct filter to the rotating filter wheel as indicated in figure 6 . [SEP]
[CLS] using the correct imaging settings the following spectra were acquired for dapi , cy3 and tppl . [SEP]
[CLS] as can be seen from figure 6 fluorescence B-property from the cell B-material nucleus corresponding to a dapi signal was clearly imaged . [SEP]
[CLS] the fluorescence B-property signal showed the characteristic spherical shape of the cell B-material nucleus , which corresponds well with the nuclear shape also imaged on the confocal ( figure 4 ) . [SEP]
[CLS] this is a strong indication that imaging of the fluorescence B-property signal corresponding to dapi was successful . [SEP]
[CLS] moreover , the cy3 fluorescence B-property appeared to be located throughout the cell B-material with a stronger intensity being observed at certain sites rather than others . [SEP]
[CLS] on the other hand a uniform tppl signal was not imaged throughout the cell B-material with a strong tppl signal imaged at one position within the cell B-material , which could correlate to clusters of nanoparticles B-nanoparticle located within multivesicular bodies or endosomes ( either early or late ) . [SEP]
[CLS] however , weaker tppl signals that may correspond to individual nanoparticles B-nanoparticle were also imaged . [SEP]
[CLS] these were located both within the cell B-material as well as extracellularly perhaps from aunps B-nanoparticle that have been exocytosed or were about to be taken up . [SEP]
[CLS] as the cy3 signal was diffused throughout the cell B-material upon mrna detection , co - localisation studies were not possible . [SEP]
[CLS] for clarity , images presented in figure 6 were also overlapped ( by taking images from one z plane ) as shown below in figure 7 . [SEP]
[CLS] apart from imaging in the x and y direction creating a 2d image , as seen in figure 7 , an image at the same position was also acquired in the z direction creating 3d images of dapi and cy3 fluorescence B-property as well as tppl as seen in figure 8 . [SEP]
[CLS] once more it can be seen that although the nucleus is well defined , the fluorescence B-property signal corresponding to flare release is diffused and observed throughout the cell B-material . [SEP]
[CLS] on the other hand , a tppl signal when analyzed in each direction did not appear to be well - dispersed throughout the cell B-material which is a strong indication that flare release is imaged within the cytoplasm leading to an almost uniform fluorescence B-property signal whereas the aunps B-nanoparticle are most likely located within endosomes leading to a very bright aggregated tppl signal with a weaker signal being imaged in areas , which may correspond to single particles located within the cytoplasm following endosomal escape [SEP]
[CLS] 1 . ( a ) tem image of 43 ± 4 nm spherical aunps B-nanoparticle . [SEP]
[CLS] ( b ) histogram showing the size distribution of spherical aunps B-nanoparticle . [SEP]
[CLS] scale bar is 100 nm . [SEP]
[CLS] 2 . uv - vis spectra of bspp coated 43 ± 4 nm spherical aunps B-nanoparticle . [SEP]
[CLS] ( a ) uv - vis spectra of oligonucleotide coated and non - coated 43 ± 4 nm spherical aunps B-nanoparticle . [SEP]
[CLS] ( b ) digital image of non - coated ( left eppendorf ) and oligonucleotide coated ( right eppendorf ) aunps B-nanoparticle in pbs . [SEP]
[CLS] 4 . confocal microscopy B-technique image of fixed mrc 5 cells B-material incubated B-technique with 43 ± 4 nm dnacoated aunps B-nanoparticle designed to detect all mature cellular mrna ( gmrna ) . [SEP]
[CLS] colour guide : greenflare release , blue - fam ( dye on sense strand ) , whitenuclear counterstain . [SEP]
[CLS] scale bar is 15 µm [SEP]
[CLS] 5 . schematic illustration of the microscopy B-technique setup used for both tppl and fluorescence B-technique imaging I-technique using the mrc 5 cell B-material line . [SEP]
[CLS] 6 . acquired images showing fluorescence B-property from the cell B-material nucleus ( dapi ) and from flare release ( cy3 ) as well as tppl from the aunps B-nanoparticle . [SEP]
[CLS] scale bars are 15 µm [SEP]
[CLS] overlayed images dapi and cy3 fluorescence B-property as well as tppl from the aunp B-nanoparticle core B-material . [SEP]
[CLS] a clear diffuse cy3 signal was imaged throughout the cell B-material ( green ) , which at points does not co - localize with any tppl signal ( red ) and is indicative of cytoplasmic dye diffusion . [SEP]
[CLS] on the other hand , bright tppl signals were imaged , which could be due to endosomal localisation . [SEP]
[CLS] furthermore , weak tppl signals were imaged extracellularly , which could be due to np B-nanoparticle exocytosis as imaging in the perimeter of another cell B-material as cy3 fluorescence B-property could also be observed at the bottom of the image . [SEP]
[CLS] scale bar is 15 µm [SEP]
[CLS] 8 . 3d images created by imaging in the z direction for dapi and cy3 fluorescence B-property as well as tppl from the aunp B-nanoparticle core B-material . [SEP]
[CLS] images were analyzed and presented as a top view , long side and short side view for each scan . [SEP]
[CLS] human bone marrow ( bm ) derived stromal cells B-material contain a population of skeletal stem cells B-material ( sscs ) , with the capacity to differentiate along the osteogenic , adipogenic and chondrogenic lineages enabling their application to clinical therapies . [SEP]
[CLS] however , current methods , to isolate and enrich sscs from human tissues remain , at best , challenging in the absence of a specific ssc marker . [SEP]
[CLS] unfortunately , none of the current proposed markers , alone , can isolate a homogenous cell B-material population with the ability to form bone , cartilage , and adipose tissue in humans . [SEP]
[CLS] here , we have designed dna - gold nanoparticles B-nanoparticle able to identify and sort sscs displaying specific mrna signatures . [SEP]
[CLS] the current approach demonstrates the significant enrichment attained in the isolation of sscs , with potential therein to enhance our understanding of bone cell B-material biology and translational applications . [SEP]
[CLS] in the last decade , progress in colloid chemistry has enabled the synthesis of advanced , functional nanoparticles B-nanoparticle ( nps B-nanoparticle ) that can survive complex biological media and perform programmed tasks including cell B-material targeting , sensing , and drug release . [SEP]
[CLS] mirkin and co - workers demonstrated how the surface of gold B-material nps B-nanoparticle could be functionalised with a shell B-material of synthetic oligonucleotides attached to the gold B-material surface via a thiol B-material bond . [SEP]
[CLS] this design of oligonucleotidecoated nps B-nanoparticle , also termed spherical nucleic B-material acids I-material ( snas ) have subsequently emerged as live cell probes for the detection of targets including rna , molecules and ions B-material . [SEP]
[CLS] the extensive use of snas within the biomedical field is primarily due to their high specificity , binding of complementary oligonucleotide strands and improved resistance towards dna enzymatic degradation as well as the enhanced uptake of snas by multiple types of cells B-material . [SEP]
[CLS] seferos et al . showed how snas could be hybridised to shorter fluorophore bearing oligonucleotides for the targeted detection of mrna . the authors demonstrated the detection of survivin mrna , where a significant increase in fluorescence B-property intensity was recorded compared to treatment with nontargeting nps B-nanoparticle . [SEP]
[CLS] following this work , several studies have demonstrated the use of snas for the detection of mrna targets with high accuracy and specificity . [SEP]
[CLS] lahm et al . demonstrated the use of snas for the detection of nanog and gdf3 mrna in embryonic stem cells B-material as well as induced pluripotent stem ( ips ) cells B-material of murine , porcine and human origin . [SEP]
[CLS] target positive cells B-material were sorted after which nanogspecific probes identified reprogrammed murine ips cells B-material . [SEP]
[CLS] the detection of nanog mrna was also taken advantage of by owen et al . for the detection and isolation of cancer stem cells B-material . [SEP]
[CLS] in contrast , wang et al . developed snas to enable selfactivating and imaging of mrna sequential expression ( tubb3 and fox 3 mrna ) during the neural stem cell B-material differentiation process . [SEP]
[CLS] recently , we have demonstrated that snas can be used to detect vimentin mrna in skin wounds as vimentin mrna is expressed during the epithelial to mesenchymal transition that occurs during wound healing . [SEP]
[CLS] in addition , we have designed snas to detect hymyc1 mrna , responsible for the regulation of the balance between stem cell B-material selfrenewal and differentiation , in real time in live hydra vulgaris . [SEP]
[CLS] we have also shown the development of snas and dimer snas , which can detect up to two different mrna targets whilst coordinating the release of two different types of drugs within the same local microenvironment . [SEP]
[CLS] recently , the sna design has evolved to include mrna detection directed by an external stimulus . [SEP]
[CLS] lin et al . demonstrated the use of such a strategy for the specific detection of manganese B-material superoxide dismutase ( mnsod ) mrna . [SEP]
[CLS] following application of oligonucleotides modified with a photoactivated linker , mrna detection was present only in select cells B-material at a desired time point via the use of two - photon illumination . [SEP]
[CLS] by illuminating specific cells B-material of interest , mrna detection was visualized only in these specific cells B-material , while neighboring cells B-material remained fluorescently B-property dormant . [SEP]
[CLS] the design of snas has been further exploited for the detection of other targets including mirnas and , recently , for the delivery of oligonucleotides or peptides B-material to activate the immune system . [SEP]
[CLS] zhai et al . reported the detection of microrna - 1246 , a breast cancer biomarker B-property , in human plasma exosomes , whereas , zhao et al . used the same design for the direct detection of microrna - 375 . [SEP]
[CLS] in contrast , ferrer et al . developed a dual targeting sna that could coordinate the delivery of a nucleic B-material acid I-material specific for toll like receptor B-material 9 ( tlr9 ) inhibition and a small molecule ( tak - 242 ) that inhibited tlr4 ; with the goal of attenuating inflammation by downregulating pro - inflammatory markers downstream of each receptor B-material . [SEP]
[CLS] undoubtedly , snas have emerged as a versatile tool , with significant potential within the biomedical field . [SEP]
[CLS] an area of research that has come to the fore in recent years is the application of stem cells B-material in regenerative medicine . [SEP]
[CLS] the ready accessibility of human bone marrow stromal cells B-material ( bmscs ) from bone marrow and their ability to differentiate into bone - forming osteoblasts when implanted in vivo , has garnered significant scientific interest and driven the application of sscs in the clinic . [SEP]
[CLS] bone marrow ( bm ) is the soft connective tissue within bone cavities that functions to produce blood cells B-material and as a store for fat . [SEP]
[CLS] traditionally bm has been seen as an organ composed of two main systems : the hematopoietic tissue and the supportive stroma . [SEP]
[CLS] hematopoietic stem cells B-material ( hscs ) sustain the generation of all blood cell B-material types and are thus invaluable for the treatment of hematopoietic disorders . [SEP]
[CLS] in contrast , the bm stroma contains mesenchymal stem / stromal cells B-material ( mscs ) . [SEP]
[CLS] these are defined as unspecialized cells B-material that lack tissuespecific characteristics and maintain an undefined phenotype . [SEP]
[CLS] the term msc was originally coined in reference to a hypothetical common progenitor of a wide range of " mesenchymal " ( non - hematopoietic , nonepithelial , mesodermal ) tissues . [SEP]
[CLS] the term represents a heterogeneous cell B-material population when cultured in vitro and are likely to include cells B-material with a wide spectrum of regenerative potential from specific terminal cell type progenitors to multipotent stem cells B-material , named skeletal stem cells ( sscs ) . [SEP]
[CLS] the ssc is used here to refer specifically to the self - renewing stem cell B-material of the bone marrow stroma responsible for the regenerative capacity inherent to bone with osteogenic , adipogenic and chondrogenic differentiation potential in vivo . [SEP]
[CLS] however , a number of challenges limit ssc application including the limited numbers of sscs in bm aspirates ( 1 in 10 - 100 , 000 ) , and the absence of specific markers limiting facile homogenous isolation of the ssc from human tissue . [SEP]
[CLS] thus , there remains limited understanding of ssc fate , immunophenotype and simple selection criteria ; all of which have proven to be limiting factors in the widespread clinical application of these cells B-material . [SEP]
[CLS] current techniques employed for ssc sorting are based on fluorescence B-property and magnetic activated cell B-material sorting ( respectively named facs and macs ) combined with plastic culture adherence . [SEP]
[CLS] however , facs and macs depend on the use of antibodies B-material to target antigens present on the cell B-material membrane , in the cytoplasm or the nucleus , with detection of internal antigens requiring the cells B-material to be fixed and therefore unable to be cultured further . [SEP]
[CLS] cells B-material separated via facs depend on the absence or presence of a fluorescence B-property tag , whereas , macs uses magnetic B-property beads attached to a primary antibody B-material . [SEP]
[CLS] tagged cells B-material are thus retained in the device by a strong magnetic B-property field . [SEP]
[CLS] the use of both techniques is hindered by the cost and time of sample processing , although , the single largest hurdle remains the lack of specific cell B-material membrane markers for sscs . [SEP]
[CLS] widely used markers , such as stro - 1 , interact with only a small percentage of bm cells B-material that contain a subpopulation that includes the ssc / progenitor population . [SEP]
[CLS] seminal work by shi and gronthos reported a 2 , 000 - fold enrichment of sscs following a combinatorial approached that used macs to sort stro - 1 + cells B-material followed by dual - colour facs to sort stro - 1 bright and cd146 + cells B-material . [SEP]
[CLS] it should be noted that the use of both facs and macs based on surface antigens make the process prone to cell B-material damage and loss , highly time - consuming , and effortful , thus impractical in a clinical setting . [SEP]
[CLS] although a range of cell B-material surface markers can enrich for sscs , none of the proposed markers , in isolation , can isolate single cells B-material with the ability to form bone , cartilage and adipose tissue in humans and a specific marker for the ssc , to date , remains elusive . [SEP]
[CLS] in this study , we take advantage of the versatile nature of snas not only to detect sscs based on mrna expression but also to rapidly isolate sscs from human bm ; a critical requirement for tissue engineering strategies harnessing sscs to aid bone regeneration . [SEP]
[CLS] as opposed to the detection of antigens present on the cells B-material ' membrane , snas detect endocellular mrna targets . [SEP]
[CLS] bm cells B-material expressing specific mrna targets were isolated via facs and subsequently used to isolate sscs , without affecting long - term skeletal cell B-property viability I-property . [SEP]
[CLS] despite the limited ssc numbers present in bm aspirate , the current study demonstrates the advantage of sna specificity for mrna detection and enrichment to 1 in 200 sscs isolated from bm aspirates . [SEP]
[CLS] this corresponds to a 50 - 500 fold enrichment , which together with the speed and simplicity afforded of such a strategy , demonstrates the potential of this method for broader applications . [SEP]
[CLS] 1 shows a schematic illustration of the experimental approach . [SEP]
[CLS] cells B-material isolated from human bmscs directly taken from patients were incubated B-technique with snas for the detection of specific mrnas . [SEP]
[CLS] for this study two mrna targets were chosen , runx2 and hspa8 . [SEP]
[CLS] runx2 is a bone specific transcription B-event factor critical in osteogenic differentiation and bone formation . [SEP]
[CLS] runx2 promotes the expression of osteogenesis related genes , regulates cell B-material cycle progression and can improve the bone microenvironment . [SEP]
[CLS] in contrast , hspa8 was selected as a target since the hspa8 gene encodes for the stro - 1 antigen . [SEP]
[CLS] stro - 1 is expressed on the surface of skeletal stem and progenitor populations and has been routinely used as a marker for ssc analysis and ssc sorting . [SEP]
[CLS] stro - 1 has shown to provide enrichment specificity for sscs , as stro - 1 positive cell B-material populations display enhanced osteogenic differentiation both in vitro and in vivo . [SEP]
[CLS] a scrambled control sequence designed to not detect any cellular mrna was used to demonstrate the specificity of our snas towards the targeted detection of mrna . [SEP]
[CLS] following a 1 h incubation B-technique period of snas with bmscs , facs was used to separate target positive from target negative cells B-material based on their fluorescence B-property signature . [SEP]
[CLS] target positive cells B-material were subsequently plated and stained to determine the number of colonies formed . [SEP]
[CLS] sscs have the ability to form colonies , termed colony forming units fibroblastic cells B-material ( cfu - f ) , when plated and cultured at limiting dilutions . [SEP]
[CLS] the cfu - f assay harnesses the ability to identify sscs and progenitor cells B-material , as each colony is derived from a single stem / early progenitor cell B-material . [SEP]
[CLS] sscs display upon cfu - f formation , a fibroblastic appearance when isolated and cultured . [SEP]
[CLS] 2 demonstrates the design of snas for mrna detection . [SEP]
[CLS] spherical aunps B-nanoparticle with a size of ~ 13 nm were functionalized with synthetic oligonucleotides bearing a terminal 3 ' thiol modification and a terminal 5 ' fam dye ( for simplicity the oligonucleotides directly attached to the aunp B-nanoparticle surface are termed ' sense ' strands ) . [SEP]
[CLS] sense strands were partially hybridized to shorter oligonucleotide complements modified with a cy5 dye at their 5 ′ end ( termed ' flare ' strands ) . [SEP]
[CLS] critically , the sequence of the sense strand was designed to bind only the mrna of interest . [SEP]
[CLS] when both the sense and flare oligonucleotide were bound to the aunp B-nanoparticle core B-material , the oligonucleotide dyes were quenched ( off state ) , given their close proximity to the aunp B-nanoparticle surface . [SEP]
[CLS] however , once the target mrna bound to the corresponding sense sequence , the resulting displacement of the flare was detected as an increase in fluorescence B-property at the specific wavelength of the cy5 fluorophore ( on state ) . [SEP]
[CLS] the fluorophore on the flare strand acted as a reporter , while the fam dye on the sense strand was introduced to monitor the integrity of the sense strand , with the fam dye released , and detectable , only if the sense strand was degraded by nucleases or detached from the nanoparticle B-nanoparticle surface . [SEP]
[CLS] in this study , the synthesis of snas was customized for the detection of bone cell B-material mrnas ( see table s1 for oligonucleotide sequences ) . [SEP]
[CLS] by adopting a gradual salt aging approach ~ 13 nm au nps B-nanoparticle ( see figure s1 ) were functionalized with a shell B-material of synthetic oligonucleotides . [SEP]
[CLS] a shift of 4 nm was observed at the visible spectrum of the nps B-nanoparticle indicating the change of the refractive B-property index I-property following attachment of the oligonucleotides ( see figure s2 ) whilst ζpotential measurements showed that the oligonucleotide coated nanoparticles B-nanoparticle had a strong net negative charge ( see figure s3 ) . [SEP]
[CLS] through the dissolution of the au np B-nanoparticle core B-material using a solution of ki / i2 and the quantitative analysis of the oligonucleotides in solution , it was determined that each au np B-nanoparticle was coated with approximately 110 ± 4 oligonucleotide strands with no significant variation between oligonucleotides of varying sequences ( see table s2 ) . [SEP]
[CLS] respectively , each spherical nucleic B-material acid I-material for the detection of mrna consisted of approximately × 60 flare strands as shown in table s2 . [SEP]
[CLS] binding studies were also performed in a tube using perfectly matched synthetic oligonucleotide targets for each sna ( see figure s5 ) together with stability tests , where the resistance towards nucleases was determined ( see figure s6 , s7 and s8 ) . [SEP]
[CLS] snas for the detection of runx2 and hspa8 mrna were incubated B-technique with human bmscs for 1 , 2 or 3 h . [SEP]
[CLS] following determination of the appropriate settings for facs separation ( see figure s9 ) , the fluorescence B-property signal corresponding to mrna detection and the subsequent flare release was monitored by observing an increase in cy5 fluorescence B-property in comparison to cells B-material incubated B-technique with no snas . [SEP]
[CLS] 1 shows that following addition of runx2 and hspa8 snas , cy5 positive cells B-material could be detected within 1 h of incubation B-technique in the different sub - populations of bm cells B-material including lymphocytes , granulocytes and monocytes , while the fluorescence B-property intensity was observed to increase with longer incubation B-technique times ( 2 - 3 h ) . [SEP]
[CLS] moreover , the absence of a signal from the fam dye conjugated to the sense strand indicated the absence of sense strand degradation ( figure s10 ) . [SEP]
[CLS] in both cases , a scrambled sna designed to not detect any intracellular mrna was also employed as a control . [SEP]
[CLS] the absence of both a cy5 and fam signal from the flare and sense strand respectively , after a 3 h incubation B-technique period , indicated not only strand stability against nuclease degradation but also the specificity of snas for the detection of mrna . [SEP]
[CLS] following cell B-material sorting of cy5 positive cells B-material for runx2 and hspa8 mrna , the recovered cells B-material were grown for 14 days then stained , and the number of colonies formed determined . [SEP]
[CLS] only cells B-material of monocyte size and granularity were collected by facs , as this gate has been shown to contain the larger fraction of sscs in a typical bm sample , whilst other cell B-material types were discarded . [SEP]
[CLS] 2a shows that isolated cells B-material produced varying levels of cfu - fs , with cells B-material sorted from snas designed for the detection of runx2 mrna producing a higher number of cell B-material colonies than hspa8 with significant enrichment observed in comparison to unsorted cells B-material . [SEP]
[CLS] in contrast , the fraction of non - fluorescent B-property cells B-material ( cy5 negative ) showed no / negligible cfu - f formation . [SEP]
[CLS] to determine if the cells B-material isolated were indeed sscs with tri - lineage potential and the capacity to generate the requisite stromal lineages , the collected cells B-material , isolated using the runx2 sna ( see figure s12 for experimental control ) , were grown and expanded and then subjected to different culture conditions to generate osteogenic , adipogenic and chondrogenic stromal populations . [SEP]
[CLS] 3 demonstrates that cells B-material maintained under osteogenic conditions produced enhanced positive staining for alkaline phosphatase ( see figure 3a ) compared to basal conditions . [SEP]
[CLS] alcian blue staining of the chondrogenic pellets revealed proteoglycan synthesis ( see figure 3b ) whereas sirius red stain retention was absent in pellets , indicating an absence of collagenous matrix formation . [SEP]
[CLS] oil - red - o staining of lipids B-material provided evidence of adipogenesis and lipid B-material droplet formation ( see figure 3c ) , with no lipid B-material visible in basal conditions . [SEP]
[CLS] the results demonstrate that the runx2 sna - isolated cells B-material displayed tri - lineage potential and generated stromal populations with osteogenic , adipogenic and chondrogenic potential thus indicating that the isolated cells B-material contained sscs . [SEP]
[CLS] the current study demonstrates the development of snas for the detection of specific mrna targets , runx2 and hspa8 , in human bm stromal populations , which enables the isolation and enrichment of human sscs . [SEP]
[CLS] following a short incubation B-technique period with runx2 and hspa8 snas with human bmscs , fluorescent B-property cells B-material were sorted using facs . [SEP]
[CLS] further plating of isolated mrna target positive cells B-material resulted in a significant enrichment in cfu - f formation . [SEP]
[CLS] using this technique , we demonstrate that despite the low abundance of sscs in human bm , up to 1 in 200 of the cy5 positive cells B-material isolated by facs formed colonies with high cfu - f levels on culture . [SEP]
[CLS] in conclusion , the current study demonstrates the potential of snas for the isolation and enrichment of skeletal stem cell B-material populations with exciting opportunities therein for translational application , one of our future research goals . [SEP]
[CLS] briefly bis ( p - sulfonatophenyl ) phenylphosphine dihydrate dipotassium salt B-material ( bspp ) coated 13 nm aunps B-nanoparticle ( 10 nm , 1 ml ) synthesised using the turkevich method were incubated B-technique with thiolmodified synthetic oligonucleotides ( 3 µm , 1 ml ) ( see table s1 for oligonucleotide sequences ) . [SEP]
[CLS] after the mixture was left shaking for 24 h , bspp ( 1mg / 20 µl , 10 µl ) , phosphate buffer ( 0 . 1 m , ph 7 . 4 ) and sodium B-material dodecyl sulphate ( sds , 10 % solution ) were added to achieve a final concentration of 0 . 01 m phosphate and 1 % sds respectively . [SEP]
[CLS] a gradual salt B-material aging was then performed by six equal additions of nacl ( 2 m ) over 8 h in order to achieve a final salt B-material concentration of 0 . 3 m in solution . [SEP]
[CLS] after shaking overnight , the resulting snas were purified by three rounds of centrifugation ( 16 , 400 rpm , 20 min ) and were stored at 4 °c in phosphate buffer saline ( pbs ) . [SEP]
[CLS] for flare hybridization , snas ( 16 nm , 500 µl ) were mixed with an excess of complementary flare oligonucleotides ( 960 nm , 500 µl ) . [SEP]
[CLS] the solution was heated to 60 °c for 5 min , followed by slow cooling to room temperature . [SEP]
[CLS] samples were purified by two rounds of centrifugation ( 16 , 400 rpm , 15 min ) and redispersed in pbs . [SEP]
[CLS] the snas were added to the bmsc suspension ( at 10 6 cells B-material per ml ) to the required final concentration of 0 . 2 nm and for the appropriate incubation B-technique time . [SEP]
[CLS] cells B-material were subsequently washed in basal medium and then resuspended in facs solution ( 0 . 5 % bsa , 2mm edta in 1x pbs ) [SEP]
[CLS] prior to facs analysis [SEP]
[CLS] the facs aria cytometer ( becton dickinson , wokingham , uk ) was used to acquire 20000 cells B-material with data analysed using the flowjo software version 10 . 6 . 1 . [SEP]
[CLS] all washing stages were at 400 x g for 5 minutes . [SEP]
[CLS] bone marrow samples were obtained from haematologically healthy patients undergoing hip replacement surgery with local ethics committee approval ( lrec194 / 99 / 1 and 18 / nw / 0231 and 210 / 01 ) and informed patient consent . [SEP]
[CLS] in brief , bone marrow was first washed at least 3 times in 50 ml α - mem medium to remove fat , then passed through a 70 μm cell B-material strainer . [SEP]
[CLS] marrow cells B-material were resuspended in 10 ml α - mem and subjected to density centrifugation using 20 ml [UNK] ( lonza ) at 800 x g for 20 minutes with no braking on the centrifuge . [SEP]
[CLS] the buffy coat B-material layer , containing bone marrow mononuclear cells B-material was washed in 5 ml basal medium ( α - mem containing 10 % fbs and 100 u ml −1 penicillin and 100 µg ml −1 streptomycin ; lonza ) . [SEP]
[CLS] all washing stages were at 400 x g for 5 minutes . [SEP]
[CLS] a facs aria was used to collect sna positive and negative cells B-material from human bone marrow samples . [SEP]
[CLS] cells B-material were gated for monocytes , single cells B-material , and cy5 fluorescence B-property . [SEP]
[CLS] positive samples were deemed to be the top 15 % of cy5 fluorescent B-property cells B-material and negative samples were deemed to be the lower 15 % of cy5 cells B-material collected . [SEP]
[CLS] cy5 positive and negative cells B-material were collected by facs sorting . [SEP]
[CLS] ten thousand cells B-material were placed into each well of 6 - well tissue culture plates containing 2 ml basal medium . [SEP]
[CLS] cells B-material were grown for 14 days , with a medium change after 7 days . [SEP]
[CLS] on day 14 , wells were washed with 3 ml pbs and then fixed with 1 ml 95 % ethanol for 10 minutes . [SEP]
[CLS] the wells were subsequently air dried and 1 ml 0 . 05 % crystal violet solution added to each well for 1 minute . [SEP]
[CLS] the wells were then washed twice with 2 ml distilled B-material water I-material and the number of visible colonies determined by eye . [SEP]
[CLS] passage 1 cells B-material were cultured at 37 °c in 5 % co2 until confluent , then seeded at 10 , 000 cells B-material per well on a 12 well plate followed by culture in basal media for 24 hours . [SEP]
[CLS] cells B-material were then cultured in osteoinductive media ( basal medium with 50 µm ascorbic acid 2 - phosphate and 10 nm vitamin d3 ) for 14 days at 37 °c in 5 % co2 with media change every 3 - 4 days . [SEP]
[CLS] cells B-material were washed in pbs , fixed in 95 % etoh , then stained with alkaline phosphatase . [SEP]
[CLS] passage 1 cells B-material were cultured at 37 °c in 5 % co2 until confluent , then seeded at 10 , 000 cells B-material per well on a 12 well plate followed by culture in basal media until 80 % confluent , after approximately 3 - 5 days . [SEP]
[CLS] cells B-material were then cultured in adipogenic media ( basal medium with 100 nm dexamethasone , 500 µm ibmx , 3 µg / ml its solution , and 1 µm rosiglitazone ) for 14 days at 37 °c in 5 % co2 with media change every 3 - 4 days . [SEP]
[CLS] cells B-material were washed in pbs , fixed in 4 % paraformaldehyde , washed in pbs again and then stained with oil red o . [SEP]
[CLS] passage 2 cells B-material were cultured at 37 °c in 5 % co2 until confluent , then diluted to 500 , 000 cells B-material per ml in chondrogenic media ( α - mem containing 100 u ml −1 penicillin and 100 µg ml −1 streptomycin , 100 µm ascorbic acid 2 - phosphate , 10 ng / ml tgf - b3 , 10 µg / ml its solution , 10 nm dexamethasone ) in a universal container . [SEP]
[CLS] cells B-material were centrifuged at 400 x g for 10 minutes to form a cell B-material pellet , and all but 1 ml of media removed . [SEP]
[CLS] cells B-material were then cultured with the tube cap loose for 14 days at 37 °c in 5 % co2 with media change every 2 days . [SEP]
[CLS] cells B-material were washed in pbs , fixed in 95 % etoh , then stained with alcian blue and sirius red . [SEP]
[CLS] wilcoxon - mann - whitney statistical analysis and anova were performed where appropriate using the spss for windows program version 23 ( ibm corp , portsmouth , hampshire , uk ) . [SEP]
[CLS] all experiments were completed at least three times and data are presented as mean ± sd . [SEP]
[CLS] significance was determined with a plevel of 0 . 05 or lower . [SEP]
[CLS] schematic illustration of the isolation of skeletal stem cells B-material and the formation of their colonies . [SEP]
[CLS] primary human bone marrow stromal cells B-material in suspension were incubated B-technique with snas designed to detect hspa8 and runx2 mrna . [SEP]
[CLS] following facs separation , fluorescent B-property cells B-material were isolated and any sscs formed colonies . [SEP]
[CLS] schematic illustration of snas that detect mrna . [SEP]
[CLS] when both sense and flare strands are in close proximity to the aunp B-nanoparticle core B-material fluorescence B-property is quenched . [SEP]
[CLS] when the target mrna is present , competitive hybridization to the sense strand leads to flare displacement and the concomitant restoration of its fluorescence B-property signature , which is observed via facs . [SEP]
[CLS] snas for the detection of hspa8 and runx2 mrna including a scrambled sna were incubated B-technique with human bone marrow stromal cells B-material for 1 , 2 or 3 hours at 0 . 2 nm final concentration . [SEP]
[CLS] after washing , the cells B-material were analyzed by flow B-technique cytometry I-technique for cy5 fluorescence B-property intensity . [SEP]
[CLS] cy5 intensity versus cell B-material number overlay plots for the different snas for different cell B-material types within the bone marrow as determined by the facs selection criteria . [SEP]
[CLS] color guide : red - 3 hour incubation B-technique , orange - 2 hour incubation B-technique , light blue - 1 hour incubation B-technique , light greenscrambled control at 3 hour incubation B-technique , dark greenno snas at 3 hour incubation B-technique . [SEP]
[CLS] 2b and c shows that cells B-material isolated using the snas for the detection of hspa8 ( b ) and runx2 ( c ) mrna displayed a fibroblastic phenotype ( see figure s11 for confocal images of isolated sscs ) . [SEP]
[CLS] through the collection of different proportions of the brightest cy5 cells B-material ( 15 - 60 % ) with the two different snas , regardless of their overall intensity , it was demonstrated cfu - fs occurred in the top 15 % of brightest cells B-material for these two targets . [SEP]
[CLS] ( a ) graph shows cfu - f count for cells B-material sorted using hspa8 and runx2 snas . [SEP]
[CLS] the statistical analysis was run comparing the number of colonies formed from cells B-material sorted using the two types of snas , vs unsorted cells B-material , which were plated to give the equivalent of 10 , 000 cells B-material in the monocyte region per well . [SEP]
[CLS] graphs show means with sd from n = 3 patients for each sna . [SEP]
[CLS] * p < 0 . 05 , * * p < 0 . 01 . [SEP]
[CLS] ( b , c ) representative images of cells B-material within individual cfu - f [SEP]
[CLS] cells B-material isolated using the runx2 sna display capacity for tri - lineage stromal cell B-material differentiation . [SEP]
[CLS] isolated populations demonstrated ( a ) osteogenic , ( b ) chondrogenic and ( c ) adipogenic induction indicating the presence of sscs . [SEP]
[CLS] † school of engineering and ‡ school of natural sciences , university of california , merced , 5200 n lake rd . , merced , california , 95343 , united states [SEP]
[CLS] abstract : the spatial arrangement of target and probe molecules on the biosensor is a key aspect of the biointerface structure that ultimately determines the properties of interfacial molecular recognition and the performance of the biosensor . [SEP]
[CLS] however , the spatial patterns of single molecules on practical biosensors have been unknown , making it difficult to rationally engineer biosensors . [SEP]
[CLS] here we have used high resolution atomic B-technique force I-technique microscopy I-technique to map closely spaced individual probes as well as discrete hybridization events on a functioning electrochemical dna sensor surface . [SEP]
[CLS] we also applied spatial statistical methods to characterize the spatial patterns at the single molecule level . [SEP]
[CLS] we observed the emergence of heterogeneous spatiotemporal patterns of surface hybridization of hairpin probes . [SEP]
[CLS] the clustering of target capture suggests that hybridization may be enhanced by proximity of probes and targets that are about 10 nm away . [SEP]
[CLS] the unexpected enhancement was rationalized by the complex interplay between the nanoscale spatial organization of probe molecules , the conformational changes of the probe molecules , and target binding . [SEP]
[CLS] such molecular level knowledge may allow one to tailor the spatial patterns of the biosensor surfaces to improve the sensitivity and reproducibility . [SEP]
[CLS] biosensors typically consist of recognition elements immobilized on the surfaces of the transducer materials . [SEP]
[CLS] for example , the electrochemical dna biosensors , which hold significant potential in point - of - care diagnostics due to their abilities in highly selective , label - free , and miniaturized detection of a range of biomarkers B-property in complex biofluids , immobilize nucleic B-material acid I-material capture probes onto the electrode surfaces . [SEP]
[CLS] molecular recognition is influenced by the interactions between the probe and the biointerface , which consists of neighboring probe molecules and captured biomarkers B-property ( target molecules ) , the passivating layer , the solid substrate ( planar or nanostructured surfaces [SEP]
[CLS] ) , and the solution [SEP]
[CLS] an outstanding challenge is that the influences of the biointerface are complex and difficult to predict , hampering the effort to form biosensors with predictable performance . [SEP]
[CLS] extensive studies have explored the effects of probe design , probe surface density , surface chemistry , and surface morphology [SEP]
[CLS] however , one aspect that has remained poorly understood is how the nanoscale lateral organization of probe and captured target molecules influences molecular recognition . [SEP]
[CLS] the spatial organization is a key aspect of the biointerface structure that ultimately determines the complex interactions and the properties of interfacial molecular recognition . [SEP]
[CLS] the heterogeneous local density of probe molecules was thought to have difficult - to - predict influences on the accessibility of target molecules to the probe molecules ( crowding interactions ) . [SEP]
[CLS] numerous studies provided indirect evidence that the impacts of the poorly controlled , often heterogeneous spatial organization of probe molecules may be profound : they may not only limit detection sensitivity but also be the root cause of the large device - to - device signal variabilities of many of these surface - based sensors devices . [SEP]
[CLS] immobilization procedures that enhance probe dispersion were found to improve reproducibility in target binding . [SEP]
[CLS] moreover , the attachment of probes to nanoscale dna tetrahedra increased the target binding rate by orders of magnitude . [SEP]
[CLS] it was proposed that the footprint of the tetrahedral structures helps maintain uniform inter - probe separations and facilitate target binding . [SEP]
[CLS] however , a definitive correlation between nanoscale spatial patterns and interfacial molecular recognition had not been possible as existing techniques cannot resolve single molecules on biosensors . [SEP]
[CLS] on a practical biosensor surface with a probe density in the range of 10 10 - 10 13 / cm 2 , 7 , 28 many of the molecules are separated by less than 10 nm , which is beyond the resolving power of existing techniques . [SEP]
[CLS] although few techniques , such as fluorescence B-technique microscopy I-technique , atomic B-technique force I-technique microscopy I-technique and surface plasmon resonance have detected single recognition events , single molecule imaging was only achieved on surfaces with extremely dilute coverages . [SEP]
[CLS] hence , almost all existing studies relied on the overall surface densities as the key parameter to describe how surface immobilization impacts molecular recognition . [SEP]
[CLS] while such studies have revealed general trends suggesting that surface crowding by probe molecules and captured targets inhibits target recognition , these ensemble averaging observables are not adequate descriptors of the crowding interactions , especially in light of growing evidence that a realistic biosensor may be highly heterogeneous in probe density . [SEP]
[CLS] in addition , the difficulty is compounded by the complex influences of other surface heterogeneities such as surface morphology , surface chemistry , and molecular conformations . [SEP]
[CLS] previously , using surfaces that can switch interactions with dna on demand , we have enabled atomic B-technique force I-technique microscopy I-technique ( afm ) to spatially resolve single dna molecules that are tethered to selfassembled monolayers ( sams ) on gold B-material . [SEP]
[CLS] however , the previous work did not shed light on the complex relationship between the surface structure of the dna probes and the hybridization , because the surface hybridization kinetics was characterized using the overall hybridization yield , which is an ensemble - averaging observable that obscures important details of an intrinsically heterogeneous system . [SEP]
[CLS] in addition , how the surface structure impacts the electrochemical signals was unknown as the imaging was not carried out on functioning electrochemical sensors . [SEP]
[CLS] to address the above challenges , we have investigated for the first time how the spatial patterns of single probe molecules impact the molecular recognition of a functioning biosensor . [SEP]
[CLS] we constructed model surfaces that serve as electrochemical dna sensors and simultaneously utilized afm to spatially resolve surface hybridization even when the inter - probe separation is less than 10 nm , allowing us to probe the regime where crowding interactions are important . [SEP]
[CLS] the surfaces , which consist of electroactive dna probes tethered to highly ordered sams , make it possible to modulate interaction with dna , and provide an ideal platform for investigating how spatial organization of single molecules alters molecular recognition as these surfaces minimize the impact of uncontrolled morphological and compositional heterogeneities . [SEP]
[CLS] moreover , by applying spatial statistical tools including ripley ' s k function , nearest - neighbor distances , local crowding indices to characterize spatial heterogeneities in target binding , our study revealed unexpected spatiotemporal patterns of surface hybridization . [SEP]
[CLS] the hybridization yields of probe molecules were observed to vary substantially with the nearest neighbor - distance . [SEP]
[CLS] hybridization of dna targets with hairpin probes preferentially occurs where the probes are clustered , suggesting that the interactions between molecules separated by ~ 10 nm may facilitate target binding . [SEP]
[CLS] the cooperative effect , in contrast with the prevailing view that increasing molecular crowding inhibits target capturing , suggests new mechanisms through which the biosensor surface can influence target binding . [SEP]
[CLS] our study provided the first direct evidence that the nanoscale spatial distribution of probe molecules exerts a major influence on surface hybridization . [SEP]
[CLS] as the sensitivity of a biosensor is directly linked to interfacial molecular recognition , our findings have ramifications in biosensor design . [SEP]
[CLS] the dramatic effect of the nearestneighbor distance on hybridization suggests a new pathway to improve the sensitivity : we may tailor the spatial patterns to favor inter - probe separations that maximize target binding efficiency . [SEP]
[CLS] it should also be noted that the nanoscale spatial organization likely has important but undetermined influences on almost all surfacebased biosensors . [SEP]
[CLS] in addition to surface hybridization , our approach of combining spatially resolved measurement on model surfaces with single molecule spatial statistical analysis may be applied to other types of molecular recognition , such as aptamer sensors that detect proteins B-material . [SEP]
[CLS] how complex intermolecular B-property interactions I-property at the surface / interface influence the pathways of target recognition remain largely unknown . [SEP]
[CLS] therefore , probing and analyzing these biointerfaces at the molecular scale will lead to valuable insight that can help improve the performance of these biosensors . [SEP]
[CLS] to investigate target recognition by electrochemical dna sensors at the single molecule level , we assembled a 11mercaptoundecanoic acid ( muda ) sam on single - crystal au ( 111 ) disk electrodes , then 23 - base stem - loop probes ( p1 ) possessing a terminal thiol B-material on the 5 ' end and a methylene blue ( mb ) redox reporter on the 3 ' end were tethered to the sam ( insertion method , see methods for details ) , which generates a relatively uniform probe distribution as opposed to the conventional backfilling method ( figure s1 ) . [SEP]
[CLS] similar negatively charged sams have been used for electrochemical dna sensors in previous studies . [SEP]
[CLS] the probes can be strongly immobilized on the muda sam in the presence of ni 2 + ( figure 1a ) , appearing as circular protrusions of ~ 8 nm diameter ( figure 1e ) . [SEP]
[CLS] importantly , these immobilized probes can return to their upright state by replacing the ni 2 + buffer with a saline tris - acetate - edta ( stae ) buffer to enable both differential pulse voltammetry ( dpv ) measurement and target hybridization ( figure 1b , c ) . [SEP]
[CLS] the sensor surface produces a well - resolved dpv peak , whereas after the addition of target dnas , the peak signal drops ( figure 1f , g ) . [SEP]
[CLS] we employed a target containing a 19 - base singlestranded sticky - end , a 2 - base spacer B-material , and 19 - bp double - stranded segment ( t1 ) . [SEP]
[CLS] the double - stranded tail was designed to facilitate afm identification of the target - probe duplexes . [SEP]
[CLS] in addition , the target can to some extent mimic the larger footprints of nucleic B-material acid I-material targets used in clinical settings , which are often 150 - 300 nt in length , i . e . , much longer than the dna capture probes ( 20 - 50 nt in length ) . [SEP]
[CLS] the radius of gyration of the p1 - t1 duplex is about 18 nm , which is similar to that of a target probe duplex that has a 20 - bp doublestranded segment and 150 - nt single - stranded segment . [SEP]
[CLS] the afm images of the surface before and after hybridization showed distinct features that correspond to unhybridized hairpin probes ( figure 1e ) and p1 - t1 duplexes ( ~ 20 nm long worm - like protrusions , figure 1h ) . [SEP]
[CLS] based on the above platform , we first explored how the probe densities can be controlled . [SEP]
[CLS] the muda sam was exposed to solutions containing different concentrations of thiolated dna probes , ranging from 100 nm to 4 m , as depicted in figure 2a ( as well as in figure s2a ) , the probe surface density steadily rises when the concentration of thiolated dna probes is increased . [SEP]
[CLS] when the concentration is 4 m , the resulting immobilized individual probe molecules are difficult to resolve due to significant overlap between the molecular features . [SEP]
[CLS] hence only a lower limit , 2 10 12 probes / cm 2 could be estimated . [SEP]
[CLS] as listed in table 1 ( supporting information ) , these values cover most of the range of probe surface densities of biosensors and microarrays used in practice , except for the high end , 10 12 - 10 13 / cm 2 . [SEP]
[CLS] while single molecule imaging can directly quantify the probe densities , we also utilized the unique ability to characterize the spatial patterns of single molecules , which are inaccessible with existing averaging techniques . [SEP]
[CLS] first , we calculated the ripley ' s k function , which can characterize the tendency for the probe molecules to cluster or disperse at different spatial scales . [SEP]
[CLS] where is the number density of the molecules , e is the number of molecules within a radius of r from the molecule of interest , w ( li , lj ) the weight function for edge correction , i is the indicator B-property function . [SEP]
[CLS] for a surface with a completely random spatial pattern , [SEP]
[CLS] statistically significant clustering or dispersion can be identified by comparing measured l ( r ) - r against monte carlo simulated l ( r ) - r curves ( figure s2 ) . [SEP]
[CLS] all l ( r ) - r curves , except for those with very low probe densities ( 1 . 33 10 10 and 2 . 91 10 10 / cm 2 , where fluctuation in l ( r ) - r is significant due to sparsity ) , lie close to the expected value of 0 for all distances up to 150 nm , implying an overall spatially random distribution of probes ( figure 2c and s2b ) . [SEP]
[CLS] an exception is that for surfaces with medium probe densities ( 5 . 90 10 10 - 1 . 04 10 11 / cm 2 ) , the l ( r ) - r values dip below the 2 . 5 % quantile of mont carlo simulated l ( r ) - r values at 10 nm ( black curve of figure 2c and red curves of figure s2b ) . [SEP]
[CLS] the [SEP]
[CLS] statistically significant deviation reveals the tendency for the probe molecules to be dispersed at this scale . [SEP]
[CLS] the dispersion may be caused by the repulsive interactions between the molecules during probe immobilization , especially under our low ionic strength conditions ( 50 mm naac ) . [SEP]
[CLS] the absence of dispersion at higher probe densities ( blue curve in figure 2c ) suggests that the repulsive interactions are overcome by a larger driving force to pack the surface with probe molecules . [SEP]
[CLS] while the ripley ' s k function characterizes dispersion / clustering for the entire surface , a spatial property that likely has a direct effect on target recognition is the nearest neighbor distance ( n . n . d . ) . [SEP]
[CLS] using the spatial coordinates , we displayed the n . n . d . of each of the probe molecules in the heat map in figure 2b . [SEP]
[CLS] the range of n . n . d . values is very broad . [SEP]
[CLS] however , as shown in ripley ' s k function ( figure 2c ) , the broad distribution of n . n . d . is a consequence of random distribution instead of clustering of molecules . [SEP]
[CLS] notably , the histograms of n . n . d . ( figure s2c ) , shows that up to 94 . 4 % of the values are below the commonly used average probe separation , < n . n . d . > lat = ( a / n ) 1 / 2 , 10 , 33 , 51 - 52 which is calculated assuming that the molecules are arranged in a square lattice , using a , the total surface area , and n , the number of molecules . [SEP]
[CLS] the discrepancy clearly shows the reliability of the average inter - probe separation as the descriptor of crowding is limited . [SEP]
[CLS] we then explored the correlation between the electrochemical signal and the overall probe density . [SEP]
[CLS] the peak current increased as the dna probe concentration increased from 100 nm to 4 µm ( figure 2d ) . [SEP]
[CLS] more importantly , figure 2e shows that the peak current scaled linearly with the probe density from 1 . 3 10 10 to 1 . 0 10 11 probes / cm 2 , then began to level off at 5 . 2 10 11 probes / cm 2 . [SEP]
[CLS] the saturation in peak current and the decline in peak current / probe density ratio suggests that the interactions between the probe molecules may induce the unfolding of the hairpin probe , separating the redox reporter from the electrode surface and reducing the rate of electron transfer . [SEP]
[CLS] alternatively , as the interprobe separations are reduced , the repulsive interactions between probe molecules led to subtle orientation / conformational changes that increased the separation between the redox label and the electrode surface . [SEP]
[CLS] it should be noted that while the imaging resolution achieved ( a few nm ) is insufficient for distinguishing between the folded and unfolded states , the results above nevertheless provide direct evidence that molecular crowding impacts the electrochemical signals . [SEP]
[CLS] we next examined the correlation between the hybridization yield and electrochemical signal suppression . [SEP]
[CLS] specifically , we measured the signal suppression of a surface with a probe density of 5 . 9 10 10 / cm 2 , when the target concentration was varied between 10 nm to 1 m ( 300 nm and 1 µm in figure s3 ) at a fixed hybridization time of 30 min . [SEP]
[CLS] afm images of the sensor surface were acquired as well ( figure 3a - d ) to directly quantify the overall hybridization yield . [SEP]
[CLS] 3e shows that the fraction of hybridized probes , i . e . , hybridization yield , increases with the increasing target concentration and reaches a plateau of 88 % at 100 nm ( red curve ) . [SEP]
[CLS] the trend tracks the curve of the dpv signal suppression ( blue curve ) . [SEP]
[CLS] the gap between the hybridization yield curve and signal suppression curve is consistent with the observation that some of the probe / target duplexes have sufficient conformational freedom to tilt toward the surface for facile electron transfer , leading to incomplete signal suppression . [SEP]
[CLS] moreover , while from electrochemical measurement alone , it is unclear whether the incomplete signal suppression is caused by the finite electron transfer rate or incomplete hybridization , single molecule afm analysis provides definitive evidence that a small fraction ( ~ 10 % ) of the probe molecules are inactive as no further enhancement in hybridization yield was observed even at higher target concentrations of 300 nm and 1 µm ( figure 3e and s3 ) . [SEP]
[CLS] a less than unity hybridization yield was also observed for hybridization in a homogeneous solution . [SEP]
[CLS] the origin is not yet clear . [SEP]
[CLS] as the yield of each of the solid phase coupling reaction steps is below 100 % , commercial synthetic oligonucleotides typically contain 10 - 15 % impurities B-property that are missing one or more nucleotides . [SEP]
[CLS] therefore , one possibility is that these truncated oligonucleotides may be responsible for incomplete hybridization . [SEP]
[CLS] in addition to concentration dependence , the hybridization yield was measured at different time intervals at a fixed target concentration of 100 nm . the hybridization yield increased over time and saturated after 30 minutes ( figure s4a ) . [SEP]
[CLS] the same trend was found in the corresponding time evolution of dpv suppression ( figure s4b ) . [SEP]
[CLS] in addition to calibration - free , quantitative measurement of the overall hybridization yield , high resolution afm imaging affords the unique opportunity to examine the spatial patterns of hybridization . [SEP]
[CLS] interestingly , we found that the probe - target duplexes tend to form clusters and the number of clusters grows with increasing target concentrations ( figure 3a - d , highlighted by green circles ) . [SEP]
[CLS] to develop a more quantitative description , we analyzed the spatial distribution of these duplexes using the ripley ' s k function . [SEP]
[CLS] the positive values in figure 3f show that the duplexes are clustered . [SEP]
[CLS] the values approach zero with increasing target concentrations , presumably due to the merging of clusters ( l ( r ) - r curves for 300 nm and 1 m targets are shown in figure s3c ) [SEP]
[CLS] the emergence of a heterogeneous spatial pattern of hybridization from a mostly random spatial distribution of probe molecules is rather surprising as crowding is thought to inhibit target binding . [SEP]
[CLS] the spatial pattern suggests target capture is instead a cooperative process . [SEP]
[CLS] to gain more mechanistic insights , we imaged the same areas of the sensor surface ( 5 . 9 10 10 / cm 2 ) to track the evolution of the spatial patterns ( figure 4a - c ) . [SEP]
[CLS] this experiment can provide direct information concerning how probe spatial organization affects the cluster distribution and the pathway of cluster formation . [SEP]
[CLS] the areas in green squares enlarged in the insets of figure 4a - c show that the cluster started to emerge at 45 min and evolved into a complete cluster at 105 min at 10 nm target concentration . [SEP]
[CLS] the nearest - neighbor distances ( n . n . d . ) map in figure 4d showed that the captured target molecules predominantly appeared in the regions where the n . n . d . are less than or equal to 15 nm ( red or orange dots ) . [SEP]
[CLS] the histograms in figure 5a and 5c further confirm that the hybridization of the probes is highly sensitive to n . n . d . . [SEP]
[CLS] the fraction of hybridized probes with n . n . d . of 10 nm is close to 50 % , while only 5 % of the probes with n . n . d . of 25 nm are hybridized . [SEP]
[CLS] these results provide evidence that under certain conditions , the crowding interactions between the molecules may enhance instead of inhibiting target binding . [SEP]
[CLS] moreover , we also used the local crowding index lci ( r ) , a parameter we introduced in a previous work , to explore if molecules that are located beyond nearest - neighbors also impact hybridization . [SEP]
[CLS] lci ( r ) counts the number of neighboring probes surrounding a particular probe , within an interaction radius r ( 20 nm which is about twice the length of probe p1 is used here ) . [SEP]
[CLS] a measure of the local probe density at a specific spatial scale , lci ( r ) enables us to assess the degree of local crowding experienced by individual probes , which can then be correlated to their hybridization efficiency . [SEP]
[CLS] compared to n . n . d . , which serves an indicator B-property of the " two - body " interactions between the probe of interest and its nearest neighbor , lci ( r ) allows us to examine our system from " many - body " perspective of neighboring probes . [SEP]
[CLS] the lci ( 20nm ) versus n . n . d . plots at 45 min and 105 min ( figure 5b , d ) show that the probability of hybridized probe molecules ( red dots ) increases at the top . [SEP]
[CLS] for probe molecules with the same n . n . d . , those with higher lcis are more likely hybridize . [SEP]
[CLS] overall , figure 5 suggests that the presence of neighboring molecules ( ~ 15 nm ) may accelerate the hybridization rate of the hairpin probes by an order of magnitude or more . [SEP]
[CLS] moreover , molecules that are located beyond the nearest - neighbors can also impose crowding interactions that accelerate hybridization . [SEP]
[CLS] to confirm that the local crowding of probe molecules is responsible for enhanced hybridization , we need to exclude the possibility that clustered probe molecules may coincide with disordered sam domains that may accelerate hybridization through nonspecific adsorption on a more hydrophobic B-property surface . [SEP]
[CLS] a previous study suggested that hydrophobic B-property surfaces increase the residence time of the dna targets and enhance the rate of hybridization . [SEP]
[CLS] no features of target molecules could be observed with afm when a muda sam without capture probes was exposed to the same target solution , indicating that only targets binding to the captured probes can remain on the surface and non - specific adsorption on sam domains is weak ( figure s5 ) . [SEP]
[CLS] moreover , we replaced the hairpin probe p1 with a linear probe p2 ( figure 6 ) . [SEP]
[CLS] interestingly , although the probe surface density and spatial pattern of immobilized p2 molecules ( figure 6b , c ) are similar to those of p1 ( figure 4d and s6 ) , the spatial pattern of p2 - t1 duplexes is substantially different ( figure 6d , e ) . [SEP]
[CLS] the clustering function reveals a random spatial pattern of p2 - t1 duplexes ( figure 6f ) . [SEP]
[CLS] the absence of target clustering on surfaces with p2 shows that nonspecific adsorption on local sam domains is not responsible for clustering of target - probe duplexes . [SEP]
[CLS] the absence also rules out the possibility that the aggregation of target molecules in the solution is responsible for the clustering of captured targets with the hairpin probe p1 . [SEP]
[CLS] another piece of supporting evidence is that clustering of captured targets is observed at target concentrations as low as 10 nm in a monovalent cation B-material na + buffer . [SEP]
[CLS] dynamic B-technique light I-technique scattering I-technique ( dls ) measurements of the target solutions used for hybridization showed no aggregation ( figure s7 ) . [SEP]
[CLS] consistent with previous studies , aggregation of dnas in a monovalent buffer solution only occurs at higher concentrations ( m or more ) . [SEP]
[CLS] therefore , we conclude that the clustering is caused by a combination of the secondary structure of the probe and a higher local probe density . [SEP]
[CLS] furthermore , we performed a hybridization experiment that used p1 to capture t2 , which is a shorter , 19 - base single - stranded counterpart of t1 . [SEP]
[CLS] ripley ' s k function analysis also revealed a random spatial pattern of the p1 - t2 duplexes under the same experimental conditions , including probe surface density , target concentration and hybridization time ( figure s8 ) . [SEP]
[CLS] the observed spatial heterogeneity can be attributed to two distinct characteristics of the surface immobilized probes . [SEP]
[CLS] first , the crowding interactions are more important for surface immobilized probe molecules than for probes in a solution . [SEP]
[CLS] the average distance between dna probes on a biosensor surface ranges from a few nanometers to tens of nanometers . [SEP]
[CLS] in contrast , the average distance in a homogeneous solution is typically 100 nm or more , as the typical probe concentration is less than micromolar . [SEP]
[CLS] second , while the hybridized and unhybridized probes undergo free brownian motion in solution hybridization , the surface tethered probes maintain their positions relative to the surface . [SEP]
[CLS] therefore , the interactions with neighboring molecules in a dilute solution are short - lived . [SEP]
[CLS] in contrast , the spatial heterogeneity of surface immobilized probes is persistent ( figure 4a - c ) and hence can affect hybridization kinetics as well as affinity substantially . [SEP]
[CLS] our study provides the first single molecule level evidence that the spatial proximity of probe molecules has a major impact on target recognition . [SEP]
[CLS] however , our findings are a departure from the prevailing assumption that surface crowding hinders target recognition and optimal hybridization kinetics is achieved at the lowest densities . [SEP]
[CLS] two factors could be responsible for the discrepancy . [SEP]
[CLS] first , as existing studies measured the overall target binding kinetics of surfaces that have highly heterogeneous interprobe seperations , it has not been possible to quantify the influences of specific inter - probe separations . [SEP]
[CLS] our single molecule resolution of hybridization represents an ideal way to address this question . [SEP]
[CLS] in addition , many existing studies focus on high surface density regimes , 10 12 / cm 2 or greater , where the accessibility to probe molecules may indeed be the limiting factor . [SEP]
[CLS] our study focuses on a less crowded regime ( ~ 10 11 / cm 2 ) , where accessibility is less an issue and enhancement mechanisms may manifest themselves . [SEP]
[CLS] the use of less perturbing imaging modes , such as noncontact afm , may allow afm to study the single molecule spatial patterns at higher probe densities , when the probes are separated by less than a few nanometers , where limited accessibility to the probe molecules may begin to inhibit target binding . [SEP]
[CLS] the counter - intuitive cooperative effect may be rationalized by examining the microscopic mechanisms of dna hybridization . [SEP]
[CLS] the hybridization of a dna hairpin probe is thought to proceed via a nucleation step that forms a stable contact between the loop and the target molecule , followed by the melting of the stem , and propagation of the base paired region to form a full probe - target duplex ( zippering ) . [SEP]
[CLS] while the rate limiting step for hybridization of unstructured probes is the formation of stable target - probe contacts , that for hybridization of hairpin probes may be stem melting [SEP]
[CLS] when the probe molecules are separated by 10 - 15 nm , there is sufficient space for a target to form contacts with a probe . [SEP]
[CLS] based on the data either directly from this work or the literature , we propose three mechanisms including ( i ) probe crowding , ( ii ) target crowding and ( iii ) crowding - induced surface trapping that may facilitate target recognition at these inter - probe distances . [SEP]
[CLS] first , these probe molecules may impose a repulsive potential that destabilizes the hairpin structure of neighboring probe molecules and accelerates target binding . [SEP]
[CLS] supporting evidence of this mechanism includes figure 5 as well as the absence of clustering of target - probe duplexes using linear probes , p2 ( figure 6d - f ) . [SEP]
[CLS] moreover , the sublinear relationship between the electrochemical signal and the probe density when the probe density exceeds 1 10 11 / cm 2 ( figure 2e ) also suggests that probe crowding interaction favors the unfolded state . [SEP]
[CLS] unfolding of densely packed dna hairpin probes was also observed in previous studies . [SEP]
[CLS] while the probe density studied , 5 . 9 10 10 / cm 2 , appears too low to cause a significant fraction of them to unfold , our spatial statistical analysis shows that 20 % of the probe molecules have n . n . d . of 10 nm or less ( figure s6 ) due to the random nature of spatial distribution . [SEP]
[CLS] it should be noted that zippering does not need to be preceded by complete unfolding . [SEP]
[CLS] even a modest destabilization of the hairpin may reduce the activation barrier B-property of concomitant stem melting and formation of target - probe duplex and accelerate the kinetics . [SEP]
[CLS] the second mechanism is the destabilization of the stem by binding of target molecules to nearby hairpins . [SEP]
[CLS] a probe - target duplex increases electrostatic repulsion within a hemisphere , due to its ability to rotate around its anchor ; the localized increase of electrostatic repulsion may also favor unfolding of neighboring hairpin probes and accelerate binding in close proximity . [SEP]
[CLS] this target crowding is supported by the more facile hybridization of t1 compared to that of the shorter target , t2 ( figure s8 ) . [SEP]
[CLS] it should be noted that as many nucleic B-material acid I-material targets for molecular diagnostics are notably longer than the capture probes , this target crowding - induced cooperative effect is likely relevant . [SEP]
[CLS] the third enhancement mechanism is that probe molecules in proximity can trap the target molecules and increase their residence time . [SEP]
[CLS] the formation of a full duplex is typically preceded by many unproductive contacts between the target and the hairpin loop . [SEP]
[CLS] a target molecule that is transiently bound may have a greater opportunity to hop onto a neighboring probe molecule and get captured . [SEP]
[CLS] so far , no single mechanism could explain all the findings . [SEP]
[CLS] while there is direct evidence supporting the first two mechanisms , probe crowding and target crowding , we cannot exclude the possibility that the third mechanism also contributes to the enhancement . [SEP]
[CLS] future studies that systematically explore the effects of target size , probe design and probe density can elucidate the relative contributions of the proposed mechanisms . [SEP]
[CLS] spatially resolved measurement of molecular recognition of an e - dna sensor revealed novel complex , heterogeneous behaviors that are difficult to capture using ensemble averaging techniques . [SEP]
[CLS] even when the probe density is relatively uniform , the wide distribution of inter - probe distances arising from random probe immobilization may lead to heterogeneous target binding . [SEP]
[CLS] therefore , the probe density alone may not be a reliable predictor of interfacial molecular recognition , given the observed major impact of inter - probe distances on target binding . [SEP]
[CLS] these findings set the stage for future studies that can explore how these probe spatial patterns help determine the sensitivity and device to device variabilities of existing electrochemical biosensors . [SEP]
[CLS] moreover , it would be intriguing to explore the extent to which the spatial patterns impact the selectivity . [SEP]
[CLS] ultimately , the mechanistic insights from spatially resolved measurements and single molecule spatial statistical analysis may help establish a predictive relationship between molecular scale spatial patterns and the performance of the biosensor , paving the way toward the rational engineering of biosensor devices . [SEP]
[CLS] preparation of e - dna sensors . [SEP]
[CLS] unless otherwise stated , all chemicals were obtained from fisher scientific co . ( pittsburg , pa , usa ) . [SEP]
[CLS] insertion method : an au ( 111 ) single crystal disk ( mateck gmbh , juelich , germany ) substrate was used for both electrochemical measurement and afm imaging . [SEP]
[CLS] the au single crystal disk was cleaned following a standard protocol and then immersed into 1 mm muda ( sigma - aldrich co . , st . louis , mo , usa ) solution in 9 : 1 ethanol ( vwr , radnor , pa , usa ) : acetic acid for 1 h . [SEP]
[CLS] the disulfide dna probes ( p1 or p2 ) were reduced in 2mm tcep ( tris - ( 2 - carboxyethyl ) - phosphate , sigma - aldrich co . , st . louis , mo , usa ) at room temperature for 20 min and then purified using a qiaquick nucleotide removal kit ( qiagen , germantown , md , usa ) and stored at - 20 o c . [SEP]
[CLS] after 1 h , the au substrate was thoroughly rinsed with 9 : 1 ethanol : acetic acid and water B-material , blown dried with filtered air , and incubated B-technique with a buffer containing the thiolated dna probes , 50 mm naac , and 2 mm tcep for 30 minutes . [SEP]
[CLS] following the insertion step , the substrate was rinsed 3 times with a tae ( 40 mm tris - acetate and 1 mm edta , ph 8 . 3 ) buffer to remove nonspecific probe adsorption . [SEP]
[CLS] backfilling method : the cleaned au single crystal disk was immersed into a buffer containing the 20 nm thiolated dna probes , 50 mm naac , and 2 mm tcep for 1 h . [SEP]
[CLS] after dna probe assembly , the au disk substrate was backfilled with 1 mm muda solution in 9 : 1 ethanol : acetic acid for 3h . [SEP]
[CLS] following the backfilling step , the substrate was thoroughly rinsed with in 9 : 1 ethanol : acetic acid and tae buffer . [SEP]
[CLS] e - dna sensor hybridization and probe / target duplex denaturation . [SEP]
[CLS] the hybridization was carried out by incubating B-technique the sensor surface with a buffer containing dna targets ( t1 or t2 ) , 1x pbs7 ( 10 mm phosphate , 1m nacl , ph 7 ) at room temperature in the dark . [SEP]
[CLS] after a predetermined hybridization time , the sensor surface was then rinsed 3 times with an stae buffer ( 1x tae , 200 mm nacl ) to remove nonspecific adsorbed targets . [SEP]
[CLS] to perform the kinetic study of the hybridization , the sensor surface was regenerated by immersion in an alkaline buffer ( 1x ab = 10 mm naoh , 330 μm edta , ph 12 ) for 5 minutes after each target incubation B-technique , followed by thorough rinsing with stae to remove the denatured targets . [SEP]
[CLS] the denatured sensor surface is shown in figure s9 . [SEP]
[CLS] electrochemical measurement . [SEP]
[CLS] all dpv measurements was performed relative to an ag / agcl ( 3m kcl ) reference electrode at room temperature using an epsilon electrochemical analyzer ( basi , west lafayette , in , usa ) . [SEP]
[CLS] the potential range was from - 0 . 4 to - 0 . 1 v and the pulse amplitude was 50 mv . [SEP]
[CLS] an au single crystal disk was used as a working electrode , together with a platinum B-material counter electrode . [SEP]
[CLS] in all experiment , the pbs7 was used as the electrolyte . [SEP]
[CLS] afm imaging . [SEP]
[CLS] all afm images were obtained using agilent / keysight 5500 afm ( keysight technologies , santa rosa , ca , usa ) and ntegra vita afm ( nt - mdt co . , moscow , russia ) equipped with snl - 10 cantilevers ( spring constants of 0 . 2 - 0 . 4 n / m , bruker , bellerica , ma , usa ) under a ni 2 + buffer ( 5 mm niac2 , 0 . 1x tae ) . [SEP]
[CLS] intermittent contact mode afm imaging with a resonant frequency of approximately 16 khz was performed . [SEP]
[CLS] after imaging , the sensor surface was thoroughly rinsed with the stae buffer to remove ni . [SEP]
[CLS] probe density and hybridization yield quantification . [SEP]
[CLS] to measure the probe densities , afm imaging was carried out in at least four different areas of the au substrate . [SEP]
[CLS] an average probe density ( molecules / area ) in the absence of targets was determined by counting the number of corresponding features ( 8 nm diameter circular protrusions ) in images using gwyddion ( http : / / gwyddion . net / ) and wsxm 67 image analysis software . [SEP]
[CLS] the number of hybridized probes after hybridization ( ~ 20 nm long worm - like protrusions ) was determined in the same manner as the above . [SEP]
[CLS] the hybridization yield was then defined as the number of hybridized probes divided by the total number of probes after hybridization . [SEP]
[CLS] spatial statistical analysis of probe distribution . [SEP]
[CLS] gwyddion image analysis software was used to extract the xy - coordinates of dna probes from afm images . [SEP]
[CLS] a mask was generated for all features that are above the minimal pixel area and height threshold . [SEP]
[CLS] minor manual editing was carried out to separate the partially overlapping features , especially for surfaces at higher probe densities . [SEP]
[CLS] the coordinates of the centroids were then used to calculate the ripley ' s k ( r ) function as well as [ k ( r ) / π ] 1 / 2 - r = l ( r ) - r . [SEP]
[CLS] the observed l ( r ) - r can be compared with the corresponding data under complete spatial randomness ( csr ) computed by monte carlo simulation . [SEP]
[CLS] 999 simulations were performed to reduce the sampling uncertainty and calculate the quantiles of l ( r ) - r for each value of r , in which 0 . 025 and 0 . 975 quantiles were directly compared with l ( r ) - r . [SEP]
[CLS] the same coordinates were also used for n . n . d . and lci analyses , where the former determines the distance between a probe and its closest neighboring probe , while the latter counts the number of surrounding probes with respect to a specific probe . [SEP]
[CLS] this material B-material is available free of charge via the internet on the acs publications website at materials and methods , summary of probe densities , images of the surface prepared using backfilling method , additional data and analysis of different probe densities and target concentrations , dynamic study of target hybridization , images of the sam surface after exposure to the targets , n . n . d . distribution histogram for unhybridized probes , dls measurement of target aggregation in solution , control experiment using small targets , and images of denatured surface ( pdf ) tao . ye @ ucmerced . edu [SEP]
[CLS] 1 . schematic of a dynamically switchable e - dna sensor surface . [SEP]
[CLS] ( a ) a stem - loop dna probe is covalently tethered at one end to a singlecrystal au electrode passivated with a muda monolayer and modified with a mb tag ( silver B-material ) at the other end . [SEP]
[CLS] the probe is pinned down to the surface after adding ni 2 + and thus can be imaged by afm . [SEP]
[CLS] ( b ) after imaging , the surface is rinsed with an stae buffer and a dpv measurement is carried out under a pbs7 buffer . [SEP]
[CLS] ( c ) upon the addition of a complementary target dna into the pbs7 buffer , the same dpv measurement is repeated . [SEP]
[CLS] ( d ) the hybridized surface is imaged following the same procedure as given in step ( a ) above . [SEP]
[CLS] afm images and dpv signals of the sensor surface ( e , f ) before and ( h , g ) after the hybridization with the targets . [SEP]
[CLS] the scale bar is 25 nm . [SEP]
[CLS] correlating probe density and dpv of the e - dna sensor surface . [SEP]
[CLS] ( a ) representative afm images of the sensor surface fabricated using dna probe concentrations at 100 nm , 500 nm and 2 . 5 µm . [SEP]
[CLS] the insertion time is fixed at 30 min . [SEP]
[CLS] the scale bar is 100 nm . [SEP]
[CLS] ( b ) the nearest - neighbor distance ( n . n . d . ) analysis of probes in panel ( a ) . [SEP]
[CLS] the color bar indicates the range of n . n . d . from 0 nm ( red ) to 120 nm ( blue ) . [SEP]
[CLS] ( c ) the ripley ' s k - function analysis of the distribution of probes . [SEP]
[CLS] ( d ) the corresponding dpv voltammograms ( from bottom to top ) . [SEP]
[CLS] ( e ) the relation between probe surface density and peak current . [SEP]
[CLS] inset , the current peak of e - dna sensor is linearly related to the probe surface density at low probe densities ( < 10 11 probes / cm 2 ) . [SEP]
[CLS] red dot represents the estimated probe density . [SEP]
[CLS] 3 . spatial patterns of surface hybridization . [SEP]
[CLS] ( a - d ) representative afm images of the sensor surface in the presence of complementary t1 target dnas at 10 nm , 30 nm , 60 nm and 100 nm . [SEP]
[CLS] the green dotted circles are used to outline the clusters . [SEP]
[CLS] the hybridization time is fixed at 30 min . [SEP]
[CLS] the scale bar is 100 nm . [SEP]
[CLS] ( e ) the relation between hybridization yield ( red curve ) and dpv signal suppression ( blue curve ) at different target concentrations . [SEP]
[CLS] ( f ) the ripley ' s k - function analysis of the distribution of hybridized probes . [SEP]
[CLS] the l ( r ) - r curve shows a decrease of clustering with increasing target concentration ( from top to bottom ) . [SEP]
[CLS] 4 . tracking evolution of heterogeneous spatial patterns of dna surface hybridization . [SEP]
[CLS] representative afm images of the sensor surface after exposed to 10 nm target dna for ( a ) 0 min , ( b ) 45 min and ( c ) 105 min . [SEP]
[CLS] the scale bar is 100 nm . [SEP]
[CLS] insets are zoom in images of green - squared areas . [SEP]
[CLS] ( d ) the nearest - neighbor distance ( n . n . d . ) analysis of unhybridized probes in panel ( a ) . [SEP]
[CLS] the color bar indicates the range of n . n . d . from 10 nm ( red ) to 60 nm ( blue ) . [SEP]
[CLS] 5 . spatial statistical analysis of probe distributions . [SEP]
[CLS] ( a ) , ( c ) the histograms of the hybridization yield at 45 min and 105 min as a function of n . n . d . . ( b ) , ( d ) plots of lci vs . n . n . d . at 45 min and 105 min for each dna probe . [SEP]
[CLS] probes with high lci and low n . n . d . are more likely to capture targets . [SEP]
[CLS] the red dots represent hybridized probes , while the black dots represent unhybridized probes . [SEP]
[CLS] control experiment using linear probes . [SEP]
[CLS] representative afm images of the linear probe surface in the absence ( a ) and presence of complementary target dnas at 30 nm ( d ) and 100 nm ( e ) . [SEP]
[CLS] the hybridization time is fixed at 15 min . [SEP]
[CLS] the scale bar is 100 nm . [SEP]
[CLS] ( b ) the nearest - neighbor distance ( n . n . d . ) analysis of unhybridized probes in panel ( a ) . [SEP]
[CLS] the color bar indicates the range of n . n . d from 10 nm ( red ) to 60 nm ( blue ) . [SEP]
[CLS] histograms of unhybridized probes in panel ( a ) as a function of n . n . d . . [SEP]
[CLS] ( f ) the ripley ' s k - function analysis of the distribution of hybridized probes . [SEP]
[CLS] the l ( r ) - r curves show uniform distributions of unhybridized probes ( black ) and hybridized probes at 100 nm ( blue ) , and a relatively dispersed distribution of hybridized probes at 30 nm ( red ) . [SEP]
[CLS] we report the development of a novel scanometric microrna ( scano - mir ) platform for the detection of relatively low abundance mirnas with high specificity and reproducibility . [SEP]
[CLS] the scano - mir system was able to detect 1 fm concentrations of mirna in serum with single nucleotide mismatch specificity . [SEP]
[CLS] indeed , it provides increased sensitivity for mirna targets compared to molecular fluorophore - based detection systems , where 88 % of the low abundance mirna targets could not be detected under identical conditions . [SEP]
[CLS] the application of the scano - mir platform to high density array formats demonstrates its utility for high throughput and multiplexed mirna profiling from various biological samples . [SEP]
[CLS] to assess the accuracy of the scano - mir system , we analyzed the mirna profiles of samples from victims of prostate cancer ( cap ) , the most common noncutaneous malignancy and the second leading cause of cancer death among american men . [SEP]
[CLS] the platform exhibits 98 . 8 % accuracy when detecting deregulated mirnas involved in cap , which demonstrates its potential utility in profiling and identifying clinical and research biomarkers B-property . [SEP]
[CLS] micrornas ( mirna , mir ) , small noncoding rna molecules , have emerged as new candidate biomarkers B-property for detecting a wide variety of cancers . [SEP]
[CLS] introductionmirnas are regulatory elements believed to control between 30 - 90 % of all protein B-material - coding genes in the human genome by binding to the 3 ' - untranslated region ( 3 ' - utr ) of target mrnas . [SEP]
[CLS] importantly , mirna expression levels have been shown to be tissue - and function - specific . [SEP]
[CLS] furthermore , mirnas in the bloodstream are protected from nuclease degradation and can be extracted for detection and downstream analyses . [SEP]
[CLS] therefore , molecular diagnostic tools that allow one to identify mirnas and measure their concentrations at biologically relevant levels in blood and tissue samples may become useful for studying and diagnosing disease . [SEP]
[CLS] individual markers rarely are unambiguously indicative of disease type and state , and mirnas are often at very low concentrations in serum and precious tissue samples , which precludes the use of conventional diagnostic tools with low sensitivity . [SEP]
[CLS] although amplification strategies such as polymerase chain reaction ( pcr ) provide a means to amplify nucleic B-material acid I-material targets , the short length of the mirna targets makes it difficult to develop multiplexed high sensitivity pcr - based quantification protocols . [SEP]
[CLS] ideally , one needs a method that allows for high sensitivity nucleic B-material acid I-material detection , which does not rely on enzymatic - based target amplification , and can be used to simultaneously profile hundreds to thousands of mirnas . [SEP]
[CLS] herein , we report the development of a novel scanometric microrna ( scano - mir ) array profiling assay that is capable of detecting low abundance mirnas from human serum , cultured human cells B-material , and cancer tissue samples with a limit of detection ( lod ) at 1 fm with single nucleotide polymorphism ( snp ) selectivity . [SEP]
[CLS] moreover , this technique is applicable to high density array formats where thousands of targets can be interrogated at once . [SEP]
[CLS] prostate cancer cell B-material lines and tissue samples are used to help validate the performance of the scano - mir assay , and also to demonstrate its potential utility in profiling and identifying prostate cancer biomarkers B-property from human prostate cancer tissue . [SEP]
[CLS] the current workhorse technologies for mirna profiling are high density microarrays ( mirna - arrays ) , quantitative real time pcr ( qrt - pcr ) , and deep sequencing methods ; however , no single technique provides the ability to study mirnas at low cost , with high selectivity , at low abundance , in a high throughput and multiplexed fashion . [SEP]
[CLS] in all cases , the detection of low abundance mirnas ( below 10 fm ) is a major obstacle because of their short length ( typically 20 - 25 nucleotides ) . [SEP]
[CLS] for microarrays , typical detection strategies are dominated by techniques that involve direct detection with molecular fluorophores as target labels ( i . e . no pcr amplification ) . [SEP]
[CLS] however , the fluorophore - based arrays are challenged with respect to lod ( typically > 1 pm ) and therefore are inherently prone to false negatives . [SEP]
[CLS] qrt - pcr - based profiling assays have been shown to provide higher sensitivity ( ~ 10 fm for mir - 141 ) and are ideal for screening a small number of mirnas , but there are challenges associated with using qrt - pcr for high - throughput mirna profiling . [SEP]
[CLS] specifically , mirnas are difficult to amplify using pcr because of their short length . [SEP]
[CLS] ligation of pcr primers at the 3 ' and / or 5 ' ends of mirnas is required to increase their length for successful amplification , which introduces challenges with regard to primer design . [SEP]
[CLS] indeed , qrt - pcr assays are limited to studying mirnas based upon the design of a combination of universal and specific primers and probes for each mirna target , which leads to a reduction in specificity . [SEP]
[CLS] deep sequencing , a more sophisticated approach to mirna detection , has emerged as an alternative to microarray - and qrt - pcrbased detection of mirnas . [SEP]
[CLS] deep sequencing of mirna libraries from biological samples has been used to perform multiplexed , highly specific , and quantitative detection of both novel and previously identified mirna sequences . [SEP]
[CLS] a limitation of deep sequencing is its lower sensitivity compared to qrt - pcr due to the need to ligate platform - specific linking sequences to the 5 ' and 3 ' ends of the mirnas because of their short length with subsequent yield - reducing purification steps . [SEP]
[CLS] moreover , deep sequencing assays rely on pcr amplification where the relative abundance of mirna species in the starting materials cannot be preserved . [SEP]
[CLS] these steps increase the complexity and cost of such assays and simultaneously decrease the sensitivity of deep sequencing techniques . [SEP]
[CLS] in 2000 , we reported the development of the scanometric assay , 14 a novel nucleic B-material acid I-material detection method based upon the use of spherical nucleic B-material acid I-material - gold B-nanoparticle nanoparticle I-nanoparticle conjugates ( sna - au nps B-nanoparticle ) . [SEP]
[CLS] the assay utilizes a low density microarray on a glass slide to capture dna target and then sandwiches it with the sna - au np B-nanoparticle probes . [SEP]
[CLS] the signal is then amplified by catalytic reduction of ag + in the presence of hydroquinone or gold B-material enhancement with tetrachloroaurate and hydroxylamine . [SEP]
[CLS] after the reduction step , the slide is used as a wave guide , and scattered light is measured from the metal B-material spots to determine target identity and concentration . [SEP]
[CLS] the lod of the method is 100 am for large dna targets and does not require pcr or related target amplification techniques . [SEP]
[CLS] because the sna - au np B-nanoparticle probes exhibit cooperative melting transitions over more narrow temperature ranges than duplexes formed from molecular fluorophore probes of the same sequence , stringency conditions can be employed to provide significantly higher target discrimination capability . [SEP]
[CLS] we rationalized that this assay might be ideal for detecting short , relatively low abundance mirnas , without the need for enzymatic amplification steps with high selectivity and sensitivity . [SEP]
[CLS] therefore , we have studied a new version of this assay aimed at profiling the expression of mirna species from human serum , cell B-material culture , and human tissue samples . [SEP]
[CLS] data demonstrate that the scano - mir assay is highly specific , sensitive , and reproducible for profiling mirnas . [SEP]
[CLS] importantly , these studies not only show this modified scanometric method can be used , for the first time , with high density arrays but also demonstrate its ability to identify mirna markers with higher sensitivity and selectivity than fluorophore based high - density array techniques . [SEP]
[CLS] microrna ( mirna ) cloning linker ii and a propylthiol - modified ssdna recognition sequence complementary to linker ii with a 10 adenosine base ( a10 ) spacer B-material ( 5 ' propylthiol - ( a ) 10 - tccttggtgcccgagtg - 3 ' ) were purchased from idt ( coralville , iowa ) . [SEP]
[CLS] the propylthiol terminated strands at a concentration of 4 µm were chemisorbed onto 13 nm gold B-nanoparticle nanoparticles I-nanoparticle ( au nps B-nanoparticle , 10 nm concentration ) by incubating B-technique the strands with the solution of citrate stabilized au nps B-nanoparticle for 1 hr at room temperature . [SEP]
[CLS] next , sodium B-material dodecylsulphate ( sds ) , phosphate buffer ( ph = 7 . 4 ) , and sodium B-material chloride I-material ( nacl ) , were added to the solution at final concentrations of 0 . 01 % , 10 mm , and 0 . 1 m , respectively , and incubated B-technique for an additional 1 hr at room temperature . [SEP]
[CLS] two aliquots of nacl were added with an incubation B-technique time of one - hour between each addition to achieve a final concentration of 0 . 3 m . the mixture was sonicated ( 10 sec ) between each salt B-material addition . [SEP]
[CLS] following this brief salt aging process , the mixture was incubated B-technique overnight at room temperature with gentle shaking ( 130 rpm ) . [SEP]
[CLS] for purification , three successive rounds of centrifugation ( 16000 × g for 20 min ) , supernatant removal , and re - suspension in phosphate buffered saline ( 137 mm nacl , 10 mm phosphate , 2 . 7 mm kcl , ph 7 . 4 ) were performed . [SEP]
[CLS] all reagents used to synthesize mirna sequences were purchased from glen research . [SEP]
[CLS] synthetic mirna species were synthesized on a mermade 6 ( bioautomation ) and purified using high performance reverse - phase liquid B-technique chromatography I-technique ( varian ) . [SEP]
[CLS] the synthetic mirna sequences used in this study are listed in table s1 . [SEP]
[CLS] synthetic mir - 16 at concentrations of 1 , 10 , and 100 fm , were ligated to the universal mirna cloning linker ii by using t4 rna ligase ii ( new england biolabs ) following the manufacturer ' s protocol . [SEP]
[CLS] briefly , truncated t4 rna ligase 2 , mirna cloning linker ii , peg 8000 , and t4 rnl2 buffer , were added to 200 ng of synthetic mir - 16 at a final concentration of 200 u , 900 ng , 12 % , and 1x , respectively . [SEP]
[CLS] this solution was allowed to incubate B-technique for 3 hrs at 37°c . [SEP]
[CLS] the ligated rna was added into 1 ml of pre - denatured serum followed by total rna extraction ( see supplementary information ) . [SEP]
[CLS] isolated total rna was re - suspended in 400 µl rnasefree 2x ssc hybridization buffer ( 0 . 3 m nacl , 0 . 03 m sodium B-material citrate , ph 7 . 0 ) , denatured at 95°c for 2 min , and incubated B-technique on ice for another 2 min . [SEP]
[CLS] the whole mixture was hybridized overnight at 52°c onto a high density complement mirna microarray ( ncode human mirna microarray v3 , invitrogen ) displaying the complement to mir - 16 as well as the appropriate mutant control with a single mismatch . [SEP]
[CLS] following the incubation B-technique , the array was washed with a pre - warmed ( 52°c ) 2x ssc for 2 min , followed by a 2 min immersion in the following rnase - free solutions at room temperature : 2x ssc , phosphate buffered saline ( 137 mm nacl , 10 mm phosphate , 2 . 7 mm kcl , ph 7 . 4 ) , and nanopure water B-material . [SEP]
[CLS] the array was then exposed to a solution of 1 nm ( final concentration ) sna - au nps B-nanoparticle in 400 µl rnase - free 2x ssc at 56°c for 1 hr . [SEP]
[CLS] unreacted sna - au nps B-nanoparticle were washed away with prewarmed ( 56°c ) 2x ssc followed by a 2 min immersion in the same rnase - free solutions as described above . [SEP]
[CLS] gold enhancing solution [ a freshly mixed 1 : 1 ( v : v ) solution of 1 mm haucl 4 and 10 mm nh 2 oh ] was used to deposit au 0 onto the immobilized sna - au nps B-nanoparticle in order to increase the light scattering of the au nps B-nanoparticle . [SEP]
[CLS] this amplification process was performed by covering the array with 1 ml of the gold B-material enhancing solution and incubating B-technique the array for 5 min at room temperature followed by washing with nanopure water B-material . [SEP]
[CLS] the array was then air - dried and imaged with a scanner ( ls reloaded , tecan , salzburg , austria ) . [SEP]
[CLS] three rounds of gold B-material deposition for 5 minutes each were performed according to literature methods in order to characterize the sensitivity of the scano - mir system . [SEP]
[CLS] the gray scale images were converted into colored ones and intensity values were calculated using genepix pro 6 software ( molecular devices ) . [SEP]
[CLS] to examine the array selectivity of this design , five synthetic mirnas ( mir - 20a , −143 , −143 * , −205 , and −210 ) were synthesized and ligated to the universal mirna cloning linker ii as described above ( see table s1 for sequences ) . [SEP]
[CLS] four of these synthetic mirnas were added in 2x ssc at 10 2 , 10 3 , 10 4 , and 10 5 fm , respectively in one pool and labeled as array 1 ( see table s2 for mixture information ) . [SEP]
[CLS] the orders of the mirna concentrations were inverted in a latin square experimental design to produce a total of five different pools , labeled as arrays 1 - 5 ( table s2 ) . [SEP]
[CLS] these five pools were hybridized to five separate mirnaarrays and analyzed with the scano - mir system as described above . [SEP]
[CLS] each mirna - array was developed once with gold B-material enhancing solution for 5 minutes and the image was recorded using the tecan scanner . [SEP]
[CLS] to perform the direct sensitivity comparison of the scano - mir assay to the fluorophorebased approach , a cy5 - labeld ssdna recognition sequence complementary to linker ii with a 10 adenosine base ( a10 ) spacer B-material was purchased from idt ( 5 ' - cy5 - ( a ) 10 - tccttggtgcccgagtg - 3 ' ) . [SEP]
[CLS] eight synthetic mirnas ( mir - 200c , −21 , −210 , −205 , −20a , −143 * , −143 , and −16 ) were synthesized , ligated to the universal mirna cloning linker ii , and added in 2x ssc at 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 , and 1 fm , respectively ( see table s1 for sequences ) . [SEP]
[CLS] the sensitivity of cy5 - labeled arrays was measured as follows . [SEP]
[CLS] a 400 µl aliquot of the mirna solutions was hybridized overnight at 40°c to a separate mirna - array . [SEP]
[CLS] following the incubation B-technique , the array was washed with a pre - warmed ( 40°c ) 2x ssc for two min , followed by 2 min immersions in the same rnase - free wash solutions described above . [SEP]
[CLS] this array was labeled by adding 1 nm of the cy5 - labeled probe in 400 µl rnase - free 2x ssc at 40°c for 1 hr . [SEP]
[CLS] unreacted cy5 - labeled probes were washed away with pre - warmed ( 40°c ) 2x ssc followed by a 2 min immersion in the same rnasefree solutions as described above . [SEP]
[CLS] labeled arrays were scanned immediately . [SEP]
[CLS] another aliquot of the mirna solutions used above ( 400 µl ) was hybridized to a separate mirnaarray and analyzed with the scano - mir system as described above and scanned after each round of gold B-material deposition to achieve a total of three rounds of gold B-material deposition , each for 5 min . [SEP]
[CLS] similar protocols were applied to obtain complete mirna profiles from 0 . 8 µg total rnas isolated from the prostate cancer cell B-material line ( pc - 3 ) . [SEP]
[CLS] the signal intensity values , calculated using genepix pro 6 software , were generated from arrays analyzed with both the scano - mir system and the cy5 - labeled arrays and used to perform the direct sensitivity comparison . [SEP]
[CLS] to investigate the ability of the scano - mir system to detect mirnas from human serum ( see table s3 ) , total rna isolated from 5 ml serum ( see supporting information ) was added to a solution of truncated t4 rna ligase 2 , mirna cloning linker ii , peg 8000 , and t4 rnl2 at final concentrations of 200 u , 900 ng , 12 % , and 1x , respectively , and incubated B-technique for 3 hrs at 37°c . [SEP]
[CLS] following incubation B-technique , the ligated species were analyzed with the scano - mir system as described above . [SEP]
[CLS] the signal intensity values , calculated using genepix pro 6 software , were generated from two scano - mir arrays after mirna profiling two replicates of 5 ml serum . [SEP]
[CLS] signal intensity values were used to calculate the correlation coefficient between the two biological replicates . [SEP]
[CLS] to realize a high throughput scanomir system , we had to create a universal probe that could be utilized to develop an array in a single set of assay steps ( scheme 1 ) . [SEP]
[CLS] in a typical assay , mirna targets in a sample mixture are ligated to a universal mirna cloning linker ( see table s1 for sequences ) using truncated t4 rna ligase ii . [SEP]
[CLS] importantly , the products of the ligation reaction are added directly to the mirna - array ( ncode human mirna microarray v3 , invitrogen ) without the need for purification steps , thereby avoiding the loss of low abundance mirnas . [SEP]
[CLS] the mirna - array consists of a pattern of multiple dna sequences that are complementary to mirna sequences of interest . [SEP]
[CLS] target mirna sequences that are present in the sample hybridize with appropriate complement sequences at unique spots on the array . [SEP]
[CLS] following hybridization , unreacted linkers , unbound mirna strands , and ligation enzymes are then easily washed away . [SEP]
[CLS] the universal sna - au nps B-nanoparticle , which contain a dna sequence that is complementary to the universal mirna cloning linker ( see table s1 for the sequence ) , are then added to the mirna - array . [SEP]
[CLS] hybridization is performed at 56 °c to prevent off - target hybridization of the sna - au nps B-nanoparticle . [SEP]
[CLS] dehybridized mirnas and unreacted sna - au nps B-nanoparticle are washed away , and the mirna - array is subjected to gold B-material enhancement with tetrachloroaurate and hydroxylamine . [SEP]
[CLS] this amplification process deposits au 0 onto the immobilized sna - au nps B-nanoparticle , and dramatically increases their light scattering properties . [SEP]
[CLS] the lod of the scano - mir assay was determined by adding synthetic mir - 16 strands into denatured human serum at known concentrations . [SEP]
[CLS] to avoid detecting endogenous mir - 16 , synthetic mir - 16 ( see table s1 for the sequence ) was pre - ligated with the universal linker prior to the synthetic mir - 16 addition step , and the endogenous mirnas were not ligated to the universal linker . [SEP]
[CLS] the lod for the synthetic mir - 16 mirna was determined to be 1 fm ( ~ 600 copies / µl serum ) without pcr amplification ( figure 1a ) . [SEP]
[CLS] the high sensitivity of the scano - mir system stems from the increased light scattering properties of the nanoparticles B-nanoparticle upon gold B-material deposition . [SEP]
[CLS] in addition to high sensitivity , high selectivity is equally important if one wants to differentiate low abundance mirnas that are closely related , including those that may only vary by a single nucleotide . [SEP]
[CLS] to test the specificity of the scano - mir system , synthetic mir - 16 was hybridized to a mirna - array that displayed two types of capture probes : one that was perfectly matched to mir - 16 , and another one with a single nucleotide mismatch ( the position of the mismatch is not disclosed by the commercial supplier of the array ) . [SEP]
[CLS] the intensities of the scattered light from both the perfectly matched capture probes ( rno - mir - 16 ) and the capture probes with a single nucleotide mismatch ( mut1 - rno - mir - 16 ) , respectively , were compared . [SEP]
[CLS] the scano - mir assay exhibits single nucleotide mismatch specificity down to an lod of 1 fm ( figure 1a ) . [SEP]
[CLS] the analytical performance of the scano - mir system was investigated through a comparative hybridization of a set of five synthetic mirnas ( table s1 ) in a latin square experimental design . [SEP]
[CLS] this design is a five - by - five matrix wherein detection of targets within pools of mirnas provides the opportunity to compare the quantitative array response for multiple targets , assess the quantitative capabilities of the assay as a function of varying target concentrations , and determine the variability of different arrays without the need to individually probe a large number of test samples . [SEP]
[CLS] in a typical experiment , test samples were created by loading hybridization buffer with four different synthetic mirnas at concentrations of 10 2 , 10 3 , 10 4 , and 10 5 fm target , respectively , and compared to buffer without target . [SEP]
[CLS] the concentrations of each of the targets were sequentially changed four more times , according to the original concentration profile , to complete the five - by - five matrix ( see table s2 for latin square design ) . [SEP]
[CLS] the five mixtures of synthetic mirnas were hybridized to five separate mirna - arrays and then studied via the scano - mir system ( figure 1b ) . [SEP]
[CLS] plotting signal intensity versus mirna concentrations provides a correlation coefficient of r 2 = 0 . 97 , which confirms the profiling reliability of the scano - mir assay and its low variability from one array to another ( figure 1b ) . [SEP]
[CLS] as a result , the scano - mir assay is able to simultaneously detect multiple mirna targets over a range of concentrations that vary by at least four orders of magnitude with high accuracy and reproducibility across arrays . [SEP]
[CLS] a comparison of the conventional microarrays , which utilize fluorophore probes to detect mirnas , with the scano - mir methodology was directly evaluated to determine the sensitivity advantage afforded by the sna - au np B-nanoparticle conjugates . [SEP]
[CLS] consistent with literature reports , the fluorophore - based approach with cy5 - labeled probes allows one to detect targets over the 10 nm to 100 pm concentration range ( figure 2a , b ) . one advantage of the scano - mir system is the ability to perform sequential metal B-material deposition steps to intensify signal in a controllable manner . [SEP]
[CLS] the scano - mir system is able to detect mirnas from 1 nm to 100 fm after one round of gold B-material deposition , resulting in a dynamic range of four orders of magnitude . [SEP]
[CLS] however , additional rounds of gold B-material deposition improve the limit of detection down to 1 fm ( figures 2a , b ) . [SEP]
[CLS] importantly , the ability to perform sequential metal B-material depositions allows the user to tune the dynamic range of the scano - mir system to detect high or low abundance mirnas over a wide overall dynamic range ( ~ 6 orders of magnitude ) ( figures 2a , b ) without the need to perform separate hybridization experiments which would require additional samples . [SEP]
[CLS] to further compare the capabilities of fluorescence B-property and cy5 - labeled probes to sna - au nps B-nanoparticle of the scano - mir approach , experiments were performed to detect mirnas isolated from a prostate cancer cell B-material line ( pc - 3 ) . [SEP]
[CLS] interestingly , the scano - mir assay identified 97 % of those targets identified using the fluorophore - based approach , and detected 88 % more mirna species than the cy5 - based detection method ( figure 2c ) . [SEP]
[CLS] taken together , these data highlight the potential of the scano - mir system to generate a more complete profile of mirna expression , especially for low - abundance mirnas . [SEP]
[CLS] after determining the scano - mir system ' s performance by directly comparing it with conventional fluorophore - based technology , we used it to examine mirnas recovered from human serum ( figure 3a ) ( see table s3 for information about donor 277 ) . [SEP]
[CLS] a rank - order list of the serum mirnas detected is provided in the supporting information ( table s4 ) . [SEP]
[CLS] these results demonstrate that the scano - mir system can profile endogenous mirnas isolated from human serum in a multiplexed and high - throughput fashion . [SEP]
[CLS] two biological replicates of serum mirna isolates were also used to hybridize mirnas onto two different mirnaarrays and detected using the scano - mir system . [SEP]
[CLS] signal intensities generated from both mirna - arrays were quantified and used to examine the extent of the scano - mir system ' s reproducibility in the presence of biological background . [SEP]
[CLS] the correlation coefficient of signal intensities between the two biological replicates was 0 . 95 ( figure 3b ) . [SEP]
[CLS] arrays are useful for profiling mirnas from samples that have been treated differently in order to identify mirnas that are being regulated by a chosen intervention . [SEP]
[CLS] accordingly , we sought to demonstrate the capability of the scano - mir technology for the semi - quantitative detection of known mirnas and compare the results using qrt - pcr . [SEP]
[CLS] we chose to compare the mirna profiles from pc - 3 cells B-material following hypoxia inducible factor 1 - alpha ( hif - 1a ) stabilization using cobalt B-material chloride B-material ( cocl 2 ) treatment . [SEP]
[CLS] the mirna expression of cocl 2treated pc - 3 cells B-material was compared to that of pc - 3 cells B-material without cocl 2 treatment . [SEP]
[CLS] more specifically , mir - 210 has been called a " hypoxamir " due to its robust increase upon hif - 1a stabilization . [SEP]
[CLS] cobalt B-material chloride B-material treatment results in a 3 . 85 fold increase in mir - 210 levels ( figure 4a ) . [SEP]
[CLS] this difference in magnitude is consistent with our qrt - pcr results ( figure 4b ) and with literature reports . [SEP]
[CLS] finally , we investigated the applicability of the scano - mir assay to tissue samples with human prostate cancer ( cap ) . [SEP]
[CLS] in the us , cap is the most frequent noncutaneous malignancy and the second leading cause of cancer - related death among men . [SEP]
[CLS] contemporary studies have demonstrated conflicting results on the utility of prostate specific antigen ( psa ) and have called into question psa screening due to the overdiagnosis of so - called insignificant cancer and its treatment . [SEP]
[CLS] cap screening includes a digital rectal exam and serum psa testing . [SEP]
[CLS] an elevated serum psa value often prompts a prostate biopsy for definitive diagnosis and to provide pathological staging based on the gleason classification system . [SEP]
[CLS] the gleason score ( range 2 - 10 ) is determined by a pathologist after microscopic analysis of the prostate tissue . [SEP]
[CLS] the gleason score is based on the sum of the two predominant microscopic gland architectures ( the microscopic morphology and the glandular differentiation patterns ) , and a higher gleason score is associated with more aggressive disease . [SEP]
[CLS] in addition to current methods that include psa measurements and pre - treatment gleason score to assess risk of aggressive cancer , mirna profiles may provide diagnostic insight into the biological potential of insignificant versus aggressive cap prior to therapy , as has been shown for other cancers , in order to optimally risk - stratify patients for treatment . [SEP]
[CLS] as a proof - of - concept , two rna samples from cap specimens were studied in order to compare the expression profiles of 706 mirnas in high gleason grade versus in low gleason grade cap samples ( samples cr562458 and cr562503 , details described in table s3 ) . [SEP]
[CLS] the rna ( 0 . 8 µg ) from each sample was analyzed by the scano - mir assay , and a total of 163 mirnas were determined to be differentially expressed as defined by 1 . 5 fold difference in signal ; 109 mirnas were downregulated and 54 mirnas were upregulated ( table s5 ) . [SEP]
[CLS] based on the identity of these 163 mirnas , target genes were retrieved and further analyzed by disease ontology gene enrichment analysis . [SEP]
[CLS] this search revealed 94 cap related target genes . [SEP]
[CLS] as validation , these cap related genes were mapped to the 163 deregulated mirnas , and 161 mirnas ( 98 . 8 % ) were obtained ( figure s1 , table s5 ) . [SEP]
[CLS] as a result , the scano - mir system was able to detect deregulated mirnas that might be important for prostatic progression of high gleason grade cap with 98 . 8 % accuracy ( 161 / 163 ) . [SEP]
[CLS] moreover , we performed functional analysis to construct a function - gene - mirna network for 35 select mirnas and 13 target genes that are involved in 5 hallmarks of cap like regulation of apoptosis B-event , epigenetic changes , cell B-material migration and differentiation , and response to hormone stimuli ( figure 5 , table s6 ) . [SEP]
[CLS] the mirna expression profiling of cap by the scano - mir assay has revealed deregulated mirnas that might play critical roles in the regulation of cap tumorigenesis . [SEP]
[CLS] while some of these mirnas may prove to be novel biomarkers B-property , a larger clinical study is essential as a follow - up to this proof - of - concept one . [SEP]
[CLS] in conclusion , we have developed , characterized , and demonstrated the utility of a novel scano - mir platform for mirna detection . [SEP]
[CLS] conclusionsmirnas are ideal targets for recognition using the scano - mir platform due to their short length , the inability to directly pcr amplify mirnas , and their presence at relatively low abundance in many biological samples . [SEP]
[CLS] this simple yet powerful scano - mir assay design allows rapid and simultaneous quantification of all known mirna sequences with minimal modification and purification steps , which provides a highly attractive alternative to current conventional methods . [SEP]
[CLS] the scano - mir system is able to detect low abundance mirna species due to the employment of a light scattering amplification protocol , which provides a lower lod than fluorophore - based detection systems . [SEP]
[CLS] the narrow melting transitions associated with duplexes formed from sna - au np B-nanoparticle conjugates leads to the ability to differentiate perfectly complementary mirna targets from ones with single base mismatches , and are responsible for the high specificity of this novel high throughput assay . [SEP]
[CLS] the scano - mir system has the potential to allow identification of novel mirna targets for a wide variety of biomedical targets , especially those that pertain to cancer diagnostic applications . [SEP]
[CLS] ( a ) detection of human serum mirnas ( from donor 277 , table s6 ) using the scano - mir system was performed after hybridizing recovered mirnas onto a high - density mirnaarray and recorded using the tecan scanner , where the gray scale image was converted into color using genepix pro 6 software ( molecular devices ) . [SEP]
[CLS] each spot corresponds to a single mirna and the intensity of the color corresponds to the mirna concentrations . [SEP]
[CLS] ( b ) biological replicates were analyzed at two different time points using the scano - mir system . [SEP]
[CLS] the signal intensity values , calculated using genepix pro 6 software , are plotted to calculate the correlation coefficient ( r 2 = 0 . 95 ) . [SEP]
[CLS] the log2 of the signal intensities are plotted . [SEP]
[CLS] a function - gene - mirna network of 35 deregulated mirnas was analyzed that target 13 prostate - cancer related genes involved in 5 gene ontology terms . [SEP]
[CLS] a large yellow node in the network represents a single gene ontology term , while the small blue nodes stand for genes . [SEP]
[CLS] green and red nodes stand for mirnas with negative and positive logr ratios , respectively . [SEP]
[CLS] the saturation is proportional to the absolute value of logr ratio . [SEP]
[CLS] for the scanometric array - based multiplexed detection of mirna species ( scano - mir ) . [SEP]
[CLS] isolated mirnas are enzymatically ligated to a universal linker followed by hybridization onto mirna microarray . [SEP]
[CLS] after washing away unbound mirna species , universal sna - functionalized gold B-nanoparticle nanoparticle I-nanoparticle conjugates ( sna - au nps B-nanoparticle ) are subsequently hybridized to detect captured mirna targets . [SEP]
[CLS] next , signal intensity is amplified by depositing gold B-material with gold B-material enhancing solution ( 1 : 1 ( v : v ) mixture of 1 mm haucl 4 and 10 mm nh 2 oh ) for 5 min and imaged with a scanner ( ls reloaded , tecan , salzburg , austria ) . [SEP]
[CLS] 1 . ( a ) synthetic mir - 16 was added into denatured human serum at different concentrations and analyzed using the scano - mir platform . [SEP]
[CLS] signal intensities generated from both perfectly matched capture probe sequences ( rno - mir - 16 ) and capture probe with a single nucleotide mismatch ( mut1 - rno - mir - 16 ) were plotted . [SEP]
[CLS] ( b ) comparative hybridization of five synthetic mirnas ( mirna - 20a , −143 , −143 * , −205 , and −210 ) in a latin square design was performed to complete a five by five matrix ( tables5 , see experimental sections for experimental details ) . [SEP]
[CLS] plotting signal intensity versus mirna concentrations generated r 2 = 0 . 97 . [SEP]
[CLS] ( a ) profile of synthetic mirnas at different concentrations and ( c ) profile of total mirnas from pc - 3 using the scano - mir assay was plotted and compared to conventional cy5 - based mirna - array . [SEP]
[CLS] ( b ) the low - end detection of ( a ) is expanded . [SEP]
[CLS] ( c ) inset : scale for the background is expanded . [SEP]
[CLS] determining the change in expression level of mir - 210 in pc - 3 cells B-material in normoxia and hypoxia conditions using ( a ) the scano - mir assay and compare the results using ( b ) qrt - pcr . [SEP]
[CLS] figure 4 . a ) u6 serves as an endogenous loading control . [SEP]
[CLS] b ) concentrations on y - axis were calculated based on known spiked - in mir - 210 concentrations . [SEP]
[CLS] cellular transfection of nucleic B-material acids I-material is necessary for regulating gene expression through antisense or rnai pathways . [SEP]
[CLS] the development of spherical nucleic B-material acids I-material ( snas , originally gold B-nanoparticle nanoparticles I-nanoparticle functionalized with synthetic oligonucleotides ) has resulted in a powerful set of construct that are able to efficiently transfect cells B-material and regulate gene expression without the use of auxiliary cationic B-material co - carriers . [SEP]
[CLS] the gold B-material core B-material in such structures is primarily used as a template to arrange the nucleic B-material acids I-material into a densely packed and highly oriented form . [SEP]
[CLS] in this work , we have developed methodology for coating B-material the gold B-material particle with a shell B-material of I-material silica I-material , modifying the silica with a layer of oligonucleotides , and subsequently oxidatively dissolving the gold B-material core B-material with i 2 . [SEP]
[CLS] the resulting hollow silica - based snas exhibit cooperative binding behavior with respect to complementary oligonucleotides and cellular uptake properties comparable to their gold B-material - core sna counterparts . [SEP]
[CLS] importantly , they exhibit no cytotoxicity B-property and have been used to effectively silence the egfp gene in mouse endothelial cells B-material through an anti - sense approach . [SEP]
[CLS] spherical nucleic B-material acid I-material ; oligonucleotide ; silica B-nanoparticle nanoparticle I-nanoparticle ; gene regulation recent work has shown that spherical nucleic B-material acids I-material ( snas ) , structures consisting of linear nucleic B-material acids I-material that are highly oriented and densely packed on the surface of a spherical B-nanoparticle nanoparticle I-nanoparticle ( np B-nanoparticle ) , exhibit the ability to efficiently enter cells B-material without a transfection agent . [SEP]
[CLS] [SEP]
[CLS] this is in contrast to free linear nucleic B-material acids I-material , which generally require a cationic B-material moiety to neutralize their negative charge in order to pass through the cellular membrane . [SEP]
[CLS] 3 however , these cationic B-material lipids B-material and polymers B-material often display cytotoxic B-property effects at high concentrations and [SEP]
[CLS] the inability to be degraded biologically [SEP]
[CLS] sna - np B-nanoparticle conjugates thus provide a unique platform for internalizing large quantities of nucleic B-material acids I-material into cells B-material under mild conditions that can subsequently be used for intracellular detection and gene regulation . [SEP]
[CLS] thus far , we have shown that scavenger receptors mediate the cellular entry of snas , and cellular uptake is dependent on the density of nucleic B-material acids I-material on the nanoparticle B-nanoparticle surface . [SEP]
[CLS] furthermore , sna - np B-nanoparticle conjugates have a unique set of properties that are advantageous for intracellular applications , including high binding coefficients for complementary dna and rna , nuclease resistance , and minimal immune response . [SEP]
[CLS] with respect to cellular internalization and activity , these observations are all based upon the hypothesis that the unique properties of the sna architecture stem from the oligonucleotide shell B-material and the density and orientation of the nucleic B-material acids I-material that comprise it as opposed to the nanoparticle B-nanoparticle core B-material . [SEP]
[CLS] importantly , we recently demonstrated a synthetic route for making hollow snas by crosslinking oligonucleotides on the surface of gold B-nanoparticle nanoparticles I-nanoparticle and subsequently dissolving the gold B-material particle template . [SEP]
[CLS] consistent with our hypothesis , these structures are capable of cellular internalization and gene regulation via anti - sense and rnai pathways . [SEP]
[CLS] the hollow structures are attractive , especially if one is concerned about the long - term toxicity B-property of the gold B-nanoparticle nanoparticle I-nanoparticle core B-material . [SEP]
[CLS] the disadvantage of the approach is that specialty oligonucleotides capable of cross - linking are required , and at present , they are prohibitively expensive . [SEP]
[CLS] these observations pose the challenge of identifying other chemical routes to hollow sna structures that possess similar properties to those derived from gold B-material particles and perhaps offer even greater capabilities . [SEP]
[CLS] herein we report a new class of core - free sna conjugate consisting of a biocompatible B-property porous silica B-material shell I-material . [SEP]
[CLS] by using a silica - coated gold B-nanoparticle nanoparticle I-nanoparticle as a template , we can easily functionalize it with nucleic B-material acids I-material using a wide variety of coupling strategies and relatively simple and readily available coupling molecules . [SEP]
[CLS] significantly , the silica B-material shell I-material acts as a cross - linked scaffold to assemble oriented oligonucleotides with a porous architecture that allows one to chemically dissolve the gold B-material core B-material . [SEP]
[CLS] the hollow silica snas maintain the unique properties of the sna gold nanoparticle I-nanoparticle conjugates and exhibit the ability to be internalized by cells B-material without a transfection agent and efficiently knock down a target mrna sequence . [SEP]
[CLS] moreover , silica is an attractive material B-material from a biological perspective since it is known to degrade into bio - inert silicic acid under physiological conditions . [SEP]
[CLS] previous studies of porous silica B-nanoparticle nanoparticles I-nanoparticle have shown a degradation rate of approximately 15 % per day in a cellular environment . [SEP]
[CLS] in principle , these new sna conjugates should degrade over time and release active oligonucleotides . [SEP]
[CLS] to prepare the silica ( sio 2 ) shells B-material , 13 nm citrate - stabilized gold B-nanoparticle nanoparticles I-nanoparticle ( au np B-nanoparticle ) were synthesized to serve as sacrificial templates . [SEP]
[CLS] the au nps B-nanoparticle were passivated with a short polyethylene glycol ( peg ) chain containing a thiol B-material functional B-material group I-material on one end and a carboxylic B-material acid I-material on the other ( sh - ( ch 2 ) 11 - ( eg ) 6 - och 2 - cooh ) and redispersed in ethanol . [SEP]
[CLS] the au nps B-nanoparticle were directly coated with a thin layer ( ~ 15 nm ) of silica using an ammoniacatalyzed hydrolysis of tetraethyl orthosilicate ( teos ) and subsequent condensation of silicic acid to give a network of tetrahedral sio 4 units with shared vertices . [SEP]
[CLS] the thickness of the silica B-material shell I-material can easily be controlled by changing the relative concentrations of au nps B-nanoparticle , water B-material , ammonia , and silicon B-material alkoxide in the reaction . [SEP]
[CLS] the resulting au core - silica B-material shell I-material ( au @ sio 2 ) particles were heated at 60°c for 24 hours to ensure a homogeneous silica B-material shell I-material ( see experimental details in supporting information ) . [SEP]
[CLS] to achieve a dense layer of dna on the silica B-material shell I-material surface , the heterobifunctional cross linker p - maleimidophenyl isocyanate ( pmpi ) was used since cross - linkers with aminereactive isocyanates have demonstrated improved retention of maleimide activity compared with nhs - ester based linkers . [SEP]
[CLS] the au @ sio 2 nps B-nanoparticle were first derivatized with ( aminopropyl ) triethoxysilane ( aptes ) and subsequently activated with amine - reactive pmpi to introduce thiol - reactive maleimide B-material groups I-material . [SEP]
[CLS] the addition of dna oligonucleotides designed to target an mrna sequence coding for enhanced green fluorescent B-property protein B-material ( egfp ) with terminal propylthiol groups ( 3 ' sh ( c 3 h 6 ) - aaaaaaaaaaggtgttcaagtcgcacaggc5 ' ) , followed by the slow addition of nacl to 0 . 3 m , led to the formation of polyvalent au @ sio 2 particles with a loading of ~ 75 strands per particle . [SEP]
[CLS] upon selective oxidative dissolution of the au np B-nanoparticle core B-material with i 2 , hollow sio 2 snas are formed ( figure 1 ) . [SEP]
[CLS] finally , the silica snas were dialyzed overnight against water B-material to remove i 2 remaining in solution . [SEP]
[CLS] the ability of i 2 to oxidatively dissolve the gold B-material core B-material indicates that the silica B-material shells I-material remain porous through the heating and dna functionalization steps . [SEP]
[CLS] the au @ sio 2 and gold B-material - free sio 2 particles were characterized by scanning B-technique transmission I-technique electron I-technique microscopy I-technique ( stem ) in scanning , z - contrast , and transmission modes ( figure 2 , a - c and d - f respectively ) . [SEP]
[CLS] indeed , the microscopy B-technique images indicate that the au np B-nanoparticle cores B-material are entirely dissolved upon the addition of i 2 and a hollow interior remains . [SEP]
[CLS] importantly , the silica B-material shells I-material remain as discrete particles and maintain their structure upon dissolution of the gold B-material core B-material , a conclusion also verified by dynamic B-technique light I-technique scattering I-technique ( dls ) . [SEP]
[CLS] initially the au nps B-nanoparticle have an average hydrodynamic radius of 17 . 3 ± 0 . 8 nm . [SEP]
[CLS] after the deposition of the silica B-material shells I-material onto the au np B-nanoparticle templates , this value increased to 47 . 7 ± 10 . 1 nm . [SEP]
[CLS] upon functionalization with dna using aptes and pmpi , the final hydrodynamic radius was measured to be 85 . 8 ± 16 . 4 nm . [SEP]
[CLS] it should be noted that the dls measurements of the hydrated particles are slightly larger than the diameters of the dry particles measured with electron B-technique microscopy I-technique . [SEP]
[CLS] however , the trend in the dls data is indicative of growth at each step in the synthesis without the formation of large aggregates . [SEP]
[CLS] the synthesis of the silica snas was also monitored with uv - vis spectroscopy B-technique ( figure 3a ) . [SEP]
[CLS] the uv - vis spectra reveal that the au @ sio 2 particles exhibit a distinct absorption at ~ 530 nm that is characteristic of dispersed gold B-nanoparticle nanoparticles I-nanoparticle albeit slightly red shifted compared to au nps B-nanoparticle due to the increase in the dielectric constant of the silica B-material shell I-material . [SEP]
[CLS] after the addition of i 2 , the absorption band at 530 nm is no longer present , consistent with removal of the au core B-material . [SEP]
[CLS] previously , we have shown that snas , when hybridized with complementary oligonucleotides , exhibit narrow melting transitions compared with free dna strands due to a high degree of cooperative binding . [SEP]
[CLS] this phenomenon is also observed for hollow snas consisting of cross - linked nucleic B-material acids I-material . [SEP]
[CLS] due to the layer of highly oriented oligonucleotides on the surface of the silica B-material shells I-material ( ~ 75 dna strands per particle when salted to 0 . 3m nacl , determined by the oligreen ® assay ) , it was hypothesized that both the au @ sio 2 particles and the hollow silica snas would exhibit cooperative binding behavior as well . [SEP]
[CLS] upon the addition of a self - complementary linker strand that binds to the oligonucleotides on the silica surface , the particles formed visible aggregates , which were then dehybridized by slowly increasing the temperature . [SEP]
[CLS] as shown in figure 3b , the aggregated sio 2 particles exhibit a sharp melting transition ( fwhm of the derivative = ~ 2°c ) , indicative of cooperative behavior . [SEP]
[CLS] once it was confirmed that the au @ sio 2 and core - free sio 2 snas retain the cooperative binding properties of au nps B-nanoparticle densely functionalized with nucleic B-material acids I-material , the ability of these particles to be transfected into cells B-material and facilitate gene regulation via the anti - sense pathway was investigated . [SEP]
[CLS] to qualitatively access the cellular uptake of the sio 2 snas , particles with and without the au np B-nanoparticle core B-material were functionalized with cy5 dye - labeled anti - egfp dna oligonucleotides . [SEP]
[CLS] the particles ( 5 nm ) were then incubated B-technique overnight with c166 mouse endothelial cells B-material stably expressing the egfp gene . [SEP]
[CLS] it is important to note that no cationic B-material transfection agent was included during the incubation B-technique step . [SEP]
[CLS] the c166 cells B-material were washed , fixed , and imaged by laser scanning confocal microscopy B-technique . [SEP]
[CLS] as shown in figure 4a , both the au @ sio 2 and the hollow sio 2 particles are taken into the cytoplasm of the c166 cells B-material . [SEP]
[CLS] the mechanism of cellular uptake of snas has previously been demonstrated to involve receptor - I-event mediated endocytosis B-event and stems from the dense , highly oriented layer of nucleic B-material acids I-material 2 . [SEP]
[CLS] it is therefore hypothesized that a similar mechanism applies for the dna functionalized au @ sio 2 and hollow sio 2 particles . [SEP]
[CLS] note that neither of these particles enters the nuclei of the cells B-material because of their size . [SEP]
[CLS] images of planes collected at various depths within the cell B-material samples ( z - stack ) further confirmed cellular uptake ( see supporting information ) . [SEP]
[CLS] the silica - based snas were next evaluated for their ability to regulate target genes . [SEP]
[CLS] au @ sio 2 particles , hollow sio 2 particles , and au nps B-nanoparticle were functionalized with anti - egfp dna oligonucleotides and incubated B-technique ( 5 nm ) with c166 cells B-material stably expressing egfp . [SEP]
[CLS] particles functionalized with non - targeting scrambled dna oligonucleotides served as a negative control . [SEP]
[CLS] the cells B-material were then collected , lysed , and analyzed for their egfp mrna levels by quantitative real - time reverse transcriptase polymerase chain reaction ( qrt - pcr ) . [SEP]
[CLS] mrna levels of cells B-material treated with anti - egfp au @ sio 2 particles and core - free sio 2 shells B-material were significantly reduced by 68 % and 50 % respectively when compared with those treated with scrambled " nonsense " dna particles ( figure 4b ) . [SEP]
[CLS] importantly , the amount of knockdown is comparable to that observed with dna functionalized au nps B-nanoparticle ( ~ 52 % ) . [SEP]
[CLS] furthermore , the au @ sio 2 particles and hollow sio 2 snas were shown to exhibit low cytotoxicity B-property toward c166 cells B-material using the 3 - ( 4 , 5 - dimethylthiazol - 2 - yl ) - 2 , 5 - diphenyl tetrazolium bromide B-material ( mtt ) assay . [SEP]
[CLS] as indicated in figure 4c , cells B-material treated with the targeted and non - targeted dna oligonucleotides for 8 hours showed up to 95 % viability compared with a non - treated control . [SEP]
[CLS] taken together , these data suggest that this new class of core - free sna retains the unique properties of the au np B-nanoparticle snas , including efficient cellular uptake without the need for conventional cationic B-material transfection agents , a high capacity for gene regulation , and low cytotoxicity B-property . [SEP]
[CLS] the present work describes a simple , scalable , and biocompatible B-property sna construct that serves to confirm our hypothesis that the emergent properties of snas are a result of the layer of oriented oligonucleotides and not the inorganic B-nanoparticle nanoparticle I-material core I-material . [SEP]
[CLS] the use of silica as the crosslinking reagent makes this construct extremely versatile ; the thickness and porosity of the silica B-material shell I-material is tunable with reaction conditions and many well - established coupling chemistries including edc / nhs ester , copper - catalyzed or copper - free click chemistry , and reductive amination B-event can be utilized to achieve a densely packed , oriented nucleic B-material acid I-material shell B-material . [SEP]
[CLS] additionally , there are potential benefits of a hollow architecture including tunable degradation profiles and the ability to load the hollow interior with biologically relevant molecules or drugs . [SEP]
[CLS] indeed , these novel core - free sio 2 snas represent a new class of sna that shows promise for a wide range of intracellular gene regulation applications and , therefore , constitutes a new class of nanotherapeutics . [SEP]
[CLS] synthesis of dna functionalized hollow sio 2 particles using gold B-nanoparticle nanoparticles I-nanoparticle as sacrificial templates . [SEP]
[CLS] ( a ) extinction spectra of au np B-nanoparticle templates , silica - coated au nps B-nanoparticle ( au @ sio 2 ) , and hollow sio 2 particles resulting from the treatment of the au @ sio 2 particles with i 2 . [SEP]
[CLS] the au @ sio 2 particles exhibit a distinct absorption at ~ 530 nm that is characteristic of au np B-nanoparticle , albeit slightly red - shifted due to the silica B-material shell I-material . [SEP]
[CLS] after treatment with i 2 , the hollow sio 2 particles do not contain this absorption band , confirming the dissolution of the au np B-nanoparticle core B-material . [SEP]
[CLS] ( b ) melting analysis of dna functionalized sio 2 particles hybridized using a selfcomplementary linker . [SEP]
[CLS] the sharp melting transition ( full - width half - max = ~ 2°c ) is indicative of cooperative binding . [SEP]
[CLS] 2 . stem images of au @ sio 2 particles ( a - c ) and hollow sio 2 particles ( d - f ) in scanning , zcontrast , and transmission modes , respectively . [SEP]
[CLS] in a - c , the au np B-nanoparticle cores B-material are visible . [SEP]
[CLS] after the addition of i 2 , the au np B-nanoparticle cores B-material dissolve leaving a hollow interior ( e - f ) . [SEP]
[CLS] scale bars are 100 nm . [SEP]
[CLS] 4 . cellular uptake of the dna functionalized au @ sio 2 and dna functionalized hollow sio 2 particles into c166 cells B-material . [SEP]
[CLS] ( a ) c166 cells B-material were treated with au @ sio 2 particles ( upper panel ) and hollow sio 2 particles ( lower panel ) functionalized with cy5 dye - labeled anti - egfp dna oligonucleotides . [SEP]
[CLS] cy5 fluorescence B-property is observed in the cytoplasm , but not in the nuclei , indicating the internalization of the particles into the cells B-material . [SEP]
[CLS] scale bar = 20 μm . [SEP]
[CLS] ( b ) egfp knockdown at the mrna level determined by qrt - pcr . [SEP]
[CLS] figure 4 . mrna levels of cells B-material treated with anti - egfp dna oligonucleotide functionalized au @ sio 2 and hollow sio 2 particles were significantly reduced when compared with those treated with scrambled - sequence dna particles . [SEP]
[CLS] ( c ) minimal cell B-material toxicity B-property of the au @ sio 2 and hollow sio 2 particles toward c166 cells B-material was detected using the mtt B-technique assay I-technique . [SEP]
[CLS] cells B-material treated with the egfp - targeted and nontargeted dna oligonucleotides showed up to 95 % viability compared with non - treated cells B-material . [SEP]
[CLS] spherical nucleic B-material acid I-material ( snas ) constructs are promising new single entity gene regulation materials capable of both cellular transfection and gene knockdown , but thus far are promiscuous structures , exhibiting excellent genetic but little cellular selectivity . [SEP]
[CLS] in this communication , we describe a strategy to impart targeting capabilities to these constructs through non - covalent functionalization with a complementary antibody - dna conjugate . [SEP]
[CLS] as a proof - of concept , we designed her2 - targeting snas and demonstrated that such structures exhibit cell type selectivity in terms of their uptake , and significantly greater gene knockdown in cells B-material overexpressing the target antigen as compared to the analogous antibody - free and off - target materials . [SEP]
[CLS] spherical nucleic B-material acids I-material ( snas ) are a novel class of non - viral gene regulation agents that are developing rapidly alongside conventional cationic B-material polymer B-material and liposomal B-nanoparticle systems . [SEP]
[CLS] typically , these structures are comprised of densely functionalized and highly oriented nucleic B-material acids I-material covalently attached to the surface of a metallic , semiconducting , or insulating inorganic or polymeric B-material core I-material material B-material . [SEP]
[CLS] they also can be core - less , hollow structures composed almost entirely of nucleic B-material acid I-material molecules . [SEP]
[CLS] 12 such constructs are capable of bypassing the natural defenses of biological systems for exogenous nucleic B-material acids I-material and inhibiting the expression of certain target genes through either antisense or sirna pathways . [SEP]
[CLS] [SEP]
[CLS] consequently , snas offer several advantages over viral vectors and many other synthetic systems , including low toxicity B-property , low immunogenicity B-property , resistance to enzymatic degradation , and more persistent gene knockdown . [SEP]
[CLS] [SEP]
[CLS] conventional approaches for transporting nucleic B-material acids I-material into the cytoplasm involve their complexation with cationic B-material polymers B-material or nanoparticles B-nanoparticle , or the use of viral capsids . [SEP]
[CLS] these structures serve two primary purposes : they protect the nucleic B-material acid I-material from degradation and facilitate cellular uptake and intracellular transport . [SEP]
[CLS] the sna , on the other hand , achieves protection and efficient delivery of nucleic B-material acids I-material utilizing unique properties arising from its densely packed , highly oriented nucleic B-material acid I-material shell B-material . [SEP]
[CLS] we have shown that such shells B-material create areas of high local salt B-material concentration , which when combined with steric inhibition , serve to reduce nuclease activity and protect the nucleic B-material acids I-material from enzymatic degradation . [SEP]
[CLS] in addition , these snas recruit scavenger proteins B-material to their surfaces from the natural extracellular environment , which facilitate endocytosis B-event . [SEP]
[CLS] this pathway seems general with respect to both sna and cell B-material type , including primary cells B-material . [SEP]
[CLS] however , this universal cell B-material entry mechanism cannot distinguish diseased cells B-material from healthy cells B-material , thus restricting the sna platform to uses that involve local delivery or systemic ones that result in preferential tumor B-material loading in the case of cancer applications . [SEP]
[CLS] therefore , to fully realize the potential of these constructs for systemic in vivo diagnostic and therapeutic applications , methods will need to be developed to target them to specific cell B-material types of interest . [SEP]
[CLS] herein , we report the design and synthesis of a new sna - nucleic acid - antibody B-material conjugate that shows outstanding selectivity for cell B-material lines with receptors recognized by the antibody B-material . [SEP]
[CLS] specifically , these sna conjugates consist of a monoclonal B-material antibody I-material ( mab ) - dna conjugate hybridized to an sna containing a gold B-nanoparticle nanoparticle I-nanoparticle ( aunp B-nanoparticle ) core B-material ( figure 1a ) . [SEP]
[CLS] the proofof - concept structure has a mab that recognizes the human epithelial growth B-material factor I-material receptor I-material 2 ( her2 ) , a member of the erbb protein B-material family , which is involved in signal transduction pathways leading to increased cell B-material growth and differentiation . [SEP]
[CLS] by using inductively coupled plasma mass spectrometry ( icp - ms ) , we show that the her2 - targeting snas are taken up by cells B-material expressing her2 to a much greater extent and at a faster initial rate compared to non - targeted particles . [SEP]
[CLS] we further demonstrate efficient antisense gene knockdown in her2 - overexpressing cell B-material lines at remarkably low particle concentrations and using short particle exposure times . [SEP]
[CLS] therefore , these novel constructs point towards a way of increasing both the selectivity and potency of the sna platform . [SEP]
[CLS] in a typical experiment , an azide - functionalized mab is initially covalently conjugated with a fluorophore - labeled sense dna sequence ( sequence : 5 ' agc acc atg gag t 5 - ( fluorescein - t ) - peg 1 - alkyne 3 ' ; mab - dna ) using the cu ( i ) catalyzed huisgen cycloaddition reaction ( click chemistry ) . [SEP]
[CLS] it is important to note that the click chemistry was performed using in situ generated cu ( i ) as the catalyst B-property and ( trishydroxypropyltriazolylmethyl ) amine ( thpta ) as the ligand . [SEP]
[CLS] thpta is necessary to prevent the cu ( i ) - induced aggregation of various proteins B-material . [SEP]
[CLS] after reaction , residues and excess dna were removed by fast protein B-material liquid B-technique chromatography I-technique ( fplc ) . [SEP]
[CLS] unreacted mabazide was removed in subsequent centrifugation - resuspension steps ( vide infra ) . [SEP]
[CLS] when 2 . 0 equivalents of dna - alkyne were reacted with 1 . 0 equivalent of mab - azide , we observed the conjugation of ~ 1 dna / mab as indicated by the shift of the antibody B-material maldi - tof peak from 154 . 2 kda to 161 . 4 kda ( figure 1b ) . [SEP]
[CLS] however , the full width at half - maximum of the primary peak increased from 4 kda to 15 kda , indicating the actual degree of functionalization ranges from 0 to 2 . [SEP]
[CLS] note that excess dna - alkyne was required to maximize the number of mabs with at least one oligonucleotide . [SEP]
[CLS] once the ab - dna conjugates have been isolated and purified , they can be hybridized to the complementary antisense sequences that comprise the surface of a sna aunp B-nanoparticle conjugate ( 5 ' ctc cat ggt gct cac - t 10 - sh 3 ' , figure 1a ; antibody B-material and dna sequence details are in the supporting information , table s1 ) . [SEP]
[CLS] hybridization of the mab - dna to the sna was achieved by co - incubation B-technique at 40 °c for 12 h in phosphate buffered saline containing 0 . 01 % tween 20 ( pbst , ph 7 . 2 ) . [SEP]
[CLS] thereafter , the conjugates were returned to room temperature and subjected to three sets of successive centrifugation - resuspension steps to remove unbound mab . [SEP]
[CLS] they were finally suspended in pbst at a particle concentration of 100 nm . to quantitatively determine the number of mab - dna strands associated with each sna , we oxidatively dissolved the aunp B-nanoparticle of the sna ( 10 nm ) in the presence of kcn , releasing the dna strands from the surface . [SEP]
[CLS] we then measured the fluorescence B-property of these unquenched strands , and compared the results to a standard curve constructed from known fluorescent B-property mab - dna concentrations . [SEP]
[CLS] we determined that on average 1 . 9 mab - dnas were hybridized to each sna ( a mab - dna : sna molar ratio of 2 : 1 was used during hybridization ) . [SEP]
[CLS] after hybridization , the sna hydrodynamic diameter increased from 19 to 23 nm as measured by dynamic B-technique light I-technique scattering I-technique ( dls ) , consistent with mabs - dna being tethered to the snas ( figure 1c ) . [SEP]
[CLS] to further verify that the mab - dna is hybridized to the snas , a melting experiment was performed in which the solution temperature was gradually increased from 20 to 80 °c while the fluorescence B-property of the fluorophore - labeled mab - dna was monitored ( excitation : 488 nm , emission : 515 nm ) . [SEP]
[CLS] below the duplex melting temperature ( t m , 44 . 9 °c ) , the fluorophore label on the mab - dna is partially quenched due to close proximity to the aunp B-nanoparticle surface . [SEP]
[CLS] as the temperature of the system is increased , a rise in the fluorescence B-property is observed , indicative of dehybrization of the two strands and release of the mab - dna strand ; when released , the fluorophore is no longer quenched ( figure s1 ) . [SEP]
[CLS] to ensure that the her2 mab - functionalized snas ( anti - her2 snas ) retain their antigen B-event binding I-event properties , we designed and performed a competitive binding assay to quantitatively measure the relative binding affinity of the conjugates with her2 . [SEP]
[CLS] in this assay , mabconjugated structures ( mab - dna and anti - her2 sna ) were each mixed at room temperature with free , biotin - conjugated mab ( b - mab ) at ratios ranging from 1 to 100 , and the mixtures were allowed to compete for surface - immobilized her2 . [SEP]
[CLS] thereafter , a streptavidin - horseradish peroxidase conjugate was used to detect the b - mab . [SEP]
[CLS] the binding affinity of the mab - dna and anti - her2 sna relative to b - mab can be derived from the binding isotherms ( table s2 and figure s2 ) . [SEP]
[CLS] we found that dna conjugation to the free mab did not significantly affect mab recognition for her2 ( k free mab / k b - mab = 0 . 37 ± 0 . 05 vs k mab - dna / k b - mab = 0 . 48 ± 0 . 08 ) . [SEP]
[CLS] when the mab - dna is hybridized to the sna , its binding affinity dropped slightly , to 0 . 11 ± 0 . 02 times k b - mab , likely due to increased steric hindrance . [SEP]
[CLS] in contrast , bovine serum albumin ( bsa ) , a negative control , shows no significant binding . [SEP]
[CLS] these data show that the anti - her2 snas are excellent binders B-property for her2 . [SEP]
[CLS] we next investigated if these materials preferentially bind to her2 - overexpressing cells B-material . [SEP]
[CLS] results from three cell B-material lines were compared : a549 ( her2 non - expressing ) , mcf - 7 ( moderate her2 expression ) , and skov - 3 ( her2 overexpression ) . [SEP]
[CLS] endogenous her2 expression levels in each type of cell B-material were confirmed by western blotting ( figure s3 ) . [SEP]
[CLS] anti - her2 snas and non - mab snas were incubated B-technique at 4 °c for 4 h with each set of cells B-material . [SEP]
[CLS] at 4 °c , cellular B-event processes I-event including endocytosis B-event are inhibited and therefore the observed cellassociated particles are primarily cell B-material surface - bound . [SEP]
[CLS] the cells B-material were harvested and lysed for gold B-material content analysis by using icp - ms . [SEP]
[CLS] when the her2 - targeted particles were used ( 10 nm ) , the skov - 3 cells B-material showed the highest amount of cell - associated particles of ca . 2 . 74 × 10 5 particles / cell B-material ( figure 2a ) . [SEP]
[CLS] this value is ca . 10 . 1 fold higher compared with the non - targeted particles . [SEP]
[CLS] for mcf - 7 cells B-material , ca . 6 . 0 × 10 4 particles were found to be associated with each cell B-material when the targeted snas were used , ca . 4 fold higher than non - targeted . [SEP]
[CLS] in contrast , a549 cells B-material did not show a significant difference for targeted and non - targeted particles , and all cells B-material incubated B-technique with the non - targeting snas only exhibit a background level of cell - associated particles of ca . 5 - 15k particles / cell B-material . [SEP]
[CLS] laser scanning confocal microscopy B-technique ( lscm ) revealed that , at 4 °c , the particles were predominantly located on the exterior of the cells B-material ( figure 3a ) . [SEP]
[CLS] while incubation B-technique at 4 °c provides information on cell - surface associated particles , it is also important to compare the cell B-material uptake at biologically relevant temperatures ( i . e . , 37 °c ) . [SEP]
[CLS] it is possible that non - specific , scavenger - mediated endocytosis B-event becomes the dominant internalization mechanism , rendering the her2 - mediated endocytotic pathway insignificant . [SEP]
[CLS] after 4 h of incubation B-technique at 37 °c , we observed an increase of cell - associated snas for all three cell B-material lines using particles with or without mab . [SEP]
[CLS] when compared to the samples at 4 °c , the numbers were much higher ( 10 - 27 fold increase ) because the energy dependent endocytosis B-event pathway is activated . [SEP]
[CLS] lcsm show that the snas are indeed primarily located within the cells B-material ( figures 3b and s4 ) . [SEP]
[CLS] the anti - her2 snas exhibited a slightly reduced , but nonetheless high specificity for skov - 3 cells B-material at 37 °c as compared to the specificity at 4 °c ( 7 . 8 fold higher cell B-material uptake compared with non - targeted snas at 10 nm vs . 10 . 1 fold for 4 °c ) . [SEP]
[CLS] in mcf - 7 cells B-material , the selective advantage over non - targeted snas is ca . 2 . 6 - 5 . 0 fold , as the cell B-material surface presents fewer copies of her2 than skov - 3 cells B-material ( figure 2b ) . [SEP]
[CLS] these data suggest that her2 - mediated endocytosis B-event of snas roughly scales with non - specific , scavenger - mediated endocytosis B-event as the temperature is increased from 4 to 37 °c . [SEP]
[CLS] the slight drop in specificity could be due to a combined effect of cellular differences for a particular endocytosis B-event pathway , and an inherent decrease in selectivity of the anti - her2 mab binding at 37 °c . [SEP]
[CLS] we also studied the cell B-material uptake of both targeted and non - targeted snas as a function of time with skov - 3 cells B-material . [SEP]
[CLS] the cell B-material selectivity diminishes as incubation B-technique time is extended beyond 24 h ( figure 4 ) . [SEP]
[CLS] in the first 6 h of incubation B-technique , targeted snas exhibit rapid cellular uptake ( initial uptake rate ca . 236 particles • s - 1 cell B-material - 1 vs . 19 particles • s - 1 cell B-material - 1 for non - targeted ) . [SEP]
[CLS] however , after 24 h of incubation B-technique , there is only a 1 . 2 fold increase in accumulated anti - her2 sna in cells B-material relative to non - targeted sna ( figure 4 ) . [SEP]
[CLS] the rate for her2 - mediated endocytosis B-event becomes significantly reduced after 8 h , to on average 7 particles • s - 1 cell B-material - 1 . [SEP]
[CLS] the drop in rate may result from the cell B-material ' s inability to rapidly replenish her2 on its surface after mab binding / endocytosis B-event . [SEP]
[CLS] nevertheless , we hypothesize that the initial cell B-material association in the first 4 hours ( 6 × 10 6 particles / cell B-material , figure 2b ) can lead to sufficient accumulation of the snas in her2 - overexpressing cells B-material for effective gene regulation . [SEP]
[CLS] we performed a set of gene knockdown experiments utilizing the anti - her2 snas . [SEP]
[CLS] in this case , the dna strands on the surface were designed to bind her2 mrna . [SEP]
[CLS] two negative controls were also included , which have either an off - target antibody B-material ( anti - his polyclonal antibody B-material ) or a scrambled antisense dna sequence ( 5 ' gag ctg cac gct gcc gtc a 3 ' ) . [SEP]
[CLS] we incubated B-technique skov - 3 cells B-material with anti - her2 snas and controls at 37 °c , using concentrations ranging from 50 pm to 10 nm , for 4 h . [SEP]
[CLS] following the incubation B-technique period , the solution was replaced with fresh growth media , and the cells B-material were allowed to grow for another 48 h before being lysed and assayed by immunoblotting . [SEP]
[CLS] strikingly , we found that using only 50 pm of the anti - her2 sna , her2 expression in skov - 3 cells B-material can be reduced to 12 . 7 % of untreated cells B-material by band density analysis , and at 1 nm , no her2 was detected ( figure 5 ) . [SEP]
[CLS] in contrast , the cells B-material treated with 1 nm of the off - target snas still show 48 % of her2 . [SEP]
[CLS] particles with a scrambled sequence did not reduce her2 expression at all concentrations tested , indicating the knockdown effect is specific . [SEP]
[CLS] these data show that hybridization - based tethering of the her2 mab to snas imparts cell B-material selectivity to these particles , and allows for faster cell B-material uptake and more efficient gene knockdown than the native sna structures of the same sequence . [SEP]
[CLS] moreover , these conjugates exhibit many of the same attributes of the non - targeted sna structures . [SEP]
[CLS] taken together , the targeted snas represent a significant step forward towards constructs that will allow for the systemic targeting of diseases with genetic bases , including many forms of cancer . [SEP]
[CLS] importantly , the general hybridization approach to functionalize snas reported herein can be extended to a wide variety of antibodies B-material and other targeting moieties such as peptides B-material , small molecules , and aptamers . [SEP]
[CLS] refer to web version on pubmed central for supplementary material B-material . [SEP]
[CLS] cell B-material uptake of 5 nm targeted vs . non - targeted snas in skov - 3 cells B-material as a function of time . [SEP]
[CLS] targeted entry is more rapid at early time points ( 0 - 6 h ) , but becomes much slower at later time points . [SEP]
[CLS] the difference in total particle uptake largely diminishes after 24 h . [SEP]
[CLS] $ watermark - text $ watermark - text $ watermark - text western blotting of her2 expression in skov - 3 cells B-material after treatment with anti - her2 snas and control samples . [SEP]
[CLS] gapdh is used as an internal reference . [SEP]
[CLS] ( a ) schematic showing the synthesis of anti - her2 snas . [SEP]
[CLS] ( b ) maldi - tof spectra of anti - her2 mab ( purple ) and mab - dna conjugate ( red ) . [SEP]
[CLS] the m / z difference between the primary peaks is 7 . 2 kda , corresponding to the mass of one sense dna strand . [SEP]
[CLS] ( c ) hydrodynamic diameters of citrate stabilized aunps B-nanoparticle ( 10 . 4 ± 1 . 2 nm ) , snas ( 18 . 5 ± 2 . 1 nm ) , and anti - her2 snas ( 23 . 3 ± 2 . 8 ) , as measured by dls . [SEP]
[CLS] 2 . number of gold B-nanoparticle nanoparticles I-nanoparticle per cell B-material after 4 h incubation B-technique at : ( a ) 4 °c and ( b ) 37 °c [SEP]
[CLS] her2 overexpressing cells B-material ( skov - 3 ) and moderate expressing cells B-material ( mcf - 7 ) show significantly higher uptake with targeted particles , while no selectivity was found for her2 nonexpressing cells B-material ( a549 ) . [SEP]
[CLS] 3 . confocal micrograph of skov - 3 cells B-material incubated B-technique with 5 nm anti - her2 snas for 4 h at : ( a ) 4 °c and ( b ) 37 °c . [SEP]
[CLS] anti - sense dna strands were labeled at the 5 ' - end with fluorescein . [SEP]
[CLS] tumors B-material are composed of highly proliferate , migratory , invasive , and therapy - evading cells B-material . [SEP]
[CLS] these characteristics are conferred by an enormously complex landscape of genomic , ( epi - ) genetic , and proteomic aberrations . [SEP]
[CLS] recent efforts to comprehensively catalogue these reversible and irreversible modifications have began to identify molecular mechanisms that contribute to cancer pathophysiology , serve as novel therapeutic targets , and may constitute biomarkers B-property for early diagnosis and prediction of therapy responses . [SEP]
[CLS] with constantly evolving technologies that will ultimately enable a complete survey of cancer genomes , the challenges for discovery cancer science and drug development are daunting . [SEP]
[CLS] bioinformatic and functional studies must differentiate cancer - driving and - contributing mutations from mere bystanders or ' noise ' , and have to delineate their molecular mechanisms of action as a function of collaborating oncogenic and tumor suppressive signatures . [SEP]
[CLS] in addition , the translation of these genomic discoveries into meaningful clinical endpoints requires the development of co - extinction strategies to therapeutically target multiple cancer genes , to robustly deliver therapeutics to tumor B-material sites , and to enable widespread dissemination of therapies within tumor B-material tissue . [SEP]
[CLS] in this perspective , i will describe the most current paradigms to study and validate cancer gene function . [SEP]
[CLS] i will highlight advances in the area of nanotechnology , in particular , the development of rna interference ( rnai ) - based platforms to more effectively deliver therapeutic agents to tumor B-material sites , and to modulate critical cancer genes that are difficult to target using conventional small - molecule - or antibody B-material - based approaches . [SEP]
[CLS] i will conclude with an outlook on the deluge of challenges that genomic and bioengineering sciences must overcome to make the long - awaited era of personalized nano - medicine a clinical reality for cancer patients . [SEP]
[CLS] personalized cancer medicine , i . e . , the design of therapeutic regimens informed by tumor B-material genotyping , has recently entered oncological practice . [SEP]
[CLS] fda - approved alk kinase inhibitor crizotinib and the braf inhibitor vemurafenib are the most recent examples of tailored cancer therapy , which have been successfully advanced for the treatment of alktranslocated lung cancer , and braf - mutated melanoma , respectively . [SEP]
[CLS] these successes demonstrate how the study of dna - associated abnormalities can guide drug development and clinical trials to pharmacologically target these tumorigenic perturbations , and to stratify patients for treatment . [SEP]
[CLS] the vast majority of the dauntingly complex genomic datasets , however , have yet to be translated into meaningful therapeutic strategies . [SEP]
[CLS] exigent barriers B-property for the rapid and cost - effective translation of the genome into clinical practice have become obvious , and are beginning to galvanize multidisciplinary teams of geneticist , computational scientists , cancer biologists , and bioengineers to develop the next generations of computational algorithms , preclinical cell B-material and animal models , and refined therapeutic conjugates . [SEP]
[CLS] in this article , i will highlight the most recent successes in translating genomic information into clinical practice ; i will describe advances in the preclinical interrogation of gene function in silico , and in cell B-material and animal models in vivo , and will summarize efforts in the bioengineering sciences to develop nanotechnological platforms that enable more effective , and less toxic targeting of multiple oncogenic lesions . [SEP]
[CLS] i will conclude with an outlook on future challenges to further advance and integrate functional cancer genomics and material B-material sciences . [SEP]
[CLS] comprehensive surveys of somatic mutations in cancer genomes have revealed a compendium of aberrations that confer growth and / or survival advantage to a tumor B-material , and have enabled the development of therapies that specifically target these oncogenic signatures . [SEP]
[CLS] the fda approval of small molecule inhibitors ( smis ) and biotherapeutic antibodies B-material targeting amplified , over - expressed , and / or mutated receptor B-material tyrosine B-material kinases ( rtks ) embodied the early promises of tailored therapies aimed at neutralizing the mitogenic , pro - invasive , pro - migratory , and anti - apoptotic activities of the cancer cell receptor B-material kinome . [SEP]
[CLS] these drugs include the anti - erbb2 ( her2 ) monoclonal B-material antibody I-material trastuzumab and the smi lapatinib for the treatment of her2 - amplified breast cancers , the abl kinase smi imatinib , which blocks the activity of a bcr - abl fusion protein B-material in chronic myeloid leukemia ( cml ) , the smi gefitinib , which targets mutated , hyperactivated epidermal growth B-material factor I-material receptor I-material ( egfr ) in advanced non - small cell lung cancers ( nsclcs ) as first - in - line treatment , and the vegf - neutralizing , anti - angiogenic monoclonal B-material antibody I-material bevacizumab ( avastin ) for the treatment of glioblastoma ( gbm ) , nsclcs , and metastatic colorectal and kidney cancer . [SEP]
[CLS] building on these early clinical successes , the past few years have witnessed an exceedingly rapid translation of genomic discoveries into clinical endpoints ( see figure 1 detailing the timeline for the translation of genomic findings into drug development ) . [SEP]
[CLS] activating mutations in the serine B-material / threonine kinase braf acting downstream of membrane - proximal rtk activation were identified in > 50 % of melanoma patients . [SEP]
[CLS] functional studies revealed that mutated braf ( braf ) could directly activate mitogen - activated kinases , thereby uncoupling mek - erk signaling from growth factor - driven rtk activation . mirroring remarkable activities in preclinical cancer models , clinical trials with the braf mut inhibitor vemurafenib revealed extraordinarily high response rates in therapy - refractory patients with metastatic , brafmutated melanoma . [SEP]
[CLS] equally dramatic clinical responses were observed with smi crizotinib , which targets an aberrant and constitutively active alk fusion protein B-material in nsclc patients . [SEP]
[CLS] the rapid preclinical and clinical validation of these targeted therapeutics , together with the development of companion diagnostic tests to assess braf mutation and alk translocations in lung and melanoma patients , represent the most important achievements in personalized cancer medicine to date ( see figure 1 ) . [SEP]
[CLS] additional kinase inhibitors currently in clinical trials target activated jak2 v617f in myelofibrosis , mutated ret in medullary thyroid carcinoma [SEP]
[CLS] , and pi3k , akt , and fgfr in various malignancies ( see review by courtney et al ) . finally , non - kinase smi currently being evaluated in the clinic include the smoothened ( smo ) smi gdc - 0449 ( vismodegib ) [SEP]
[CLS] , and smis targeting the dna repair enzyme poly ( adp ) ribose polymerase i ( parp1 ) . smo becomes hyperactivated and triggers constitutive activation of the hedgehog pathway in basal cell B-material carcinomas and a subset of medulloblastomas due to lossof - function mutations of the tumor B-material suppressor and smo inhibitor ptch1 ( see review by rubin and sauvage ) . [SEP]
[CLS] parp smis are effective in breast and ovarian cancers with incapacitated homologous recombination ( hr ) due to loss - of - function of two critical dna repair enzymes , brca1 or brca2 . [SEP]
[CLS] hr - deficient cancers are dependent on parp - driven alternative mechanisms of dna repair , and consequently , parp inhibitors show synthetic lethality in the setting of brca1 / 2 mutation . [SEP]
[CLS] recent genome sequencing efforts identified additional and ' druggable ' mutations , such as recurrent activating mutations in the heterotrimeric g - protein B-material α - subunit gnaq , which triggers mapk activation in uveal melanoma , activating notch1 mutations in chronic lymphocytic leukemia ( cll ) , and various mutations within several genes of the nf - κb pathway critical for the development of multiple myeloma . [SEP]
[CLS] currently available mek , notch , and nf - κb signaling inhibitors can readily be enrolled into ( pre - ) clinical testing for the treatment of these malignancies . [SEP]
[CLS] in addition , gain - and loss - of - function mutations of enzymes implicated in chromatin modification , e . g . , histone ( de ) methyltransferases and components of the swi - snf complex , ( see review by albert and helin ) , dna methylation ( e . g . , dnmt3a ) , and pathways generating important metabolites critical for the function of these enzymes ( e . g . , isocitrate dehydrogenase 1 ( idh1 ) , or ten - eleventranslocation gene 2 ( tet2 ) ) , have emerged as additional drug targets in lymphoid , myeloid and solid tumors B-material . [SEP]
[CLS] while a more detailed understanding of their roles in tumorigenesis is still pending , these epigenetic regulators define a novel class of cancerassociated aberrations , and may drive the development of pathway - specific drugs for the treatment of genomically defined cancers . [SEP]
[CLS] the rapidly growing field of cancer genomics has , thus , identified myriad genetic and epigenetic perturbations within cancer genomes . [SEP]
[CLS] drugs targeting some of these mutations have already been translated into oncological practice with clear benefits for genomically defined patient populations . [SEP]
[CLS] where do we go from here ? [SEP]
[CLS] the confluence of several areas of cancer discovery science , i . e . , genome surveys , medicinal chemistry processes , computational science approaches , and high - throughput genome - scale interrogation of cancer gene function will be critical for prognostication and advancing personalized drug design in the near future . [SEP]
[CLS] these efforts will address important questions in basic and clinical cancer sciences : which genes with aberrant copy number and / or expression are critical for tumorigenesis ? how do cancer - associated mutations dictate phenotypic hallmarks of proliferation , angiogenesis B-event , migration , invasion , and therapy resistance ? [SEP]
[CLS] what are the most important molecular markers to match individual patients with the most appropriate therapy to achieve the best possible outcome ? [SEP]
[CLS] surveys of oncogenomes have delivered extensive lists of candidate cancer genes ( ccgs ) with increasing accuracy and resolution . [SEP]
[CLS] the cancer genome atlas project ( tcga ) is the most recent and most impressive example describing the landscape of genetic and epigenetic changes in gbm and ovarian cancers . [SEP]
[CLS] some ccgs have well - annotated functions in disease pathogenesis and are characterized by high - level recurrent copy number alterations and expression ; consequently , these are classified as important ' driving ' mutations . [SEP]
[CLS] examples include amplification and mutation of egfr , deletions of the tumor B-material suppressors cdkn2a or pten , and mutations in tp53 , ras , braf , or pi3k , which are found across many different solid tumors B-material ( see review by chin and gray ) . [SEP]
[CLS] other ccgs with lowamplitude genomic and genetic perturbations , including non - coding rnas , have yet to be mechanistically characterized . [SEP]
[CLS] some of these less prevalent lesions may represent drivers or contributors , while the vast majority constitute ' passenger ' mutations randomly acquired during the lifespan of the tumor B-material . [SEP]
[CLS] differentiating between drivers / contributors and genomic ' noise ' is a dauntingly difficult task that continues to challenge multi - disciplinary research teams of computational scientists and cancer biologists . [SEP]
[CLS] the integration of several multidimensional cancer datasets in concert with comparative interspecies cancer genomics provide the substrate for knowledge - based pathway and epistasis analyses , and for subsequent functional cell B-material culture - based and animal studies to document the roles of ccgs in cancer pathogenesis . [SEP]
[CLS] 2 illustrates the workflow of functional genomics to identify , characterize , and therapeutically target cancer - associated mutations . [SEP]
[CLS] analysis of the cancer genome using multiple ' omic ' dimensions is critical to understanding how ccg function is compromised ( figure 2a ) . [SEP]
[CLS] is a ccg crippled by mutations of its coding or non - coding sequences , by dna or histone modifications , by changes to dna structure , or by expression of antagonizing non - coding rnas ? [SEP]
[CLS] because of their importance in malignant transformation , drivers are often inactivated via several mechanisms . [SEP]
[CLS] the tumor B-material suppressor ink4a ( encoded by cdkn2a ) is inactivated by homozygous deletion , epigenetic silencing ( via promoter methylation ) , and by point mutation ( see review by ) ; hyperactivation of oncogenic pi3ca is driven by amplification , overexpression and mutation ; and tie - 2 / tek , a critical effector of endothelial cell B-material survival and vascularization , is regulated at the levels of copy number , promoter methylation , mutation , and mrna expression ( stegh and kesari , unpublished observation ) . [SEP]
[CLS] consequently , integrative analyses assessing genomic , genetic , and epigenetic regulation across multiple tumor B-material types represent a critical first step in assessing the pathobiological importance of a given ccg , and in evaluating its potential as a therapeutic target or prognostic biomarker B-property ( figure 2b ) . [SEP]
[CLS] murine and human tumors B-material harbor orthologous genomic lesions , and many human oncogenes are transforming in murine cells B-material and vice versa despite low - level evolutionary conservation . [SEP]
[CLS] consequently , more advanced computational approaches use murine cancer models in crossspecies , comparative analyses . [SEP]
[CLS] as critical cancer - driving genes and pathways are likely conserved across different species , evolutionary conservation is used in these approaches as yet another criterion to assess disease relevance of a given ccg . [SEP]
[CLS] conserved genomic signatures were found upon k - ras activation in lung cancers , and notch1 mutations were identified in t cell acute lymphoblastic leukemia of both human and murine origin . [SEP]
[CLS] in addition , comparison of human and murine gene signatures also uncovered novel oncogenes . [SEP]
[CLS] chin and colleagues analyzed genomic profiles of metastatic human and murine k - ras - driven melanoma , and identified nedd9 as a novel pro - migratory and pro - invasive gene that exhibited amplification - driven overexpression in these tumors B-material . [SEP]
[CLS] similar analyses in human and murine hepatocellular and mammary carcinomas identified recurrent coamplification of oncogenic yap and ciap - 1 in liver , and grb7 and 14 - 3 - 3 - σ as contributors to erbb2 - driven carcinogenesis in breast cancer . [SEP]
[CLS] although integrative inter - species analyses of oncogenomes have identified critical ccgs , and have illuminated a path toward diagnostics and therapy development , the prioritization of the vast majority of ccgs buried in highly rearranged and mutated human oncogenomes remains a major challenge for cancer discovery sciences , and fervidly demands a systematic approach to interrogate gene function on a genome - scale . [SEP]
[CLS] such mechanistic studies not only provide information on the modus operandi of a given ccg in a particular cancer cell B-material lineage , they also aid in identifying collaborating non - mutated genes with essential functions within ccg - driven pathways that may serve as important drug targets . [SEP]
[CLS] stunning advances in the development of cdna , rnai , and chemical libraries over the past few years have made genome - scale functional studies an attainable reality . [SEP]
[CLS] when combined with the structural characterization of oncogenomes , and further improved in terms of sensitivity , specificity , cost , and throughput , these emerging technologies will continue to identify and validate novel ccgs as putative drug targets . [SEP]
[CLS] here , i will focus on the most recent examples of near genome - scale functional studies that resulted in the identification of critical ccgs across many different malignancies ; retrovirus - / transposon - based mutagenesis studies , the development , technical specifications and applications of orf , cdna , mirna , and chemical smi - based libraries are described and reviewed in detail elsewhere . [SEP]
[CLS] using a lentiviral shrna library with more than 54 , 000 shrnas targeting ~ 11 , 000 genes , hahn and colleagues assessed functional consequences of rnai - mediated gene knockdown on cellular growth and survival . [SEP]
[CLS] screening more than 100 human cell B-material lines including 25 ovarian cancer lineages , and integrating these functional data with genomic surveys of oncogenomes revealed strikingly different oncogene dependencies across several tumor B-material types . [SEP]
[CLS] in - depth analyses of genetic vulnerabilities of ovarian cancers identified 54 genes critical for ovarian cancer cell B-material survival . [SEP]
[CLS] these genes also showed amplification and overexpression in primary ovarian tumors B-material and cells B-material . [SEP]
[CLS] one of these genes , paired box gene ( pax ) 8 , is focally amplified in 16 % of high - grade serous ovarian cancers , and its rnaimediated knockdown induces apoptosis B-event in ovarian cancer cells B-material , suggesting that pax8 represents a lineage - specific survival factor in ovarian tumorigenesis . [SEP]
[CLS] similarly , an rnai screen in 34 breast cancer cell B-material lines using kinome - targeting pooled sirna oligonucleotides revealed selective rnai - induced lethality in genomically defined cell B-material lineages . [SEP]
[CLS] for example , viability B-property of I-property pten - deficient breast cancer cells B-material was dependent on genes controlling the mitotic spindle assembly checkpoint , e . g . , the checkpoint kinase ttk / msp1 , and consequently , rnai - mediated knockdown or smi - mediated inhibition of ttk showed synthetic lethality with pten loss - of - function [SEP]
[CLS] using a comparable screening strategy , barbie et al . and elledge and colleagues identified the non - canonical iκb kinase tbk1 , and regulators of mitosis B-event ( e . g . , the mitotic kinase plk1 , the anaphase - promoting complex / cyclosome , and the proteasome ) as genes and gene networks that when inhibited pharmacologically or via rnai resulted in death B-event of I-event kras I-event mutant I-event cells I-event and derived xenografts . [SEP]
[CLS] additional studies pointed to sgk2 and pak3 kinases as essential genes in p53 - deficient cells B-material , and identified met , cdk6 , and mek1 as critical survival factors in cells B-material lacking vhl . [SEP]
[CLS] rather than focusing on the identification of individual ccgs displaying synthetic lethal interactions with oncogenic drivers , several groups began to construct comprehensive gene interaction maps using high - throughput ( ht ) , combinatorial rnai approaches to generate double - deletion mutants in yeast . [SEP]
[CLS] comparing such interaction maps under normal and dnadamaging conditions using differential epistasis mapping lead to the identification of dynamic genetic interactions and ascribed novel roles of slt2 kinase , pph3 phosphatase , and histone variant htz1 in dna repair . [SEP]
[CLS] feasibility studies in drosophila targeting 96 genes with two independent sirna oligonucleotides per gene generated approximately 18 , 000 possible double - deletion mutants . [SEP]
[CLS] this co - extinction approach revealed that combinatorial knockdown of > 600 genes triggered phenotypic changes that could not be predicted from perturbations of individual genes . [SEP]
[CLS] while the generation of such interaction maps for mammalian genomes represents an enormous challenge , the systematic probing of genetic interactions will be critical to understanding cancer cell B-material circuitry , and to unraveling genetic co - dependencies . [SEP]
[CLS] these approaches promise to answer the question of which combination of genes has to be neutralized in order to impact cell B-material growth and survival , and what is the most effective combinatorial treatment regimen for a specific , genomically , genetically and epigenetically characterized cancer . [SEP]
[CLS] to further test and validate ccg function , over the past few years a wide spectrum of different model systems has been developed , which have added levels of sophistication to the standard arrays of transformed human cancer cell B-material lines . [SEP]
[CLS] these include primary , patientderived cells B-material , most importantly cancer stem cells B-material ( cscs ) , csc - derived orthotopic xenograft models , and genetically engineered mouse models ( gemms ) , along with their primary and transformed cell B-material derivatives , and secondary syngeneic explants ( see review by chin et al ) . [SEP]
[CLS] if enrolled into the most adequate applications / assays , each of these cell B-material and in vivo tumor B-material models represents a powerful experimental system to study ccg activity and / or anti - ccg drugs . [SEP]
[CLS] transformed and primary patient - derived cells B-material are invaluable tools for studying gene function in low - and high - throughput applications . [SEP]
[CLS] these cell B-material culture models are relatively inexpensive , easy to maintain , and easy to manipulate . [SEP]
[CLS] in addition , efforts to verify cellular effects of rnai - mediated loss - of - function or cdna complementation across a spectrum of genetically diverse cells B-material also minimize the risk that an observed phenotype is cell B-material - line specific . [SEP]
[CLS] on the other hand , cell B-material lines grown on plastic do not capture the intricacies of human cancers , such as tumor B-material - microenvironment interactions , and are often only partially characterized on genomic levels . [SEP]
[CLS] in addition , the oncogenome of transformed , long - term cultured cells B-material does not necessarily capture the genomic aberrations seen in primary tumors B-material , and the genotype of patient - derived lineages represents only a limited and often less prevalent combination of genetic and epigenetic aberrations resident in clinical samples . [SEP]
[CLS] given the importance of genetic context to understand ccg function , re - enforcing complementary data from other cancer models are always required to validate initial cell B-material culture - based mechanistic studies . [SEP]
[CLS] primary , non - transformed , genetically engineered cells B-material have emerged as a powerful cell B-material system that in the absence of full transformation could be transfected with ccgs to survey their cancer - relevant functions in the context of a defined mutational spectrum . [SEP]
[CLS] these minimally transformed cells B-material also permit the generation of isogenic cell B-material lines that mirror mutational profiles in specific tumor B-material subtypes . [SEP]
[CLS] finally , gemms represent the most elaborate model system to assess the role of ccgs in tumorigenesis . [SEP]
[CLS] inducible knockout and transgenic alleles crossed onto tumor B-material - prone strains can determine the role of a ccg in tumor B-material maintenance , and consequently , these models represent decisive tools to identify driver mutations ( reviewed by sharpless and depinho ) . [SEP]
[CLS] in addition , refined gemm provide faithful pheno - and genocopies of human malignancies , and consequently , have emerged as invaluable tools for interspecies oncogenomics . [SEP]
[CLS] it is important to consider , however , that murine tumors B-material are characterized by few , if any , chromosomal gains or losses , and exhibit less complex profiles of point mutations . [SEP]
[CLS] consequently , the low - level genomic instability of murine tumors B-material limits the application of large - scale comparative approaches to prioritize extensive atlases of human mutations . [SEP]
[CLS] in contrast , murine tumor B-material models associated with telomerase dysfunction more faithfully replicates the complex genomic profiles seen in human cancers , as telomere - based crises cause high - level non - reciprocal translocations and regional copy number variations . [SEP]
[CLS] importantly , these perturbations non - randomly overlap with chromosomal aberrations observed in several human oncogenomes . [SEP]
[CLS] these findings point to a similar malignant evolution of human and murine cancers , and underline the usefulness of telomerasedeficient murine cancer models as critical filters for the prioritization of human oncogenomic datasets . [SEP]
[CLS] the extensive time frame and high costs associated with the generation and characterization of gemms , however , has limited their application for high throughput assays . [SEP]
[CLS] nongermline gemms , i . e . , genetically engineered , tissue - restricted stem and progenitor cells B-material orthotopically implanted into a syngeneic host , represent a valid alternative ( reviewed by heyer et al ) . [SEP]
[CLS] here , primary , minimally transformed cells B-material can be transfected with orf or rnai libraries and implanted into recipients . [SEP]
[CLS] the subsequent development of syngeneic tumors B-material can then be analyzed for the selection of cooperating events that in concert with signature mutations drive explant growth . [SEP]
[CLS] examples include the stem cell B-material transgenesis system developed by bachoo et al . using minimally transformed murine cortical astrocytes and neural stem cells B-material , and a hematopoietic progenitor cell B-material system derived from eμ - myc transgenic mice , which has been used successfully in ht rnai applications to identify haploinsufficient tumor B-material suppressors driving lymphomagenesis . [SEP]
[CLS] ht surveys of cancer genomes , e . g . , the tcga , have provided a periodic table of key genetic elements that drive and contribute to cancer development . [SEP]
[CLS] so far , 474 genes have been described as professional cancer genes ( cancer gene census ) encoding 2 % of the human proteome . [SEP]
[CLS] with advances in genomic technologies , this number will increase by several fold , and will likely trigger an equally impressive evolution of technologies to mechanistically understand and ultimately prosecute these genes as putative targets . [SEP]
[CLS] as pointed out above , these functional studies have to provide a deep mechanistic understanding of selected ccgs as a function of cooperating oncogenic and tumor suppressive signatures . [SEP]
[CLS] such studies have to explain ccg relevance for cancer development in the context of a complex , intertwined tumor B-material circuitry , rather than as linear , isolated pathways . [SEP]
[CLS] they also have to consider that cancers represent highly heterogeneous neoorgans , which are composed of cells B-material with different genomic profiles , a multitude of histopathological phenotypes , and staggering heterotypic interactions with the host microenvironment . [SEP]
[CLS] currently identified , clinically validated , and most importantly , ' druggable ' targets , such as braf or alk , are ' low - hanging ' fruits for drug development . [SEP]
[CLS] the vast majority of ccgs , however , will likely represent unprecedented , non - enzymatic targets with unknown modi operandi . [SEP]
[CLS] how can multiple cooperating , undruggable , and uncharacterized genes be functionally compromised ? [SEP]
[CLS] while it is conceivable that the rapid evolution of genomics can address the challenges posed by high - level plasticity of cancers , the most critical question becomes whether ' conventional ' drug development pipelines focusing on smi and biotherapeutic antibodies B-material are equipped to tackle the challenge of drugging the undruggable . [SEP]
[CLS] in addition , many targeted therapies have failed in the clinic because they cannot be effectively delivered to tumors B-material sites , and exhibited suboptimal intratumoral dissemination . [SEP]
[CLS] effective drug delivery is a critical challenges for solid organ tumors B-material , in particular cancers of the brain and pancreas . [SEP]
[CLS] here , tailored therapies bound for the central nervous system have to negotiate passage through the blood - brain barrier B-property ( bbb ) , the blood - cerebrospinal fluid barrier B-property ( bcsf ) , and the blood - tumor barrier B-property ( btb ) , and must withstand the substantial dynamic force in the brain interstitium caused by csf flow , edema - associated intratumoral , and tumor B-material mass - related pressure ( reviewed by ) . [SEP]
[CLS] similarly , pancreatic cancer drugs have to extravasate from the tumor B-material vasculature and permeate thick fibrotic tissue to target tumor B-material cells B-material . [SEP]
[CLS] finally , cancers can compensate for functional ablation of one genetic element by activating other circuitry components , suggesting that co - extinction strategies are required to halt tumor B-material progression . [SEP]
[CLS] combinatorial therapies utilizing multiple smi - or antibody B-material - based agents have to consider significant drug - drug interactions , systemic toxicity B-property due to pronounced off - target effects , and the emergence of target gene mutations leading to drug resistance . [SEP]
[CLS] nanotechnology , i . e . , the science of engineering materials on a molecular , nanometer scale , provides fundamentally different approaches to cancer therapy . [SEP]
[CLS] nano - drug delivery systems with a size of < 100 nm can accumulate at higher concentrations within tumors B-material compared to unconjugated therapeutics through the enhanced permeability and retention ( epr ) effect , and consequently have lower dose - limiting adverse side effects , and higher therapeutic efficacies ( reviewed by maeda et al and petros and desimone ) . [SEP]
[CLS] several formulations have been described , including liposomes B-nanoparticle , polymeric B-nanoparticle nanoparticles I-nanoparticle , dendrimers B-nanoparticle , metal B-nanoparticle nanoparticles I-nanoparticle , and molecular targeted nanoparticles B-nanoparticle . [SEP] B-nanoparticle
[CLS] importantly , several nano - conjugates have successfully been used to deliver small interfering rnas ( sirnas ) to tumor B-material sites , and have been found to harness the great promise of rnai - mediated biotherapeutic gene silencing to neutralize virtually any gene , including undruggable oncogenes , and to overcome the major challenges of rna interference ( rnai ) - based therapy , i . e . , poor cellular uptake , lack of intracellular stability , and rapid renal clearance following systemic administration ( reviewed by dillon et al and reischl and zimmer ) . [SEP]
[CLS] i will describe the most prominent nano - rnai approaches , i . e . , chemically modified sirnas , lipid - , polymer - based delivery strategies , and rnai - functionalized metal B-nanoparticle nanoparticles I-nanoparticle , and their potential to drive the concept of personalized cancer medicine toward clinical opportunity . [SEP]
[CLS] rnai is a potent mechanism of gene silencing capable of blocking the translation of mrna , and thus reducing the expression of pathological proteins B-material , in particular those that are difficult to target by traditional pharmacological approaches . [SEP]
[CLS] the rnai approachsirnas are generated by cleavage of long double - stranded ( ds ) rnas into ~ 20 nucleotide - containing sirnas by the enzyme dicer . [SEP]
[CLS] unwinding of sirnas into two single - stranded ( ss ) rnas , incorporation of the guide strand into the rna - induced silencing complex ( risc ) , and binding of sirnas to complementary mrna triggers the degradation of endogenous mrna by argonaute , the catalytic component of the risc complex ( figure 3 ; reviewed by hannon and rossi ) . [SEP]
[CLS] sirna oligonucleotides can silence expression of various cancer genes implicated in growth , apoptosis B-event , migration , and invasion , and consequently , have motivated myriad preclinical studies to assess the potential of rnai as anti - cancer therapeutics . [SEP]
[CLS] due to the negative charge of the rna backbone , sirna oligonucleotides require delivery systems to overcome negatively charged membranes , and to counteract electrostatic repulsion . [SEP]
[CLS] in addition , systemic delivery strategies have to prevent rapid renal and hepatic clearance and degradation of sirnas by nucleases , and most importantly , have to display favorable safety profiles . [SEP]
[CLS] while local delivery may overcome some of these difficulties , the cancerous tissue to be targeted , however , may not be easily accessible or may be too extensive for local treatment , e . g . , in the case of metastatic disease . [SEP]
[CLS] the first rnai - based viral vectors faced challenges relating to significant adverse side effects , high costs associated with the production of sufficient viral stocks , and suboptimal dissemination . [SEP]
[CLS] while late - generation viral platforms for nucleic B-material acid I-material delivery may overcome some of these shortcomings , the development of non - viral , less toxic B-property alternatives for rnai delivery is highly desirable ( reviewed by akhtar and benter ) . [SEP]
[CLS] with physico - biological properties significantly dependent on particle size , charge , and hydrophobicity B-property ( reviewed by petros and desimone ) , nanomaterials B-material at the submicron scale have features that make them ideal carrier systems for rnai delivery . [SEP]
[CLS] in addition to intra - and extracellular stability and biocompatibility B-property , the design of these nanoconstructs has to consider several critical aspects of conjugate chemistry , i . e . , particle aggregation , endosomal escape of particles into the cytosol to gain access to the risc signaling machinery , and off - target effects of sirnas . [SEP]
[CLS] aggregation of sirna particles can be counterbalanced by reducing surface charge , e . g . , by co - functionalization with poly ( ethylene glycol ) ( peg ) , sugar molecules ( e . g . , cyclodextrin ) , or hyaluronic acid ( ha ) . [SEP]
[CLS] ph - or reduction - sensitive polymers B-material can enhance endosomal escape , as sharp ph differences and a redox potential exist between the intra - endosomal and the cytosolic compartment . [SEP]
[CLS] consequently , cationic B-material polymers B-material with a pk a slightly below physiological ph , e . g . , poly ( ethylene imine B-material ) ( pei ) , the most widely used ph - responsive polymer B-material ( reviewed by kim and kim , schaffert and wagner , and midoux et al ) can absorb protons , increase the intra - endosomal osmotic pressure , and ultimately rupture endosomal membranes - a phenomenon referred to as the " proton sponge effect " [SEP]
[CLS] other strategies to enhance endosomal escape have utilized ( a ) polymers B-material undergoing hydrophilic B-property - to - hydrophobic B-property transitions , [SEP]
[CLS] ( b ) fusogenic peptides B-material aimed at disrupting endosomal membranes , and ( c ) protein transduction peptides B-material that facilitate membrane penetration of sirnas through an as yet only partially characterized mechanism . [SEP]
[CLS] lastly , the most critical aspect of rnai biology and therapeutic application is the reduction of off - target effects , i . e . , the unspecific binding of sirnas to non - target mrnas , which may result in increased cellular toxicity B-property and immunogenicity B-property ( reviewed by aigner ) . [SEP]
[CLS] chemical modification of the sirna guide strand represents one strategy to limit off - target effects . [SEP]
[CLS] here , the modified guide strand anneals to a passenger strand and abrogates off - target effects mediated by passenger strand complementarity ( see reviews by rao and colleagues ) . [SEP]
[CLS] while the delivery of naked sirna oligonucleotides via hydrodynamic injection and electroporation is not suited for in vivo ( systemic ) delivery due to rna instability in serum , conjugation of the guide strand with small molecules , peptides B-material , or polymers B-material can increase rna stability . [SEP]
[CLS] standard modifications of sirnas include 2 ′ - o - methyl , 2 ′ - fluoro , 2 ′ - omethoxyethyl and phosphorothioate ( reviewed by wilson and keefe ) . [SEP]
[CLS] additional approaches involving conjugation of sirnas with cell B-material - penetrating peptides B-material ( cpps ) , peg , cholesterol and its derivatives , long - chain fatty acids ( > c18 ) , bile - salt B-material derivatives , and acidresponsive polymers B-material containing peg and an n - acetylgalactosamine ( nag ) have been described ( reviewed by de paula et al ) . [SEP]
[CLS] these constructs resulted in potent gene silencing in cell B-material culture in vitro and in organ systems in vivo but may be associated with increased offtarget effects , attenuated therapeutic efficacy , and production of toxic B-property metabolites due to degradation of rna - modifying compounds . [SEP]
[CLS] cationic B-material polymers B-material , e . g . , the pei - derivatives pei - peg36 , cyclodextrin - containing polycations , polylysine , chitosan B-material , and cationic B-material peptides B-material and proteins B-material such as cady , mpg - 8 , and cpp - modified proteins B-material have been used to neutralize the negatively charged phosphate backbone to stabilize sirnas and enhance cellular delivery via binding to the negatively charged plasma membranes ( reviewed by kim and kim ) . [SEP]
[CLS] co - functionalization of cationic B-material polymers B-material , e . g . , via pegylation , is critical for reducing material B-material toxicity B-property and unspecific binding caused by the positive charge . [SEP]
[CLS] additional strategies include crosslinking of pei via reversible disulfide bonds , amine B-material modification , e . g . , acetylation and introduction of negatively charged propionic acid or succinic acid groups , or ketalizing branched pei . [SEP]
[CLS] importantly , such nano - constructed pei derivatives demonstrated high transfection efficiencies and substantial target knockdown in vivo coupled with robust endosomal escape , but displayed marked , albeit lower , toxicity B-property compared to the unmodified polymeric B-material cation I-material . [SEP]
[CLS] chitosan B-material particles , in contrast , effectively and safely delivered sirnas in vivo upon intranasal and intravenous administration ( reviewed by andersen et al ) . [SEP]
[CLS] additional rationally designed cationic B-material polymers B-material with significant knockdown efficacies and biocompatibility B-property include mpeg45 - b - pcl100 - b - ppeea12 , an amphiphilic B-property and cationic B-material triblock copolymer consisting of monomethoxy peg , poly ( 3 - caprolactone ) , and poly ( 2aminoethyl ethylene phosphate ) , a transferrin - conjugated β - propionamide - cross - linked oligoethylenimine , a polyplex composed of poly ( amido ethylenimine ) and linear pei , a poly ( β - amino ester ) ( pbae ) / pei / peg hybrid conjugate , and a poly ( amino ester glycol urethane ) ( paegu ) / sirna polyplex [SEP]
[CLS] lipid B-nanoparticle nanoparticles I-nanoparticle ( lnp ) are one of the most widely used platforms to deliver sirnas to cells B-material , tissue and tumors B-material . [SEP]
[CLS] lnp platforms vary significantly with regard to lipid B-material composition , lipid B-material ratios , particle size , and overall structure . [SEP]
[CLS] 1 , 2 - dioleoyl - 3 - trimethylammonium - propane ( dotap ) represents one of the first cationic B-material lipids B-material engineered for in vivo delivery of sirnas , and unlike polymers B-material , results in faster sirna decomplexation into the cytosol , and consequently , in more effective gene knockdown ( reviewed by kim and kim ) . [SEP]
[CLS] the stable nucleic acid lipid particle ( snalp ) represent a prominent class of lnps . [SEP]
[CLS] this platform has a diameter of approximately 80 nm , and contains four different lipids B-material , i . e . , a peg moiety ( mpeg2000 - c - dma ) to stabilize and to prevent aggregation of the construct , cholesterol , a neutral helper lipid B-material ( dppc ) , and the ionizable B-property lipid B-material dlindma , which triggers fusion with endosomal membranes to release the sirnas into the cytosol , and mediates condensation of lipid B-material and anionic rnai components during particle formation . [SEP]
[CLS] importantly , modification of the cationic B-material lipid B-material moiety of snalp had dramatic impact on the in vivo efficacy of the conjugate : snalp based on the ester - containing lipid B-material dlindap exhibited reduced knockdown efficacy in vivo when compared to particles containing the alkoxy - containing lipid B-material dlindma , and insertion of one additional methylene group into the headgroup ( dlin - kc2 - dma ) resulted in a 4 - fold increase in potency compared to dlin - k - dma [SEP]
[CLS] in addition , akinc et al . developed a high - through put synthesis scheme to rapidly generate a library of amino - alkyl - acrylate and - acrylamide materials termed ' lipidoids ' , which were tested in cell B-material and animal models for rnai delivery and efficacy . [SEP]
[CLS] similar to snalp formulations , lipoids required sirna dosages greater than 1 mg / kg . [SEP]
[CLS] using epoxide chemistry , love et al . generated a lipid B-material library that enabled systemic delivery of sirnas at doses below 0 . 01 mg / kg , and is capable of silencing multiple hepatic genes simultaneously after only one i . v . injection . [SEP]
[CLS] specifically , epoxide - derived lipidoids formulated with five sirnas targeting genes implicated in cholesterol metabolism , i . e . , apob , pcsk9 , xbp1 , and sort1 , triggered gene knockdown greater than 65 % in murine liver tissue in vivo upon i . v . injection , demonstrating the high - level efficacy and the potential to target entire oncogenic signatures in cancer in the near future . [SEP]
[CLS] clinical proof - of - concept studies of the snalp platform include a phase i single - dose study of snalp - apob ( tekmira pharmaceuticals ) in patients with elevated ldl cholesterol ( reviewed by barros and gollob ) , and of aln - ttr01 ( alnylam pharmaceuticals ) for the treatment of attr ( i . e . , amyloidosis triggered by mutations in the transthyretin ( ttr ) gene ) . [SEP]
[CLS] attr is characterized by the accumulation of pathogenic deposits of mutant and wild - type ttr protein B-material in liver and in multiple extra - hepatic tissues , including the peripheral nervous system , heart , and the gastrointestinal tract . [SEP]
[CLS] aln - ttr01 was well - tolerated , and lead to substantial and persistent knockdown of serum ttr protein B-material ( reviewed by barros and gollob ) . [SEP]
[CLS] aln - vsp ( alnylam ) is a second snalp - based conjugate , which carries two types of sirna oligonucleotides targeting vegf and kinesin family member 11 ( kif11 , or ksp ) , genes with critical function in tumor B-event angiogenesis I-event and mitotic spindle formation . [SEP]
[CLS] multi - dose phase i clinical trials in patients with advanced solid tumors B-material revealed that aln - vsp was well tolerated , without signs of liver toxicity B-property , only modest adverse side effects including fatigue , nausea and fever , and transient immunogenicity B-property . [SEP]
[CLS] dose - limiting side effects in a small number of patients included transient grade 3 thrombocytopenia , and grade 3 hypokalemia . [SEP]
[CLS] importantly , rnai - mediated cleavage of vegf and ksp mrnas within tumor B-material biopsies confirmed activity of delivered sirnas , and translated into partial responses , including stable disease greater than 2 months , and reduction in tumoral B-event angiogenesis I-event ( see alnylam . com for more information ) . [SEP]
[CLS] additional snalp conjugates include snalp - plk1 , which utilize sirnas targeting polo - like kinase 1 ( plk1 ) , a cell B-material cycle kinase with critical functions in mitosis B-event and cytokinesis . [SEP]
[CLS] results from multi - dose phase - i clinical trials in patients with advanced solid tumors B-material and primary liver cancers or liver metastases B-event are expected in late 2012 . [SEP]
[CLS] finally , the cationic B-material liposomal B-nanoparticle conjugate atu - 027 ( silence therapeutics ) , comprised of the cationic B-material lipid B-material atufect01 , a neutral fusogenic dphype helper lipid B-material , peg - , and sirna targeting protein B-material kinase n3 ( pkn3 ) , is another example of successful implementation of the lipid B-nanoparticle nanoparticle I-nanoparticle platform into early clinical trials . [SEP]
[CLS] atu - 027 was well tolerated in the absence of dose - limiting toxicities B-property , with few patients exhibiting disease stabilization and partial regression . [SEP]
[CLS] ( for a detailed review of these and additional cationic B-material lipids B-material , see huang and liu and schroeder et al . [SEP]
[CLS] neutral liposomes B-nanoparticle encapsulate sirnas , protect them from nuclease degradation , and deliver them to cells B-material and tissue via membrane fusion or receptor - I-event mediated endocytosis B-event . [SEP]
[CLS] prominent examples include 1 , 2 - dioleoyl - sn - glycero - 3 - phosphatidylcholine ( dopc ) - based nanoliposomes ( mean size < 65 nm ) , which have been used for the in vivo delivery of sirna sequences targeting epha2 , fak , 109 neutrophilin - 2 , or il - 8 in various xenograft cancer models . [SEP]
[CLS] these constructs have resulted in 10 to 30 - fold increases in intratumoral sirna levels compared to unconjugated dotap or naked sirnas , and have induced substantial tumor B-material shrinkage in the absence of toxicity B-property in normal cell B-material lineages , e . g . , fibroblasts , bone marrow and hematopoietic cells B-material . [SEP]
[CLS] preclinical studies of polyelectrolyte complex ( pec ) micelles B-material loaded with vegfr - specific sirnas showed similar results , including potent local or systemic delivery of sirnas , robust intratumoral target knockdown , and suppression of tumor B-material growth . [SEP]
[CLS] finally , dotap engineered with an outer lipid B-material bilayer I-material of 1 , 2 - distearoyl - sn - glycero - 3 - phosphoethanolamine - n - ( polyethylene glycol - 2000 ) and egg phosphatidylcholine ( egg - pc ) ( peg - dspe ) also document the potency of this platform , resulting in serum availability of sirnas 20 hrs post injection . [SEP]
[CLS] additional non - cationic B-material polymeric nanostructures include poly ( isobutyl cyanoacrylate ) based liposomes B-nanoparticle with sirnas targeting the ewsyfli1 transcriptional B-event activator and poly ( lactic - co - glycolic acid ) ( plga ) - based liposomes B-nanoparticle loaded with erc / mesothelin - specific sirnas . [SEP]
[CLS] in addition to encapsulation of sirnas into liposomal B-nanoparticle structures , sirnas can also be directly conjugated to polymer B-material chains , including peptides B-material , cholesterol , and aptamers . [SEP]
[CLS] this strategy is exemplified by pbave , an amphipathic poly ( vinyl ether B-material ) functionalized with sirnas targeting hepatocellular apolipoprotein b ( apob ) and peroxisome proliferatoractivated receptor B-material alpha ( pparα ) . [SEP]
[CLS] with approximately 10 , 000 articles published over the past decade , polyvalent gold B-nanoparticle nanoparticles I-nanoparticle ( au - nps B-nanoparticle ) densely functionalized with highly oriented dna or sirna oligonucleotides represent one of the most prominent nucleic acid - based nanoconjugates . [SEP]
[CLS] after the introduction of dna - au - nps B-nanoparticle in 1996 and demonstration of their relatively straightforward synthesis , their capacity to penetrate cells B-material and their ability to silence gene expression via an antisense mechanism , several sirna - au - np B-nanoparticle platforms were developed using gold B-material - thiol chemistry or electrostatic au - rna interactions to decorate gold B-material particles with rnas ( reviewed by cutler et al ; see figure 4 for a schematic overview of gold - rnai nanoconjugates ) . [SEP]
[CLS] using thiol - functionalized sirnas , 15 nm au - nps B-nanoparticle , together with peg 5000 - pama 7500 as passivating and stabilizing ligands , oishi et al . reported the first sirna - based gold B-nanoparticle nanoparticle I-nanoparticle , which triggered robust and persistent gene knockdown of luciferase expression in huh - 7 . [SEP]
[CLS] giljohann et al . used a similar approach decorating 13 nm au - nps B-nanoparticle with thiolated sirna and peg 400 ( hereafter referred to as spherical nucleic B-material acids I-material , snas ) . [SEP]
[CLS] despite their large negative charge ( zeta B-property potential I-property < −30 mv ) , these snas showed highly efficient cellular uptake , significant serum stability without the use of auxiliary transfection strategies or chemical modifications , and consequently efficient , specific , and persistent gene knockdown . [SEP]
[CLS] cellular uptake is mediated by the nucleic B-material acid I-material moiety of the particle , as coreless or iron B-material oxide I-material snas exhibit similar internalization rates . [SEP]
[CLS] uptake is reduced several fold when au - nps B-nanoparticle without dna / rna are passivated with bsa . it has been suggested that cellular uptake is mediated by scavenger receptor - driven endocytosis B-event . [SEP]
[CLS] this class of pattern - recognition receptors binds to serum proteins B-material absorbed by the nucleic B-material acid I-material moiety of snas , which , upon their internalization and endosomal escape , accumulate within the cytoplasm to silence gene expression . [SEP]
[CLS] robust intracellular and serum stability of snas has been attributed to the high local sodium B-material ion B-material concentration , which potently deactivates nucleases . [SEP]
[CLS] nucleic B-material acid I-material degradation is 4 times slower in aqueous solution compared to free duplexes , and serum stability is further increased by the aforementioned absorption of serum proteins B-material , which limit access of nucleases to the gold B-material - bound rna . [SEP]
[CLS] due to highlevel intracellular stability , snas are potent , and achieve highly persistent gene knockdown at picomolar concentrations . [SEP]
[CLS] due to potent penetration of snas into tissue in vivo , and minimal systemic toxicity B-property and immunogenicity B-property ( as measured by quantification of β - ifn levels ) in rodents , rnai - functionalized gold B-nanoparticle nanoparticles I-nanoparticle represent a powerful therapeutic platform to combat cancers and other diseases of genetic etiology . [SEP]
[CLS] in addition , the stability of the nucleic B-material acid I-material shell B-material allows for co - functionalization of snas with chemotherapeutics or corresponding pro - drugs , e . g . , platinum ( iv ) prodrug , which upon internalization is reduced to a cytotoxic B-property platinum ( ii ) species , and released into the cytosol through reductive elimination of their axial ligands resulting in a pt - sna species that is more effective than cisplatin B-material or the pro - drug . [SEP]
[CLS] in addition , drug conjugation can increase the solubility B-property of chemotherapeutic drugs such as paclitaxel B-material , resulting in 4 - 10 fold lower ic50 values . [SEP]
[CLS] the ability to generate these single - entity , highly effective hybrid ( h ) - snas capable of biotherapeutic gene silencing and chemotherapy - induced tumor B-material cell B-material killing illustrates the enormous potential of snas as anti - cancer compounds . [SEP]
[CLS] further harnessing the stability of the nucleic B-material acid I-material shell B-material , along with efforts to co - functionalize snas with imaging agents such as gadolinium B-material ( gd ( iii ) ) , may aid in tracking intramural accumulation of particles via mri . [SEP]
[CLS] most clinical studies rely on indirect measures to quantify tumor B-material uptake of compounds such as smis or antibodies B-material , e . g . , drug responses or measurements of drug levels in body fluids . [SEP]
[CLS] however , if a drug does not elicit a response , it is rarely determined if the drug was ineffective or if it simply failed to reach its intended target . [SEP]
[CLS] in addition to the 13 nm peg 400 - snas discussed in detail above , a different conjugation strategy utilized 15 nm au - nps B-nanoparticle decorated with thiolated peg 1000 - nh 2 , an sirna corona attached via a disulfide crosslinker ( spdp ) to the terminal amine B-material group I-material on the peg , and further coated with poly ( β - amino ester ) s ( pbaes ) to enhance cellular uptake and endosomal escape . [SEP]
[CLS] the pbae moiety appears to be critical to the capacity of the nano - conjugate to elicit significant gene knockdown , as particles without pbaes are ineffective . [SEP]
[CLS] it remains uncertain whether the ester conjugation triggers more efficient cellular uptake and endosomal escape , and / or impacts sirna accessibility by the risc complex . [SEP]
[CLS] in addition to using gold B-material particles of various diameters , 40 nm hollow gold B-material nanoshells B-nanoparticle ( aunss ) have emerged as yet another core B-material structure for delivery of sirnas into cells B-material . [SEP]
[CLS] conjugation of aunss with sh - sirna - peg 300 and sh - peg 300 resulted in constructs with > 100 sirna oligonucleotides per particle . [SEP]
[CLS] coating B-material with transactivator of transcription B-event ( tat ) lipids B-material to enable efficient internalization , and irradiation with near - infrared ( nir ) light to trigger endosomal escape and decomplexation of the sirna moiety , resulted in profound knockdown of a gfp reporter in c166 mouse endothelial cells B-material . [SEP]
[CLS] aunss co - functionalized with sh - sirna and ta - peg 5000 - f ( ta , thioctic acid required for conjugation of sirnas ; f , folic B-material acid I-material required for cellular targeting ) , triggered potent knockdown of endogenous levels of the nfκb subunit p65 in hela cells B-material . [SEP]
[CLS] gene silencing activity was strictly dependent on nir irradiation and on the presence of the folate group . [SEP]
[CLS] in vivo administration of these conjugates via tail vein injections resulted in knockdown of p65 in subcutaneous hela cell - derived tumors B-material and increased sensitivity of tumor - bearing mice to irinotecan . [SEP]
[CLS] taking advantage of electrostatic interactions between negatively charged sirnas and the au - np B-nanoparticle surface , several constructs have emerged that use 15 nm au - nps B-nanoparticle coated with layers of positively charged pei and sirna oligonucleotides . [SEP]
[CLS] the resulting particles can have either a terminal rna or a pei layer , which impact cellular uptake and endosomal escape . [SEP]
[CLS] importantly , the surface density of sirna oligonucleotides is significantly higher ( 780 vs . 30 - 50 oligonucleotides on au - sh - nps B-nanoparticle ) , likely due to the strong electrostatic interactions of the pei and rna moieties . [SEP]
[CLS] prominent constructs include au - nps B-nanoparticle decorated with layers of pei , an anionic charge - reversal polyelectrolyte ( pah - cit = cis - aconitic anhydridefunctionalized poly ( allylamine ) ) , and sirnas . [SEP]
[CLS] during acidification within the endosomal compartment , pah - cit undergoes a charge reversal resulting in the release of the sirnas . [SEP]
[CLS] additional constructs include ( a ) au - nps B-nanoparticle with pei 25000 chains replacing the pah - cit compound to create a positively charged au - np B-nanoparticle ; 129 ( b ) au - nps B-nanoparticle coated with a stabilizing and cationic B-material block polymer B-material poly ( n - 2 - hydroxypropyl methacrylamide - block - n - [ 3dimethylamino ) propyl ] methacrylamide p ( hpma 70 - b - dmapma 24 ) and sirna ; ( c ) au - nps B-nanoparticle synthesized in the presence of cysteamine hydrochloride and functionalized with sirna - peg 5000 ; 131 and ( d ) cationic B-material gold B-material nanorods B-nanoparticle ( aunrs ) without the addition of stabilizing peg chains . all of these constructs triggered significant gene silencing in vitro , and pointed to specific ligand requirements for cellular uptake and endosomal escape ; all are awaiting detailed in vivo efficacy studies . [SEP]
[CLS] additional nanoconjugates for rnai delivery include hyaluronic acid ( ha ) nanoparticles B-nanoparticle , also referred to as nanogels , poly ( d , l - lactic - co - glycolic ) acid ( plga ) nanoparticles B-nanoparticle , and dendrimer - conjugated magnetofluorescent nanoworms ( dendriworms ) , all of which can effectively penetrate cells B-material and silence gene expression in vivo , and in the case of dendriworms , show prominent intracellular and intratumoral accumulation , which can be followed by fluorescent B-property tracking ( reviewed by davis et al ) . [SEP]
[CLS] materials on the nanoscale preferentially accumulate in tumor B-material elements due to the epr of a compromised tumor B-material vasculature . [SEP]
[CLS] tumor B-material vessels are characterized by poorly aligned endothelial cells B-material with wide fenestration , which together with absent intratumoral lymphatic drainage results in highly distorted dynamics of molecular and fluid transport ( reviewed by hirsjarvi et al ) . [SEP]
[CLS] to further enhance tumor - specific uptake and tumoricidal activities , and to reduce systemic toxicity B-property in non - tumors organ sites , a spectrum of nanoconstructs has been functionalized with ligands recognizing surface elements of cancerous tissue , foremost the transferrin , folate , and integrin receptors which display soaring overexpression in a variety of tumor B-material tissues . [SEP]
[CLS] specifically , folate - conjugated cholesteryl - 3 - beta - carboxyamidoethylene - n - hydroxyethylamine , and peg - distearoylphosphatidyl ethanolamine ( dspe ) nanoparticles B-nanoparticle with her2 - specific sirnas showed significant in vivo efficacy in xenograft models , and pegylated transferrin receptor - targeted nanoparticles B-nanoparticle composed of a β - cyclodextrincontaining polycation loaded with sirnas against the m2 subunit of ribonucleotide reductase triggered significant reduction in target mrna in cell B-material and animal models , and showed a dose - dependent accumulation in tumor B-material cells B-material of melanoma patients in early phase i clinical trials [SEP]
[CLS] additional targeting B-material ligands I-material have included antibody - protamine fusion proteins B-material for the targeted delivery of sirnas to silence hiv - 1 capsid gene ( gag ) to block hiv replication in primary t cells , anti - b7 integrin antibodies B-material to deliver polymeric B-nanoparticle nanoparticles I-nanoparticle with cyclin d - specific sirnas to leukocytes , [SEP]
[CLS] tumor - targeted nanoparticlesshort peptides B-material derived from rabies virus glycoprotein ( rvg ) to enable sirna to cross the blood - brain - barrier B-property and accumulate within neurons , pegylated rgd peptide B-material targeting pei - sirna nanoconstructs to tumor B-material vasculature to silence vegf - 2 , 141 aptamers recognizing prostatespecific membrane antigen ( psma ) to deliver sibcl - 2 and siplk1 oligonucleotides , and small molecules , e . g . , lactose , and nag to drive sirna - mediated gene silencing in hepatocytes . [SEP]
[CLS] strikingly , targeted particles have yet to make the leap from promising concept to effective anti - cancer compound , as the vast majority of nanoconjugates in advanced preclinical testing and early phase clinical trials do not contain targeting B-material ligands I-material . [SEP]
[CLS] the reasons for the apparent shortcoming of past efforts are complex : ( 1 ) systematic auditing of biological and physico - chemical variables of targeted materials critical for cellular / tissue uptake and pharmacokinetical properties are often suboptimal . [SEP]
[CLS] in addition , reproducible synthesis and consequently large - scale production of targeted conjugates is difficult ; ligand density and activity often vary , impacting biodistribution and half - life ( reviewed by farokhzad and langer ) . [SEP]
[CLS] ( 2 ) surface molecule used as entry sides for targeted therapies via receptormediated endocytosis B-event often display a highly heterogeneous intratumoral distribution , making robust tumor B-material dissemination challenging . [SEP]
[CLS] ( 3 ) different receptors show different rates of endocytosis B-event in different tissue . [SEP]
[CLS] thus , a detailed understanding of endosomal trafficking pathways in different cancers is required to choose the most optimal ligand / receptor B-material system . [SEP]
[CLS] ( 4 ) intratumoral accumulation is a passive process , and requires the extravasation of nanodrugs through the structurally and functionally compromised tumor B-material vasculature into the tumor B-material mass . [SEP]
[CLS] thus , the improvement of biological , and physicochemical properties of the conjugate rather than the addition of a targeting B-material ligand I-material can increase circulating half - lives , and can lead to conjugates that tend to amass in cancerous tissue more effectively . [SEP]
[CLS] while circulation and consequently intratumoral accumulation represent parameters that are independent of targeting B-material ligands I-material , retention and specific cellular uptake of conjugates , however , are processes driven by the targeting moiety of the nanoparticle B-nanoparticle , and can result in more potent anti - tumorigenic effects of targeted versus untargeted therapies . [SEP]
[CLS] john f . kennedy ' s quote " the greater our knowledge increases the more our ignorance unfolds " summarizes the journey cancer genomics began more than 15 years ago . [SEP]
[CLS] cancer initiation , progression , dissemination , and therapy responses are driven by a vast and unpredictably complex spectrum of mutations . [SEP]
[CLS] copy number alterations , nucleotide changes , and translocations can alter protein B-material abundance and protein B-material function , or can create novel proteins B-material . [SEP]
[CLS] collaborations between academic and industrial centers around the world ( e . g . , the tcga , and the international cancer genome consortium , icgc ) have resulted in the maturation and expansion of whole - genome , exome , and transcriptome sequencing , and have released partial cancer genome data sets pointing to more than 7 , 500 putative cancer genes ( icgc dataset version 6 ; http : / / www . icgc . org ) . [SEP]
[CLS] exemplified by the most recent introduction of alk and braf inhibitors into clinical practice , this novel technological paradigm has already begun to revolutionize cancer medicine . [SEP]
[CLS] our advanced understanding of genomic perturbations has allowed us ( a ) to select specific patient populations for clinical trials testing specific target agents , e . g . , trastuzumab for her2 - positive breast cancer , and ( b ) to predict clinical response , e . g . , the ineffectiveness of egfr mab for the treatment of kras - mutated colon cancers ( reviewed by ) , or the failure of raf inhibitors to block progression of cancers with mutated ras alleles ( ' the raf paradox ' ; reviewed by cox and der and by ) . [SEP]
[CLS] together with advances in the functional interrogation of cancer genomes and the development of rnai - based nanotechnological strategies to target emerging driving and contributing oncogenes , the implementation of personalized cancer nanomedicine is an attainable and , most of all , ethically imperative goal . [SEP]
[CLS] the rise of genomic medicine and nanotechnology , however , also point to significant challenges in translating genomic information and nanodrug development into clinical endpoints . [SEP]
[CLS] some of the most relevant questions for genomic and drug development pipelines are : which are the most critical driving and contributing mutations , and how can they be distinguished from genomic noise ? [SEP]
[CLS] what is the genetic and tumor B-material biological context of a given driving or contributing ccg ? [SEP]
[CLS] how can the efficacy , specificity , and biocompatibility B-property of rnai nanotherapeutics be improved ? [SEP]
[CLS] the equally important challenges for bioinformatics , pathology , imaging , biospecimen repositories , and clinical trial management to enable personalized medicine are summarized elsewhere in detail . [SEP]
[CLS] the number of mutations in a cancer cell B-material can range from thousands to hundreds of thousands . [SEP]
[CLS] the majority of these perturbations result from genomic instability and dna damage ( i . e . , passengers ) , with a relatively small number of aberrations having causal roles in disease progression ( drivers and contributors ) . [SEP]
[CLS] among these , only a subset can be therapeutically targeted ( ' druggable ' ) and / or has prognostic or diagnostic significance ( ' actionable ' ) ( reviewed by dancey et al ) . [SEP]
[CLS] in addition , cancer is a highly heterogeneous disease with mutational profiles differing between cancer types , between tumors B-material arising from the same cancer lineage , and between cells B-material within a tumor B-material . [SEP]
[CLS] cancer development and progression is also a highly dynamic process , as tumors B-material can continuously acquire additional mutations due to increasing genomic instability ( intrinsic selection ) , and due to extrinsic pressures conferred by therapy and environmental cues . [SEP]
[CLS] finally , ccgs do not function in linear pathways , but rather exist as part of a multidimensional circuitry , where the activity of one ccg is influenced by the mutational status and function of other ccgs . [SEP]
[CLS] such convoluted wiring of signaling pathways , together with heterotypic tumor - stroma interactions , and the overshadowing impact of patient - specific germline mutations on tumor B-material development , add yet additional layers of complexity to an already intricate disease ( reviewed by chin and gray and by chin et al ) . [SEP]
[CLS] in particular , a recent study by todd golub and colleagues revealed that that the tumor B-material stroma significantly contributes to therapy susceptibility of cancer cells B-material via non - tumor autonomous mechanisms . [SEP]
[CLS] one specific mechanism identified in this study involved the stromal secretion of hepatocyte growth factor ( hgf ) , subsequent activation of the hgf receptor B-material met , and downstream mapk and pi3k pathways in braf mutant melanoma cells B-material , triggering resistance of melanomas toward braf inhibitors . [SEP]
[CLS] how can we make sense of the cancer genome ? [SEP]
[CLS] the continuous and rapid evolution of genomic technologies will likely unravel a complete list of cancer - relevant genes . [SEP]
[CLS] these structural analyses have to be complemented by genome - wide , systematic and integrative approaches to characterize function of ccgs and their context dependencies ( figure 2 ) . [SEP]
[CLS] as described above , these efforts have already led to the identification of several novel cancer genes and pathways . [SEP]
[CLS] it will require , however , further development and refinement of such functional assays to enable true genome - wide functionalization . [SEP]
[CLS] specifically , genome coverage of mammalian orf , cdna , and rnai libraries is incomplete , splice variants are poorly characterized , one - dimensional readouts in most cases are only focused on cell B-material survival , and screening is usually executed in a limited number of cell B-material lines not reflecting the high - level heterogeneity seen in cancers . [SEP]
[CLS] consequently , larger collections of cell B-material cultures have to be used in a wider spectrum of phenotypic assays , e . g . , migration and invasion experiments , as critical cancer genes might not be involved in cellular growth , but may play important roles in other processes , e . g . , metastasis B-event . [SEP]
[CLS] in addition , rnai or cdna complementation screens can produce false positives due to off - target effects . [SEP]
[CLS] here , the integration of functional data obtained from over - and underexpression screens in different cell B-material lineages , derived from different cancer types , together with structural genomic information can provide additional levels of validation . [SEP]
[CLS] finally , the majority of ht screens have focused on the characterization of genes localizing to chromosomal regions with copy number variability . [SEP]
[CLS] to study mutations and translocations , additional alleles encoding for mutant and fusion genes have to be generated to probe their impact on the biology of cancers . [SEP]
[CLS] once ccg lists have been created via ht gain - and loss - of - function screens , triangulated with structural genomic information , and analyzed in vitro for their impact on growth , cell B-event death I-event , migration , invasion , and angiogenesis B-event , then sophisticated xeno - and syngeneic orthotopic models and gemms representing the final step in ccg validation can be conducted . [SEP]
[CLS] as outlined below , these mouse models will not only aid in describing the role of a ccg in tumorigenesis , but will also serve as more refined in vivo testing platforms for novel therapeutics , and will provide important preclinical information preceding clinical trials . [SEP]
[CLS] importantly , such rigorous biological interrogation has to be an iterative process , where deep biological analyses or even early clinical trials have to inform structural and ht functional analyses . [SEP]
[CLS] it is plausible that in - depth characterization of ccgs may contradict early - stage genomic and ht analyses , and may reveal that our definition of a given ccg as a driver or contributor is limited , has to be substantially revised , or is simply incorrect . [SEP]
[CLS] much ink has been devoted to weighing the advantages and disadvantages of murine models for preclinical drug testing . [SEP]
[CLS] many cancer biologists and geneticists would agree that graft and gem models have contributed considerably to our understanding of oncogenes and tumor B-material suppressor function over the past decades . [SEP]
[CLS] in addition , rodent models are the most tractable mammalian systems for pharmacokinetic , pharmacodynamic , and toxicology studies . [SEP]
[CLS] much disagreement , however , exists on the usefulness of xenograft models in cancer drug discovery . [SEP]
[CLS] i argue that the poor correlation between drug efficacy in subcutaneous xenograft studies versus human clinical trials has proven that these models are largely ineffective for preclinical drug evaluation , and are better classified as human - inmouse systems , in which established human cancer cells B-material with limited genotypic resemblance to primary cancer specimens are grown in an immunocompromised host with the support of murine stroma and vasculature . [SEP]
[CLS] due to their cost - effectiveness , ease of generation , and suitability for ht applications , however , graft models are highly useful for drug testing , but have to be significantly refined ( reviewed by sharpless and depinho ) . [SEP]
[CLS] first , patientderived primary cells B-material , e . g . , tumor - initiating cells B-material ( tics ) enriched for cancer stem cells B-material , should be used to generate xenogeneic grafts that more faithfully recapitulate genotypic and phenotypic hallmarks of the human disease compared to transformed cancer cell B-material lines . [SEP]
[CLS] second , the numbers of genomically fully characterized cell B-material lines have to be significantly increased , such that xenografts more adequately represent the genotypic diversity of human tumor B-material specimens so that pharmacogenomic correlates of drug responses can be cataloged . [SEP]
[CLS] third , xenograft systems with more physiological tumor B-material - stroma interactions have to be further developed by orthotopically injecting cells B-material into the organ site of interest , by coimplanting tumor B-material and stromal elements , or by using gemm - derived cancer cells B-material in syngeneic explant studies . [SEP]
[CLS] notwithstanding the continuing evolution of xenograft models , their inability to faithfully model tumor B-material progression ( i . e . , the development of hyperplastic to dysplastic to more malignant stages ) , and to adequately represent the genetic diversity of human cancers limits their applicability , and point to gemms with spontaneously and stochastically developing tumors B-material as superior drug testing platforms . [SEP]
[CLS] in this regard , the costs and ease of generating and managing multi - allelic models , tumor B-material latencies , penetrance , and faithful recapitulation of human tumor B-material characteristics are critical parameters to consider . [SEP]
[CLS] while short latencies and high penetrance of tumor B-material development are preferred , rapidly evolving cancers driven by strong oncogenes with concomitant loss of potent tumor B-material suppressors may not acquire stochastic secondary genetic or epigenetic lesions , but may develop multi - focal tumors B-material that may not properly co - evolve with their stromal microenvironment . [SEP]
[CLS] consequently , we have to define the right balance between practicability and faithful recapitulation of cancer hallmarks . [SEP]
[CLS] although conditional gene targeting remains a lengthy and involved process , gemms can most certainly overcome shortcomings of explant models , as the tumor B-material emerges in an immunocompetent environment , and displays functional stromal interactions . [SEP]
[CLS] simple breeding schemes can be developed to generate the desired multi - allelic cohort , as multiple controls , such as mice harboring individual mutant alleles , are typically not required for drug testing . [SEP]
[CLS] high - penetrance and short latency allow for speedy , cost - effective ht - testing of drugs . [SEP]
[CLS] induction of conditional alleles is straightforward by administering tamoxifen to mice with somatically inducible cre - ert2 alleles . [SEP]
[CLS] finally , tumor B-material development can be followed non - invasively by mr imaging , palpation , quantification of serological markers , or assessment of intratumoral luciferase activity by in vivo imaging system ( ivis ) - based analyses . [SEP]
[CLS] these technological advancements will allow for the facile , yet conclusive preclinical evaluation of drugs . [SEP]
[CLS] several model systems for a variety of different cancer types are available through the mouse models of human cancer consortium ( mmhc ) ( reviewed by sharpless and depinho ) . [SEP]
[CLS] a recent study by wong and colleagues demonstrated the predictive power of gemm for clinical drug testing . [SEP]
[CLS] they conducted synchronous ' co - clinical ' trials in lung cancer patients and gemms harboring different , lung - cancer specific genetic signatures ( kras mutation with concomitant p53 and lkb1 deletions ) . [SEP]
[CLS] drug testing in kras - driven gemms successfully predicted which genetically defined patient populations would benefit from combinatorial treatment regimens consisting of docetaxel and the mek inhibitor selumetinib ( azd6244 ) . [SEP]
[CLS] the discovery and validation of novel oncogenes can immediately inform the design of rnai nanomaterials B-material to specifically target these cancer genes ( figure 5a ) . [SEP]
[CLS] several nanomaterials B-material , such as snas , have emerged as fundamentally novel classes of therapeutics that can overcome the shortcomings of rnai - based therapy , i . e . , delivery , intracellular stability , off - target effects , systemic toxicity B-property , and immunogenicity B-property . [SEP]
[CLS] in particular , snas with densely packed , highly oriented , sirna oligonucleotides can be recognized and endocytosed by scavenger receptors , and following endosomal escape , can potently and persistently neutralize gene expression due to increased resistance toward nuclease - driven degradation ( reviewed by cutler et al ) . [SEP]
[CLS] however , further mechanistic and biological studies are required to fully understand some of the fundamental properties underlying cellular entry , tissue dissemination , low - level immunogenicity B-property , and systemic toxicity B-property . [SEP]
[CLS] furthermore , the modification of snas and other nano - rnai conjugates with ligands or antibodies B-material enabling more robust tumor B-material - specific uptake beyond the epr effect has to be optimized to further increase conjugate efficacy while reducing the potential for adverse side effects associated with systemic administration . [SEP]
[CLS] finally , targeted nano - constructs together with nanomaterials B-material functionalized with multiple sirna sequences enabling concomitant silencing of several oncogenes have to be enrolled into detailed in vivo validation , pharmacokinetic , pharmacodynamic , and toxicology studies in relevant graft and perhaps gem models to drive these platforms toward early - phase clinical trials . [SEP]
[CLS] 5b exemplifies a validation scheme for rnai - based nanoconstructs , starting with detailed expression analyses of ccgs to be targeted , followed by a detailed characterization of nano - rnai - triggered knockdown in cells B-material , animals , and humans . [SEP]
[CLS] collectively , these nanoconstructs provide radically novel treatment options for cancer patients . [SEP]
[CLS] our capacity to design particles functionalized with multiple sirna sequences targeting entire oncogenic signatures , together with the speed of nanodrug development and nanodrug testing in established xeno - , allo - and gem models makes rnai - based nanotechnology a highly attractive anti - cancer approach suitable for co - extinction strategies . [SEP]
[CLS] the foreseeable progress in the development of targeting B-material ligands I-material through ht yeast , phage , and ribosomal display methodologies coupled with a better understanding of how particle size , geometry , and interactive forces between sirna cargo and nanomaterial B-material impact delivery , intracellular release , toxicity B-property , and immunogenicity B-property will most certainly drive the further implementation of nanopharmaceuticals into oncology practice . [SEP]
[CLS] with the signing of the national cancer act of 1971 by then u . s . president richard nixon , and a > 200 billion usd investment in cancer research over the past decades , long - term survival of cancer patients has considerably improved ; advanced - stage malignancies , however , still culminate in death within five years post diagnosis . [SEP]
[CLS] why did we fall short ? why are drug discovery and development processes so ineffective with a near 95 % failure rate in gaining fda approval ? and why is the attrition rate of compounds most prominent in late - stage clinical trials , the most expensive phase of drug testing ? [SEP]
[CLS] major contributing factors are the lack of stringent target gene identification and validation , ineffective preclinical drug testing in physiologically relevant model systems , and only moderate progress in enrolling novel therapeutic concepts , first and foremost nanodrugs , into preclinical and clinical pipelines . [SEP]
[CLS] we have to move past the many proof - of - principle studies of nanoconstructs aimed at targeting an investigator ' s favorite oncogene in standard cell B-material and xenograft models , and move toward genome - scale , unbiased identification of rate - limiting oncogenic networks , and the systematic design of nano - rnai conjugates targeting these oncogenic lesions . [SEP]
[CLS] furthermore , it will be critical to preclinically characterize nanomaterials B-material neutralizing these networks in the most physiologically relevant cell B-material and animal models . [SEP]
[CLS] we have to develop smart combinatorial treatment regimes that combine the power of nano - rnai constructs to neutralize virtually any oncogenic lesions , and the proven efficacy of conventional chemotherapy ( e . g . , dna - damage - inducing agents ) and targeted pharmaceuticals that inhibit critical driving oncogenes , such as rtks . [SEP]
[CLS] it will be critical to determine the molecular mechanisms that act as roadblocks preventing chemo - and rtktargeted therapies from inducing tumor - specific apoptosis B-event and regression . [SEP]
[CLS] we then can target these roadblocks using rnai - based nanomaterials B-material , and can envision using hybrid conjugates , such as h - snas co - functionalized with chemotherapeutics and sirna oligonucleotides to concomitantly target driving oncogenes and downstream anti - apoptotic roadblocks . [SEP]
[CLS] lastly , we have to integrate teams of specialists with expertise in the areas of cancer biology , clinical cancer sciences , and bioengineering to enable deep biological and clinical characterization of nanomaterials B-material . [SEP]
[CLS] larger platform grants , such as centers for cancer nanotechnology excellence ( ccnes ) will continue to be instrumental in driving this integration . [SEP]
[CLS] together with more effective collaborations of academia and industry , the establishment of novel academic constructs and infrastructures that combine multileveled biological and clinical validation with milestone - driven drug development may aid in overcoming the ' valley of death ' separating bench from bed side . [SEP]
[CLS] the past few years have given us a glimpse of the potential of personalized cancer nanomedicine . [SEP]
[CLS] now , cancer geneticists and bioengineers are poised to build on these most recent successes , and to develop bold and ambitious plans to further translate basic genomic findings into clinical endpoints . [SEP]
[CLS] it will be the amalgamation of chemistry and basic and clinical cancer research that will not only increase our knowledge , but also will also help us realize and overcome our ignorance to launch the most audacious attack on cancer yet . [SEP]
[CLS] high - throughput characterization and functional interrogation of cancer genomes has unraveled a complex landscape of genetic and epigenetic modifications , and has initiated the implementation of personalized cancer medicine into clinical practice . [SEP]
[CLS] the sheer complexity of genomic information , and the difficulty to concomitantly modulate the action of multiple ' undruggable ' targets , however , pose significant challenges to drug development . [SEP]
[CLS] here , i provide an overview of the current state and future challenges of genomics - driven cancer medicine . [SEP]
[CLS] i discuss the role that nanotechnology may play in overcoming some of the most critical barriers B-property to clinical progress , foremost the targeting of the undruggable oncogenome , and highlight the importance of integrative , crossdisciplinary approaches bridging cancer genetics , cancer biology , and bioengineering to enable personalized ( nano ) drug design . [SEP]
[CLS] clinical translation of more recent discoveries of alk translocation and braf mutations in non - small cell B-material lung carcinoma ( nsclc ) and melanoma patients , respectively , has been translated into clinical endpoints much faster . [SEP]
[CLS] here , crizotinib , originally discovered as a cmet inhibitor , has entered clinical phase i / ii trials 3 years after the discovery of alk translocations , and the braf inhibitor plx4032 has been enrolled into clinical proof of concept ( poc ) studies in melanoma patients 8 years after the initial discovery of braf mutations . [SEP]
[CLS] in addition , the more rigorous mapping of cancer - associated driving and collaborating mutations enabled prognostication . [SEP]
[CLS] specifically , her2 overexpression ( oe ) has been correlated with favorable responses toward her2 - targeting herceptin , and lead to the development of the diagnostic herceptest . [SEP]
[CLS] similarly , the presence of brac1 / 2 mutations dictates responses toward parp inhibitors . [SEP]
[CLS] tsg , tumor B-material suppressor gene . [SEP]
[CLS] ( a ) multi - dimensional analysis of human and murine cancers include identification of copy number variations ( assessed by fluorescence B-property in situ hybridization and arrayed comparative genomic hybridization , acgh ) , dna mutation ( evaluated by dna sequencing ) , promoter methylation ( determined by chromatin immunoprecipitation and microarray analysis of immunoprecipitated dna ) , and aberrant mrna and protein B-material expression ( assessed by microarray analysis and e . g . , by antibody B-material arrays ) . [SEP]
[CLS] each of these genomic , genetic , epigenetic , and proteomic changes can alter the function , the splicing patterns , or the expression levels of candidate cancer genes ( ccgs ) that potentially can drive cancer initiation , progression , and dissemination . [SEP]
[CLS] additional model systems not depicted here include zebrafish , nematodes , fruitflies and yeast . [SEP]
[CLS] ( b ) integrative , cross - species bioinformatics , and genome - scale interrogation of gene function inform mutation - specific drug design and evaluation . [SEP]
[CLS] here , the assessment of genomic , genetic and proteomic aberrations , cross - species , comparative analyses , and the identification of similarities between different tumor B-material types enable the identification of critical ccgs . [SEP]
[CLS] subsequently , ccgs will be functionally interrogated in massively parallel high - through put ( ht ) gainand loss - of - function ( gof / lof ) studies to assess the modus operandi and impact on tumorigenic signaling cascades . [SEP]
[CLS] subsequently , validated ccgs are enrolled into detailed clinicopathological analyses ( i . e . , cgc expression analyses in primary tumors B-material ) , and cell B-material culture - and animal model - based experiments . [SEP]
[CLS] ccgs with strong cancer - promoting activities can be used as therapeutic targets and / or biomarkers B-property , prompting further clinical evaluation . [SEP]
[CLS] importantly , this cascade has to be an iterative process , where the result from each step can inform and refine preceding analyses to help improve entire drug development process . [SEP]
[CLS] tics , tumor initiating cells B-material ; gemms , genetically engineered mouse models . [SEP]
[CLS] discovery of cancer mutations and its translation into drug development cancer genetics accelerated the clinical development of mutation - specific targeted drugs . [SEP]
[CLS] highlighted examples include targeted therapies that inactivate bcr - abl , egfr , parp , erbb2 , braf and alk ( left panels ) . [SEP]
[CLS] on the right , seminal discoveries in cancer biology and cancer genetics are highlighted , e . g . , boveri ' s discovery of the genetic basis of cancer in 1902 , and the characterization of human glioblastoma and ovarian cancer genome in 2008 . [SEP]
[CLS] gleevec received fda - approval more than 40 years after the initial discovery of bcr - abl translocation in chronic myelogenous leukemia ( cml ) , and the egfr inhibitor gefitinib was finally approved in 2003 , almost 25 years after the cloning of the egfr gene , and more than 20 years after the identification of egfr overexpression as a cancer - driving event . [SEP]
[CLS] mechanism of rnailong double - stranded ( ds ) rna is processed by the rnase iii enzyme dicer to 21 - 23 nucleotide sirna - duplex - like intermediates B-property . [SEP]
[CLS] the duplex is unwound ( mediated by the rna helicases armitage and r2d2 ) , and the risc complex with single - stranded sirnas is formed to mediate cleavage of target mrnas [SEP]
[CLS] 4 . different rnai - based gold B-nanoparticle nanoparticles I-nanoparticle based on gold - thiol chemistry and electrostatic interactions peg , poly ( ethylene glycol ) ; pama , poly ( alkyl methacrylate ) ; f , folate ; pbae , poly ( βamino ester ) ; pei , poly ( ethyleneimine ) ; pah - cit , cis - aconitic anhydride - functionalized poly ( allylamine ) [SEP]
[CLS] integrative nano - drug development ( a ) amalgamation of cancer biology / genetics , bioengineering , i . e . , the design and synthesis of nanotechnological platforms , and nanodrug development to enable the preclinical and clinical development of rna - based nanoconjugates . [SEP]
[CLS] ( b ) validation scheme for the ( pre - ) clinical characterization of iccg nano - conjugates . [SEP]
[CLS] initial studies aim to assess ccg expression in a panel of cancer cells B-material to stratify lineages with high , low , or absent expression for subsequent functional studies to assess siccg - nanoconjugates . [SEP]
[CLS] animal studies , together with toxicology , pharmacokinetic and - dynamic studies will enable early phase 0 / i / ii clinical studies in humans . [SEP]
[CLS] of note , model systems to validate iccg nanoconstructs show different degrees of cost - and time effectiveness , and complexity . [SEP]
[CLS] although many different nanomaterials B-material have been tested as substrates for laser desorption and ionization B-property mass spectrometry ( ldi - ms ) , this emerging field still requires more efficient multifuncional nanomaterials B-material for targeting , enrichment and detection . [SEP]
[CLS] here , we report the use of gold B-material - manganese B-material oxide B-material ( au @ mno ) hybrid nanoflowers as an efficient matrix for ldi - ms . [SEP]
[CLS] the nanoflowers were also functionalized with two different aptamers to target cancer cells B-material and capture adenosine triphosphate ( atp ) , respectively . [SEP]
[CLS] these nanoflowers were successfully used for metabolite extraction from cancer cell B-material lysates . [SEP]
[CLS] thus , in one system , our multifunctional nanoflowers can 1 ) act as an ionization B-property substrate for mass spectrometry , 2 ) target cancer cells B-material , and 3 ) detect and analyze metabolites from cancer cells B-material . [SEP]
[CLS] purpose uses [SEP]
[CLS] the high surface area provided by hybrid nanomaterials B-material can simultaneously convey more than one functional B-material group I-material ( e . g . , nucleic B-material acids I-material or aptamers , small chemical molecules , antibodies B-material , peptides B-material ) , and / or carry multi - cancer drugs ( watersoluble or insoluble ) in cancer - specific delivery , and / or combine several imaging agents ( fluorescence B-property dyes and radionuclides ) for multi - modal imaging . [SEP]
[CLS] particularly , multifunctional hybrid nanomaterials B-material have been developed to overcome the limitations of conventional techniques used for diagnosis and therapy in biomedical applications . [SEP]
[CLS] for example , fe 3 o 4 / mno hybrid nanoparticles B-nanoparticle can be used for magnetic B-property resonance imaging ( mri ) , and the iron B-material or manganese B-material domain can be functionalized with targeting B-material ligands I-material to specifically recognize target cells B-material . [SEP]
[CLS] the cancer drugs or imaging agents can simultaneously be attached on the second domain of the same particles . [SEP]
[CLS] there are no significant interferences observed between functional groups on hybrid nanoparticles B-nanoparticle due to the presence of two discrete domains . [SEP]
[CLS] these heterostructured materials composed of two or more different nanocomponents have been prepared in dumbbell , peanut , flower and star shapes . [SEP]
[CLS] for example , sun and coworkers developed and synthesized core - shell , dumbbell - like and flower - shaped au @ fe 3 o 4 nanoparticles B-nanoparticle . [SEP]
[CLS] these heterostructures have distinct advantages compared to individual au and fe 3 o 4 nanoparticles B-nanoparticle . [SEP]
[CLS] their magnetic B-property ( fe 3 o 4 ) and optical ( au ) active domains have been used , respectively , for magnetic B-property imaging and optical detection purposes . two - component dumbbell - like nanoparticles B-nanoparticle are also very suitable for selective immobilization of two different biofunctional groups for target capturing , imaging and drug delivery . [SEP]
[CLS] chen et al . and tremel et al . have respectively reported hybrid flower - like au @ fe 3 o 4 and au @ mno nanoparticles B-nanoparticle which were used for simultaneous optical and magnetic B-property resonance imaging ( mri ) and therapy . [SEP]
[CLS] in addition , gold B-material nanostars with magnetic B-property cores B-material have been developed by wei and co - workers to demonstrate magnetomotive imaging by using the nir scattering properties of the gold B-material shell B-material and the magnetic B-property properties of the iron B-material oxide I-material cores B-material . [SEP]
[CLS] thus , the limitations possessed by single component nps B-nanoparticle have been surpassed with development of hybrid nps B-nanoparticle . [SEP]
[CLS] in addition to the aforementioned uses , nps B-nanoparticle have recently been employed in a relatively new field called nanostructure assisted laser desorption ionization mass spectrometry ( nano - ldi ) . [SEP]
[CLS] in parallel with the development of mass spectral instrumentation , such as tandem ms and imaging mass spectrometry , matrix - assisted laser desorption / ionization B-property mass spectrometry ( maldi - ms ) has become a well - established analytical tool for ionization B-property and analysis of biomolecules . [SEP]
[CLS] for instance , the prominent work was done by deng and coworkers by using titanium dioxide decorated graphene composite ( tio 2 / graphene ) for the selective enrichment of phosphor from mixture of peptide B-material with matrix - assisted laser desorption ionization - time of flight mass spectrometry ( maldi - tof - ms ) . [SEP]
[CLS] however , signal suppression effects , shot - to - shot reproducibility problems and the need for careful optimization of sample preparation still limit the use of maldi - ms . [SEP]
[CLS] because the ionization B-property process in maldi is also very complex and still poorly understood , progress in the development of new methods to improve ionization B-property efficiency is slow . [SEP]
[CLS] to overcome these problems , several nanomaterials B-material have been developed and applied as matrices for laser desorption ionization mass spectrometry ( ldi ) . [SEP]
[CLS] essentially , the use of nanomaterials B-material as energy - absorbing materials dates back to the early days of ldi development , in which ultrafine cobalt B-nanoparticle nanoparticles I-nanoparticle dispersed in glycerol were used to promote ionization B-property . [SEP]
[CLS] however , because of the difficulty of handling glycerol and the resulting contamination in ms systems , the use of nanomaterials B-material was largely overlooked until the development of desorption ionization B-property on porous silicon B-material ( dios ) , which initiated a new area called nanomaterial - based surface - assisted laser desorption / ionization B-property . [SEP]
[CLS] following this seminal work , various nanomaterials B-material have been tested as substrates for ionization B-property , including plasmonic particles ( au and ag spheres and clusters ) , semiconductor crystals ( cdse , cds , and cdte quantum B-nanoparticle dots I-nanoparticle ) , magnetic B-property particles ( fe 3 o 4 , coo , and mno ) , other metallic particles ( pt and pd ) , carbon - based materials ( swcnts , mwcnts , go and combinations ) and silicon B-material wafers . [SEP]
[CLS] chang [SEP]
[CLS] and [SEP]
[CLS] coworkers [SEP]
[CLS] used hgte nanostructure as a new matrix to analyze protein B-material , protein - drug complexes and peptides B-material as small biological molecules with technique of surface - assisted laser desorption / ionization B-property mass spectrometry ( saldi - ms ) . [SEP]
[CLS] hgte nanostructure which was employed as a matrix provided high analyst signal and low background due to soft absorption and desorption process under the low laser power . [SEP]
[CLS] the use of nanomaterials B-material in ms has several additional advantages over organic matrices , including easy sample preparation , high salt B-material tolerance , absence of self - ionization B-property , and rapid data collection . [SEP]
[CLS] while nanomaterials B-material themselves are very useful as a matrix for ionization B-property , their use as a tool to detect biomolecules in complex biological environments is difficult , as their intrinsic capturing ability is quite limited . [SEP]
[CLS] so far only a handful of examples exist in the literature where nps B-nanoparticle were used both as a capturing probe and at the same time as an ionization B-property matrix in a dual " catch and detect " format . [SEP]
[CLS] this task can only be achieved by surface functionalization of nanoparticles B-nanoparticle . [SEP]
[CLS] in the elegant approach of the rotello group , the surfaces of au nps B-nanoparticle were functionalized with cationic B-material ligands and these " mass barcodes " were then used to assess the cellular uptake of au nps B-nanoparticle by ldi - ms . [SEP]
[CLS] this method has proven to be very successful in demonstrating the intracellular stability of nps B-nanoparticle and excellent matrix properties of au nps B-nanoparticle for ms . [SEP]
[CLS] the next best option is the use of tethering affinity ligand on np B-nanoparticle surfaces . [SEP]
[CLS] siuzdak ' s group has demonstrated nanostructured dios surfaces can be selective by avidin - biotin chemistry or through antibody B-material immobilization . [SEP]
[CLS] antibodies B-material are very useful affinity probes however oriented chemical immobilization of them onto solid supports still represents a big hurdle . [SEP]
[CLS] generally complex chemical strategies which deteriorate the antibody B-material activity are needed . [SEP]
[CLS] aptamers have emerged as very attractive candidates for affinity tailoring of np B-nanoparticle surfaces with enhanced recognition capabilities . [SEP]
[CLS] aptamers are dna - or rna - based molecules selected by an in vitro iterative process called selex ( systematic evolution of ligands by exponential enrichment ) . [SEP]
[CLS] aptamers can bind to their targets with high affinity and specificity , and have been generated for a wide variety of targets , including small molecules and ions B-material , large proteins B-material , and even biological cells B-material [SEP]
[CLS] various different functional groups can easily be introduced onto aptamers without affecting their affinities . [SEP]
[CLS] therefore , aptamer surface immobilization onto nps B-nanoparticle is quite easy and efficient compared to antibody B-material immobilization . [SEP]
[CLS] we and others have shown that aptamer conjugated nps B-nanoparticle can be used for specific cell B-material targeting , rare protein B-material capture and enrichment , drug delivery and therapy . [SEP]
[CLS] despite all the efforts invested using nps B-nanoparticle in ms applications , the choice of nanoparticles B-nanoparticle has largely been made by trial and error and very few studies have compared the performance of different nanoparticles B-nanoparticle for their performance for ldi - ms . [SEP]
[CLS] however , based on previous studies [SEP]
[CLS] noble metals B-material , such as au and pt nanoparticles B-nanoparticle , and metallic B-material oxides I-material have been reported to have better performance . [SEP]
[CLS] it has also been reported very recently that bringing two nanostructures together results in better ms performance due to the synergistic effect . [SEP]
[CLS] however , to the best of our knowledge , hybrid au - metal B-material oxide I-material nanomaterials B-material have not previously been tested for ldi - - ms . [SEP]
[CLS] herein we report the use of a novel au @ mno nanoflower - shaped nanoparticle B-nanoparticle matrix , and compare its performance to similar , but differently shaped , nanoparticles B-nanoparticle , such as fe 3 o 4 , fe 3 o 4 @ au core B-material / shell B-material , au - fe 3 o 4 dimerlike and au @ fe 3 o 4 flower - like nps B-nanoparticle ( supporting information , figure s2 ) . [SEP]
[CLS] furthermore , by using benzyl pydridinium chloride B-material thermometer ion B-material , we have compared the survival yield of 5 different particles with different shapes and geometries , and we have demonstrated that au @ mno nanoflowers serve as the matrix for ldi - ms among these particles . [SEP]
[CLS] ( supporting information , figures s3 - s4 ) [SEP]
[CLS] after selecting au @ mno nanoflowers as a material B-material for ionization B-property , we analyzed a series of different small molecules to show that this particle is indeed an efficient substrate for ionization B-property which gives little or no background ions B-material ( supporting information , figures s5 - s8 ) . [SEP]
[CLS] au @ mno nanoflowers were tested in the negative and positive ionization B-property mode , as well with model compounds , to show the proper mode for reduction of salt B-material effects , which is important in the analysis of peptides B-material , fatty acids , as well as nucleosides and nucleotides ( supporting information , figures s9 - s10 ) . [SEP]
[CLS] the major goal of this study is to demonstrate the synergistic effect of two domains in one nanoflower particle for ionization B-property and to also show how aptamers can improve the selectivity of this system . [SEP]
[CLS] we have also functionalized the surfaces of au @ mno nanoflowers to combine the selectivity of aptamers and the matrix capabilities of au @ mno nanoparticles B-nanoparticle and used these as a proof of concept for selective isolation of atp from cancer cells B-material . [SEP]
[CLS] apart from its biological importance , atp was chosen as a model target molecule for several reasons . [SEP]
[CLS] first , a very good aptamer is already available for atp targeting . [SEP]
[CLS] second , atp is an abundant , metabolite for ms analysis . [SEP]
[CLS] third , the molecular weight of atp falls in the m / z range where nanoparticles B-nanoparticle show high ionization B-property efficiency . [SEP]
[CLS] the aptamer - functionalized au - mno nanoflowers developed in this study can act as a targeting and sensing platform for selective metabolite extraction from cancer cell B-material lysates followed by mass spectral analysis . [SEP]
[CLS] the au @ mno nanoflowers are functionalized with two different aptamers : sgc8 to target ccrf - cem cancer cells B-material and an anti - atp aptamer to sense and capture atp . [SEP]
[CLS] thus , in one system , these multifunctional au @ mno nanoflowers can target cancer cells B-material , capture metabolites , and serve as a mass spectrometry ionization B-property substrate . [SEP]
[CLS] the presence of two different domains , mno and au , simplified the surface modification and eliminated any interference between the two aptamers . [SEP]
[CLS] 1 shows that the synthesized au @ mno nanoflowers were first made water B-property - I-property soluble B-property using dopamine - peg - cooh biopolymer B-material . [SEP]
[CLS] the dopamine side of the biopolymer B-material has two hydroxyl B-material groups I-material that react with the mno component of the nanoflowers through strong catechol reactions to transfer nanoflowers to an aqueous environment for bioapplications . [SEP]
[CLS] to activate the carboxyl B-material group I-material of the biopolymer B-material , edc / nhs chemistry was applied to the dopamine - peg - cooh biopolymer - attached nanoflowers . [SEP]
[CLS] the amine B-material ( - nh 2 ) group - terminated sgc8 aptamers were introduced into the activated dopamine - peg - cooh nanoflower solution and incubated B-technique for 1 hr . [SEP]
[CLS] secondary , thiol modified anti - atp aptamers were immobilized on the surface of the au domain of nanoflowers through thiol - gold B-material surface conjugation . [SEP]
[CLS] synthesis of the heterobifunctional ligand , dopamine - peg - cooh , was carried out with the three following steps : 1 ) synthesis of 3 , 4 - dihydroxyhydrocinnamic acid - pentafluorophenol ester , 2 ) synthesis of nh 2 - peg - cooh , and 3 ) synthesis of dopamine - peg - cooh ( supporting information , figure s1 ) . [SEP]
[CLS] au @ mno nanoflowers were characterized using tem , hr - tem , energy dispersive x - ray ( edx ) analysis and uv - vis spectrometry , respectively . [SEP]
[CLS] 2 shows the tem image of au @ mno nanoflowers ( fig . 2a ) with large and discrete mno petals by high - resolution transmission I-technique electron I-technique microscopy I-technique ( hr - tem ) ( insert ) . [SEP]
[CLS] several nucleation sites on the gold B-nanoparticle nanoparticles I-nanoparticle resulted in the formation of three or four mno petals on au seeds . [SEP]
[CLS] the use of the optimum molar ratio ( 20 : 1 ) of the mn ( acac ) 2 / au ( oocch 3 ) 3 precursors resulted in the absence of single au or mno np B-nanoparticle formation . [SEP]
[CLS] the mno and au elemental composition of nanoflowers can be observed on the edx spectrum ( fig . 2b ) . [SEP]
[CLS] au nanospheres B-nanoparticle exhibit a single and sharp absorption peak in the visible range between 505 nm and 560 nm based on size . [SEP]
[CLS] in particular , au nanospheres B-nanoparticle in the size range of 10 - 30 nm show a characteristic peak around 525 nm , which corresponds to the frequency of the surface plasmon oscillations . [SEP]
[CLS] it is well known that the position of the absorption peak of au nanoparticles B-nanoparticle varies with shape , size , and surface coating B-material , as well as the concentration of other metals B-material . [SEP]
[CLS] moreover , the nucleation of mno petals on au spheres creates an electron junction between the au core B-material and mno petals and a high local dielectric effect around the au core B-material . [SEP]
[CLS] both effects lead to a remarkable red shift of wavelength to 565 - 580 nm and broadening of the peak ( fig . 2c ) . [SEP]
[CLS] s 2d and 2e confirm that fitc - labeled sgc8 aptamer and tmr - labeled anti - atp aptamer were successfully bound to the surface of the mno and au domains of nanoflowers , respectively . [SEP]
[CLS] 2d shows that the fluorescence B-property intensity of fitc - labeled aptamers attached to the surface of mno petals is enhanced with increasing numbers of nanoflowers . [SEP]
[CLS] because of the corresponding increase in the number of fitc - labeled sgc8 aptamers , tmrlabeled anti - atp aptamers immobilized on nanoflower au nanoparticles B-nanoparticle also exhibit higher fluorescence B-property intensity with increasing numbers of nanoflowers ( fig . 2d ) . [SEP]
[CLS] no effective quenching was observed because the tmr - labeled atp aptamers were not sufficiently close to the au surface to initiate fluorescence B-property resonance energy transfer ( fret ) , considering the length of the anti - atp aptamer ( 29 mers length with two ends modifications , ~ 10 nm ) ( fig . 2e ) . [SEP]
[CLS] the effective fret generally takes place in less than 10 nm distance , particular distance is ~ 7 nm , between fluorophore and quencher or plasmonic nps B-nanoparticle . [SEP]
[CLS] these two fluorescence B-property spectra show that heterobiaptameric nanoflower conjugates can be prepared without significant interference between the aptamers . [SEP]
[CLS] binding and internalization studies were performed at 4°c and 37 °c , respectively . [SEP]
[CLS] ccrf - cem cells B-material were prepared in six separate tubes and divided into two sets of three tubes each . [SEP]
[CLS] one hundred μl of 20 ng / ml , 200 ng / ml , and 800 ng / ml nanoflowers were added to each set . [SEP]
[CLS] one set of tubes was incubated B-technique on ice at 4 °c for 30 minutes for a surface binding study , and the other set was incubated B-technique in incubator B-technique shaker at 250 rpm ( thermo electron corporation , forma orbital shaker refrigerate B-property , nc , usa ) at 37°c for 2 hours for an internalization study . [SEP]
[CLS] s 3a and 3b show the flow B-technique cytometry I-technique results of the surface binding ( fig . 3a ) and internalization ( fig . 3b ) . [SEP]
[CLS] two million ccrf - cem cells B-material were incubated B-technique with 20 , 200 , and 800 ng of scg8 - nanoflowers at 4°c ( surface binding ) or at 37°c ( internalization ) . [SEP]
[CLS] the nanoflowers ( 800 ng ) showed optimum signal shift for surface binding and internalization in the flow B-technique cytometry I-technique experiment . [SEP]
[CLS] a significant increase in the fluorescence B-property signal occurred between the lowest ( 20 ng / ml ) and highest ( 800 ng / ml ) concentration of nanoflowers used for the cell B-material uptake study . [SEP]
[CLS] however , the fluorescent B-property signal at 37 °c was lower than that reported at 4 °c , possibly because of particle aggregation in the cell B-material , resulting in partial quenching of fitc dye , or the weak fluorescence B-property nature of fitc dye attached to the sgc8 aptamer . [SEP]
[CLS] cell B-material internalization was verified by confocal microscopy B-technique utilizing the tmr label on the anti - atp aptamer . [SEP]
[CLS] s 3c and 3d show no tmr fluorescence B-property from the ccrf - cem cells B-material or cells incubated B-technique with bare nanoparticles B-nanoparticle . [SEP]
[CLS] cells B-material incubated B-technique with tmr - labeled anti - atp aptamer - functionalized nanoflowers ( 800 ng ) displayed intense fluorescence B-property signals ( fig . 3e ) . [SEP]
[CLS] the flow B-technique cytometry I-technique results of the surface binding and internalization using 800 ng / ml of nanoflowers are highly consistent with the confocal microscopy B-technique results . [SEP]
[CLS] after verification of nanoflower internalization , the next step was to test different cell B-material lysis conditions to obtain the most efficient detection of metabolites by ldi - ms . [SEP]
[CLS] extraction of metabolites from cell B-material lysates is an area of great interest , especially in the growing area of metabolomics . [SEP]
[CLS] in the past five years , several methods have been used with different conditions to release the metabolites from the biological mixtures , but success still depends on trial - and error - based optimization . [SEP]
[CLS] organic solvents , such as cold acetone , methanol , and ethanol , have been generally used for metabolite extraction . [SEP]
[CLS] in our first attempt , we used cold methanol for cell B-material lysis . [SEP]
[CLS] after treatment with cold methanol for 10 min , the final cell B-material lysis mixture was brought to room temperature , and particles were centrifuged and removed from the cell B-material lysate . [SEP]
[CLS] after several cycles of washing , an aliquot of the particles was spotted onto the maldi plate and analyzed . [SEP]
[CLS] however , as shown in figure 4a , no signal for atp or its secondary metabolites could be observed . [SEP]
[CLS] lack of atp signal could have resulted from ( 1 ) an insufficient amount of internalized particles and instability of the particles , ( 2 ) nonfunctional atp aptamer within the cells B-material because of cellular complexity or cleavage by endonucleases , or ( 3 ) disruption of aptamer - target interaction by cell B-material lysis with organic solvents . [SEP]
[CLS] the first two reasons are very unlikely because aptamers can be easily internalized and can recognize their targets , even in cells B-material . [SEP]
[CLS] although it is possible that aptamers may be cleaved by cellular nucleases , recent studies showed that nanoparticles B-nanoparticle provide a strong barrier B-property against such unwanted enzymatic reactions . [SEP]
[CLS] a notable example is the nanoflare method developed to monitor mrna activity . [SEP]
[CLS] this approach relies on immobilizing an oligonucleotide and a reporter complementary strand on a gold B-nanoparticle nanoparticle I-nanoparticle surface , then reporter complementary strand fluoresces B-property upon target binding . [SEP]
[CLS] the authors showed that endonucleases did not cleave nanoparticle - immobilized oligonucleotides . this has also been demonstrated in a similar work on " aptamer nanoflares " . [SEP]
[CLS] moreover , by careful choice of the surface chemistry , recent studies have clearly demonstrated that gold B-nanoparticle nanoparticles I-nanoparticle are highly stable in cells B-material . [SEP]
[CLS] the last , and most likely , reason is the use of an organic solvent during cell B-material lysis and metabolite extraction . [SEP]
[CLS] compared to antibodies B-material , aptamers are more stable molecules against changes in ph , temperature , and ionic strength , but target recognition still depends on their 3d conformations . [SEP]
[CLS] it is therefore very plausible that the cold methanol treatment irreversibly disrupts the aptamer - target interaction . [SEP]
[CLS] therefore , in the next set of experiments , we replaced cold methanol with boiling water B-material for metabolite extraction and cell B-material lysis . [SEP]
[CLS] while aptamers can be denatured with heat , they can easily refold to their proper 3d conformation upon cooling . [SEP]
[CLS] moreover , previous studies have shown that boiling water B-material is a better solvent for extracting nucleotides from cells B-material . [SEP]
[CLS] we repeated the same experiments using water B-material at 65°c as the metabolite extraction solvent . [SEP]
[CLS] however , this time we were challenged by the rapid turnover of atp molecules to adp and amp . [SEP]
[CLS] because the anti - atp aptamer also recognizes adp and amp , signals by these secondary metabolites were observed ( fig . 4b ) . [SEP]
[CLS] to overcome this problem , we have slightly modified the metabolite extraction step by incorporating another step using cold nh 4 hco 3 buffer and dipping the cells B-material into liquid n 2 to quench the metabolism prior to metabolite extraction and cell B-material lysis . [SEP]
[CLS] using this method , the results in figure 5 clearly show that atp can be detected with no significant atp decomposition . [SEP]
[CLS] the respective control experiments show , to a great extent , that atp can be captured , extracted and detected only by the dual aptamer - modified au @ mno nanoflowers . [SEP]
[CLS] in conclusion , our study demonstrated that flower - shaped au @ mno nanoparticles B-nanoparticle could be efficiently used as a multifunctional platform to specifically target ccrf - cem cells B-material and capture atp molecules from cell B-material lysate . [SEP]
[CLS] atp as stated in our work was used as a model analyte but potentially this method can also be utilized for other intracellular targets where high affinity aptamers are available . [SEP]
[CLS] the nanoflowers can also serve as an efficient ionization B-property substrate for laser desorption / ionization B-property without an additional matrix . [SEP]
[CLS] therefore , single - platform nanoflower conjugates containing mno and au components provide an ideal " all - in - one system " for selective functionalization with the desired biofunctional groups , as well as for use as an ionization B-property substrate for ldi - ms . [SEP]
[CLS] this method can potentially be used for other important biological targets where high affinity aptamers are available . [SEP]
[CLS] all chemicals were used as received without further purification . [SEP]
[CLS] manganese ( ii ) acetylacetonate , [ fe ( co ) 5 ] 98 % , oleic acid , oleylamine , 1 - octadecene 90 % , hexane , toluene > 99 % , 1 , 2 , 3 , 4 - tetrahydronaphthalene 99 % , tert - butylamine - borane 97 % , and phenyl ether B-material 99 % , were obtained from sigma aldrich . [SEP]
[CLS] gold ( iii ) acetate > 99 . 9 % was purchased from alfa aesar . [SEP]
[CLS] hexane was purchased from fisher . [SEP]
[CLS] for the synthesis of dopamine - peg - cooh , succinic anhydride , n , n , dicyclohexylcarbodiimide ( dcc ) , 3 , 4dihydroxyhydrocinnamic acid , and ( 3 - ( 3 , 4 - dihydroxyphenyl ) - propionic acid were obtained from alfa aesar . [SEP]
[CLS] o , o - bis ( 2aminopropyl ) polypropylene - glycol - block - polyethylene glycolblockpolypropyleneglycol , 2 , 3 , 4 , 5 , 6 , pentafluorophenol ( pfp ) , 1 , 4 dioxane and dithiothreitol ( dtt ) were purchased from sigma - aldrich . [SEP]
[CLS] one drop of au @ mno nanoparticles B-nanoparticle dispersed in hexane was deposited on carbon B-material - coated copper B-material grids and allowed to dry for at least two hours . [SEP]
[CLS] transmission B-technique electron I-technique microscopy I-technique ( tem ) on a hitachi h - 7000 transmission electron microscope with a working voltage of 100 kv was used to obtain images of the nanoflowers . [SEP]
[CLS] a jeol jem - 2010f field emission electron microscope coupled with spatially resolved energy dispersive x - ray spectroscopy B-technique ( edx ) was also used for magnified images and further characterization of nanoflowers . [SEP]
[CLS] an 1800 uv - vis spectrophotometer ( shimadzu scientific instruments , columbia , md ) was used for the absorption spectra of nanoflowers and to determine the concentrations of dna aptamers . [SEP]
[CLS] fluorescence B-property measurements were carried out on a fluoromax - 4 ( horoba jobin - yvon , edison , nj , usa ) , which was also used for validation of the dye - labeled aptamernanoflower conjugates . [SEP]
[CLS] an olympus fv - 500 - ix81 confocal microscope ( olympus , center valley , pa , usa ) having a 40x - oil - dispersion objective was used to image cancer cells B-material incubated B-technique with nanoparticles B-nanoparticle . [SEP]
[CLS] binding of functionalized nanoparticles B-nanoparticle to target cells B-material was demonstrated with a facscancytometer ( becton dickinson immunocytometry systems , san jose , ca , usa ) . [SEP]
[CLS] all mass spectra were acquired using a maldi - tof / tof mass spectrometer ( abi / sciex 5800 , applied biosystems , foster city , ca ) with a nd : yag laser at 355 nm . [SEP]
[CLS] the spectra were recorded in reflection mode in either positive or negative mode using an accelerating voltage of 20 kv , a 66 % grid voltage , and a 50 - 200 ns delay extraction . [SEP]
[CLS] typically , 10 - 60 laser shots were collected per spectrum . [SEP]
[CLS] applied biosystems calibration mixture 1 was used to calibrate the mass spectrometer . [SEP]
[CLS] data analysis was performed using data explorer software . [SEP]
[CLS] ccrf - cem cells B-material ( ccl - 119 t - cell , human acute lymphoblastic leukemia ) were obtained from atcc ( american type culture collection ) . [SEP]
[CLS] rpmi medium supplemented with 10 % fetal bovine serum ( fbs ) and 100 iu / ml penicillin - streptomycin was used for cell B-material culture ( 5 % co 2 , 37 °c ) . [SEP]
[CLS] a hemocytometer was used to determine the cell B-material density before each experiment . [SEP]
[CLS] two million cells B-material suspended in rpmi cell B-material media were centrifuged at 970 rpm for 5 min and redispersed in 2 ml of washing buffer ( dulbecco ' s pbs with calcium B-material chloride B-material and magnesium B-material chloride supplemented with 4 . 5 g / l glucose and 5 mm mgcl 2 ) for incubation B-technique . [SEP]
[CLS] an abi3400 dna / rna synthesizer ( applied biosystems , foster city , ca ) was used for the synthesis of the aptamers at the 1 μmol scale : tamra - modified aptamer sequences were deprotected in 0 . 05m potassium B-material carbonate B-material in methanol at 65 °c for 3 - 4 hr , and all other sequences were deprotected in ama ( ammonium hydroxide B-material / 40 % aqueous methylamine 1 : 1 ) at 65 °c for 30 min . [SEP]
[CLS] deprotected sequences were purified by reversedphase hplc ( prostar - varian , walnut creek , ca ) with a c18 column ( econosil , 5 μm , 250 - 4 . 6 mm ) from alltech ( deerfield , il ) using the mixture of 100 mm triethylamine - acetic acid buffer ( teaa , ph 7 . 5 ) and acetonitrile ( 0 - 30min , 10 - 100 % ) . [SEP]
[CLS] synthesis of aptamersas the mobile B-property phase . [SEP]
[CLS] after hplc purification , the purified dna solution was dried in acid - resistant centrivap centrifugal vacuum concentrators ( labconco , kansas city , mo ) . [SEP]
[CLS] the dried dna was dissolved in 50 μl dna grade water B-material , and the concentration of each sequence was determined based on the absorbance value at 260 nm using a uv - 1800 uv - vis spectrophotometer ( shimadzu scientific instruments , columbia , md ) . [SEP]
[CLS] au @ mno nanoparticles B-nanoparticle were synthesized using a modified method with standard schlenk line techniques . [SEP]
[CLS] a solution containing diphenyl ether B-material ( 10 ml ) , oleic acid ( 1 ml , 3 mmol ) and oleylamine ( 2 ml , 4 mmol ) was degassed at room temperature for 45 min and backfilled with argon B-material at least three times to remove any trace of air and moisture . [SEP]
[CLS] the degassed solution containing diphenyl ether B-material ( 10 ml ) , oleic acid ( 1 ml , 3 mmol ) and oleylamine ( 2 ml , 4 mmol ) was injected into a 100ml three - necked flask containing 1mm of manganese B-material ( ii ) acetylacetonate and 0 . 05 mm of gold ( iii ) acetate ( 20 : 1 mn ( acac ) 2 / au ( oocch 3 ) 3 ) placed in a glove box . [SEP]
[CLS] the resulting mixture was rapidly heated to 265°c with vigorous stirring . [SEP]
[CLS] after refluxing for 60 min , the mixture was allowed to cool to room temperature under argon B-material flow . [SEP]
[CLS] ethanol was added to the mixture to precipitate a dark - purple product , which was collected by centrifuging at 10 , 000 rpm for 15 min . [SEP]
[CLS] the precipitated product was redispersed in toluene or hexane and reprecipitated by addition of ethanol . [SEP]
[CLS] this purification process was repeated three times . [SEP]
[CLS] finally , the product was dissolved in toluene or hexane , flushed with argon B-material and stored at 4 °c for further use . [SEP]
[CLS] aqueous phase transfer of au @ mno nanoflowers from toluene or hexane to ultrapure water B-material or pbs buffer was achieved using heterobifunctional ligand ( see suppoting information scheme s1 for structure and synthesis ) . [SEP]
[CLS] the hydroxyl ( oh ) groups on the dopamine side of the peg ligand preferentially bind the surface of the mno domain of nanoflowers , and the carboxyl B-material group I-material on the other side contributes water B-material - solubility B-property and is free to react with aptamers in the presence of 1 - ethyl - 3 - ( 3 - dimethylaminopropyl ) carbodiimide ) , edc and n - hydroxysulfosuccinimide ( sulfo - nhs ) . [SEP]
[CLS] a 10 mg sample of au @ mno nanoflowers was dissolved in 5ml of anhydrous terahydrofuran ( thf ) and flushed with argon B-material for 15 min . [SEP]
[CLS] after degassing , the solution was transferred to a dropping funnel . [SEP]
[CLS] heterobifunctional dopamine - terminated peg ( 150 mg ) was dissolved in 20 ml of anhydrous thf in a 100ml three - necked flask . [SEP]
[CLS] the nanoflower solution was added dropwise to the peg ligand solution . [SEP]
[CLS] the resultant mixture was stirred under argon B-material for 15 min and kept under an argon B-material blanket at 50 °c overnight . [SEP]
[CLS] the nanoflower - peg conjugates were precipitated via addition of ~ 3ml of hexane and collected by centrifugation at 10000 rpm for 20 min . [SEP]
[CLS] the supernatant was discarded , and the product , a nanoflower - peg conjugate at the bottom of centrifuge tube , was washed with ethanol and centrifuged twice to remove excess unreacted peg ligands . [SEP]
[CLS] finally , the nanoflower precipitate was dispersed in 2 ml of ultrapure water B-material or pbs buffer for aptamer conjugation . [SEP]
[CLS] conventional protein B-material labeling chemistry was utilized to attach two different aptamers , sgc8 and anti - atp , to the surfaces of two different nanoflower domains , mno and au . [SEP]
[CLS] a 30 μl aliquot of 0 . 1 m 1 - ethyl - 3 - ( 3 - dimethylaminopropyl ) carbodiimide ( edc ) was added to 150 μg / ml nanoflowers in 10mm phosphate buffered saline ( pbs ) to activate the carboxyl B-material group I-material on the peg ligand for 20 minutes . [SEP]
[CLS] after gently shaking for 20 minutes , 13 . 1 μl of 228 μm sgc8 aptamer and 30 μl of 0 . 12 m n - hydroxysuccinimide ( nhs ) in 10 mm pbs buffer were added and incubated B-technique for 1 hour , while gently shaking to functionalize the mno domain of nanoflowers . [SEP]
[CLS] after 1 hour incubation B-technique , 7 . 45 μl of 300 μm anti - atp aptamer and 77 μl of 10 mm pbs buffer were added , and the mixture was placed on a shaker for incubation B-technique overnight at room temperature to functionalize the au domain . [SEP]
[CLS] before immobilization of the anti - atp aptamer , the thiol B-material modified on the 5 ′ - end was deprotected by dithiothreitol ( dtt ) . [SEP]
[CLS] one mm 5 ′ prime thiol - modified anti - atp aptamer in 1 ml of 10 mm pbs buffer ( ph 8 . 3 ) was incubated B-technique with 1 m dtt for 30 min at room temperature to deprotect the thiol B-material functionality . [SEP]
[CLS] excess dtt was removed by washing five times with 0 . 5 ml ethylacetate . [SEP]
[CLS] after immobilization of aptamers onto nanoflowers , the resultant mixture was washed three times with 10 mm pbs buffer and finally redispersed in 300 μl of 10 mm pbs buffer . [SEP]
[CLS] 1 . functionalization of nanoflowers . [SEP]
[CLS] schematic illustration of functionalization of au @ mno nanoflowers with two different aptamers : fitc - labeled - scg8 ( green ) and tmr - labeled anti - atp ( red ) for cell B-material recognition and metabolite target sensing , respectively . [SEP]
[CLS] 2 . characterization of au @ mno nanoflowers and fluorophore - labeled aptamer binding . [SEP]
[CLS] ( a ) tem images of au @ mno nanoflowers ; inset : hr - tem image of nanoflowers . [SEP]
[CLS] ( b ) analysis of au @ mno nanoflowers by dispersive x - ray spectroscopy B-technique ( edx ) . [SEP]
[CLS] ( c ) uv / vis spectra of au nanoparticles B-nanoparticle and au @ mno nanoflowers . [SEP]
[CLS] fluorescence B-property emission spectra of ( d ) fitclabeled aptamer - nanoflower conjugates ( fitc : ex / em , 488nm / 520nm ) and ( e ) tmrlabeled aptamer - nanoflowers ( tmr : ex / em , 565nm / 580nm ) with two different concentrations of nanoflowers . [SEP]
[CLS] 3 . flow cytometric analysis and in vitro confocal imaging . [SEP]
[CLS] ( a ) flow cytometric assay to monitor the binding of fitc - sgc8 - conjugated nanoflowers ( 20 , 200 , 800 ng / ml ) to ccrf - cem cells B-material ( target cells ) at 4°c with 30 - min incubation B-technique for surface binding and ( b ) at 37°c , 2 - hr incubation B-technique for cell - surface binding . [SEP]
[CLS] ( c ) confocal microscopy B-technique images of cancer cells B-material only . [SEP]
[CLS] ( d ) cancer cells B-material incubated B-technique with bare nanoflowers without aptamer functionalization . [SEP]
[CLS] ( e ) cancer cells B-material incubated B-technique at 37 °c for 2 hr with aptamer - functionalized nanoflowers ( 800ng ) . [SEP]
[CLS] 4 . ldi - ms of cem cell B-material lysate after metabolite extraction . [SEP]
[CLS] ( a ) with cold methanol using nanoflowers and ( b ) with hot water B-material using nanoflowers . [SEP]
[CLS] ldi - ms of cem cell B-material lysate after cell B-material metabolite extraction with hot water B-material followed by quenching with cold nh 4 hco 3 buffer and liquid n 2 to quench the metabolism . [SEP]
[CLS] ( a ) cell B-material lysate and sgc8 - conjugated nanoflowers ; ( b ) cell B-material lysate and sgc8 - and random dnaconjugated nanoflowers ; ( c ) cell B-material lysate and sgc8 - atp - conjugated nanoflowers . [SEP]
[CLS] herein , we describe a polymeric B-nanoparticle micellar I-nanoparticle nanoparticle B-nanoparticle capable of rendering nucleic B-material acids I-material resistant to nuclease digestion . [SEP]
[CLS] this approach relies on utilizing dna as the polar head group of a dnapolymer amphiphile B-property in order to assemble well - defined , discrete nanoparticles B-nanoparticle . [SEP]
[CLS] dense packing of dna in the micelle B-material corona allows for hybridization of complementary oligonucleotides while prohibiting enzymatic degradation . [SEP]
[CLS] we demonstrate the preparation , purification and characterization of the nanoparticles B-nanoparticle , then describe their resistance to treatment with endo - and exonucleases including snake - venom phosphodiesterase ( svp ) a common , general dna digestion enzyme . [SEP]
[CLS] strategy is based on the hypothesis that single - stranded oligonucleotides , consisting of natural nucleotide structures , may be made resistant to nuclease attack by densely packing them as organic polymeric micellar nanoparticles B-nanoparticle . [SEP]
[CLS] the design is predicated on the idea that steric hindrance through dense packaging limits the accessibility of dna to selective and non - selective nucleases . [SEP]
[CLS] we sought to demonstrate this approach to resistant nucleic B-material acids I-material by utilizing a polymerization strategy that is known to be highly functional B-material group I-material tolerant , would allow efficient end - terminus functionalization , and provides polymers B-material of low polydispersity . [SEP]
[CLS] nucleic B-material acids I-material were packed as dna - polymer B-material amphiphiles B-property ( dpas ) into micellar nanoparticles B-nanoparticle consisting of a high - density ssdna corona with a hydrophobic B-property organic B-material polymer I-material core B-material . [SEP]
[CLS] we demonstrate that this morphology allows free access to additional complementary dna strands while preventing and / or inhibiting the activity of various types of nucleases . [SEP]
[CLS] dpas were prepared via conjugation of a hydrophobic B-property polymer B-material ( prepared via ring - opening metathesis polymerization ) , terminally modified with a carboxylic B-material acid I-material moiety , to a 5 ′ amino - modified oligonucleotide on solid support ( figure 1 ) . [SEP]
[CLS] the resulting dna - polymer B-material conjugate was separated from unmodified polymer B-material by thoroughly rinsing the support . [SEP]
[CLS] subsequent cleavage and dialysis gave a mixture of spherical micellar I-nanoparticle nanoparticles B-nanoparticle and free , non - conjugated single - stranded dna ( ssdna , figure 1a , lane 1 ) . [SEP]
[CLS] the particles were separated from ssdna via size - exclusion chromatography B-technique ( sec - fplc ) to give purified material B-material ( figure 1a , b ) . [SEP]
[CLS] this procedure was utilized in the preparation of two micellar nanoparticles B-nanoparticle , p1 and p2 , both exhibiting low polydispersity with diameters on the order of 20 nm , as determined by tem ( figure 1c ) , and dynamic B-technique light I-technique scattering I-technique ( dls , see supporting information figure s8 ) . [SEP]
[CLS] static light scattering ( sls ) was utilized to confirm aggregation numbers ( n agg ) on the order of ~ 200 dna strands per particle ( see supporting information figure s8 ) . [SEP]
[CLS] therefore , dna is at exceptionally high densities of approximately 0 . 2 dna strands / nm 2 on the surface of the micelles B-material . [SEP]
[CLS] the two particles ( p1 and p2 ) were engineered to incorporate ssdna sequences ( ssdna - 1 and ssdna - 2 ) as substrates for a selective endonuclease and also a pair of indiscriminate exonucleases . [SEP]
[CLS] ssdna - 1 and ssdna - 2 differ only in the location of dye - and quencherlabels . [SEP]
[CLS] ssdna - 1 consists of a dabcyl - modifier located towards the 3 ′ - terminus , and a fluorescein - modifier 13 bases away towards the 5 ′ - terminus . [SEP]
[CLS] by contrast , ssdna - 2 has the reverse arrangement with a fluorescein - modifier towards the 3 ′ - terminus . [SEP]
[CLS] this pair of sequences was designed to detect nuclease activity via a fret assay in which the enzymetriggered release of a dabcyl - ( ssdna - 1 ) , or fluorescein - ( ssdna - 2 ) modified fragment from the oligonucleotide sequence results in an increase in fluorescence B-property signal ( vide infra ) . [SEP]
[CLS] additionally , to serve as independent controls , ssdna - 1 and ssdna - 2 were purified as nonpolymer conjugated oligonucleotides . [SEP]
[CLS] to examine how dpa - nanoparticles B-nanoparticle respond to sequence - selective endonucleases , we incorporated a substrate for nicking endonuclease nt . [SEP]
[CLS] cvipii [SEP]
[CLS] cca [SEP]
[CLS] , see supporting information figure s4 ) between fluorescein - and dabcyl - labeled thymidine moieties of the oligonucleotide . [SEP]
[CLS] nt . [SEP]
[CLS] cvipii is a nicking endonuclease that recognizes double - stranded dna ( dsdna ) and introduces a single - strand break on the 5 ′ side of the recognition site ( 5 ′ … * ccx … 3 ′ , x = a , g , or t ) . [SEP]
[CLS] the system was designed such that nucleolytic cleavage occurs on the sequence of the dpa - nanoparticle B-nanoparticle or ssdna analogue while leaving the complementary sequence of the duplex fully intact . [SEP]
[CLS] we reasoned that this design would facilitate a catalytic degradation of both the nanoparticle B-nanoparticle and ssdna in response to small quantities of complementary dna in the presence of the enzyme . [SEP]
[CLS] specifically , we programmed the nick site between bases 10 and 11 of the 20 bp duplex such that the melting temperature ( t m ) of the nicked product would drop to approximately half that of the full 20 bp duplex ( from ~ 60 to 30 °c ) . [SEP]
[CLS] through subsequent , thermodynamically favorable strand invasion , intact ssdna or nanoparticle dna would then be allowed to hybridize to its complement in order to recycle the target . [SEP]
[CLS] to monitor nt . cvipii activity , we employed two complementary analytical techniques ; a fluorescence B-property assay and an assessment of dna melting temperature with and without enzyme treatment ( figure 2 ) . [SEP]
[CLS] the first method involves a fluorescence B-property dequenching experiment wherein the particles or ssdna sequences are allowed to hybridize to complementary dna and subsequently introduced to the endonuclease . [SEP]
[CLS] fluorescein fluorescence B-property was monitored over time in order to assess the activity B-property of I-property the I-property enzyme I-property . [SEP]
[CLS] in this case , an increase in fluorescein fluorescence B-property corresponds to a nick in the oligonucleotide sequence and a dissociation of the quencher - and fluorophore - labeled fragments . [SEP]
[CLS] indeed , after hybridization to complementary dna , the labeled ssdna sequence is readily destroyed in the presence of the nicking endonuclease ( figure 2b , c ) . [SEP]
[CLS] on the contrary , the dpa - nanoparticles B-nanoparticle show virtually no activity via fluorescence B-property under identical conditions ( figure 2a , c ) . [SEP]
[CLS] notably , this observation is independent of the dye and quencher arrangement in the nanoparticle B-nanoparticle substrates . [SEP]
[CLS] this is a critical observation , because for p1 there is the possibility that the fluorescein - labeled fragment may be quenched by neighboring , fully intact strands within the particle shell B-material , whereas this is not possible for p2 , as the fluoresceinlabeled fragment should be free in solution following nicking ( t m ~ 37 °c ) . [SEP]
[CLS] alternatively , we reasoned that perhaps the lack of fluorescence B-property increase for both p1 and p2 could be due to the possibility that a nicked sequence on the particle may not dissociate into solution due to the density of dna in close proximity to the cleaved product . [SEP]
[CLS] to rule out these possibilities , we analyzed the t m of both single - stranded and nanoparticle - based systems following nuclease treatment ( figure 2d , e ) . [SEP]
[CLS] this analysis confirms that the activity of the endonuclease on the ssdna - complement duplex is accompanied by a significant decrease in the t m of the duplex ( δ = −26 . 1 °c for ssdna - 1 and ssdna - 2 ) , consistent with complete nicking of the oligonucleotide . [SEP]
[CLS] by contrast , the t m of the nanoparticle - complement duplex remains consistent ( δ = −0 . 5 °c for p1 , −1 . 6 °c for p2 ) after nuclease treatment , thus indicating the presence of an intact 20 - base oligonucleotide shell B-material on the dpa nanoparticle B-nanoparticle . [SEP]
[CLS] we note that unlike previously reported dna - functionalized gold B-nanoparticle nanoparticle I-nanoparticle systems we do not see an enhanced melting temperature on the initial dpa - nanoparticles B-nanoparticle , which would have been indicative of cooperative hybridization of complementary dna . [SEP]
[CLS] rather , we observe a slight depression in the melting temperature ( figure 2d , e ) ; an observation consistent with steric hindrance at the interface between dna and the hydrophobic B-property polymer B-material . [SEP]
[CLS] this type of effect has been noted by others with respect to unusual dna hybridization characteristics at interfaces . [SEP]
[CLS] given that dpa - nanoparticles B-nanoparticle exhibit a high level of resistance against a sequence - specific nicking endonuclease , we were interested in determining how they would respond as substrates to a non - specific 3 ′ exonuclease ( figure 3 ) . [SEP]
[CLS] exonuclease iii ( exo iii , from e . coli ) is reported to catalyze the stepwise removal of mononucleotides from the 3 ′ - hydroxyl termini of duplex dna with preferred substrates being blunt or recessed 3 ′ termini . [SEP]
[CLS] however , in our hands , the enzyme exhibits indiscriminate activity on both ssdna and dsdna substrates ( see supporting information figure s9 ) . [SEP]
[CLS] therefore , we analyzed the activity of exo iii against ssdna and corresponding dpa - nanoparticles B-nanoparticle in the absence of any additional complementary dna . [SEP]
[CLS] exo iii activity against ssdna and dpa - nanoparticles B-nanoparticle was monitored via fluorescein fluorescence B-property dequenching over time . [SEP]
[CLS] upon initial observation , p1 appears to be highly resistant to exo iii digestion , while p2 appears to be degraded at a substantial rate . [SEP]
[CLS] indeed , a detailed kinetic analysis of p2 with respect to exo iii reveals that it is a substrate , albeit a significantly poorer one than ssdna - 2 with a 3 - fold difference in the magnitude of the second order rate constant and a greater than 4 - fold difference in initial rates ( figure 3c , table 1 ) . [SEP]
[CLS] however , these kinetic data , derived from fluorescence B-property measurements ( see supporting information figure s10 ) , reveal only that p2 is indeed a substrate with respect to removal of the first few bases at the 3 ′ - terminus ; that is , at the location where fluorescein is liberated and hence detectable . [SEP]
[CLS] therefore , we reasoned that the apparent discrepancy between exo iii activity on p1 and p2 ( as monitored by fluorescence B-property ) is most likely due to the fact that for p2 , the fluorescein - labeled nucleotide is located only one base from the 3 ′ hydroxyl terminus . [SEP]
[CLS] therefore , liberation of the fluorescent B-property product into solution ( i . e . detection of fluorescence B-property ) only requires the removal of two bases . [SEP]
[CLS] in the case of p1 , the liberation of a dabcyl - labeled nucleotide does not have the same effect . [SEP]
[CLS] here , we conclude the fluorescein - labeled nucleotide is not liberated into solution but remains in an environment surrounded by dabcyl quencher molecules still present on intact , neighboring dna strands , as well as neighboring guanosine bases , which are also known to quench fluorescein fluorescence B-property . [SEP]
[CLS] therefore , the discrepancy between p1 and p2 response to exo iii is consistent with the nuclease digesting , or " shaving " away a limited fraction of 3 ′ - terminal bases . [SEP]
[CLS] to confirm observations and conclusions drawn from fluorescence B-property kinetic studies ( table 1 ) , and to determine the extent of digestion , a hybridization study via dna duplex melting analyses was required . [SEP]
[CLS] briefly , dpa - nanoparticles B-nanoparticle or ssdna analogues were allowed to react with exo iii for one hour before deactivating the enzyme with edta and heat . [SEP]
[CLS] following enzyme deactivation , an equimolar quantity of complementary dna was allowed to hybridize to the nanoparticle B-nanoparticle or ssdna . [SEP]
[CLS] thermal denaturation analysis reveals the absence of a melting transition in the case of both ssdna strands indicating complete degradation following enzyme treatment ( figure 3d , e , see supporting information figure s11 , s13 ) . [SEP]
[CLS] by contrast , in the case of enzyme - treated dpa - nanoparticles B-nanoparticle , p1 and p2 , we observe a sharp melting transition of the particle - duplex indicative of an intact dpananoparticle . [SEP]
[CLS] however , we do observe a slight decrease in the particle - duplex t m ( δ = −3 °c in each case , figure 3d , e , see supporting information figure s13 ) , consistent with fluorescence B-property evidence suggesting that the enzyme digests several bases of the nanoparticle B-nanoparticle nucleic B-material acid I-material shell B-material at the outer edge and is subsequently sterically hindered thus preventing complete digestion . [SEP]
[CLS] indeed , the data following this partial digestion are consistent with a duplex on the order of approximately 18 base pairs compared to 20 base pairs for the fulllength sequence without enzymatic treatment . [SEP]
[CLS] indeed , encouraged by our results demonstrating dpa - nanoparticle B-nanoparticle resistance to exo iii , we aimed to determine whether a nuclease routinely used for complete digestion of synthetic oligonucleotides would yield similar results . [SEP]
[CLS] in addition , we sought to answer whether dpa nanoparticles B-nanoparticle serve to protect dna against general exonuclease digestion and not just specifically exo iii digestion . [SEP]
[CLS] therefore , we subjected the single - stranded particles and corresponding ssdna analogues to snake venom phosphodiesterase ( phosphodiesterase i from crotalus adamanteus ) , an enzyme known for its aggressive 3 ′ - exonuclease activity and routinely utilized for complete digestion of synthetic oligonucleotides . [SEP]
[CLS] indeed , based on fluorescence B-property dequenching experiments identical to those for exo iii activity analysis , the dpa - nanoparticles B-nanoparticle exhibit exceptional resistance consistent with observations made utilizing exo iii ( figure 4 ) . [SEP]
[CLS] although the relative initial rates differ between svp and exoiii depending on substrate , it is clear that the trends in activity are consistent between the two nucleases , that is , p2 is resistant compared to ssdna - 2 and p1 is resistant compared to ssdna - 1 . [SEP]
[CLS] in summary , we have described a novel approach for rendering dna resistant to two key classes of nuclease that are otherwise capable of rapidly degrading substrates in a sequenceselective or non - selective fashion . [SEP]
[CLS] we propose steric hindrance due to dense packing is the simplest explanation for the observation that the endonuclease has undetectable activity on p1 and p2 , whereas 3 ′ - nucleases show some activity , but only at the outer few bases . [SEP]
[CLS] inspiration for this investigation is drawn from the increasing interest in novel approaches for packaging and delivering nucleic B-material acids I-material for in vivo applications . [SEP]
[CLS] this interest has led to an array of materials designed to facilitate potent and selective communication with important cellular machinery . [SEP]
[CLS] our approach is predicated on the idea that a key requirement for any enabling technology of this type is a well - defined nucleic - acid based material B-material that maintains the integrity of the base - sequence in nuclease - rich environments . [SEP]
[CLS] the utilization of dpa - nanoparticles B-nanoparticle for targeted delivery of intact hybridization competent nucleic B-material acids I-material in vitro and in vivo is currently underway in our laboratories . [SEP]
[CLS] this approach , together with other well - defined dna - based nanomaterials B-material , constitutes a concerted effort to move away from amorphous , poorly defined , multicomponent , and cytotoxic B-property polyplexed transfection agents . [SEP]
[CLS] finally , we note that this approach is likely general in terms of particle core B-material chemistry , as other polymerization strategies are amenable to the incorporation of dna , and potentially the preparation of resistance micellar particle systems . [SEP]
[CLS] synthesis of ( n - benzyl ) - 5 - norborene - exo - 2 , 3 - dicarboximide ( 1 ) - see supporting information figure s1 for chemical structure . [SEP]
[CLS] compound 1 was prepared according to a modification of a previously reported procedure . [SEP]
[CLS] to a stirred solution of n - benzylamine ( 2 . 85 g , 26 . 6 mmol ) in dry toluene ( 125 ml ) was added 5 - norbornene - exo - 2 , 3 - dicarboxylic anhydride ( 4 . 10 g , 25 . 0 mmol ) and triethylamine ( 3 . 83 ml , 27 . 5 mmol ) . [SEP]
[CLS] the reaction was heated to reflux overnight under an atmosphere of n 2 . [SEP]
[CLS] the reaction was cooled to room temperature and washed with 10 % hcl ( 3 × 50 ml ) and brine ( 2 × 50 ml ) . [SEP]
[CLS] the aqueous layers were combined and extracted with ethyl acetate ( 60 ml ) . [SEP]
[CLS] the combined organic layers were dried with mgso 4 , filtered and concentrated to dryness yielding a pale yellow solid that was then recrystallized from ethyl acetate / hexanes to give 1 ( 4 . 98 g , 79 % ) as white crystals . [SEP]
[CLS] supporting information figure s1 for chemical structure . [SEP]
[CLS] to a stirred solution of ethyl 4hydroxybenzoate ( 5 . 5 g , 33 . 1 mmol ) in 100 ml dry dmf was added potassium B-material carbonate B-material ( 7 . 28 g , 52 . 7 mmol ) . [SEP]
[CLS] to this stirred suspension was added cis 1 , 4 - dichlorobutene ( 2 . 0 g , 16 mmol ) . [SEP]
[CLS] the solution turned brown within minutes and the reaction was allowed to stir under an atmosphere of n 2 at 90 °c overnight . [SEP]
[CLS] the mixture was then cooled to room temperature , filtered , and concentrated to dryness . [SEP]
[CLS] the resulting solid was dissolved in ethyl acetate and washed three times with h 2 o . [SEP]
[CLS] the organic layer was dried over magnesium B-material sulfate and concentrated to dryness to yield solid white crystalline needles . [SEP]
[CLS] this solid was recrystallized from ether B-material to yield the pure diester ( 2 . 18 g , 35 % ) . [SEP]
[CLS] h nmr ( cdcl 3 ) : δ ( ppm ) 1 . 38 ( t , 6h , 2 x ch 3 ) , 4 . 35 ( q , 4h , 2 x ch 2 ) , 4 . 74 ( d , 4h , 2 x ch 2 ) , 5 . 96 ( t , 2h , ch = ch ) , 6 . 92 ( d , 4h , 4 x arh ) , 8 . 0 ( d , 4h , 4 x arh ) . [SEP]
[CLS] the diester ( 2 . 18 g , 5 . 66 mmol ) was dissolved in 95 % ethanol and potassium B-material hydroxide B-material was added ( 12 . 0 g , 215 mmol ) . [SEP]
[CLS] the reaction was heated to reflux for 5 hours , cooled to room temperature , diluted with an equal volume of h 2 o and acidified with hcl to form a white precipitate . [SEP]
[CLS] the precipitate was filtered off to yield 2 as an orangetan solid ( 1 . 78 g , 100 % ) . [SEP]
[CLS] s1 for chemical structure . [SEP]
[CLS] monomer B-material 1 ( 870 mg , 3 . 4 mmol ) was dissolved in 5 ml cdcl 3 and cooled to −78 °c . [SEP]
[CLS] ruthenium B-material catalyst B-property ( imesh 2 ) ( c 5 h 5 n ) 2 ( cl ) 2 ru = chph ( 124 mg , 0 . 17 mmol ) was added as a powder , followed by 1 ml additional cdcl 3 to solubilize the catalyst B-property . [SEP]
[CLS] the reaction was then allowed to warm to room temperature and stir under n 2 for 35 minutes ( nmr confirms the absence of the original olefin resonance from monomer B-material 1 at 6 . 28 ppm , and the presence of broad cis and trans polymer backbone olefin resonances at 5 . 45 and 5 . 71 ppm ) . [SEP]
[CLS] at this point , 200 μl of the reaction mixture was removed and quenched with an excess of ethyl vinyl ether B-material to provide a homopolymer for molecular weight determination ( sec - mals : m n = 5221 g / mol , pdi = 1 . 075 , figure s2 ) . [SEP]
[CLS] termination agent 2 ( 111 mg , 0 . 34 mmol ) was dissolved in 2 . 0 ml dmf - d7 , added to the reaction mixture , and the mixture was allowed to stir at room temperature for 20 minutes . [SEP]
[CLS] the ruthenium B-material alkylidene proton resonance was monitored in order to track the completion of the polymer B-material termination event ( figure s3 ) . [SEP]
[CLS] once termination was determined to be complete , excess ethyl vinyl ether B-material was added to quench the ruthenium B-material catalyst B-property . [SEP]
[CLS] the crude polymer B-material was precipitated from cold methanol and further purified by column chromatography B-technique in order to eliminate any traces of unreacted termination agent . [SEP]
[CLS] the crude precipitated polymer B-material was dry loaded onto a silica column , the column was washed with 200 ml ch 2 cl 2 , and the polymer B-material was eluted with 3 % methanol in ch 2 cl 2 to yield a glassy yellow - brown solid as the pure polymer B-material ( 905 mg , 97 % , rf = 0 . 56 ) . [SEP]
[CLS] oligonucleotides ssdna - 1 and ssdna - 2 were synthesized in house using automated phosphoramidite chemistry and saccharin 1 - methylimidazole as an activator . [SEP]
[CLS] standard 2cyanoethyl protected phosphoramidites include da ( n - bz ) , dg ( n - dmf ) , ( n - acetyl ) dc , and t . [SEP]
[CLS] oligonucleotides were synthesized on a 1 . 0 μmol scale using columns packed with 1000 a cpg beads . [SEP]
[CLS] a 5 ′ - amino modifer was incorporated into each synthetic oliognucleotide through use of 5 ′ - amino modifer c12 phosphoramidite ( glen research ) . [SEP]
[CLS] in the case of ssdna - 1 and ssdna - 2 , the 5 ′ - amino terminus was acetylated on solid support using the automated synthesizer . [SEP]
[CLS] the mmt group was removed by treatment with 3 % trichloroacetic acid in ch 2 cl 2 for two minutes ( until the yellow color due to the mmt + cation B-material was no longer visible in the eluting deblock solution ) followed by a standard capping cycle to acetylate the free amine B-material with acetic anhydride . [SEP]
[CLS] fluorescein and dabcyl labels were incorporated into the oligonucleotides via use of fluorescein dt and dabcyl dt phosphoramidites ( glen research ) . [SEP]
[CLS] oligos were cleaved from solid support and deprotected by treatment with ama ( concentrated nh 4 oh / 40 % methylamine , 1 : 1 v / v ) at 55 ° c for 20 minutes , purified by hplc , and characterized by maldi - tof ms . [SEP]
[CLS] complement dna was purchased from integrated dna technologies ( purified by hplc , confirmed by esi - ms ) . [SEP]
[CLS] detailed sequences and enzyme recognition / cleavage sites are shown in figure s4 . s5 for corresponding spectra . [SEP]
[CLS] a maldi target plate was spotted with 1 μl of matrix solution a for each sample to be analyzed and allowed 20 minutes to dry completely ( matrix solution a was prepared by dissolving 50 mg of 3 - hpa in 500 μl of hplc acetonitrile / nanopure h 2 o ( 1 : 1 v / v ) . [SEP]
[CLS] 454 μl of this solution was mixed with 45 μl of 100 mg / ml diammonium hydrogen B-material citrate in nanopure h 2 o ) . [SEP]
[CLS] oligonucleotide samples were prepared for maldi - tof ms analysis using zip - tip c18 pipette tips . [SEP]
[CLS] oligos were loaded onto the c18 tips from concentrated stock solutions ( ca . 50 - 100 μm ) and eluted with matrix solution b ( matrix solution b was prepared as follows : dissolve 50 mg of thap in 500 μl of hplc grade acetonitrile , assist dissolution by sonication and centrifuge the resulting solution to pellet any solid remaining , mix 250 μl of the supernatant with 250 μl of 23 mg / ml diammonium hydrogen B-material citrate in nanopure h 2 o ) . [SEP]
[CLS] 1 μl of the oligonucleotide in matrix solution b was mixed with 1 μl of oligonucleotide calibration standard ( bruker ) dissolved in nanopure h 2 o . [SEP]
[CLS] 1 μl of this solution was then spotted onto the maldi plate on top of crystallized matrix a . the samples were allowed to dry for 15 - 30 minutes before analyzing via maldi - tof ms . [SEP]
[CLS] hplc purification of oligonucleotides - see supporting information figure s5 for corresponding chromatograms . [SEP]
[CLS] synthetic oligonucleotides ssdna - 1 and ssdna - 2 were purified via reverse - phase hplc using a binary gradient as indicated on each chromatogram in figure s5 ( solvent a : 10 % methanol in 50 mm triethylammonium acetate ( teaa ) ph 7 . 1 , solvent b : methanol ) . [SEP]
[CLS] for ssdna - 2 , weak anion B-material - exchange ( wax ) hplc was also necessary to purify the oligonucleotide . [SEP]
[CLS] a quaternary gradient was used for wax hplc analyses and purification ( solvent a : nanopure h 2 o , solvent b : methanol , solvent c : 100 mm tris ( hydroxymethyl ) aminomethane ( tris ) ph 8 . 0 , solvent d : 2 m nacl ) . [SEP]
[CLS] oligonucleotides were desalted post wax hplc purification using sep - pak plus c18 environmental cartridges . [SEP]
[CLS] to a solution of polymer B-material ( 1 20 - 2 ) ( 150 mg , 27 . 8 μmol ) dissolved in 250 μl dmf was added n , n - diisopropylethylamine ( 48 μl , 280 μmol ) and hatu ( 10 . 6 mg , 28 μmol ) . [SEP]
[CLS] the solution was vortexed for 10 minutes at room temperature in order to activate the polymer B-material carboxylic B-material acid I-material terminus . [SEP]
[CLS] at this point , 5 ′ - amino modified dna on cpg solid support ( ca . 1 μmol , mmt deprotected ) was added . [SEP]
[CLS] the mixture was allowed to vortex at room temperature overnight . [SEP]
[CLS] the cpg beads were filtered away from the solution using an empty synthesis column and then washed with dmf ( 2 × 20 ml ) and chcl 3 ( 2 × 20 ml ) . [SEP]
[CLS] the dna - polymer B-material amphiphile B-property ( dpa ) was cleaved from solid support via treatment with ama at 65 ° c for 30 minutes . [SEP]
[CLS] the cpg beads were filtered off using glass wool and subsequently washed consecutively with h 2 o ( 2 . 0 ml ) , dmso ( 2 . 0 ml ) , formamide ( 2 . 0 ml ) , h 2 o ( 3 . 0 ml ) , and dmso ( 1 . 0 ml ) . [SEP]
[CLS] to form micellar nanoparticles B-nanoparticle this solution of dpa was transferred to 3 , 500 mwco snakeskin dialsysis tubing ( thermo scientific ) and 2 . 0 ml h 2 o , used to wash the filtrate container , was added . [SEP]
[CLS] the resulting solution was dialyzed against 2 . 0 l of nanopure h 2 o overnight . [SEP]
[CLS] this dialyzed solution was then concentrated to 3 . 0 ml via speed vac evaporation . [SEP]
[CLS] the resulting crude dpa - nanoparticle B-nanoparticle / ssdna mixture was analyzed by denaturing page and agarose B-technique gel I-technique electrophoresis I-technique to confirm the presence of dpa conjugates and free ssdna . [SEP]
[CLS] it is important to note that low molecular weight ssdna impurities B-property ( ≤8295 g / mol ) remained present despite extensive dialysis attempts ( 20k mwco slide - a - lyzer dialysis cassette ) . [SEP]
[CLS] therefore , the crude mixture was purified via sec fplc ( mobile B-property phase : 10 mm tris , 0 . 5 mm edta ph 8 . 3 , flow rate : 2 ml / min , λ abs = 260nm ) . [SEP]
[CLS] the dpa - nanoparticles B-nanoparticle ( p1 / p2 ) elute at ca . 50 minutes ( figure s6 ) . [SEP]
[CLS] crude p1 / p2 samples were purified using hiload 16 / 60 superdex 200 prep grade sec media and exhibit a retention time differing from that of pure p1 / p2 ( figure 6a , inset ) as subsequent purifications and reinjections of pure material B-material were performed using hiprep 26 / 60 sephacryl s - 200 high resolution sec media . [SEP]
[CLS] gel B-technique electrophoresis I-technique - denaturing page was accomplished using bio - rad criterion 15 % tbe - urea precast gels ( # 345 - 0091 ) and loading 200 ng of dna per lane for each sample to be analyzed . [SEP]
[CLS] in the case of crude conjugate , 400 ng of dna was loaded per lane . [SEP]
[CLS] samples were prepared to load by mixing 1 : 1 ( v / v ) with tbe - urea sample buffer ( # 161 - 0768 , bio - rad ) and heating to 90 °c for 2 minutes followed by rapid cooling on ice . [SEP]
[CLS] the gels were run in 1x tris / boric acid / edta ( tbe ) buffer ph 8 . 4 at 200v for 70 minutes , stained with ethidium bromide B-material ( 200 ng / l ) for 30 minutes and visualized using a bio - rad fluor - s multiimager . [SEP]
[CLS] dna concentration determination - nucleic B-material acid I-material concentrations were determined by uv absorbance at 260 nm using a quartz cuvette ( fisher # 14 - 385 - 928a , pathlength = 10 mm ) . [SEP]
[CLS] an extinction coefficient of 294 , 554 . 58 l / mol • cm was used for ssdna - 1 , ssdna - 2 , p1 , and p2 . [SEP]
[CLS] this coefficient was calculated as the extinction coefficient of the entire sequence without the two thymine modified bases ( 226 , 654 . 58 l / mol • cm , oligocalc ) plus the extinction coefficients for each dye - lableled base at 260 nm ( 38 , 800 l / mol • cm for fluorescein dt , and 29 , 100 l / mol • cm for dabcyl dt , glen research ) . [SEP]
[CLS] due to the fact that p1 and p2 contain additional aromatic groups capable of absorbing uv radiation , a slight correction factor was introduced . [SEP]
[CLS] this correction factor was calculated as the ratio of absorbance of ssdna - 1 or ssdna - 2 at 492 nm versus 260 nm ( a 260 / a 492 ) . [SEP]
[CLS] this correction factor was multiplied by p1 or p2 absorbance at 492 nm in order to calculate what the absorbance at 260 nm would be if the system behaved as the standard ssdna analogues . [SEP]
[CLS] this corrected absorbance at 260 nm was then averaged with the actual dpa - nanoparticle B-nanoparticle absorbance at 260 nm and used to determined nucleic B-material acid I-material concentration . [SEP]
[CLS] for example , p1 a 260 = 0 . 168 ( 0 . 57 μm ) and a 260 corrected = 0 . 130 ( 0 . 44 μm ) . [SEP]
[CLS] therefore , a 260 average = 0 . 149 ( 0 . 50 μm ) . [SEP]
[CLS] copper B-material grids ( formvar / carbon B-material - coated , 400 mesh copper B-material , ted pella # 01754 ) were prepared by glow discharging the surface at 20 ma for 1 . 5 minutes followed by treatment with 3 . 5 μl 250 mm mgcl 2 in order to prepare the surface for dpa nanoparticle adhesion . [SEP]
[CLS] the mgcl 2 solution was wicked away with filter paper and 3 . 5 μl of dpa nanoparticle B-nanoparticle ( ca 50 μm dna in 10 mm tris ph 8 . 5 ) solution was deposited on the grid surface . [SEP]
[CLS] this solution was allowed to sit for 5 minutes before being washed away with 4 drops of glass distilled h 2 o and subsequent staining with 3 drops of 1 % w / w uranyl acetate . [SEP]
[CLS] the stain was allowed to sit for 30 seconds before wicking away with filter paper . [SEP]
[CLS] all grid treatments and sample depositions were on the dark / shiny / glossy formvar - coated face of the grid ( this side face up during glow discharge ) . [SEP]
[CLS] samples were then imaged via tem . [SEP]
[CLS] each experiment was measured in triplicate and plotted as a normalized average ( ie time point zero was set to zero fluorescence B-property ) with standard deviation plotted as error bars . [SEP]
[CLS] sigmoidal fits were performed for each data set . [SEP]
[CLS] fluorescein fluorescence B-property dequenching was monitored over time using a plate - reader and a 96 well plate ( corning , flat bottom nonbinding surface # 29110009 ) . [SEP]
[CLS] time points were collected in 15 - second intervals integrating three flashes per measurement . [SEP]
[CLS] identical gain and filter settings were used in every case . [SEP]
[CLS] for measuring nt . cvipii activity , the following conditions were used in each experiment : 5 μm ssdna or dpa - nanoparticle B-nanoparticle , 300 nm ssdna complement , 25 mm nacl , 1x ne buffer 4 , 10 mm tris ph 8 . 5 , and 5 units of nt . [SEP]
[CLS] cvipii ( 100 units in 20 μl was diluted to 100 μl with 80 μl of diluent a , 5 μl of this solution was used per reaction ) all in 50 μl total volume . [SEP]
[CLS] preparation of dna - polymer B-material amphiphiles B-property ( dpas ) and assembly of micelles B-material . [SEP]
[CLS] synthesis : i ) a hydrophobic B-property polymer B-material , terminally modified with a carboxylic B-material acid I-material moiety was mixed with a coupling agent and reacted with a 5 ′ - amino modified oligonucleotide on solid support ( controlled pore glass , cpg ) . [SEP]
[CLS] figure 1 . ii ) deprotection and cleavage of the resulting dna - polymer B-material conjugate from solid support . iii ) dialysis of cleaved dpa into deionized B-material water I-material to form a mixture of micelles B-material and free , non - conjugated nucleic B-material acid I-material . [SEP]
[CLS] t f and t d correspond to fluorescein - and dabcyl - modified thymidine phosphoramidites . [SEP]
[CLS] a ) page analysis . [SEP]
[CLS] lane 1 : crude material B-material post - micelle B-material ( p1 ) formation showing conjugate ( top band ) and free ssd - na ( lower band ) . [SEP]
[CLS] lane 2 : hplc purified sample of ssdna - 1 . [SEP]
[CLS] lane 3 : purified p1 , isolated via size - exclusion chromatrography ( sec ) . [SEP]
[CLS] b ) sec trace of purified p1 ( λ abs = 260 nm ) . [SEP]
[CLS] c ) transmission electron micrograph of p1 . [SEP]
[CLS] see supporting information figure s7 for p2 data . [SEP]
[CLS] endonuclease resistance of dpa nanoparticles B-nanoparticle . [SEP]
[CLS] figure 2 . figure 3 . a ) scheme depicting dpa - nanoparticle B-nanoparticle ( p2 ) resistance to nicking endonuclease ( nt . cvipii ) and consequently intact , quenched duplex dna at the particle surface . [SEP]
[CLS] b ) scheme depicting dsdna degradation by nt . cvipii and consequently a decrease in duplex melting temperature and increase in fluorescein fluorescence B-property . c ) nt . cvipii activity over time , monitored via fluorescein fluorescence B-property dequenching ( λ ex = 485 nm , λ em = 535 nm ) . [SEP]
[CLS] d ) thermal denaturation analysis with and without nt . [SEP]
[CLS] cvipii treatment for p1 and ssdna - 1 . λ abs = 260 nm . [SEP]
[CLS] sample subjected to enzyme for 100 minutes at 37 °c . [SEP]
[CLS] ssdna - 1 + complement : t m = 63 . 9 °c ; ssdna - 1 + nt . cvipii + complement : t m = 37 . 8 °c ; p1 + complement : t m = 58 . 8 °c ; p1 + nt . cvipii + complement : t m = 58 . 3 °c ) . [SEP]
[CLS] e ) thermal denaturation analysis with and without nt . [SEP]
[CLS] cvipii treatment for p2 and ssdna - 2 . [SEP]
[CLS] λ abs = 260 nm . [SEP]
[CLS] sample subjected to enzyme for 100 minutes at 37 °c . [SEP]
[CLS] ssdna - 2 + complement : t m = 63 . 9 °c ; ssdna - 2 + nt . [SEP]
[CLS] cvipii + complement : t m = 37 . 8 °c ; p2 + complement : t m = 56 . 9 °c ; p2 + nt [SEP]
[CLS] cvipii + complement : t m = 55 . 3 °c ) [SEP]
[CLS] see supporting information figure s12 for derivative plots of melting temperatures . [SEP]
[CLS] complement : 5 ′ - tattatatctttagacactga ctggacatgactct - 3 ′ [SEP]
[CLS] 4 . svp activity over time monitored by fluorescein fluorescence B-property dequenching ( λ ex = 485 nm , λ em = 535 nm ) . [SEP]
[CLS] h nmr ( cdcl 3 ) : δ ( ppm ) 1 . 07 ( d , 1h , ch 2 , j = 9 . 6 hz , ) , 1 . 42 ( d , 1h , ch 2 , j = 9 . 6 hz ) , 2 . 69 ( s , 2h , 2 x ch ) , 3 . 26 ( s , 2h , 2 x ch ) , 4 . 61 ( s , 2h , ch 2 ) , 6 . 28 ( s , 2h , ch = ch ) , 7 . 25 - 7 . 40 ( m , 5h , ar ) . [SEP]
[CLS] 13 c nmr ( cdcl 3 ) : δ ( ppm ) 42 . 18 , 42 . 28 , 45 . 13 , 47 . 62 , 127 . 74 , 128 . 48 , 135 . 76 , 137 . 76 , 177 . 48 . [SEP]
[CLS] lrms ( ci ) , 253 . 99 [ m + h ] + . [SEP]
[CLS] hrms , theo : 254 . 1176 [ m + h ] + , found : 254 . 1175 [ m + h ] + . [SEP]
[CLS] exonuclease iii kinetics on ssdna - 2 and p2 substrates [SEP]
[CLS] dna and poly ( n - isopropylacrylamide ) are co - assembled onto gold B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] the dna sequences can be reversibly exposed or hidden from the polymer B-material surface in response to temperature cues , thereby translating the temperature trigger to the on - off switching of the surface chemistry and function . [SEP]
[CLS] when exposed by heating ( ~ 30 °c ) , the dna rapidly hybridizes to complementary strands , and chain - end biotin groups become readily accessible , while at lower temperatures these activities are largely blocked . [SEP]
[CLS] chemically dynamic nanoparticle B-nanoparticle systems are of great interest . [SEP]
[CLS] these systems are capable of switching between at least two distinct structural or chemical states , which allow them to possess unusual properties such as on - demand activation and autonomous regulation [SEP]
[CLS] indeed , many systems are being gradually realized in a number of fields spanning biomedical research , sensing , self - healing materials , and energy research , and can incorporate sensitivity to a variety of environmental cues , such as temperature , ph , light , redox potential , the presence or absence of small molecules , and mechanical stress . [SEP]
[CLS] poly ( n - isoproylacrylamide ) ( pnipam ) often has been utilized as a temperatureresponsive material B-material , due to its ability to undergo a reversible lower critical solution temperature ( lcst ) phase transition from a swollen , hydrated state to a shrunken , hydrophobic B-property state , during which the polymer B-material loses 90 % of its volume . [SEP]
[CLS] because the lcst can be engineered through copolymerization to be near physiological temperature , pnipam and its derivatives have often been employed in tissue engineering and controlled drug release applications . [SEP]
[CLS] it has been previously shown that spherical nucleic B-material acid I-material - gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) conjugates ( termed snas ) 28 can undergo rapid endocytosis B-event , which is facilitated by engaging with membrane - bound scavenger proteins B-material . [SEP]
[CLS] this cell B-material entry mechanism appears to be general with respect to many cell B-material types ( almost all cells B-material internalize snas , albeit with different rates and to different extents ) , and cannot effectively distinguish many diseased tissues from healthy ones . [SEP]
[CLS] in order to achieve selective cell B-material entry , one strategy would be to mask the dna temporarily until an environmental trigger is applied at the region of interest . [SEP]
[CLS] in this study , we report the synthesis of a dynamic nanoparticle B-nanoparticle system consisting of pnipam and dna co - assembled onto aunps B-nanoparticle , and demonstrate that access to dna is significantly retarded at temperatures below the lcst of pnipam . [SEP]
[CLS] above this temperature , pnipam becomes dehydrated , revealing the dna for rapid binding with complementary strands ( scheme 1 ) . [SEP]
[CLS] the chain end of dna is also exposed , as demonstrated with biotinterminated dna , which binds to streptavidin only at elevated temperatures . [SEP]
[CLS] therefore , the current study potentially paves the way for novel nucleic B-material acid I-material structures capable of being reversibly activated by temperature for selective and / or localized cell B-material entry . [SEP]
[CLS] to realize such temperature - activated dna nanostructures , several design parameters must be established . [SEP]
[CLS] first , the aunp B-nanoparticle surface should have appropriate pnipam coverage such that all dna molecules are adequately masked at temperatures below the lcst . [SEP]
[CLS] similarly , the pnipam should have a molecular weight high enough to offer sufficient blockage of the dna . [SEP]
[CLS] however , the pnipam molecular weight cannot be too high as to dominate the surface while in a dehydrated state above lcst , in which case hydrophobicity - induced aunp B-nanoparticle aggregation is expected . [SEP]
[CLS] to estimate these parameters , a molecular dynamics simulation has been implemented ( see supporting information , figure s1 ) . [SEP]
[CLS] the simulation suggested that a combination of a 10 kda pnipam and a 12 - mer dna at 1 : 1 dna : polymer molar ratio would be feasible . [SEP]
[CLS] to prepare the target structures , we first synthesized two dna strands whose 3 ′ or 5 ′ terminus are modified with propyl thiol B-material groups ( sequence : 5 ′ - gag ggt aag gag - sh - 3 ′ and 5 ′ - hs - gga aag gtt agt - 3 ′ ) . [SEP]
[CLS] each of the sequences was mixed with 10 kda α - thiol ω - cooh pnipam ( pdi : 1 . 1 ) at various dna : polymer B-material molar ratios ( 1 : 2 , 1 : 1 , 5 : 1 ) in [UNK] water B-material . [SEP]
[CLS] a longer pnipam ( 30 kda , a pdi of 1 . 2 ) was used as a control . [SEP]
[CLS] immediately prior to mixing , the polymers B-material and dna strands were treated with dithiolthreitol ( dtt ) and dialyzed or passed through a size - exclusion column to ensure that free thiol B-material groups were present . [SEP]
[CLS] thereafter , the mixture containing the thiol - modified ligands was added to citrate - stabilized aunps B-nanoparticle ( 10 nm , 13 nm , synthesized by a modified frens - turkevich method ) with 0 . 01 % tween 20 to give a total ligand concentration of 5 μm . [SEP]
[CLS] over 24 h , sodium B-material chloride I-material was added , and its concentration was gradually brought up to 0 . 5 m , a process necessary for charged species ( i . e . dna ) to assemble in a densely packed fashion on the aunp B-nanoparticle surface . [SEP]
[CLS] thereafter , successive centrifugation - resuspension steps were used to remove unbound dna and pnipam . [SEP]
[CLS] finally , aunps B-nanoparticle were suspended in phosphate buffered saline with 0 . 01 % tween 20 ( pbst ) at a particle concentration of 10 nm . [SEP]
[CLS] note that the dna : polymer B-material feed ratios do not equal their actual ratios on the nanoparticles B-nanoparticle . [SEP]
[CLS] using fluorophore - labeled pnipam ( supporting information ) , we determined the actual dna : polymer molar ratios and the total number of ligands ( dna and pnipam ) present on the aunps B-nanoparticle surface ( table 1 ) . [SEP]
[CLS] once the dna - and pnipam - functionalized aunps B-nanoparticle were prepared and purified , we examined whether they would aggregate at temperatures above the lcst due to hydrophobic B-property interactions I-property , or remain stable in solution . [SEP]
[CLS] for aunps B-nanoparticle with 30 kda pnipam ( dna : polymer B-material 1 . 4 : 1 ) , visible aggregation can be observed within seconds when the solution temperature is heated above 30 °c . [SEP]
[CLS] uv - vis spectroscopy B-technique monitoring absorption at 524 nm ( aunp B-nanoparticle plasmon resonance maximum ) as the temperature was ramped up at 0 . 5 °c / min shows complete aggregation within ~ 1 °c ( figure 1 ) , indicating that the aunp B-nanoparticle surface is predominantly hydrophobic B-property when the lcst is reached . [SEP]
[CLS] in comparison , aunps B-nanoparticle with 10 kda pnipam ( dna : polymer B-material 1 . 1 : 1 ) only show slight reduction in absorption ( 5 % ) when heated to 50 °c , suggesting that the surface composition had switched from pnipam to predominantly dna , which stabilizes the aunps B-nanoparticle in solution . [SEP]
[CLS] the hydrodynamic diameter initially decreased from 21 . 0 ± 2 . 6 nm to 16 . 3 ± 0 . 8 nm as measured by dynamic B-technique light I-technique scattering I-technique ( dls ) when the temperature was increased from 25 to 40 °c , consistent with pnipam shrinkage ( figure s2 ) . [SEP]
[CLS] however , after 1 h incubation B-technique at 50 °c , particle size increased to 44 nm , suggesting that a small degree of aggregation had occurred . [SEP]
[CLS] to verify that the dna is available for binding with complementary sequence once pnipam is dehydrated , we mixed two particles ( with 10kda pnipam , dna : polymer B-material 1 . 4 : 1 ) and a common complementary linker strand ( sequence : 5 ′ - ctc ctt acc ctc act aac ctt tcc - 3 ′ ) in pbst buffer , and monitored aunp B-nanoparticle absorption at 524 nm by uv - vis spectroscopy B-technique as the temperature was increased from 25 to 80 °c at 0 . 5 °c / min . [SEP]
[CLS] at temperatures below 30 °c , absorption values stay largely unchanged , suggesting effective blockage of dna hybridization ( figure 2 ) . [SEP]
[CLS] as the temperature is increased , a rapid drop in absorption is observed at ~ 30 °c due to particle assembly , which can result from either hydrophobic B-property interactions I-property of pnipam , or dna hybridization . [SEP]
[CLS] as the temperature continued to increase , a sharp rise in absorption occurred at 53 - 55 °c , returning absorption values to nearly that before assembly . [SEP]
[CLS] the second transition is attributed to dna melting , which indicates that the initial aggregation is due to dna hybridization . [SEP]
[CLS] in contrast , dna - aunp B-nanoparticle conjugates without pnipam show immediate aggregation upon mixing , and only exhibit the dna melting transition , and particles with pnipam but matched with a noncomplementary linker show neither the aggregation nor the melting transition . [SEP]
[CLS] these data indicate that the dna strands can be blocked from hybridization below the lcst and become exposed for hybridization in response to temperature . [SEP]
[CLS] to further validate that the terminus of the dna chain is hindered from accessing the surface at lower temperatures and exposed upon heating , we designed and performed a biotin - streptavidin pull - down assay . [SEP]
[CLS] briefly , we synthesized a dna sequence with 3 ′ biotin and 5 ′ propyl thiol modifications ( sequence : 5 ′ - sh - gga aag gtt agt - biotin - 3 ′ ) . [SEP]
[CLS] the biotin group is linked with the dna through a triethylene glycol spacer B-material , which minimizes the steric hindrance between the biotin and the oligonucleotide . [SEP]
[CLS] it was co - assembled on the aunp B-nanoparticle surface with pnipam ( 10 kda ) at dna : polymer feed molar ratios ranging from 1 : 5 to 5 : 1 . [SEP]
[CLS] a dna strand without the biotin is co - assembled with pnipam at 1 : 1 feed molar ratio and used as a control . [SEP]
[CLS] thereafter , the modified aunps B-nanoparticle were mixed with dynabeads® coupled with streptavidin in the presence of 0 . 01 % tween 20 , and incubated B-technique at either room temperature or at 40 °c . [SEP]
[CLS] following 5 min incubation B-technique , the beads were pulled down by a neodymium magnet , and the supernatant was analyzed for absorption at 524 nm by uv - vis spectroscopy B-technique . [SEP]
[CLS] if the biotin group at the chain end of the dna is accessible , it leads to aunp B-nanoparticle capture by the dynabeads® and a drop in optical absorption of the supernatant . [SEP]
[CLS] when incubated B-technique at room temperature , we found that significant amounts of particles ( > 85 % relative to non - treated aunps B-nanoparticle ) remained in solution for particles with dna : polymer B-material feed ratios of 1 : 5 or 1 : 2 ( figure 3 ) . [SEP]
[CLS] when a 1 : 1 feed ratio was used , ~ 60 % aunps B-nanoparticle were bound to streptavidin . [SEP]
[CLS] further decreases in pnipam content ( at 2 : 1 or 5 : 1 ) led to high degrees of capture by the dynabeads® ( > 90 % ) , consistent with our hypothesis that sufficient pnipam coverage is important for dna blockage . [SEP]
[CLS] on the other hand , when incubated B-technique at 40 °c , all particles containing biotin are almost completely captured , with < 3 % remaining in solution even for particles with low biotin - dna contents , indicating that the dna 3 ′ termini are exposed . [SEP]
[CLS] a control involving non - biotinylated aunp B-nanoparticle was not captured under otherwise same conditions , ruling out the possibility of non - specific binding at 40 °c . [SEP]
[CLS] these data suggest that elevation of temperature is highly efficient in exposing dna molecules that are blocked by the pnipam in its hydrated state . [SEP]
[CLS] in summary , we have demonstrated a strategy to allow the surface chemistry of aunps B-nanoparticle to interchange between synthetic polymers B-material and nucleic B-material acids I-material in response to a temperature trigger . [SEP]
[CLS] this dynamic system allows dna accessibility only above the lcst of pnipam , and therefore points to the possibility of creating novel nucleic B-material acid I-material constructs capable of selective activation in the vicinity of specific materials and tissues . [SEP]
[CLS] importantly , the general uv - vis measurement at 524 nm for pnipam - dna aunps B-nanoparticle as a function of temperature . [SEP]
[CLS] high molecular weight pnipam ( 30 kda ) leads to aunp B-nanoparticle aggregation above lcst even for particles with low polymer B-material content , while particles having 10 kda pnipam remain stable . [SEP]
[CLS] schematic drawing of pnipam - and dna - functionalized gold B-nanoparticle nanoparticles I-nanoparticle ( aunp B-nanoparticle ) and response to temperature . [SEP]
[CLS] increasing solution temperature above the polymer B-material lcst results in a reduction of polymer B-material hydration and an increase in dna accessibility . [SEP]
[CLS] a ) a divalent linker dna strand hybridizes with dna - aunps B-nanoparticle when the temperature is raised above the lcst of pnipam , causing aunp B-nanoparticle assembly . [SEP]
[CLS] a further increase in temperature leads to the melting of double - stranded dna and re - dispersion of the aggregates . [SEP]
[CLS] b ) uv - vis temperature scan is performed at 0 . 5 °c / min , monitoring aunp B-nanoparticle absorption at 524 nm . [SEP]
[CLS] 3 . a ) [SEP]
[CLS] a magnetic B-property pull - down assay for determining if dna chain ends can be hidden / revealed by 10 kda pnipam . [SEP]
[CLS] b ) uv - vis measurements of np B-nanoparticle solutions at 524 nm after incubation B-technique with streptavidin - coated dynabeads® at 25 °c and 40 °c ( bead removed using a neodymium magnet ) . [SEP]
[CLS] dna : polymer B-material molar ratio on aunp B-nanoparticle surface . [SEP]
[CLS] the ability to self - assemble one - dimensional dna building blocks into two - and threedimensional nanostructures via dna / rna nanotechnology has led to broad applications in bioimaging , basic biological mechanism studies , disease diagnosis and drug delivery . [SEP]
[CLS] however , the cellular uptake of most nucleic B-material acid I-material nanostructures is dependent on passive delivery or the enhanced permeability and retention effect , which may not be suitable for certain types of cancers , especially for treatment in vivo . [SEP]
[CLS] to meet this need , we have constructed a multifunctional aptamer - based dna nanoassembly ( aptna ) for targeted cancer therapy . [SEP]
[CLS] in particular , we first designed various y - shaped functional dna domains through predesigned base pair hybridization , including targeting aptamers , intercalated anticancer B-property drugs and therapeutic antisense oligonucleotides . [SEP]
[CLS] then these functional dna domains were linked to an x - shaped dna core B-material connector , termed a building unit , through the complementary sequences in the arms of functional domains and connector . [SEP]
[CLS] finally , hundreds ( ~ 100 - 200 ) of these basic building units with 5 ′ modification of acrydite groups were further photocrosslinked into a multifunctional and programmable aptamer - based nanoassembly structure able to take advantage of facile modular design and assembly , high programmability , excellent biostability and biocompatibility B-property , as well as selective recognition and transportation . [SEP]
[CLS] with these properties , aptnas were demonstrated to have specific cytotoxic B-property effect against leukemia cells B-material . [SEP]
[CLS] moreover , the incorporation of therapeutic antisense oligonucleotides resulted in the inhibition of p - gp expression ( a drug efflux pump to increase excretion of anticancer B-property drugs ) , as well as a decrease in drug resistance . [SEP]
[CLS] therefore , these multifunctional and programmable aptamer - based dna nanoassemblies show promise as candidates for targeted drug delivery and cancer therapy . [SEP]
[CLS] although nanoparticle - and polymeric nanomaterial - based therapeutic strategies have been widely introduced into drug delivery and cancer theranostics , challenges still remain in their efficacy and complexity , as well as their biocompatibility B-property . [SEP]
[CLS] to develop a new generation of drug delivery platforms , the emergence of dna / rna nanotechnology has allowed the elegant self - assembly of one - dimensional nucleic B-material acid I-material molecules into two - and threedimensional nanostructures through specific molecular recognition and programmable molecular design , such as hydrogen B-material bonding and π - stacking . [SEP]
[CLS] these 3d nucleic B-material acid I-material nanostructures , which self - assemble by predictable and programmable nucleic B-material acid I-material molecules containing different functional moieties , have attracted increasing attention in the fields of biosensing and disease diagnosis , especially as a potential cellular carrier for drug delivery and disease treatment . [SEP]
[CLS] for example , a self - assembled short interference rna ( sirna ) microsponge was produced via a rolling circle transcription B-event to achieve high loading capacity and delivery efficiency for targeted gene silencing in vivo . [SEP]
[CLS] welldefined , uniformly sized tetrahedral dna nanoparticles B-nanoparticle functionalized with sirna or immunostimulatory cpg oligonucleotides exhibited excellent intracellular biostability and bio - compatibility , thus facilitating the efficacy of gene therapy and immunoregulation . [SEP]
[CLS] in addition , self - assembled dna tubular and triangular origami nanostructures loaded with the anti - cancer drug doxorubicin B-material circumvented the drug resistance of cancer cells B-material by the inhibition of lysosomal acidification . [SEP]
[CLS] in contrast to 1d dna / rna molecules , the power of self - assembled 3d nucleic B-material acid I-material nanostructures lies in their excellent biostability , high drug payloads , and passive delivery into living cells B-material , even tumors B-material . [SEP]
[CLS] however , self - assembled nucleic B-material acid I-material nanostructures pose challenges that remain to be solved . [SEP]
[CLS] first , most 3d nucleic B-material acid I-material nanostructures enter cancer cells B-material or tumors B-material via passive delivery . [SEP]
[CLS] while such passive delivery is satisfactory for prostate or breast cancers with leaky vasculatures , it may not be suitable for other types of cancers , such as leukemias and lymphoma , thereby impeding broad application in vivo . [SEP]
[CLS] for active targeting of leukemias , specific recognition ligands aimed at cell B-material receptors or cancer biomarkers B-property are prerequisite for providing enhanced , selective diagnosis and treatment . [SEP]
[CLS] second , the complicated modification of tumor targeting B-material ligands I-material , including antibodies B-material or small molecules , into 3d nucleic B-material acid I-material nanostructures has been reported . [SEP]
[CLS] the difficulties in precisely and programmably controlling functional ligands in 3d nucleic B-material acid I-material nanostructures for clinical trials have not yet been fully explored . [SEP]
[CLS] therefore , a facile approach for conjugation of targeting B-material ligands I-material into nucleic B-material acid I-material nanostructures in a controlled manner is still in demand . [SEP]
[CLS] third , the disassembly of watson - crick base pairings in nucleic B-material acid I-material nanostructures during cellular delivery results from the alternation of nanostructure with in vivo environment . [SEP]
[CLS] maintaining biostability of nucleic B-material acid I-material nanoassemblies is a prerequisite for cellular drug delivery . [SEP]
[CLS] more importantly , instead of having a single functionality on each self - assembled nanostructure , the ability to construct a multifunctional nucleic B-material acid I-material complex capable of active recognition , efficient transportation and elevated therapeutics would be highly desirable . [SEP]
[CLS] through complementary strategies , multifunctional and programmable nucleic B-material acid I-material nanostructures will provide efficient and reliable point - of - care platforms for rapid disease diagnosis , targeted drug delivery and cancer therapy . [SEP]
[CLS] using a bottom - up modular approach , we report the construction of a multifunctional and programmable aptamer - based dna nanoassembly ( aptna ) to address these challenges . [SEP]
[CLS] as shown in figure 1 , multifunctional dna sequences , including aptamer , acrydite - modified single - stranded dna and antisense oligonucleotide , are self - assembled to form y - shaped functional domains , which are then linked to x - shaped connectors to create building units . [SEP]
[CLS] different functional elements can be incorporated into the domains of each building unit , including antisense oligonucleotides capable of suppressing the expression of specific cellular proteins B-material , chemical anticancer B-property drugs intercalated in specific dna base pairs , and aptamers , consisting of single - stranded dnas derived from cell - based systematic evolution of ligands by exponential enrichment ( selex ) for specific recognition of certain cancer cells B-material . [SEP]
[CLS] by integrating all functional domains into one nanoassembly system , the aptamer moieties can act as a guidance system to target specific cancer cells B-material . [SEP]
[CLS] hundreds of these basic building units are then photocrosslinked into multifunctional and programmable nanoassembly structures with controllable diameters . [SEP]
[CLS] the bulky nanoassembly provides many sites available for high - capacity loading of therapeutics or bioimaging agents . [SEP]
[CLS] in addition , aptnas show excellent biostability in the physiological environment ( ph 7 . 4 ) , thus avoiding unnecessary leaking of intercalated drugs during delivery . [SEP]
[CLS] the working principle of our multifunctional and programmable nanoassembly structure , as shown in figure 1 , is explained in more detail as follows . [SEP]
[CLS] first , four single - stranded dnas were self - assembled to form an x - shaped core B-material connector via predesigned base - pair hybridization ( table 1 ) . [SEP]
[CLS] each connector had three distinct toehold sequences , called arms , at the ends of different branches . [SEP]
[CLS] to achieve multiple functionalities , y - shaped dna functional domains with arms complementary to those of the connectors were designed to link with the core B-material connector to form a building unit . [SEP]
[CLS] various functional groups , e . g . , targeted aptamers , intercalated anti - cancer drugs and therapeutic antisense oligonucleotides , were incorporated in different domains . [SEP]
[CLS] thus , each nanoassembly building unit consists of one core B-material connector and three functional domains . [SEP]
[CLS] acrydite groups were included in two of the functional domains of each building unit based on the 5 ′ - modification of the oligonucleotides during dna synthesis . [SEP]
[CLS] consequently , the acrydite - modified building units can be further photocrosslinked to form different nanostructures with controllable diameters . [SEP]
[CLS] because of the precise design of arm sequences in each building unit , an accurate ratio between different functional moieties ( aptamers or antisense oligonucleotides ) , as well as programmable self - assembled functional domains , can be achieved in the entire nanoassembly . [SEP]
[CLS] to demonstrate the precise self - assembly of this modular approach , polyacrylamide B-technique gel I-technique electrophoresis I-technique was used to examine the formation of a single building unit . [SEP]
[CLS] the smaller mobility B-property of functional domains was observed relative to that consisting of ssdna ( figure s1a ) . [SEP]
[CLS] mixed in equimolar amounts , all three functional domains were anisotropically and simultaneously linked to the connector with the aid of prede - signed arm sequences ( figure s1b , c ) . [SEP]
[CLS] compared to any one of its constituent domains , the self - assembled building unit had less mobility B-property as a result of its collective increased molecular weight . [SEP]
[CLS] however , the application of photopolymerization changed the geometry and size of these building units to create nanoassemblies composed of many hundreds of building units . [SEP]
[CLS] specifically , dynamic B-technique light I-technique scattering I-technique ( dls ) revealed that the building units with photoresponsive acrydites formed nanostructures with an initial hydrodynamic diameter of 17 nm , but further increasing to 218 nm after photoillumination for 10 min ( figure s2 ) . [SEP]
[CLS] because of the highly efficient photo - polymerization , no peak corresponding to a single building unit was observed in the dls results , and nanoassemblies displayed spherical structures , as confirmed by transmission B-technique electron I-technique microscopy I-technique ( tem , figure 2a ) , such that the diameter of the spherical nanoassembly could be controlled by simply changing the concentration of the building units ( figure 2b ) . [SEP]
[CLS] for example , a higher concentration of building units will induce a smaller overall nanostructure diameter because of an elevated ratio of polymeric units over photoinitiators . [SEP]
[CLS] to more perfectly control the number of building units in each nanoassembly , an alternative crosslinking approach , dendrimerization , can be also employed to form size and shape controllable dna nanostructures . [SEP]
[CLS] thus , this modular , photopolymerization approach can be used to generate size - controllable nano - assembly structures . [SEP]
[CLS] the aptamer targeting domains enable the nanoassembly to specifically recognize target cancer cells B-material . [SEP]
[CLS] as a proof of concept , sgc8 aptamer , which targets ccrf - cem ( t cell acute lymphoblastic leukemia cell line ) cancer cells B-material , but not ramos ( b cell human burkitt ' s lymphoma ) , was conjugated into one functional domain of each building unit . [SEP]
[CLS] after photocrosslinking , the selective binding affinity of the sgc8 - nas was verified by flow B-technique cytometry I-technique , which showed a 100 - fold fluorescence B-property signal shift over library dna - nas for cem cells B-material ( figure 3a ) , while no significant shift was observed for ramos cells B-material treated with sgc8 - and library dna - nas ( figure 3b ) . [SEP]
[CLS] as a result of the multiple building units in the nanoassembly structures , the sgc8 - nas showed an amplified fluorescence B-property signal intensity compared to a single sgc8 aptamer because of signal amplification from multiple signal molecules in each sgc8 - nas . [SEP]
[CLS] the generality of aptamer - based nanoassemblies was verified using a different aptamer , kk1b10 , which can specifically recognize dox - resistant leukemia cells B-material ( k562 / d ) , but not control ramos cells B-material ( figure s3 ) . [SEP]
[CLS] the cellular trafficking of dna - assembled nanoparticles B-nanoparticle has been reported by labeling with quantum B-nanoparticle dots I-nanoparticle or organic dyes . [SEP]
[CLS] in this study , a dna intercalated dye , sybr green , was used to stain sgc8 - nas to investigate their specific transport into target cancer cells B-material . [SEP]
[CLS] a strong green fluorescence B-property signal was observed by confocal B-technique laser I-technique scanning I-technique microscopy I-technique ( clsm ) after incubating B-technique sgc8 - nas with cem cells B-material at 37°c for 2 h ( figure 4a ) . [SEP]
[CLS] however , a slightly green fluorescence B-property signal was also observed for ramos cells B-material , possibly because of the nonspecific internalization of nanoassemblies or the leakage of dyes into nontarget cells B-material ( figure 4b ) . [SEP]
[CLS] previous work suggested that the sgc8 aptamer specifically enters cem cells B-material through receptor - I-event mediated endocytosis B-event ; therefore , a colocalization assay was used to demonstrate the final destination of sgc8 - nas in living cem cells B-material . [SEP]
[CLS] most green fluorescence B-property signals from sybr green - intercalated nanoassemblies were overlapped with the red fluorescence B-property generated by transferrin alexa - 633 ( a marker for endosomes ) , which was not seen for ramos cells B-material . [SEP]
[CLS] thus , this aptamer - based nanoassembly has properties of a large nanostructure , including selective targeting and internalization , making it a potential platform for targeted cancer therapy . [SEP]
[CLS] as a consequence of the large number of packed hybridized dna base pairs , this dnaassembled nanostructure is spatially well equipped for cargo loading , especially for such chemical anticancer B-property drugs as doxorubicin B-material ( dox ) which can preferentially intercalate into double - stranded gc base pairs . [SEP]
[CLS] comparing the molecular weight of a single nanoassembly structure to that of a single building unit , it was determined each aptna contained 100 ~ 200 building units that were able to provide more than 220 dox loading sites . [SEP]
[CLS] dox fluorescence B-property after intercalation into sgc8 - nas was dramatically quenched with a molar ratio of 1000 / 1 , indicating a high loading capacity of 10 nm sgc - nas with ~ 10 μm dox . [SEP]
[CLS] we next evaluated the release kinetics of dox loaded in sgc8 - nas by using a drug diffusion method with mini dialysis units . [SEP]
[CLS] the sgc8 - nas ( 50 nm ) with a drug payload of 50 μm displayed less than 7 % cumulative release in a physiological environment ( ph 7 . 4 ) in contrast to rapid diffusion of in acidic solution ( 50 ~ 60 % release ) probably due to the influence of the low ph on dna base pair hybridization and charge effect on dox - dna interaction ( figure s4 ) . [SEP]
[CLS] thus , sgc8 - nas exhibited excellent stability with high drug payload under physiological conditions , thus effectively preventing drug leaking , while , at the same time , facilitating drug release in an endosome - like environment . [SEP]
[CLS] to investigate selective anticancer B-property drug transport into target cancer cells B-material , the uptake of doxloaded sgc8 - nas and distribution of intercalated drugs were studied with cem and ramos cells B-material using free dox as a control . [SEP]
[CLS] for free dox , confocal imaging results show that a strong fluorescence B-property signal was produced in both cem and ramos cells B-material ( figure 5a , 5b ) . [SEP]
[CLS] in contrast , the dox fluorescence B-property was restored by release from sgc8 - nas in cem cells B-material , but not ramos cells B-material , indicating the selective delivery of dox via the aptamer - based nanoassembly structures ( figure 5c , 5d ) . [SEP]
[CLS] since it has been demonstrated that dox offloading from internalized sgc8 - nas is influenced by intracellular location and the corresponding ph environment , we believe that the transported sgc8 - nas - dox complex first enters cells B-material via endocytosis B-event , followed by residence in endosomes , which then , by their acidic environment , facilitate the rapid release of loaded anticancer B-property drugs , with dox finally escaping from the endosomes and further widening its distribution in the cytoplasm , and even nucleus . [SEP]
[CLS] the in vitro cytotoxicity B-property of dox - loaded sgc8 - nas and free dox was evaluated by the 3 - ( 4 , 5dimethylthiazol - 2 - yl ) - 5 - ( 3 - carboxymethoxyphenyl ) - 2 - ( 4 - sulfophenyl ) - 2h - tetrazolium ( mts ) assay . [SEP]
[CLS] free dox presented dose - dependent cytotoxicity B-property behavior in both cem and ramos cells B-material ( figure s5 ) ; however , dox - loaded sgc8 - nas induced an efficient and dosedependent cytotoxicity B-property only in targeted cem cells B-material , but not nontarget ramos cells B-material ( figure 6 ) . [SEP]
[CLS] regarding to different dose dependent response of cancer cells B-material to dox , the largest cytotoxic B-property enhancement was evaluated to be ~ 3 . 8 - fold with a payload of 0 . 5 μm dox in sgc8 - nas for cem cells B-material ( figure s5 ) . [SEP]
[CLS] in addition , sgc8 - nas without dox exhibited negligible cytotoxicity B-property and more than 90 % cell B-property viability I-property , even in a 1 μm concentration of building units ( figure s6 ) . [SEP]
[CLS] these results all indicate that this aptamer - based dna nanoassembly system possesses potential cancer therapeutic properties , including excellent biocompatibility B-property and highly selective killing efficacy to target cancer cells B-material . [SEP]
[CLS] having established the drug loading capability of aptnas for targeted cancer therapy , the functionalization of therapeutic antisense ( as ) oligonucleotides was explored to overcome the obstacle of multidrug resistance ( mdr ) in chemotherapy . [SEP]
[CLS] as proof of concept , a doxloaded and mdr1 - as - incorporated kk1b10 aptamer - modified nanoassembly structure was constructed to selectively kill drug - resistant myelogenous leukemia , k562 / d . mdr1 antisense oligonucleotides have been reported to specifically inhibit the overexpression of pglycoprotein ( p - gp ) , a membrane glycoprotein which acts as a drug efflux pump to increase excretion of structurally related drugs from cells B-material and to reduce intracellular drug accumulation . [SEP]
[CLS] particularly , the mdr1 - as / kk1b10 / nas - dox complex was crosslinked with building units consisting of one kk1b10 aptamer and two mdr - as domains ; thus , each nanoassembly had a highly localized concentration of antisense oligonucleotides ( ~ 400 ) . [SEP]
[CLS] after specific recognition and uptake by target drug - resistant k562 cells B-material , the recovery of dox sensitivity to k562 / d was verified by mts assay . [SEP]
[CLS] mdr1 - as / kk1b10 / nas - dox nanostructures showed dose - dependent cytotoxicity B-property and 34 % cell B-property viability I-property at 20 μm dox payload in k562 / d cells B-material , ( figure 7 ) in contrast to ~ 70 % metabolically active k562 / d cells B-material using 20 μm free dox , suggesting that the antisense oligonucleotides transported by kk1b10 - nas played a role in inhibiting p - gp expression and decreasing drug resistance . [SEP]
[CLS] neither kk1b10 nas - dox modified with random oligonucleotides nor sgc8 - na dox complexes modified with md r1 as led to substantial inactivity of k562 / d cells B-material , even with a 20 μm dox payload ( 76 % and 78 % ) , indicating that the selective killing of drug - resistant cancer cells B-material was induced only by kk1b10 nas - dox nanostructures modified with mdr1 - as . [SEP]
[CLS] the viability B-property of I-property k562 I-property / d cells I-property initiated a rapid decrease after treatment with either 5 μm free dox or mdr - as kk1b10 nas - dox complex , essentially because k562 / d cells B-material were induced and cultured with a high concentration of dox ( 3 μm ) . [SEP]
[CLS] the kk1b10 nas - dox complexes modified with mdr1 - as provided high drug payload capacity and the synergistic effects of combined chemotherapy and gene therapy for target drug - resistant cancer cells B-material . [SEP]
[CLS] based on a modular and photocrosslinking strategy , we have molecularly constructed a multifunctional and programmable aptamer nanoassembly that can be utilized for the specific recognition and selective cytotoxicity B-property of target cancer cells B-material , as well as act as a drug carrier . [SEP]
[CLS] this aptamer - based nanoassembly platform exhibits several remarkable features : ( 1 ) easy modular design , facile assembly and preparation . [SEP]
[CLS] all the nanoassemblies mentioned in this study are first self - assembled with basic building blocks in a modular manner , followed by further photopolymerization to form size - controllable nanostructures . [SEP]
[CLS] ( 2 ) integrated multifunctionality . [SEP]
[CLS] different functional domains , including targeted aptamers , intercalated anticancer B-property drugs and therapeutic antisense oligonucleotides , enable this nanoassembly to act as a potential platform for targeted cancer therapy . [SEP]
[CLS] ( 3 ) high programmability . [SEP]
[CLS] the basic building unit provides precise control of the ratio of functional moieties , as well as programmable assembly of functional domains based on the therapeutic purpose . [SEP]
[CLS] the hybridized double - stranded dna configuration also allows for thousand - fold loading of anticancer B-property drugs or bioimaging agents in a single nanoassembly . [SEP]
[CLS] ( 4 ) good biostability . [SEP]
[CLS] nanoassemblies exhibit enzymatic resistance ( figure s7 ) and loading stability under physiological conditions . [SEP]
[CLS] ( 5 ) excellent biocompatibility B-property . [SEP]
[CLS] the nanoassemblies themselves show negligible cytotoxicity B-property , while demonstrating targeted cytotoxicity B-property when modified with the appropriate aptamers . [SEP]
[CLS] thus , our multifunctional aptamer - based nanoassemblies will find potential applications for point - of - care diagnosis , efficient drug transportation , and improved targeted cancer therapeutics . [SEP]
[CLS] all oligonucleotides were synthesized based on solid - phase phosphoramidite chemistry at a 1 μmol scale using the abi3400 dna / rna synthesizer ( applied biosystems , foster city , ca ) . [SEP]
[CLS] acrydite was directly coupled at the 5 ′ - end of oligonucleotides with an extended coupling time . [SEP]
[CLS] a prostar hplc ( varian , walnut creek , ca ) with a c18 column ( econosil , 5 , 250 mm ) from alltech ( deerfield , il ) was used to purify all fabricated dna . [SEP]
[CLS] the collected sequences were vacuum - dried and quantified using a cary bio - 300 uv spectrometer ( varian , walnut creek , ca ) . [SEP]
[CLS] each basic building unit was prepared by incubating B-technique equimolar amounts of connector , functional domain 1 , functional domain 2 and functional domain 3 at 30 °c for 1 h ( 10 mm tris buffer , 15 mm mgcl2 , ph 8 ) . [SEP]
[CLS] the aptamer - based nanoassembly was photopolymerized in aqueous solution containing 0 . 5 % mw ciba® irgacure® 2959 photoinitiator edition 2 . 4 . 98 using a portable uv lamp ( 350 nm ) for 10 min . [SEP]
[CLS] ccrf - cem ( human acute lymphoblastic leukemia ) , ramos ( human burkitt ' s lymphoma ) , and k562 ( chronic myelogenous leukemia ) cell B-material lines [SEP]
[CLS] cell B-material culturewere purchased from atcc . [SEP]
[CLS] the doxorubicin B-material - resistant k562 cell B-material line ( k562 / d ) was induced and cultured by our lab . [SEP]
[CLS] all cells B-material were cultured in rpmi 1640 medium ( atcc ) supplemented with 10 % fbs and 100 iu / ml penicillin - streptomycin ( cellgro ) . [SEP]
[CLS] polymerized nas were washed and concentrated using an amiconultra - 0 . 5 ( 50k mwko ) concentrator ( millipore ) . [SEP]
[CLS] the molecular weight of single nanoassembly was measured by a zetapals dls detector ( brookhaven instruments , holtsville ) at 25 °c , ( 2 . 4±0 . 8 ) × 10 7 g / mol . [SEP]
[CLS] the molar mass of each building unit was calculated including the modified chemical group , 1 . 7 × 10 5 g / mol . [SEP]
[CLS] the ratio between the molecular weight of single nanoassembly and building unit was the estimated number of building units in polymerized nas , ~ 150 ± 50 . [SEP]
[CLS] ccrf - cem , ramos , and k562 / d cells B-material were grown at a concentration of 2 × 10 6 ml −1 before the experiments were conducted . [SEP]
[CLS] cells B-material ( 10 6 ml −1 ) were first washed with washing buffer ( 500 μl ) at 4°c , followed by staining on ice with different nanoassemblies in binding buffer ( 200 μl ) containing 10 % fbs for 20 min . [SEP]
[CLS] then , cells B-material were washed with washing buffer ( 500 μl ) three times and incubated B-technique with streptavidin pe - cy5 . 5 dye solution ( 100 μl ) for another 20 min . [SEP]
[CLS] finally , washed cells B-material were suspended in 200 μl of binding buffer for fluorescence B-property detection on a facscan cytometer ( becton dickinson immunocytometry system , san jose , ca ) . [SEP]
[CLS] the fluorescence B-property was determined by counting 10 000 events , and data were analyzed with flowjo software . [SEP]
[CLS] all cellular fluorescent B-property images were collected on the fv500 - ix81 confocal microscope ( olympus america inc . , melville , ny ) with a 40 × oil immersion objective ( na = 1 . 40 , olympus , melville , ny ) . [SEP]
[CLS] excitation wavelength and emission filters : dox : 488 nm laser line excitation , emission bp ( 580 ± 20 ) nm filter ; transferrin - alexa 633 : 633 nm laser line excitation , emission bp ( 670 ± 20 ) nm filter . [SEP]
[CLS] cells B-material ( 10 6 ml −1 ) were incubated B-technique at 37°c with dox or dox - loaded nanoassembly for 2 h , followed by washing with washing buffer ( 1 ml ) twice and suspension in binding buffer ( 200 μl ) before imaging . [SEP]
[CLS] each experiment was analyzed with fluoview software . [SEP]
[CLS] the cytotoxicity B-property of nanoassemblies or dox - loaded nanoassemblies for each individual type of cell B-material was determined by mts assay using a celltiter 96 cell proliferation assay ( promega , madison , wi , usa ) . [SEP]
[CLS] after seeding in 96 - well plates and culturing overnight , the cells B-material were incubated B-technique with nanoassemblies or dox - loaded nanoassemblies for 2 h , washed with pbs , and then cultured with fresh medium for future cell B-material growth ( 48 h ) . [SEP]
[CLS] after removing the cell B-material medium , celltiter reagent ( 20 μl ) diluted in fresh medium ( 100 μl ) was added to each well and incubated B-technique for 1 - 2 h . the absorbance ( 490 nm ) was recorded by using a plate reader ( tecan safire microplate reader , ag , switzerland ) . [SEP]
[CLS] schematic illustration of the multifunctional self - assembled nanoassembly building units and photocrosslinked nanoassembly structure . [SEP]
[CLS] multifunctional dna sequences , including aptamers , acrydite - modified single - stranded dna and antisense oligonucleotides are selfassembled to form y - shaped functional domains , which further link via x - shaped connectors to form building units through the complementary arm sequences . [SEP]
[CLS] hundreds of these basic building units are then photocrosslinked into a multifunctional and programmable nanoassembly structure . [SEP]
[CLS] mts assay was performed to assess the selective cytotoxicity B-property of cem and ramos cells B-material treated with sgc8 - na complexes . [SEP]
[CLS] 2 . characterization of aptamer - based dna nanoassembly structures . [SEP]
[CLS] ( a ) transmission B-technique electron I-technique microscopy I-technique ( tem ) image of spherical photocrosslinked nanostructures . [SEP]
[CLS] scale bar : 200 nm . [SEP]
[CLS] ( b ) size - dependent ( in diameter ) distribution of nanoassemblies based on controllable concentration of building unit . [SEP]
[CLS] 3 . specific cancer cell B-material recognition via sgc8 - nas . [SEP]
[CLS] analytical flow B-technique cytometry I-technique shows the selective binding of sgc8 - modified nanoassemblies to target ccrf - cem cells B-material ( a ) , but not nontarget ramos cells B-material ( b ) . [SEP]
[CLS] black peak : cells B-material only ; green peak : library dna - nas ; blue peak : sgc8 aptamer only ; red peak : sgc8 - nas . [SEP]
[CLS] 4 . confocal B-technique laser I-technique scanning I-technique microscopy I-technique images of the colocalization of sybr green - stained sgc8 - nas and tf - alexa 633 ( endosome marker ) , indicating the specific internalization of sgc8 - nas into cem cells B-material ( a ) rather than ramos cells ( b ) . [SEP]
[CLS] scale bar : 20 μm . [SEP]
[CLS] 5 . selective cytotoxicity B-property of dox - loaded sgc8 - nas against targeted cancer cells B-material . [SEP]
[CLS] ( a - d ) confocal fluorescence B-technique imaging I-technique shows dox transport via dox - loaded sgc8 - nas relative to the same concentration of free dox to target cem ( a and c ) and nontarget ra - mos cells B-material ( b and d ) . [SEP]
[CLS] cem and ramos cells B-material were treated with free dox ( a and b ) and sgc8 - nas dox complex ( c and d ) . [SEP]
[CLS] scale bar : 20 μm . [SEP]
[CLS] 7 . selective cytotoxicity B-property of drug - resistant k562 cells B-material treated with free dox , kk1b10 - as na - dox , kk1b10 - r na - dox and sgc8 - as na - dox . [SEP]
[CLS] advancement of rnai therapies is mainly hindered by the development of efficient delivery vehicles B-material . [SEP]
[CLS] the ability to create small size ( < 30 nm ) oligonucleotide nanoparticles B-nanoparticle is essential for many aspects of the delivery process but is often overlooked . [SEP]
[CLS] in this report , we describe di - block star polymers B-material that can reproducibly complex double - stranded oligonucleotides into monodisperse nanoparticles B-nanoparticle with 15 , 23 or 30 nm in diameter . [SEP]
[CLS] the polymer B-material - nucleic B-material acid I-material nanoparticles B-nanoparticle have a core - shell architecture with dense peg brush coating B-material . [SEP]
[CLS] we characterized these nanoparticles B-nanoparticle using itc , dls , fret , fcs , tirf and tem . [SEP]
[CLS] in addition to small size , these nanoparticles B-nanoparticle have neutral zeta B-property potentials I-property making the presented polymer B-material architecture a very attractive platform for investigation of yet poorly studied polyplex size range for sirna and antisense oligonucleotide delivery applications . [SEP]
[CLS] the ability to down - regulate genes via rna interference ( rnai ) was reported in 1998 and its great potential as a therapeutic approach , especially for cancer , was quickly recognized . [SEP]
[CLS] however , delivery of sirna to the site of interest remains the major roadblock for clinical applications of rnai therapy . [SEP]
[CLS] nanoparticles B-nanoparticle ( nps B-nanoparticle ) hold the promise to solve this longstanding problem . [SEP]
[CLS] to achieve targeting , nps B-nanoparticle should avoid renal filtration and the clearance by mononuclear phagocyte system . [SEP]
[CLS] many studies suggested that the size of nps B-nanoparticle has a direct correlation with their systemic and intratumoral distribution and np B-nanoparticle in the size range of 10 - 30 nm may achieve deeper tumor B-material penetration , avoid accelerated blood clearance , and be best suited for targeting . [SEP]
[CLS] but due to the difficulty in controlling organic - based nanoparticle B-nanoparticle size precisely , the size influence on performance of np B-nanoparticle is still elusive . [SEP]
[CLS] cationic B-material polymers B-material are often used to complex sirna into nanoparticles B-nanoparticle ( " polyplexes " ) for delivery purpose . [SEP]
[CLS] however , this process usually leads to polydisperse nps B-nanoparticle that typically are above 60 nm in size , more often above 100 nm . [SEP]
[CLS] print technology developed by desimone et al . elegantly solved this long - standing polydispersity problem and is able to produce monodisperse particles for nucleic B-material acid I-material delivery . [SEP]
[CLS] however , the current development of print technology has not yet allowed production of nanoparticles B-nanoparticle under 50 nm . [SEP]
[CLS] fabrication of ultrasmall ( < 30 nm ) nanoparticles B-nanoparticle has been realized by a few groups using inorganic particles , polymer and lipid based particles , nucleic B-material acid I-material conjugates , or self - assembly into oligonucleotide particles . [SEP]
[CLS] most of the polymer - based systems inherently suffer from polydispersity . [SEP]
[CLS] fabrication of the nucleic B-material acid I-material nanoparticles B-nanoparticle with precisely controlled small size and homogeneity is not a trivial task and is rarely addressed in the literature . [SEP]
[CLS] in this report , we describe core - shell star polymers B-material that can complex 2 , 16 and 53 molecules of oligo nucleic acid ( na ) resulting in nanoparticles B-nanoparticle with diameters of 15 , 23 and 30 nm , respectively . [SEP]
[CLS] these nanoparticles B-nanoparticle are monodisperse ( polydispersity indexes ( pdis ) < 0 . 08 ) , have neutral ζ - potentials and are colloidally stable for days in phosphate buffered saline . [SEP]
[CLS] the formation of these nanoparticles B-nanoparticle is very straightforward and is accomplished by mixing the star polymers B-material with na , which is amenable to be quickly adapted by non - experts in drug delivery . [SEP]
[CLS] with the great cost reduction of next - generation genomic sequencing and the daunting complexity of patientdependent tumor B-material environment , personalized medicine is expected to significantly improve the outcome of cancer treatment . [SEP]
[CLS] our convenient fabrication method for na nanoparticles B-nanoparticle has the potential to take personalized nanomedicine one step closer to being practical . [SEP]
[CLS] we envision that our star polymers B-material may have the potential to serve as a platform for future development of targeted na delivery to overcome the common problems associated with nanoparticle B-nanoparticle sizes , heterogeneity , stability , and cancer targeting . [SEP]
[CLS] in this report , we present a strategy based on star polymers B-material with an uncrowded cationic B-material core B-material for hosting na and dense corona that consists of peg brush polymers B-material ( figure 1a ) . [SEP]
[CLS] we employed reversible addition - fragmentation chain B-property transfer I-property ( raft ) polymerization using tetravalent chain B-property transfer I-property agent ( cta ) and n - ( 2 - aminoethyl ) methacrylamide hydrochloride ( aema • hcl ) to synthesize star polymers B-material with four cationic B-material arms ( star polymer cores B-material ) , followed by polymerization of α - methoxy - ω - methacrylamido poly ( ethylene glycol ) ( peg - ma , m n = 2kda ) to obtain final structures . [SEP]
[CLS] we started by making per - tosylated pentaerythritol 2 , which was subsequently used to obtain boc - protected tetraamine 3 that contains thioether B-material linkages B-property . [SEP]
[CLS] in addition to molecular weight determination by size exclusion chromatographystatic light scattering ( sec - sls ) , incorporation of the thioether B-material group allowed us to conveniently determine number - averaged molecular weight ( m n ) of the first block using nmr ( see si for more details ) . [SEP]
[CLS] boc - groups were then removed and produced tetraamine was converted into tetravalent cta 6 in a reaction with nhs - ester 5 . [SEP]
[CLS] polymerizations of the aema • hcl were conducted at 70° c in deoxygenated dmso using 4 , 4 ′ - ( diazene - 1 , 2 - diyl ) bis ( 4 - cyanopentanoic acid ) ( v - 501 ) as initiator and cta 6 to yield three star polymers B-material ( cationic B-material cores B-material ) with degrees of polymerization 14 , 31 and 47 per arm ( table 1 ) . [SEP]
[CLS] these polymers B-material were purified by dialysis against ultrapure water B-material , lyophilized and used as macroctas in the polymerization of peg - ma . [SEP]
[CLS] chain extension with peg - ma was conducted in a similar fashion , using water B-material / dmso mixture as a solvent and v - 501 as initiator ( see si for details ) . [SEP]
[CLS] star - polymers B-material were purified by dialysis and finally lyophilized to yield desired materials . [SEP]
[CLS] polymers B-material were analyzed by sec - sls , nmr , uv - vis and dynamic B-technique light I-technique scattering I-technique ( dls ) ( see table 1 for summary and si for details ) . [SEP]
[CLS] aema • hcl polymerization resulted in polymers B-material with expected molecular weights and narrow polydispersity indexes ( pdis < 1 . 04 ) . [SEP]
[CLS] number - averaged molecular weights ( m n ) obtained by sec matched those obtained by the end - group analysis using 1 h - nmr . [SEP]
[CLS] polymerization of peg - ma was less controlled and resulted in final structures with pdis ranging from 1 . 27 to 1 . 43 . [SEP]
[CLS] the slight loss of the control can be attributed to the bulky nature of the peg - ma monomer B-material and even bulkier nature of the propagating peg bottle brush chain ; therefore elevated pdis were expected . [SEP]
[CLS] dense peg coating B-material , however , is essential for good antifouling properties and resistance to opsonization ; therefore use of high molecular weight peg was necessary . [SEP]
[CLS] to investigate the ability of star polymers B-material to complex na , we performed isothermal titration calorimetry ( itc ) measurements . [SEP]
[CLS] since the goal for this paper is to establish a fabrication method for small na nanoparticles B-nanoparticle , we focus our studies on 22 base - pair double - stranded dna ( dsdna ) for cost - effective reasons . [SEP]
[CLS] because the complexation process between dsdna and the star polymers B-material is driven by charge - charge interactions , we expect dsdna would be a reasonable surrogate for sirna in this report . [SEP]
[CLS] star - 1 will be taken as an example to demonstrate our analysis of the itc data . [SEP]
[CLS] in the itc experiments , dsdna and the star polymer B-material were dissolved in the same buffer and dsdna was titrated into the star polymer B-material solution at 37 °c . [SEP]
[CLS] the apparent heat produced during the complexation is a result of two processes : 1 ) heat of the complexation between dsdna and the star polymer B-material ( δh cmplxtn ) and 2 ) heat of neutralization of n p moles of released by buffer ( n p δh 0 buffer ) . [SEP]
[CLS] in order to obtain the pure heat of complexation δh cmplxtn without the contribution from the buffer neutralization process , we conducted titrations in two buffers , namely tris and hepes both at ph 7 . 4 ( figures 2a and 2b , see si for experimental details ) . [SEP]
[CLS] by solving a system of linear equations in two variables for each injection , where δh intris and δh inhepes are apparent heats measured by itc and δh 0 tris and δh 0 hepes are standard heats of neutralization equal to 11 . 33 and 4 . 87 kcal • mol −1 , respectively , we extracted the heat of complexation δh cmplxtn between the star polymers B-material and na ( figure 2c , see si for an calculation example ) . [SEP]
[CLS] overall , the complexation process was found to be enthalpy driven , similar to the previously reported polyamine / na binding ( e . g . pamam ) . [SEP]
[CLS] interestingly , there were two separate equilibria observed . [SEP]
[CLS] one of them is strongly exothermic and defines the overall negative enthalpy of the complexation process . [SEP]
[CLS] on the contrary , the second accompanying process is endothermic , which can be concluded from the positive heats observed beyond 1 . 25 eq of added na , and is driven by entropy ( figure 2d ) . [SEP]
[CLS] based on the established composition for star - 1 / na nanoparticle B-nanoparticle ( vide infra ) the two equilibria are : at this point we were unable to obtain separate values for k1 and k2 as both equilibria overlap . [SEP]
[CLS] we used one - site binding model to fit the data , which cannot distinguish between two equilibria ; however it allowed us to obtain the number of na equivalents that is incorporated into nanoparticles B-nanoparticle and get an estimate about the overall order of binding ( k = k1×k2 , table 2 ) . [SEP]
[CLS] we used heats produced during the initial injections to obtain the enthalpy values . [SEP]
[CLS] as shown in table 2 , the enthalpy increases with the increase in size of the cationic B-material core B-material , which suggests that the polymer B-material with the largest core B-material can undergo greater conformational changes and maximize charge - charge interactions compared to a star polymer B-material with a smaller core B-material . [SEP]
[CLS] in addition , because two separate processes can be observed with itc and the star - 1 nanoparticle B-nanoparticle has a known composition , this system is an interesting model to study polymer B-material / nucleic B-material acid I-material complexation process in details and will be reported in a separate investigation . [SEP]
[CLS] we formulated nanoparticles B-nanoparticle using the equivalence values obtained from the itc experiments and determined their sizes with dls . [SEP]
[CLS] all three polymers B-material , star - 1 , star - 2 and star - 3 , produced monodisperse particles with 15 , 23 and 30 nm in hydrodynamic diameter , respectively ( figure 3 ) . [SEP]
[CLS] particle fabrication is highly reproducible as evident from the small standard deviation values ( figure 3c ) . [SEP]
[CLS] importantly , no change in particle hydrodynamic diameters was observed for all three nanoparticles B-nanoparticle over a 7 - day time period , attesting to their colloidal stability ( figure 3b , see si for histograms ) . [SEP]
[CLS] it is interesting to note that all three star polymers B-material have similar hydrodynamic diameters ( table 1 ) , but the sizes of the corresponding nanoparticles B-nanoparticle increase with the increase in the size of the star polymer B-material cores B-material . [SEP]
[CLS] in a free , un - complexed state , the star polymer B-material core B-material exists in a relaxed conformation , however upon complexation of the rigid na the cationic B-material core B-material may adopt a more extended conformation to maximize enthalpic gain from the electrostatic interactions . [SEP]
[CLS] as the steric bulk possessed by peg brush is moved farther away from the star polymer B-material focal point with increase in cationic B-material core B-material size , it enables the accommodation of more polymer B-material molecules inside the nanoparticle B-nanoparticle ( vide infra ) . [SEP]
[CLS] ζ - potential measurements of na nanoparticles B-nanoparticle resulted in neutral zeta B-property potentials I-property , however it should be noted that the quality of the ζ - potential measurements was suboptimal even when nanoparticles B-nanoparticle were formulated using high concentration na solution ( 150 μm , see si for details ) . [SEP]
[CLS] this is understandable as the electrophoretic B-property mobility I-property becomes indistinguishable from the brownian motion at neutral zeta B-property potential I-property . [SEP]
[CLS] neutral ζ - potential is another property desired for delivery applications as it helps to avoid non - specific interactions with biological molecules and cells B-material . [SEP]
[CLS] recently , the groups of radler and chen independently reported single sirna nanoparticles B-nanoparticle , which have hydrodynamic diameters of 30 and 25 nm , respectively . [SEP]
[CLS] we were curious to know how many na molecules there are in our smallest nanoparticle B-nanoparticle derived from star - 1 ( d h = 15 nm ) . [SEP]
[CLS] in addition , it was predicted by wittrup et al . that the size of an igg immunoglobulin B-material ( ~ 15 nm ) is the optimal size for systemic targeting , which balances renal filtration and tumor B-material tissue penetration . [SEP]
[CLS] therefore , nanoparticles B-nanoparticle derived from polymer B-material star - 1 possess a special interest as a platform for na delivery . [SEP]
[CLS] star - 1 / na B-nanoparticle nanoparticle B-nanoparticle was visualized using transmission B-technique electron I-technique microscopy I-technique ( tem ) with uranyl acetate staining . [SEP]
[CLS] as shown in figure 4 , star - 1 / na nanoparticles B-nanoparticle reveal spherical shapes and uniform distribution . [SEP]
[CLS] the diameter obtained by tem is 8 . 6±2 . 0 nm based on the analysis of more than 100 nanoparticles B-nanoparticle . [SEP]
[CLS] it is smaller than the hydrodynamic diameter obtained by dls in part due to the sample preparation ( drying ) and poor staining of peg brush by uranyl acetate ( as opposed to the complexed na that stains well ) . [SEP]
[CLS] the poor staining of peg brush , however , allows to observe a distinct core B-material and corona ( figures 1c and 4b ) , revealing the desired core - shell architecture of the particle with neutral peg corona coating B-material the polyplex . [SEP]
[CLS] the hydrodynamic diameter of star - 1 / na nanoparticle B-nanoparticle is only 3 . 6 nm greater than the hydrodynamic diameter of the polymer B-material itself and the uniform size distribution remains virtually unchanged ( figures 3d and 3e ) . [SEP]
[CLS] additionally , the size of the core B-material as evident by tem is less than 10 nm , which was used previously as an argument by chen et al . to conclude the presence of only one na molecule in the complex . [SEP]
[CLS] to test if this hypothesis stands in the case of our system , we prepared a nanoparticle B-nanoparticle between star - 1 polymer B-material and a mixture of equal amounts of cy3 - labeled oligo and cy5 - labeled oligo ( note : all the dye labeled dsdna oligos used in the following experiments were hplc - purified and contain only one dye molecule per oligo ) . [SEP]
[CLS] when a solution of this nanoparticle B-nanoparticle was excited at 520 nm , a significant increase in fluorescence B-property at 665 nm and decrease in fluorescence B-property at 565 nm was observed ( figure 5 , blue trace ) compared to the negative control - solution of oligos alone at the same total oligo concentration ( figure 5 , black trace ) . [SEP]
[CLS] this change in fluorescence B-property is a result of forster resonance energy transfer ( fret ) between two oligos , and it indicates that complexation with star - 1 brings oligos with two different dyes together and thus the nanoparticles B-nanoparticle derived from star - 1 contain at least two oligos per particle . [SEP]
[CLS] this result motivated us to determine the exact number of na molecules in nanoparticles B-nanoparticle , which was accomplished using fluorescence B-technique correlation I-technique spectroscopy I-technique measurements . [SEP]
[CLS] similar to dls for non - fluorescent B-property species , fcs measures the intensity fluctuation of the fluorescent B-property species in order to determine the diffusion coefficients and the number of fluorescent B-property species in a fixed small volume . [SEP]
[CLS] the number of fluorescently B-property labeled na molecules per nanoparticle B-nanoparticle can be calculated based on the change in a number of the fluorescent B-property species upon complexation . [SEP]
[CLS] 6a shows the autocorrelation curves for alexa - 488 dsdna oligo solution at 40 nm ( black ) and the solution of its nanoparticle B-nanoparticle with star - 1 at the same oligo concentration ( blue ) . [SEP]
[CLS] solid lines represent the best fits with monoexponential decays and the residuals for these fits are shown in figure 6b . [SEP]
[CLS] the number of the observed fluorescent B-property species is inversely proportional to the intercept of the autocorrelation function fit with y - axis . [SEP]
[CLS] as shown in figure 6a , the number of fluorescent B-property species decreased 2 fold , indicating that there are two oligonucleotide molecules in each star - 1 / na nanoparticle B-nanoparticle . [SEP]
[CLS] in a similar fashion we have analyzed the nanoparticles B-nanoparticle formed with star - 2 and star - 3 polymers B-material ( figure 3c , see si for more details ) . [SEP]
[CLS] thus , 8 molecules of star - 2 polymer B-material assemble into nanoparticles B-nanoparticle that contain 16 molecules of oligo , while the star - 3 polymer B-material , that has the steric bulk of peg brush moved the farthest away from the star polymer B-material focal point , assembles in np B-nanoparticle that contains 14 polymer B-material molecules and 53 molecules of oligo . [SEP]
[CLS] in addition , the smallest nanoparticle B-nanoparticle was also analyzed by total internal reflection fluorescence B-property ( tirf ) microscopy B-technique using the step - wise single - molecule photobleaching method as previously described . [SEP]
[CLS] when nanoparticles B-nanoparticle containing dsdna oligos that are labeled with a fluorophore cy - 5 were continuously illuminated with the laser , individual cy - 5 molecules photobleached stochastically and the total number of photobleaching steps indicated the number of dsdna - cy5 in a particular nanoparticle B-nanoparticle . [SEP]
[CLS] fluorescence B-property images ( fig . 7a ) show a representative nanoparticle B-nanoparticle undergoing a two - step photobleaching process , which is evident in its corresponding integral fluorescence B-property intensity vs . time plot ( figure 7b ) . [SEP]
[CLS] the result indicates that the photobleached nanoparticle B-nanoparticle contains two cy5 - dsdnas . [SEP]
[CLS] we find , by analyzing approximately 600 particles , that polymer star - 1 complexes with 1 . 7±0 . 8 dsdnas on average . [SEP]
[CLS] in addition to the desired physical properties , nanoparticles B-nanoparticle must be nontoxic in order to serve as a platform for oligonucleotide delivery . [SEP]
[CLS] we evaluated the viability B-property of I-property hela I-property cells I-property incubated B-technique with various concentrations of the nanoparticles B-nanoparticle using mtt B-technique assay I-technique . [SEP]
[CLS] as shown in figure 8 , cell B-property viability I-property remained unchanged when cells B-material were exposed to nanoparticles B-nanoparticle at up to 100 μg / ml ( concentrations shown based on polymer B-material concentration ) . [SEP]
[CLS] this concentration corresponds to 0 . 7 and 2 . 0 μm of na in the case of star - 1 and star - 3 nanoparticles B-nanoparticle , respectively , which is significantly higher than the na concentrations used for delivery applications . [SEP]
[CLS] in addition , no cell B-material morphology changes were observed ( data not shown ) at all the concentrations tested , supporting the results of the mtt B-technique assay I-technique . [SEP]
[CLS] in conclusion , the investigations of the polymer B-material - based oligonucleotide delivery vehicles B-material in a particularly attractive size range ( < 30 nm ) has been hindered by difficulties in obtaining particles of these sizes . [SEP]
[CLS] herein , we reported star polymers B-material with architecture that allows to form such particles , which are not only small , but also have low polydispersity , neutral zeta B-property potentials I-property and are colloidally stable . [SEP]
[CLS] the fabrication of these nanoparticles B-nanoparticle is simple and highly reproducible and the size of nanoparticles B-nanoparticle can be controlled within 15 - 30 nm range . [SEP]
[CLS] neither polymers B-material nor derived nanoparticles B-nanoparticle have any appreciable cellular toxicity B-property . [SEP]
[CLS] in addition , trithiocarbonate groups available on the distal side of these polymers B-material ( and nanoparticles B-nanoparticle ) are excellent handles for decorating the nanoparticles B-nanoparticle with targeting B-material ligands I-material . [SEP]
[CLS] consequently , these star polymers B-material will serve as a great platform for future explorations in applications of oligonucleotide delivery . [SEP]
[CLS] synthesis of tetra - valent chain B-property transfer I-property agent and star polymers B-material . [SEP]
[CLS] polymer na , eq δh , kca • mol −1 k , m −1 star - 1 0 . 9 −112±2 10 8 [SEP]
[CLS] star - 2 1 . 9 −133±4 10 7 [SEP]
[CLS] star - 3 3 . 7 −155±2 10 7 [SEP]
[CLS] ( a ) schematic illustration of nucleic B-material acid I-material ( na ) complexation by star polymer B-material . [SEP]
[CLS] ( b ) structure of the star polymers B-material . [SEP]
[CLS] ( c ) tem image of a star - 1 / na nanoparticle B-nanoparticle with visibly distinguishable polyamine / na core B-material ( black ) and peg polymer B-material brush corona ( dark gray ) . [SEP]
[CLS] isothermal titration calorimetry ( itc ) . [SEP]
[CLS] ( a ) star - 1 titration with dsdna in a tris buffer . [SEP]
[CLS] ( b ) star - 1 titration with dsdna in hepes buffer . [SEP]
[CLS] ( c ) calculation of the heat of complexation based on titrations in two buffers for each data point . [SEP]
[CLS] ( d ) extracted heat of complexation of na by star - 1 polymer B-material . [SEP]
[CLS] 3 . dls analysis of polymers B-material and nanoparticles B-nanoparticle . [SEP]
[CLS] ( a ) histograms of na nanoparticles B-nanoparticle of star - 1 ( blue ) , star - 2 ( green ) , star - 3 ( red ) . [SEP]
[CLS] ( b ) hydrodynamic diameters of nanoparticles B-nanoparticle over time in phosphate buffer saline . [SEP]
[CLS] star - 1 ( blue ) , star - 2 ( green ) , star - 3 ( red ) . [SEP]
[CLS] error bars represent sd of three formulations . [SEP]
[CLS] ( c ) histogram of star - 1 / na B-nanoparticle nanoparticle B-nanoparticle . [SEP]
[CLS] error bars represent sd of three formulations . [SEP]
[CLS] ( d ) comparison of the traces between star - 1 polymer B-material ( gray ) and star - 1 nanoparticle B-nanoparticle ( blue ) . [SEP]
[CLS] ( e ) hydrodynamic diameters of polymers B-material and corresponding nanoparticles B-nanoparticle at time 0 . [SEP]
[CLS] characterization of star - 1 nanoparticles B-nanoparticle . [SEP]
[CLS] ( a ) tem image of star - 1 / na nanoparticle B-nanoparticle stained with uranyl acetate . [SEP]
[CLS] scale bar equals to 100 nm . [SEP]
[CLS] average diameter±sd was calculated with imagej software by analysis of 112 particles . [SEP]
[CLS] ( b ) close up tem image of star - 1 / na B-nanoparticle nanoparticle B-nanoparticle . [SEP]
[CLS] scale bar equals to 20 nm . [SEP]
[CLS] peg brush corona can be observed as understained ring surrounding polyamine / na core B-material . [SEP]
[CLS] 5 . observation of fret in star - 1 derived nanoparticle B-nanoparticle . [SEP]
[CLS] ( a ) correlation functions of alexa488 - dsdna oligo ( black ) and its nanoparticle B-nanoparticle with star - 1 polymer B-material ( blue ) . [SEP]
[CLS] both measurements were conducted at 40 nm oligo concentration . [SEP]
[CLS] best fits with monoexponential decay are represented by solid lines . [SEP]
[CLS] ( b ) residuals from the fits for alexa488 - dsdna oligo ( black ) and its nanoparticle B-nanoparticle with star - 1 polymer B-material ( blue ) . [SEP]
[CLS] ( c ) number of oligos per particle ( # na / np B-nanoparticle ) and number of polymers B-material per particle ( # polymer / np B-nanoparticle ) . [SEP]
[CLS] d h and pdi were obtained with dls measurements . [SEP]
[CLS] 7 . step - wise single - molecule photobleaching of star - 1 / cy - 5na complex . [SEP]
[CLS] ( a ) tirf images of a representative particle undergoing photobleaching after continuous laser illumination of indicated times . [SEP]
[CLS] scale bars : 500 nm . [SEP]
[CLS] ( b ) fluorescence B-property intensity of the particle shown in panel a is plotted as a function of laser illumination time , showing a two - step photobleaching profile . [SEP]
[CLS] 8 . viability B-property of I-property hela I-property cells I-property upon 24h incubation B-technique with nanoparticles B-nanoparticle at various concentrations . [SEP]
[CLS] error bars represent standard deviation between 6 wells . [SEP]
[CLS] physical characterization of star polymers B-material by size exclusion chromatography B-technique - static light scattering and dynamic B-technique light I-technique scattering I-technique . [SEP]
[CLS] data and materials availability : sna conjugates and bcl2l12 - related biologicals are available upon request from c . a . m . and a . h . s . with an executed materials transfer agreement . [SEP]
[CLS] supplementary materialswww . sciencetranslationalmedicine . org / cgi / content / full / 5 / 209 / 209ra152 / dc1 methods [SEP]
[CLS] s1 . [SEP]
[CLS] bcl2l12 is a gbm oncogene . [SEP]
[CLS] s2 . [SEP]
[CLS] sna uptake into murine astrocytes and mutns depends on scavenger receptors . [SEP]
[CLS] s3 . [SEP]
[CLS] synthesis scheme for snas . [SEP]
[CLS] s1 . [SEP]
[CLS] characterization of snas . [SEP]
[CLS] s2 . [SEP]
[CLS] evaluation of serum cytokines in mice . [SEP]
[CLS] s3 . [SEP]
[CLS] body weight and blood chemistry after systemic sna administration to sprague - dawley rats . [SEP]
[CLS] movie s1 . [SEP]
[CLS] 3d reconstruction of mr images after intracranial injection of gd ( iii ) - functionalized snas . [SEP]
[CLS] references author contributions : designed the experiments : s . a . j . , e . s . d . , c . h . k . , c . a . m . , and a . h . s . synthesized and characterized the nanomaterials B-material : e . s . d . , c . h . k . , j . i . c . , w . l . d . , a . w . s . , m . w . r . , d . a . g . and t . j . meade conducted in vitro experiments : s . a . j . , c . h . k . , j . p . l . , and d . a . g . conducted in vivo experiments : s . a . j . , e . s . d . , c . h . k . , l . a . h . , j . p . l . , and f . m . k . performed data analysis : s . a . j . , e . s . d . , c . h . k . , l . a . h . , and j . p . l . obtained icp data : c . h . k . , a . j . l . , and p . c . p . obtained pharmacokinetic and toxicology data : t . j . merkel prepared the manuscript : s . a . j . , e . s . d . , c . h . k . , c . a . m . , and a . h . s . all authors edited the manuscript . [SEP]
[CLS] competing interests : d . a . g . , c . a . m . , w . l . d . , a . h . s . , and p . c . p . have interest in aurasense therapeutics , which develops snabased technologies . [SEP]
[CLS] the content is solely the responsibility of the authors and does not necessarily represent the official views of the sponsors or government , and no official endorsement should be inferred . [SEP]
[CLS] glioblastoma multiforme ( gbm ) is the most prevalent and lethal form of malignant brain tumors B-material and considered to be one of the deadliest human cancers . [SEP]
[CLS] multimodal treatment regimens combining radiation with the dna B-property alkylating I-property agent I-property temozolomide have only incrementally increased median patient survival by 2 . 5 months to 14 . 6 months ; recurrence is nearly universal , and salvage therapies to impede further progression are ineffective . [SEP]
[CLS] disease management is complicated by the coactivation of multiple mitogenic and cell B-material death - inhibitory pathways , resulting in rapid disease progression and intense resistance of tumors B-material toward therapy - induced apoptosis B-event . [SEP]
[CLS] coextinction strategies using multiple small molecule - or antibody B-material - based agents , however , are often hampered by drugdrug interactions , systemic toxicity B-property due to pronounced off - target effects , and the emergence of drug resistance . [SEP]
[CLS] additional challenges in gbm drug development include ineffective systems for drug delivery to intracerebral tumor B-material elements and the lack of imaging methodologies to quantify intratumoral drug concentrations . [SEP]
[CLS] conventional and tailored therapies bound for the central nervous system ( cns ) have to negotiate passage through the blood - brain barrier B-property ( bbb ) , the blood - cerebrospinal fluid barrier B-property ( bcsf ) , and the blood - tumor barrier B-property ( btb ) , and also withstand the substantial dynamic force in the brain caused by csf flow , brain edema , and tumor B-material mass - related pressure . [SEP]
[CLS] moreover , they must be able to disseminate throughout cancerous tissue . [SEP]
[CLS] rna interference ( rnai ) - based biotherapeutic gene silencing [SEP]
[CLS] has emerged as a promising approach to target multiple " undruggable " oncogenes implicated in growth , apoptosis B-event , migration , and invasion . [SEP]
[CLS] the lack of efficient delivery to tumor B-material sites , limited biological activity , and unfavorable safety profile , however , have prevented the implementation of many rnai - based therapeutics in the clinic . [SEP]
[CLS] recently , we have developed spherical nucleic B-material acid I-material ( sna ) nanoparticle B-nanoparticle conjugates , which are nanostructures consisting of densely packed , highly oriented small interfering rna ( sirna ) oligonucleotides surrounding an inorganic B-nanoparticle gold I-nanoparticle nanoparticle I-nanoparticle core B-material as a platform for gene silencing . [SEP]
[CLS] snas act as singleentity agents capable of simultaneous transfection and gene regulation without the need for auxiliary carriers or cationic B-material transfection agents . [SEP]
[CLS] snas are remarkably stable in physiological environments , resist nuclease degradation , and , in comparison to conventional rnai delivery platforms , result in more efficient and persistent gene knockdown in cells B-material and tissues without triggering a significant immune response and off - target effects . [SEP]
[CLS] snas can be cofunctionalized with fluorochromes or gadolinium B-material [ gd ( iii ) ] - based magnetic B-property resonance imaging ( mri ) contrast B-technique agents I-technique to track their accumulation in cells B-material and , in this study , in tumors B-material . [SEP]
[CLS] to evaluate snas as potential anti - glioma therapeutics , we elected the bcl2like12 ( bcl2l12 ) oncogene as a target for sna - mediated gene silencing . [SEP]
[CLS] bcl2l12 localizes to chromosome 19q13 , a region frequently amplified in gbm . [SEP]
[CLS] furthermore , cell B-technique - I-technique based I-technique assays I-technique and expression analyses have identified bcl2l12 as a putative oncogene with consistent and prevalent mrna and protein B-material up - regulation in gbm relative to normal brain . [SEP]
[CLS] mechanistically , bcl2l12 inhibits apoptosis B-event by neutralizing the activity of effector caspase - 3 and caspase - 7 downstream of mitochondrial dysfunction and apoptosome activity and blocks the transactivational activity of p53 , a pivotal tumor B-material suppressor that is frequently deregulated in primary gbm . [SEP]
[CLS] given the impact of bcl2l12 on key nodes of cytoplasmic and nuclear cell B-material cycle and apoptosis B-event signaling cascades , as well as the low - level expression of bcl2l12 in the adult brain , we describe here an in - depth preclinical characterization of bcl2l12 - targeting snas ( sil12 - snas ) . [SEP]
[CLS] we report that these nanoconjugates cross the bbb after systemic administration , selectively accumulate in and disseminate throughout tumor B-material tissue , and potently neutralize bcl2l12 expression , without the use of auxiliary transfection agents , thereby enhancing therapy - induced apoptosis B-event in glioma cells B-material , reducing tumor B-material burden , and significantly delaying cancer progression in glioma - bearing mice , without adverse side effects . [SEP]
[CLS] bcl2l12 is a potent caspase and p53 inhibitor that is overexpressed in the vast majority of human primary gbm specimens , yet is at low or undetectable levels in cells B-material of glial origin , in normal brain surrounding tumor B-material tissue , and in low - grade astrocytoma . [SEP]
[CLS] in addition , our analysis of 343 glioma patients in the repository of molecular brain neo - plasia data ( rembrandt ) , a web - based data mining and analysis platform , identified bcl2l12 as a potential prognostic factor , because gbm patients with high - level overexpression of bcl2l12 have shorter progression - free survival compared to patients with " intermediate B-property " ( 0 . 5 - to 4 - fold ) expression or underexpression of bcl2l12 [ p < 0 . 001 , log - rank p value calculated by rembrandt using the mantel - haenszel procedure ] ( fig . s1 ) . [SEP]
[CLS] therefore , we decided to explore the use of bcl2l12 - targeted snas as a novel gbm therapeutic . [SEP]
[CLS] to neutralize bcl2l12 expression in glioma cells B-material , we first investigated the effectiveness of sna uptake into human glioma cell B-material lines ( u87mg ) , primary murine cortical astrocytes , and patient - and mouse - derived tumor B-material neurospheres [ human ( hu ) and murine ( mu ) tns , respectively ] in vitro . [SEP]
[CLS] confocal fluorescence B-technique microscopy I-technique for snas conjugated to cyanine5 ( cy5 ) fluorochromes showed highly efficient cellular uptake ( ~ 99 % of the cell B-material population ) in glioma cell B-material lines , murine cortical astrocytes , and in both hutns ( fig . 1a ) and mutns ( fig . s2 , a to c ) , with widespread diffusion of particles throughout the core B-material of the tns ( fig . 1a and fig . s2c ) . [SEP]
[CLS] cy5 - sirnas without snas were unable to enter cells B-material ( fig . s2d ) . [SEP]
[CLS] our previous studies have shown that the accumulation of snas depends on the activity B-property of I-property scavenger I-property receptors I-property ( srs ) , in particular class a ( sr - a ) . [SEP]
[CLS] in agreement with these studies , inductively coupled plasma mass spectrometry ( icp - ms ) revealed reduced accumulation of snas in human u87mg cells B-material preincubated with known sr inhibitors , polyinosinic acid ( poly i ) and fucoidan ( fig . s2e ) . [SEP]
[CLS] to determine whether cellular uptake of snas translates into effective neutralization of endogenous bcl2l12 mrna and protein B-material expression , we next assessed the knockdown efficacies of sil12 - snas in glioma cell B-material lines and hutns . [SEP]
[CLS] we screened six sil12 - sna constructs targeting different regions of the bcl2l12 mrna backbone for their ability to down - regulate endogenous bcl2l12 levels in comparison to scrambled control sequences ( sico - sna ) . [SEP]
[CLS] through this process , we identified five conjugates that were able to knock down bcl2l12 and pursued two of these for subsequent studies : sil12 - 1 - sna and sil12 - 2 - sna . [SEP]
[CLS] s3 provides the synthesis scheme for these sirna - sna constructs . [SEP]
[CLS] sna synthesis is standardized , so their physicochemical properties are highly reproducible for optimal therapeutic application ( table s1 ) . [SEP]
[CLS] s4 ( a and b ) displays sequence information and alignment to bcl2l12 mrna . [SEP]
[CLS] these conjugates reduced bcl2l12 mrna by 40 % and protein B-material expression by up to 90 % in patient - derived tns and transformed glioma cell B-material lines u87mg , lnz308 , and lnz235 ( fig . 1 , b and c , and fig . s4c ) . [SEP]
[CLS] sna concentration as low as 0 . 1 nm was sufficient to neutralize bcl2l12 protein B-material levels in ln235 glioma cells B-material ( fig . 1b ) , comparable to bcl2l12 knockdown triggered by lipoplex - delivered sil12 oligonucleotides ( fig . s4d ) . [SEP]
[CLS] next , we used 5 ′ rna ligand - mediated rapid amplification of complementary dna ( cdna ) ends ( 5 ′ - rlm - race ) polymerase chain reaction ( pcr ) to identify the mrna cleavage product that results from sil12 - 2 - sna - triggered , rna - induced silencing complex ( risc ) - mediated rnai . [SEP]
[CLS] upon ligating rna from sil12 - 2 - sna - treated u87mg cells B-material to a generacer oligo ( fig . 1d ) , nested pcr using gene - specific primers amplified a bcl2l12 mrna cleavage fragment ( fig . 1e ) harboring a 5 ′ end identical to the predicted cleavage site ( fig . 1f ) . [SEP]
[CLS] thus , sil12 - 2 - sna - mediated bcl2l12 gene knockdown was achieved through rnai . [SEP]
[CLS] to verify the functionality of sna - mediated bcl2l12 knockdown , we surveyed bcl2l12regulated downstream signaling upon sna administration to glioma cells B-material . [SEP]
[CLS] here , snadriven knockdown of bcl2l12 increased effector caspase and p53 activation upon pretreatment of cells B-material with the pan - kinase inhibitor staurosporine , the dna methylation agent temozolomide , and dna damage - inducing doxorubicin B-material . [SEP]
[CLS] sna - driven bcl2l12 knockdown increased the levels of active caspase - 3 and caspase - 7 ( fig . 2 , a and b , and fig . s4e ) , raised p53 protein B-material levels ( fig . 2c ) , and augmented the protein B-material and mrna levels of the p53 transcriptional B-event target p21 ( fig . 2 , c to e ) . [SEP]
[CLS] bcl2l12 - targeting snas therefore sensitized glioma cells B-material to therapy - induced apoptosis B-event by enhancing caspase and p53 activities . [SEP]
[CLS] after in vitro studies , we assessed whether snas could penetrate glioma tissue in vivo using local ( intracranial ) and systemic ( intravenous ) administration . [SEP]
[CLS] glioma - bearing severe combined immunodeficient ( scid ) mice received intracranial injections of cy5 - or gadolinium B-material [ gd ( iii ) ] - labeled snas , and sna distribution was studied by icp - ms , mri , and confocal fluorescence B-property . [SEP]
[CLS] ( note that gd - snas are conjugated to dna instead of sirna ; however , we have not observed differences in uptake efficiency between dna - based snas and sirna - based snas . [SEP]
[CLS] ) icp - ms analysis indicated 10 - fold greater accumulation of snas in tumor B-material versus nontumor brain regions ( fig . 3a ) . [SEP]
[CLS] this selective intratu - moral sna accumulation may be due to the enhanced permeability and retention ( epr ) effect [SEP]
[CLS] correspondingly , mri studies using gd ( iii ) - snas ( fig . 3b ) revealed pervasive intratumoral dissemination ( fig . 3c ) . [SEP]
[CLS] here , gold B-material and gd ( iii ) distribution within corresponding coronal brain sections localized to tumor B-material elements , as determined by laser ablation ( la ) - icp - ms analysis ( fig . 3d ) . [SEP]
[CLS] confocal fluorescence B-property of serial coronal brain sections further revealed effective dispersion of cy5 - snas in tumor B-material tissue ( fig . 3e and movie s1 ) . [SEP]
[CLS] this further substantiated intratumoral dispersion of sna conjugates and demonstrated that the tumor - associated gd ( iii ) - sna signal resulted from predominant accumulation of conjugates within the extravascular tumor B-material parenchyma . [SEP]
[CLS] next , we tested whether snas could cross the bbb / btb to target intracranial lesions in an in vitro bbb model and in xenogeneic explants in vivo ( fig . 4 and fig . s5 ) . [SEP]
[CLS] for the in vitro study , we used a previously established model ( 27 ) consisting of a noncontact coculture of human primary brain microvascular endothelial cells B-material ( hubmecs ) coating B-material a semipermeable filter insert with human astrocytes cultivated in a cell B-material culture well on the opposite side of the filter ( fig . 4a ) . [SEP]
[CLS] for this model , the conductivity and composition of tight junctions have been shown to recapitulate an in vivo bbb . [SEP]
[CLS] because the astrocytes were physically separated from the hubmecs , we were able to independently monitor sna uptake into the endothelial and , upon transcytosis , the astrocytic compartment by confocal fluorescence B-technique microscopy I-technique . [SEP]
[CLS] at early stages of the experiment , cy5 . 5 - snas accumulated within hubmecs when added on top of the endothelial layer and , upon passage through the hubmec layer and filter , rapidly entered the astrocytes ( fig . 4a ) . [SEP]
[CLS] similar to sna uptake into cell B-material monolayers ( fig . 1a and fig . s2 ) , accumulation of snas in astrocytes was blocked by pretreatment of the hubmecs with poly i , suggesting that srs may play a role in bbb transcytosis . [SEP]
[CLS] to study the bbb penetration of snas in vivo , we injected glioma - bearing mice intravenously with cy5 . 5 - snas and monitored them by near - infrared fluorescence B-property ( nirf ) using an in vivo imaging system ( ivis ) . [SEP]
[CLS] consistent with sna penetration of intracerebral gliomas upon local delivery , analysis of intracranial sna distribution by ivis ( fig . 4 , b and c ) and subsequent histological analysis using hematoxylin and silver B-material stainings ( fig . 4 , d and e ) confirmed higher accumulation of snas in tumor B-material versus normal brain tissue ; specifically , quantification of radiant efficiency in excised brains using living image software revealed 1 . 8 - fold higher signal in hutns tumor - bearing mice than in mice that received a sham tumor inoculation . [SEP]
[CLS] high - resolution imaging of silver - stained brain sections revealed a speckled , cytoplasmic distribution of snas within the extravascular tumor B-material parenchyma ( hashed box in fig . 4f ) , together with robust localization of snas to cd31 + endothelial cells B-material ( fig . 4f ) . [SEP]
[CLS] selective sna infiltration of the glioma mass in comparison to adjacent noncancerous brain tissue was further confirmed by icp - ms analysis ( fig . s5a ) and is likely due to a functionally compromised bbb / btb in glioma - bearing mice and the epr effect of the glioma - associated vasculature . [SEP]
[CLS] specifically , sna accumulation in brain tissue of nontumor - bearing mice was extensive , with about 1 × 10 10 sna particles per gram of tissue . [SEP]
[CLS] tumor B-material and adjacent normal brain in glioma - bearing mice , however , revealed an almost three orders of magnitude higher sna accumulation ( fig . s5a ) , suggesting that bbb / btb compromise is a major driver of selective sna accumulation in glioma - bearing brain tissue . [SEP]
[CLS] increased bbb / btb permeability in tumor B-material - bearing mice was confirmed by evans blue ( a bbb - impermeable dye ) extravasation , documenting dye accumulation selectively in areas of tumor B-material growth as early as 7 days after tumor B-material cell B-material implantation ( fig . s6 ) . [SEP]
[CLS] bio - distribution analysis revealed that up to 1 % of the total sna amount injected was found within the tumor B-material , with most of the snas accumulating in the liver and spleen 24 hours after the last injection ( fig . s5b ) . [SEP]
[CLS] pharmacokinetic analyses ( fig . s7a ) showed that the circulation time for the bulk of a single dose was short , with more than 90 % distributing to tissues in the first 5 min after intravenous injection ( half - life for distribution , αt 1 / 2 , ~ 1 min ; fig . s7b ) . [SEP]
[CLS] the elimination phase for these particles was much slower ( half - life for elimination , βt 1 / 2 , ~ 8 . 5 hours ) ( fig . s7b ) , with low levels of snas in the blood through the 72 - hour course of the pharmacokinetics study . [SEP]
[CLS] similar to glioma - bearing mice , tissue distribution in healthy animals was primarily in the liver and spleen , with small accumulation in the lungs , kidneys , heart , brain , and intestines ( fig . s8 ) . [SEP]
[CLS] by 72 hours after injection , the liver contained most of the remaining gold B-material , and the gold in other tissues was reduced to near background levels ( fig . s8 ) . [SEP]
[CLS] we next evaluated the potential toxicity B-property of bcl2l12 - targeting snas . [SEP]
[CLS] to begin to determine potential adverse side effects of snas , we looked for induction of inflammatory cytokines in blood samples collected from xenografted mice treated with sico - sna or sil12 - 2 - sna in comparison to blood samples from saline - treated control mice 21 days after cell B-material inoculation . [SEP]
[CLS] these animals received seven injections of snas , given every other day , for a total dose of ~ 10 mg / kg ( sirna / body weight ) . [SEP]
[CLS] we did not observe any significant differences between the groups using one - way anova followed by dunn ' s multiple comparison test ( table s2 ) . [SEP]
[CLS] in addition , we administered sil12 - 2 - snas ( 10 mg / kg ) or an equivalent volume of phosphate - buffered saline ( pbs ) as a single dose intravenously into sprague - dawley rats . [SEP]
[CLS] assessment of toxicity B-property was based on mortality , clinical signs , body weights , blood chemistry , complete blood counts , and detailed histopathology . [SEP]
[CLS] we did not observe sil12 - 2 - sna - related differences in histology ( fig . s9 ) or body weight ( table s3 ) 1 or 14 days after sna administration . [SEP]
[CLS] complete blood counts ( hematology ) and blood chemistry panels were within normal range ( table s3 ) , confirming the absence of acute or long - term toxicity B-property at therapeutic dosages [SEP]
[CLS] in addition , histopathological evaluation of major organs failed to detect abnormal lesions or signs of pathological B-event processes I-event , and findings were consistent with organ structures in clinically normal , age - and strain - matched rats ( fig . s9 ) . [SEP]
[CLS] to assess the impact of sil12 - 2 - snas on gbm progression in vivo , we determined bcl2l12 protein B-material knockdown , tumor B-material burden , histology , and survival of xenografted mice upon intravenous administration of sil12 - 2 - snas . [SEP]
[CLS] animals were treated per the scheme in fig . 5a . [SEP]
[CLS] bcl2l12 - targeting snas triggered intraglioma mrna ( 26 % ) and protein B-material ( 40 % ) knockdown relative to sico - snas ( fig . 5b ) and impaired tumorigenicity as measured by reduced tumor B-material burden in u87mg - derived xenogeneic mice ( fig . 5c ) . [SEP]
[CLS] bcl2l12 knockdown significantly increased intratumoral apoptosis B-event , as evidenced by enhanced levels of dna strand breaks and active caspase - 3 ( fig . 5 , d and e ) . [SEP]
[CLS] this cellular - level effect elicited by sil12 - 2 - snas translated into a significant improvement in survival in a hutns - derived xenogeneic gbm model ( fig . 5f ) . [SEP]
[CLS] this model recapitulated important phenotypic characteristics of the human disease , including hypercellularity , micro - vascular proliferation , extensive necrosis , and diffuse and widespread invasion , including the presence of single invasive cells B-material separated from the bulk tumor B-material ( fig . s10 ) . [SEP]
[CLS] anticancer B-property drugs must disseminate throughout tumor B-material parenchyma and accumulate in therapeutic concentrations to effectively inhibit neoplastic growth . [SEP]
[CLS] intratumoral pressure gradients , abnormal tumor - associated vasculature , and , perhaps most importantly , the bbb serve as physical and physiological barriers B-property for drug delivery to brain tumors B-material . [SEP]
[CLS] tight junctions , lack of fenestration , and a dense layer of pericytes characterize endothelial cells B-material of the bbb . [SEP]
[CLS] together with high electrical resistance and abundant expression of efflux transporters , anticancer B-property compounds are forced back into systemic circulation , and these structural barriers B-property prevent the extravasation of drugs into the cns . [SEP]
[CLS] indeed , most chemotherapeutics for brain cancers are delivered orally or intravenously , and their inadequate bbb penetration limits drug efficacy . [SEP]
[CLS] additional approaches have focused on local drug delivery via implanted polymers B-material , catheters , or convection - enhanced delivery ( ced ) . [SEP]
[CLS] these locally delivered agents exhibit limited intratumoral dispersion , require surgery for wafer and catheter implementation , and , in the case of ced , depend on high infusion rates that may be associated with increased risk of neuro - toxicity B-property caused by elevated intracranial pressure . [SEP]
[CLS] intraglioma delivery of sirna is particularly challenging because rna is rapidly degraded by nucleases and entrapped in endosomes and inadequately penetrates extravascular tumor B-material tissue beyond perivascular regions . [SEP]
[CLS] such low - level penetration stems from poor blood perfusion of tumor B-material elements and high interstitial pressure . [SEP]
[CLS] in contrast , we show that snas penetrate the bbb / btb and disseminate throughout extravascular glioma tissue . [SEP]
[CLS] consequently , sna technology represents an important therapeutic paradigm for systemically targeting undruggable oncoproteins that are important for gbm progression and therapy responses via passive , epr - mediated tumor B-material targeting . [SEP]
[CLS] furthermore , the evaluation of intratumoral drug delivery , concentration , and tissue dissemination are critical for the ( pre ) clinical assessment of antineoplastic drugs . [SEP]
[CLS] here , we showed that intratumoral sna levels and distribution could be precisely measured by la - icp - ms in resected tissue , allowing one to track the biodistribution and tissue distribution of the snagold nanoparticle B-nanoparticle therapeutic . [SEP]
[CLS] notably , cellular uptake and possibly bbb penetration depend on sr - mediated endocytosis B-event . [SEP]
[CLS] in agreement with our previous studies using pharmacological sr inhibitors , we found that inhibition of sr - a with poly i and fucoidan blocked sna uptake into glioma cells B-material and bbb transcytosis . [SEP]
[CLS] the mechanism of cellular uptake of snas into gbm cells B-material and tumors B-material is not yet clear ; however , our previous studies suggest that sna uptake is dependent on the 3d architecture of the conjugates because hollow , gold - free snas , but not single - stranded dnas , effectively compete with snas to block their uptake into cells B-material . [SEP]
[CLS] in addition , we discovered that rnai - mediated knockdown of sr - a reduced the accumulation of snas in cells B-material and , consequently , snas chemically engaged with sr - a and lipid raft proteins B-material , suggesting that sr - a and lipid rafts are important factors mediating sna endocytosis B-event . [SEP]
[CLS] srs are highly expressed within endothelial cells B-material that make up the bbb , suggesting that the robust penetration of snas into brain and tumor B-material elements that we observed may involve sr - dependent transcytosis . [SEP]
[CLS] this observation has potential implications for the delivery of snas to the cns to treat other neurological diseases and cns malignancies . [SEP]
[CLS] other than successful intravenous delivery and penetration of bbb , sna conjugates triggered efficient , specific , and persistent bcl2l12 gene knockdown , which translated into a significant reduction in tumor B-material burden in mice without adverse effects or toxicity B-property . [SEP]
[CLS] snas have an ion B-material cloud associated with the high - density oligonucleotide shell B-material and steric inhibition at the particle surface , which creates a unique micro - environment that inhibits enzymatic nucleic B-material acid I-material degradation and , in turn , results in an increased stability of sirnas and potentially longer therapeutic lifetimes . [SEP]
[CLS] although the sna platform enables specific genetic knockdown upon systemic delivery , their efficacy could be further improved by potentially increasing circulation times and accumulation in targeted tissues . [SEP]
[CLS] one way to increase targeting is through the use of conjugated monoclonal antibodies B-material , an avenue that has shown promise in vitro for her2 in breast cancer cells B-material but has yet to be evaluated in vivo . [SEP]
[CLS] additionally , circulation time and sna stability could be extended through small adjustments to the physical properties of snas , for example , the length of the spacer B-material between the sirna and the gold B-material and / or the amount and length of peg used for backfill . [SEP]
[CLS] finally , this proof - of - concept study has explored the effect that neutralization of a single oncogene has on an overall disease state . [SEP]
[CLS] targeting multiple oncogenes , such as driving mutations of gbm implicated in aberrant growth B-material factor I-material receptor I-material signaling , may be possible with single sna constructs composed of multiple sirna reagents . [SEP]
[CLS] finally , the sna platform provides a tool for discovery science to validate additional genes implicated in gbm pathogenesis as therapeutic targets , including receptor B-material tyrosine B-material kinases and downstream ras - mapk ( mitogen - activated protein B-material kinase ) and pi3k ( phosphatidylinositol 3 - kinase ) - akt - mtor ( mammalian target of inhibitor ) signaling , additional antiapoptotic members of the bcl - 2 protein B-material family , genes implicated in metabolic control of gbm progression , and micrornas . [SEP]
[CLS] current strategies focusing on viral delivery of short hairpin rnas ( shrnas ) are often time - consuming , require manipulation of cells B-material in culture , and necessitate extensive optimization of shrna induction in cell B-material and animal models . [SEP]
[CLS] thus , sna - based systemic delivery of sirnas may represent a more effective way to target cancer genes in cell - based and in tumor B-material regression studies in vivo . [SEP]
[CLS] in summary , this work provides proof of concept that systemically administered snas can cross the bbb / btb in gbm cell B-material and mouse models , infiltrate tumor B-material parenchyma , silence genetic lesions of established gbm in vitro and in vivo , and effectively reduce tumor B-material burden . [SEP]
[CLS] more generally , this work establishes snas as a promising new class of therapeutic gene regulation agents capable of treating disease through systemic injection . [SEP]
[CLS] systemic delivery of bcl2l12 - specific snas to intracranial lesions exhibited both selectivity and favorable toxicity B-property and pharmacokinetic profiles that may enable sirna delivery to gbm patients . [SEP]
[CLS] bcl2l12 may also be a good target for other cancers such as melanoma and non - cns malignancies , considering its implication as a bio - marker for early - stage gastric cancer and colon cancer , and for the prediction of short - term relapse in nasopharyngeal carcinoma . [SEP]
[CLS] toward translation , further development of the sna platform will be required , including optimization of circulation times , two - species toxicity B-property studies , and efficacy studies in additional models of human gbm . [SEP]
[CLS] together , snas harness the great promise of biotherapeutic gene silencing as a personalized medicine approach to neutralize many genes , including undruggable oncogenes , and can overcome some of the major challenges associated with cns - directed drug delivery and rnai - based therapy . [SEP]
[CLS] this study aimed to evaluate sna nanoparticle B-nanoparticle conjugates as an rnai - based therapy for glioblastoma . [SEP]
[CLS] the ability of bcl2l12 - specific snas ( sil12 - snas ) to enter multiple cell B-material types , penetrate tumor B-material and normal tissue , neutralize target gene expression , and affect downstream apoptotic signaling was determined both in vitro and in vivo . [SEP]
[CLS] sna uptake into cells B-material or tissues was assessed qualitatively and quantitatively with microscopy B-technique , mri , icp - ms , and ivis . [SEP]
[CLS] the subsequent impact on bcl2l12 expression and downstream apoptotic signaling was determined by western blotting and qrt - pcr . [SEP]
[CLS] additionally , the pharmacokinetics and toxicity B-property of snas were evaluated in both rats and mice . [SEP]
[CLS] for animal studies to determine sna efficacy in vivo , the number of mice per group was calculated with a power of 0 . 8 , and animals were randomized by weight before tumor B-material inoculation with studylog ' s stratified sampling randomization to establish a p value of 0 . 99 between groups . [SEP]
[CLS] animals were then further randomized to different treatment groups within each cage , and ear tags were used to identify mice so that researchers could remain blinded to which treatment each animal had received . [SEP]
[CLS] all experiments were conducted under an approved protocol of the institutional animal care and use committee of northwestern university . [SEP]
[CLS] rna synthesis , gold B-nanoparticle nanoparticle I-nanoparticle functionalization and characterization , and synthesis of gd ( iii ) - snas are provided in the supplementary materials . [SEP]
[CLS] snas were added directly to the medium of subconfluent glioma and a patient - derived tns line at varying concentrations ( 0 . 1 to 10 nm ) . [SEP]
[CLS] to visualize sna uptake , we plated the cells B-material on chamber slides and treated them with snas for 12 to 16 hours . [SEP]
[CLS] cells B-material were counterstained with dapi and fitc - labeled cytochalasin to visualize nuclei and actin filaments , respectively , and subjected to confocal fluorescence B-technique microscopy I-technique ( n = 3 cultures per cell B-material line ) . [SEP]
[CLS] to assess functionality of sna - mediated knockdown of bcl2l12 , we treated the cells B-material with the pan - specific kinase inhibitor staurosporine ( 0 . 5 μm , sigma ) , temozolomide ( 100 μm , sigma ) , or doxorubicin B-material ( 0 . 4 μg / ml , sigma ) for the indicated time points , and we collected protein B-material lysate or total cellular rna . [SEP]
[CLS] the degree of apoptosis B-event was determined by western blotting for cleaved caspase - 3 and caspase - 7 in response to staurosporine treatment , by western blotting for p53 , p - p53 ser16 , and p21 , and by qpcr for p21 in response to doxorubicin B-material treatment ( supplementary methods ) . [SEP]
[CLS] the in vitro bbb model was adapted from with modifications . [SEP]
[CLS] in vitro bbb modelhubmecs ( sciencell ) were maintained in fibronectin - coated t - 75 flasks in endothelial cell B-material medium ( sciencell ) , and primary human astrocytes ( sciencell ) were cultivated in dulbecco ' s modified eagle ' s medium and 10 % fetal bovine serum . [SEP]
[CLS] primary human astrocytes ( 1 × 10 5 ) were plated in 24well plates and allowed to grow for 4 days . [SEP]
[CLS] hubmecs ( 4 × 10 5 ) were plated onto the upper side of 6 . 4 - mm collagen i - coated , 0 . 4 - μm pore size cell B-material culture inserts ( bd biosciences ) and were transferred to the 24 - well plates containing the primary astrocytes . [SEP]
[CLS] hubmecs and human astrocytes were cocultured for 12 days to allow for formation of the in vitro bbb . [SEP]
[CLS] cy5 . 5 - labeled snas ( 5 nm ) were then added into the insert well and incubated B-technique for 24 hours , and penetration across the hubmecs layer and into primary astrocytes below was followed by fluorescence B-technique microscopy I-technique . [SEP]
[CLS] cells B-material on coverslips or collagen - coated inserts were washed in pbs and fixed in 4 % paraformaldehyde for 5 min at 37°c , blocked for 1 hour at 37°c in 5 % bovine serum albumin , and incubated B-technique overnight with primary anti - gfap ( cell B-material signaling , 1 : 100 ) , anti - occludin ( santa cruz biotechnology , 1 : 200 ) , or anti - vimentin antibodies B-material ( thermo fisher scientific , 1 : 400 ) . [SEP]
[CLS] s4 . [SEP]
[CLS] evaluation of additional snas targeting bcl2l12 . [SEP]
[CLS] s5 . biodistribution of snas in glioma - bearing mice after systemic administration . [SEP]
[CLS] s6 . [SEP]
[CLS] the bbb / btb is compromised in tumor B-material - bearing mouse brains . [SEP]
[CLS] s7 . [SEP]
[CLS] pharmacokinetics of snas . [SEP]
[CLS] s8 . [SEP]
[CLS] biodistribution of snas in tumor - free mice . [SEP]
[CLS] s9 . [SEP]
[CLS] histological evaluation of sil12 - 2 - snas in major tissues . [SEP]
[CLS] s10 . [SEP]
[CLS] histological analysis of hutns - derived mouse tumors B-material . [SEP]
[CLS] snas penetrate glial cells B-material and down - regulate bcl2l12 in vitro ( a ) uptake of cy5 - labeled snas ( red ) into hutns and the human glioma cell B-material line u87mg . [SEP]
[CLS] cells B-material were colabeled with fluorescein B-material isothiocyanate I-material ( fitc ) - conjugated cytochalasin ( green ) and 4 ′ , 6 - diamidino - 2 - phenylindole ( dapi ) ( blue ) to visualize actin filaments and nuclei , respectively . [SEP]
[CLS] scale bars , 40 μm . [SEP]
[CLS] bf , bright field . [SEP]
[CLS] ( b ) effect of bcl2l12 - targeting snas sil12 - 1 and sil12 - 2 on bcl2l12 protein B-material levels in hutns , ln235 , and lnz308 glioma cells B-material relative to control ( sico - sna ) cultures as assessed by western blot using the noted concentrations of snas . [SEP]
[CLS] hsp70 served as a loading control . [SEP]
[CLS] bar graphs represent a densitometric analysis of western blots . [SEP]
[CLS] band intensity was normalized to hsp70 and then expressed relative to bcl2l12 levels in sico - treated samples . [SEP]
[CLS] ( c ) bcl2l12 knockdown [SEP]
[CLS] bcl2l12 - specific snas promote apoptotic signaling in glioma cells B-material ( a and b ) bcl2l12 knockdown by sil12 - snas sensitizes cells B-material to apoptosis B-event ( measured by caspase - 3 and caspase - 7 cleavage ) upon treatment with staurosporine ( sts ) and temozolomide ( tmz ) . [SEP]
[CLS] ls , large subunit ; ls + n , large subunit plus n - peptide . [SEP]
[CLS] ( c ) effect of bcl2l12 knockdown on p53 protein B-material stability , phosphorylated p53 ( p - p53 ser15 ) , and the p53 target p21 upon doxorubicin B-material ( doxo ) treatment for the indicated times . [SEP]
[CLS] ( d ) p53 target gene induction of p21 on the mrna level as measured by quantitative reverse transcription B-event polymerase chain reaction ( qrt - pcr ) . [SEP]
[CLS] ( e ) bcl2l12 mrna knockdown was assessed in parallel and expressed as fold change relative to sico - sna - treated cells B-material at 0 , 4 , 8 , and 16 hours after application of doxorubicin B-material . [SEP]
[CLS] data are means ± sd ( n = 3 ) . [SEP]
[CLS] snas disseminate throughout glioma tissue ( a ) icp - ms quantification of sna uptake into orthotopic u87mg tumor B-material and adjacent normal tissue after 48 hours . [SEP]
[CLS] data are means ± sem ( n = 3 glioma - bearing mice , n = 5 normal mice ) . [SEP]
[CLS] p value was calculated with one - way analysis of variance ( anova ) . [SEP]
[CLS] ( b ) schematic overview of the synthesis of gd ( iii ) - functionalized snas . [SEP]
[CLS] ( i ) gd ( iii ) - sna conjugates were prepared from alkyne - modified t bases and azide - labeled gd ( iii ) complexes through click chemistry . [SEP]
[CLS] ( ii ) gd ( iii ) - conjugated dna was then functionalized onto gold B-nanoparticle nanoparticle I-nanoparticle ( au - np B-nanoparticle ) surface to form gd ( iii ) - sna following the same procedure as fig . s3 , except without oligoethylene glycol / polyethylene glycol ( peg ) backfill . [SEP]
[CLS] ( c ) mr images of tumor - bearing mouse brains injected intracranially with gd ( iii ) - snas . [SEP]
[CLS] two representative coronal sections imaged 24 hours after gd ( iii ) - sna injection ( upper panel ) show localization of gd ( iii ) - sna within the intracerebral lesion . [SEP]
[CLS] gd ( iii ) signal is white and outlined in black dotted line . [SEP]
[CLS] also shown are corresponding hematoxylin and eosin ( h & e ) sections showing the location of the tumor B-material ( darker purple , outlined in light gray dotted line ) , and three - dimensional ( 3d ) reconstruction of mr images [ gd ( iii ) signal in red ] . [SEP]
[CLS] see movie s1 . [SEP]
[CLS] ( d ) la - icp - ms shows localization of au , fe , and gd ( iii ) contents in coronal brain sections of mice injected intracranially with gd ( iii ) - snas . [SEP]
[CLS] the heat map indicates the [SEP]
[CLS] snas cross the bbb / btb and selectively accumulate in glioma tissue ( a ) noncontact in vitro bbb model using a coculture of hubmecs and human astrocytes . [SEP]
[CLS] representative confocal fluorescence B-technique microscopy I-technique images demonstrate cy5 . 5 - sna ( red ) distribution in endothelial and astrocytic cells B-material . [SEP]
[CLS] endothelial and astrocytic cells B-material stained positively for occludin ( a marker for tight junctions ) and glial fibrillary acidic protein B-material ( gfap ) , respectively . [SEP]
[CLS] anti - vimentin and dapi stained cytoplasm and nuclei , respectively . [SEP]
[CLS] ( b and c ) ivis analysis of brains with or without u87mg ( b ) or hutns ( c ) tumors B-material 48 hours after systemic delivery of saline or cy5 . 5 - snas . [SEP]
[CLS] sna accumulation is indicated by increased fluorescence B-property ( yellow ) . [SEP]
[CLS] quantification of radiant efficiency is shown as relative signal amount under the images . [SEP]
[CLS] ( d and e ) selective accumulation of cy5 . 5 - snas within u87mg ( d ) or hutns ( e ) tumors B-material , but not normal brain elements . [SEP]
[CLS] coronal brain sections were silver - stained and counterstained with hematoxylin . [SEP]
[CLS] yellow arrows indicate sites of sna accumulation , which appear as dark brown regions . [SEP]
[CLS] t , tumor B-material ; n , normal brain . [SEP]
[CLS] ( f ) hutns - derived accumulated snas in vascular and extravascular tumor B-material elements as revealed by silver B-material staining and anti - cd31 immunohistochemistry on adjacent coronal sections . [SEP]
[CLS] arrows point to areas of sna and cd31 colocalization . [SEP]
[CLS] hashed box shows snas that extravasated into the tumor B-material parenchyma . [SEP]
[CLS] 5 . systemic administration of snas reduces intratumoral bcl2l12 and decreases tumor B-material burden in mice ( a ) scheme of cell B-material and sna injections . [SEP]
[CLS] mice were orthotopically injected with either u87mg or hutns cells B-material for tumor B-material burden , histology , and survival analyses ( day 0 ) followed by seven intravenous ( i . v . ) injections of sico - or sil12 - snas . [SEP]
[CLS] ( b ) bcl2l12 knockdown in u87mg - derived intracranial tumors B-material treated with sil12 - snas . [SEP]
[CLS] hsp70 was used as a loading control . [SEP]
[CLS] histograms represent a densitometric analysis of the western blots . [SEP]
[CLS] the intensity of bcl2l12 bands was normalized to hsp70 and ex - pressedasrelativebcl2l12 expression . [SEP]
[CLS] ( c ) weight of xenografted tumors B-material 21 and 28 days after sna injection . [SEP]
[CLS] data points display tumor B-material weight from individual mice . [SEP]
[CLS] p value was calculated with two - tailed student ' s t test . [SEP]
[CLS] ( d and e ) intratumoral apoptosis B-event in mice injected with sil12 - 2 - sna . [SEP]
[CLS] ( e ) the amount of activated caspase - 3 ( acasp - 3 ) and terminal deoxynucleotidyl transferase - mediated deoxyuridine triphosphate nick end labeling ( tunel ) was quantified from ( d ) in peripheral [SEP]
[CLS] design of materials to aid intracellular delivery of agents can greatly improve medical treatments . [SEP]
[CLS] while dna is a molecule difficult to introduce into cells B-material , dna can be engineered into devices capable of intracellular delivery . [SEP]
[CLS] yet , transport mediated by dna - devices void of other structural material B-material , with size greater than that associated with non - specific penetration , and with targeting capacity enough to overcome non - specific pathways has not been achived . [SEP]
[CLS] this study demonstrates that this is possible . [SEP]
[CLS] submicrometer ( 200 - nm ) dendrimers B-nanoparticle built of dna ( nucleodendrimers ( nds ) ) are coupled to antibodies B-material against selected cell - surface receptors and compared to polymer B-material nanoparticles B-nanoparticle ( nps B-nanoparticle ) . [SEP]
[CLS] nds and nps B-nanoparticle bind specifically to cells B-material expressing these targets and efficiently enter cells B-material via the pathway associated with the selected receptor B-material . [SEP]
[CLS] while nps B-nanoparticle traffic to perinuclear endo - lysosomes , nds remain scattered throughout the cell B-material , suggesting endosomal escape . [SEP]
[CLS] this is confirmed in vitro , where nds disrupt membranous vesicles at endosomal - like ph and in cell B-material culture , where they : provide endosomal escape of model drugs , sugars , proteins B-material , and nucleic B-material acids I-material ; allow access to other intracellular compartments ; result in measurable effects of cargoes ; and do not cause cytotoxicity B-property . [SEP]
[CLS] therefore , these dna - nanodevices B-nanoparticle can be used to selectively overcome intracellular barriers B-property , underscoring the growing range of applications of dna materials . [SEP]
[CLS] safe and efficient delivery of research tools , diagnostic probes , and therapeutic agents to the interior of cells B-material remains a major challenge . [SEP]
[CLS] this is due to the fact that most of these molecules cannot transverse cellular membranes , including the plasmalemma and membranes of endocytic vesicles . [SEP]
[CLS] this prevents access to the cell B-material cytosol and other intracellular compartments , hindering our ability to exploit intracellular delivery of compounds for basic research and , primarily , translational applications . [SEP]
[CLS] numerous strategies aim to overcome this limitation , including those that enhance permeation across the plasmalemma or rely on endocytosis B-event and transport through the endo - lysosomal route to then permeate acidic endosomal compartments . [SEP]
[CLS] most of these strategies employ cellpenetrating or fusogenic peptides B-material , peptidomimetics , toxins , synthetic or natural polycationic polymers B-material or lipids B-material , capitalize on the proton sponge effect or volume changes within endosomes , etc . [SEP]
[CLS] yet , despite this considerable advancement , intracellular delivery remains elusive . [SEP]
[CLS] aspects requiring optimization relate to the safety and mechanism of action of these approaches since often they : ( a ) are inherently toxic B-property , ( b ) disturb the plasma membrane ( instead of or concomitantly to the endosomal membrane ) , ( c ) involve materials that cannot be fully degraded and / or their products cannot be completely eliminated , ( d ) recognize broadly displayed signatures ( e . g . cell B-material - surface charge , etc . ) , suffering from lack of specificity , ( e ) are limited in their loading or transport potential to certain types of cargo molecules , and / or ( f ) have poor applicability beyond local administration . [SEP]
[CLS] dna represents an alternative material B-material toward this goal . [SEP]
[CLS] while in nature dna exists as a linear or circular polymer B-material , it can also be engineered into branched , dendritic , or networked forms , such as the case of y - , t - , and x - shaped dna , tiles , origami , nanocages B-nanoparticle , etc . [SEP]
[CLS] dna synthesis and assembly , as well as dna disassembly and degradation can be controlled by chemical , enzymatic , and physical methods . [SEP]
[CLS] indeed , dna - devices have been proven valuable in multiple applications , including molecular detection , diagnosis , biotechnology , and others . [SEP]
[CLS] although dna represents the most classical example of a molecule difficult to introduce into cells B-material , it also has properties amenable for drug delivery . [SEP]
[CLS] for instance , dna is water soluble , biodegradable B-property , can interact with positively - charged carriers and cargoes , can solubilize hydrophobic B-property drugs between its bases , has 5 ' - phosphate and 3 ' - hydroxyl ends for conjugation , and can be modified with other functional elements . [SEP]
[CLS] while delivery of compounds by dna - based devices has been less investigated , recent publications have shown considerable promise also on this regard . [SEP]
[CLS] as examples of this potential , dna spherical particles , origami , cages , tubes , hydrogels , and liposomes B-nanoparticle - like dnasomes can carry and release hydrophobic B-property drugs , oligonucleotides , proteins B-material , and imaging particles . [SEP]
[CLS] however , most previous studies on this application have employed dna - devices which : ( a ) contained an additional material B-material as a part of their structural scaffold ( lipidic , polymeric , inorganic , etc . ) , [SEP]
[CLS] introductionb ) had sizes small enough so that they may not be able to avoid non - specific penetration through certain tissues ( ≤ 20 - nm ) or were too large to enter non - immune cells B-material by endocytosis B-event ( macro - assemblies ) , and / or ( c ) were shown to bind and gain access into cells B-material largely by non - specific pathways ( scavenger receptors , macropinocytosis , etc . ) even when targeted to selected cell - surface I-material receptors I-material . [SEP]
[CLS] the study presented here has explored intracellular delivery of diverse compounds ( model drugs and biologicals ) mediated by dna - devices void of other structural materials , large enough to avoid broad penetration through tissues yet still small enough to allow endocytosis B-event , and with enough selectivity toward molecular targets as to overcome nonspecific receptors and endocytic pathways . [SEP]
[CLS] to determine if dna - devices can provide cell B-material targeting and endocytosis B-event via selected cellsurface receptors and pathways , commercial dna dendrimers B-nanoparticle were tested . [SEP]
[CLS] these dendrimers B-nanoparticle consist of dna modules ( figure 1a ) , where each module has two dna strands that form a central dsdna region ( waist ) with four ssdna ends ( arms ) . [SEP]
[CLS] driven by complementarity between the arms of individual dna modules , these dendrimers B-nanoparticle selfassemble to contain a core B-material , a selected number of layers ( generations ) , and a tunable number of outer arms for functionalization . [SEP]
[CLS] these devices are available as scaffolds to build signal amplifiers by conjugation to molecular probes , but had not been tested for drug delivery . [SEP]
[CLS] generation 4 dna dendrimers B-nanoparticle bearing ~ 20 anti - mouse igg molecules ( a secondary antibody B-material to which targeting antibodies B-material could be coupled by affinity ) were selected as a model . [SEP]
[CLS] these dendrimers B-nanoparticle are referred to as nucleodendrimers ( nds ) thereafter . [SEP]
[CLS] the size of these commercial nds was 175 . 4±9 . 2 - nm in diameter and 0 . 3±0 . 1 polydispersity index ( pdi ) , measured by dynamic B-technique light I-technique scattering I-technique ( dls ) . [SEP]
[CLS] binding of mouse igg , used as a model antibody B-material , increased nd size ( p = 0 . 04 ) to 211 . 3±13 . 9 - nm and 0 . 4±0 . 01 pdi . [SEP]
[CLS] this result indicates that a coat B-material of antibodies B-material ( which could be chosen to bind to selected targets ) could be similarly incorporated . [SEP]
[CLS] to then evaluate if specific targeting and endocytosis B-event is possible , nds were coupled to an antibody B-material recognizing intercellular adhesion molecule - 1 ( icam - 1 ) [SEP]
[CLS] this is a glycoprotein overexpressed on several cell B-material types in many pathologies , which has been well - studied in the context of targeting and transport of drug delivery systems . [SEP]
[CLS] anti - icam nds were incubated B-technique for 1 h at 37°c with human umbilical vein endothelial cells B-material ( huvecs ) activated with tnfα , which induces icam - 1 expression mimicking a pathological state . [SEP]
[CLS] using an established protocol ( see experimental section and ) that allows fluorescence B-technique microscopy I-technique quantification of total cell - associated , surface - bound , and internalized carriers ( figure 1b ) , anti - icam nds were observed to bind to cells B-material with high specificity : ~ 190 nds / cell B-material ( ~ 190 - fold over igg nds ) . [SEP]
[CLS] binding was blocked by co - incubation B-technique with anti - icam ( 89 % inhibition ; p < 0 . 001 ) , supporting further targeting specificity . [SEP]
[CLS] this level and specificity of binding is comparable to previously described anti - icam nanoparticles B-nanoparticle ( anti - icam nps B-nanoparticle ) built of polymers B-material ( e . g . non - degradable polystyrene or degradable poly ( lacticco - glycolic acid ) , implying that the dna scaffold of nds does not impact this parameter . [SEP]
[CLS] with regard to transport into cells B-material , anti - icam nds were efficiently internalized after 1 h incubation B-technique at 37°c ( figure 1b ) : ~ 82 % of total cell - associated nds , while this parameter was undetectable for control igg nds . [SEP]
[CLS] internalization was inhibited at 4°c ( ~ 20 % uptake ; figure 1b ) , suggesting that this is an energy dependent event , likely and endocytic process . [SEP]
[CLS] in accord , uptake by cells B-material was impaired upon incubation B-technique at 37°c in the presence of inhibitors of endocytosis B-event mediated by icam - 1 ( amiloride ; ~ 20 % uptake ) , but not inhibitors of different pathways such as caveoli ( filipin ; ~ 71 % uptake ) , clathrin ( monodansylcadaverine ; ~ 70 % uptake ) , or macropinocytosis ( wortmannin ; ~ 85 % uptake ) ( figure 1c is notmalized to control ) . [SEP]
[CLS] this set of data suggests that the rate and mechanism of uptake of icam - 1 - targeted nds is similar to other icam - 1 - targeted systems , such as anti - icam nps B-nanoparticle , previously reported . [SEP]
[CLS] therefore , the dna scaffold of nds does not seem to impact this parameter . [SEP]
[CLS] indeed , comparison of anti - icam nds to anti - icam nps B-nanoparticle of similar size ( 220±18 - nm ; 0 . 2±0 . 02 pdi ) showed similar uptake by huvecs ( ~ 80 - 90 % ; figure 2 ) . [SEP]
[CLS] however , despite the similarities with regard to binding , uptake , and endocytic mechanism , intracellular distribution of anti - icam nds was found to be different from that of anti - icam nps B-nanoparticle ( figure 2 ) . [SEP]
[CLS] anti - icam nps B-nanoparticle largely accumulated in the perinuclear region by 1 h ( ~ 70 % of all cell - associated carriers ) , in agreement with their known lysosomal routing . [SEP]
[CLS] in contrast , anti - icam nds appeared scattered throughout the cell B-material body even after 3 h ( ~ 40 % perinuclear ) . [SEP]
[CLS] this suggets that nds provide for a different trafficking route , likely escape from endosomal compartments , as it has been reported for other dna devices . [SEP]
[CLS] to explore if anti - icam nds mediate disruption of endocytic vesicles , this formulation was incubated B-technique for 15 min at 37°c with cells B-material concomitantly to the presence of texas - red dextran B-material in the cell B-material medium ( figure 3 and supplementary figure s1 ) . [SEP]
[CLS] this protocol has been previously shown to allow dextran B-material incorporation within the fluid - phase ( lumen ) of endocytic vesicles engulfing anti - icam carriers . [SEP]
[CLS] since mammalian cells B-material cannot degrade dextran B-material , this polysaccharide B-material can be used as a trackable marker regardless of its intracellular destination . [SEP]
[CLS] after uptake , unbound materials were washed and internalized materials were allowed to traffic intracellularly . [SEP]
[CLS] texas - red dextran B-material was similarly incorporated into endocytic vesicles for both anti - icam nps B-nanoparticle and anti - icam nds : ~ 60 vesicles / cell B-material in 1 h ( figure 3 and supplementary figure s1a ) . [SEP]
[CLS] yet , the total area occupied by dextran B-material was greater in cells B-material incubated B-technique with anti - icam nds ( 1 . 5 - fold ; supplementary figure s1b ) , which paires well with the more scattered distribution of nds through the cell B-material body ( figure 2 ) . [SEP]
[CLS] the number of dextran - positive vesicles and area occupied by this marker were stable over time for anti - icam nps B-nanoparticle ( δ ~ 1 comparing 3 h over 1 h ; figure 3 ) , indicating that dextran B-material does not leave endocytic vesicles . [SEP]
[CLS] this was expected based on previous reports showing trafficking of anti - icam nps B-nanoparticle to endo - lysosomal compartments . [SEP]
[CLS] in contrast , with time , anti - icam nds caused dextran B-material to appear less punctate and more diffuse throughout the cell B-material body : by 3 h the number of dextran - positive vesicles was reduced by ~ 2 - fold and the area occupied by this marker approximately doubled ( figure 3 and supplementary figure s1a - b ) . [SEP]
[CLS] these results indicate that anti - icam nds may allow dextran B-material to escape into the cytosol following endocytosis B-event , as the case for other dna - based systems . [SEP]
[CLS] to validate this , nds where incubated B-technique in vitro with membranous artificial vesicles ( liposomes B-nanoparticle ) at ph 4 . 5 to mimic interactions with membranous endo - lysosomes of cells B-material . [SEP]
[CLS] liposomes B-nanoparticle were degraded in the presence of nds ( a detergent was used as a positive control ) , which was dependent on the dose of both liposomes B-nanoparticle and nds ( figure 4 ) and did not occur at neutral ph ( supplementary figure s2 ) . [SEP]
[CLS] to explore if nds provide intracellular delivery of molecules other than dextran B-material , a series of tests were performed ( figure 5 and supplementary figure s3 ) . [SEP]
[CLS] first , cells B-material were co - incubated B-technique for 30 min with anti - icam nds and phalloidin , a drug known to interact with f - actin in permeabilized cells B-material but unable to penetrate intact cell B-material membranes per se , as verified in supplementary figure s3a . [SEP]
[CLS] in agreement with nds mediating endosomal escape following endocytosis B-event , phalloidin allowed visualization of f - actin when co - incubated B-technique with anti - icam nds at 37°c ( arrows in figure 5a ) , but not at 4°c , a temperature precludes endocytosis B-event ( supplementary figure s3a ) . [SEP]
[CLS] in contrast , a lack of f - actin staining was found for anti - icam nps B-nanoparticle ( figure 5a ) , since they do not provide endo - lysosomal escape . [SEP]
[CLS] next , similar experiments were conducted to examine if agents delivered into the cytosol by mediation of nds could be re - directed to other subcellular compartments . [SEP]
[CLS] for this purpose , cells B-material were co - incubated B-technique with anti - icam nds and a recombinant protein B-material ( albumin ) , which was tagged by a nuclear localization sequence ( nls ) . [SEP]
[CLS] as a positive control , this recombinant protein B-material could label the cell B-material nucleus of permeabilized cells B-material ( supplementary figure s3b ) , since this grants direct access across the plasmalemma . [SEP]
[CLS] as a negative control , no nuclear label was found in cells B-material incubated B-technique at 37°c with anti - icam nps B-nanoparticle ( figure 5b ) , since these carriers do not exit endo - lysosomal compartments . [SEP]
[CLS] lack of nuclear localization was observed for anti - icam nds incubated B-technique at 4°c ( supplementary figure s3b ) , since no endocytosis B-event occurs at this temperature ( figure 1b ) . [SEP]
[CLS] however , co - incubation B-technique with anti - icam nds at 37°c to allow endocytic transport resulted in albumin - nls reaching the cell B-material nucleus ( arrows in figure 5b ) . [SEP]
[CLS] next , functional activity after redistribution from the cytosol to the nucleus was assessed by evaluating the transfection potential of nds . [SEP]
[CLS] as shown in figure 5c , co - incubation B-technique of a plasmid encoding for egfp - rhoa with anti - icam nds at 37°c ( but not 4°c ; supplemental figure s3c ) provided expression of this recombinant protein B-material , while transfection was undetectable in the case of anti - icam nps B-nanoparticle ( figure 5c ) or plasmid alone ( supplementry figure s3c ) . [SEP]
[CLS] to demonstrate activity of a model therapeutic ( biological ) agent , intracellular delivery of catalase was tested . [SEP]
[CLS] catalase is an antioxidant B-property enzyme unable to transverse cellular membranes . [SEP]
[CLS] indeed , incubation B-technique of cells B-material with catalase for 3 h did not prevent cell B-event death I-event caused by h 2 o 2 - mediated oxidative injury after catalase delivery ( supplelentary figure s3d ) . [SEP]
[CLS] neither cells B-material co - incubated B-technique with anti - icam / catalase nps B-nanoparticle were protected against this pathological insult ( figure 6 ) , which was expected since catalase is known to traffic and degrade within lysosomes 3 h after delivery by anti - icam nps B-nanoparticle . [SEP]
[CLS] however , co - delivery with anti - icam nds resulted in significant antioxidant B-property protection ( ~ 90 % ) , in agreement with endosomal escape into the cytosol . [SEP]
[CLS] to evaluate whether this strategy could be adapted to target other cells B-material types , anti - icam nds were incubated B-technique with human mesothelioma ren cells B-material , human skin fibroblasts , or human colorectal adenocarcinoma caco - 2 cells B-material ( figure 7a ) . [SEP]
[CLS] as expected based on known icam - 1 expression on these cell B-material types under pathological - like conditions , anti - icam nds specifically associated to these cells B-material compared to control igg nds ( 22 - , 38 - , and 47fold , respectively ) . [SEP]
[CLS] furthermore , nds could also be targeted to other cell B-material - surface molecules ( figure 7b ) . [SEP]
[CLS] these included platelet - endothelial adhesion molecule 1 ( pecam - 1 ) , another ig - like cam expressed on the endothelium . [SEP]
[CLS] pecam - 1 is functionally involved in leukocyte extravasation and associated with cam - mediated endocytosis B-event . [SEP]
[CLS] targeting nds to pecam - 1 resulted in a 60 - fold increased binding compared to control igg nds ( shown in figure 1b ) . [SEP]
[CLS] this was also the case when targeting nds to alterative receptors , including the transferrin receptor B-material ( tfr ; 32 - fold over igg nds ) and mannose - 6 - phosphate receptor B-material ( m6pr ; 24 - fold over igg nds ) , both of which are structurally and functionally unrelated to one another or to endothelial cams , and are associated with clathrin - mediated uptake . [SEP]
[CLS] thus , specific interaction of nds with receptors and cell B-material types can be tailored by selecting appropriate targeting B-material ligands I-material . [SEP]
[CLS] finally , binding , uptake , or endosomal escape by targeted nds did not seem to affect the cell B-property viability I-property . [SEP]
[CLS] this was demonstrated by lack of an effect on endothelial cell B-material monolayer appearance , the number of cells B-material , or the cell viability I-property measured 48 h after incubation B-technique with these carriers for 5 h , which was comparable to that of anti - icam nps B-nanoparticle ( figure 8 ) . [SEP]
[CLS] this study demonstrates that targeted nds can provide receptor B-material / pathway - specific and efficient cytosolic delivery of a variety of cargoes , which can then be re - directed to other subcellular compartments without apparent cytotoxicity B-property . [SEP]
[CLS] specific binding and endocytosis B-event seems ruled by the targeting moiety coupled to the carrier , since nds behaved similar to nps B-nanoparticle regarding these parameters . [SEP]
[CLS] however , intracellular transport appears to be provided by the carrier itself , where nds allow endosomal contents to escape into the cytosol compared to nps B-nanoparticle , where contents remain within vesicular compartments . [SEP]
[CLS] the degree of endosomal escape is relevant considering that the examples studied here as a proof - of - concept represent suboptimal configurations : cargoes were not linked to nds and their incorporation into cells B-material simply relied on passive co - internalization within endocytic vesicles induced by nds . [SEP]
[CLS] however , it is possible to directly couple cargoes to a nd . [SEP]
[CLS] this can be achieved either by chemical linkage B-property to outter or inner dna branches , by coupling cargoes to oligonucleotides whose sequence is complementary to single - stranded dna branches on the nd , or certain drugs can intercalate within dna . [SEP]
[CLS] in this regard , an important question to optimize the system is whether nds themselves escape endocytic vesicles or whether they distabilize the vesicle membrane but remain within its interior , which must to be investigated . [SEP]
[CLS] nevertheless , even if nds would remain within vesicular compartments , cargoes could be linked to nds cleavable linkers , separating both components and allowing the cargo to escape into the cytosol , similar to non - linked cargoes shown here . [SEP]
[CLS] this endosomal escape property provided by nds is likely due to the nature of the material B-material composing these carriers , dna . [SEP]
[CLS] indeed pristine ( not targeted ) nds disrupted membranous liposomes B-nanoparticle in vitro when incubated B-technique at endo - lysosomal - like ph . this is in accord with the fact that several dna - devices have recently shown potential to bypass membranous cellular barriers B-property . [SEP]
[CLS] this is the case for elegant studies employing dna origami , tetrahedric cages , or dnasomes to deliver doxorubicin B-material in drug resistant cancer cells B-material , as well as spherical - dna particles or dnasomes proven to deliver oligonucleotide payloads intracellularly . [SEP]
[CLS] this is yet a poorly understood property of these dna - devices , which may arise as a consequence of the display and packing of dna in these architectures : it is known that dna strands " packed " in an oriented brush border - like conformation display pka shifts toward ph 4 - 6 and , hence , could protonate at physiological endolysosomal ph . [SEP]
[CLS] dna protonation changes pairing of bases and strand conformation , it causes volume changes , and also leads to interaction with lipid B-material membranes I-material , altogether providing a possible mechanism for endosomal escape . [SEP]
[CLS] while previous studies showed endosomal escape ability in the context of delivery of oligonucleotides or doxorubicin B-material , oligonucleotides were a part of the dna component of those carriers and doxorubicin B-material is a dna intercalating agent . [SEP]
[CLS] hence , the amenability of dna to be used as a material B-material for intracellular delivery of other cargo molecules had been a question . [SEP]
[CLS] the present study expands this knowledge , showing the potential of dna - devices to deliver a broad spectrum of cargoes within cells B-material with no apparent disruption of the plasmalemma and bypassing endocytic compartments . [SEP]
[CLS] highlighting the proterties of dna as a promising material B-material for drug delivery , several dna structures have been reported capable of selective loading and release of cargo molecules , including dna hydrogel networks , dnasomes , origami , nanotubes B-nanoparticle , nanospheres B-nanoparticle , pyramidal or tetrahedric cages , etc . [SEP]
[CLS] some of these strategies have not been tested in biological systems and their potential to interact with cells B-material in ways amenable to intracellular transport are unknown , yet they seem promising . [SEP]
[CLS] on the other hand , although still very scarce , some studies have evaluated dna - based systems for intracellular transport in biological systems , providing fundamental information to this relatively new area of research . [SEP]
[CLS] this is the case for dna origami , tetrahedric cages , dnasomes , or dna assembled on spherical B-nanoparticle nanoparticles I-nanoparticle , which were shown to enter cells B-material . [SEP]
[CLS] the approach shown here expands previous knowledge in several ways . [SEP]
[CLS] for instance , with the exception of silica B-nanoparticle nanoparticles I-nanoparticle displaying a dna shell B-material or liposome - like dnasomes which formed structures ~ 80 - 120 - nm in diameter , all other studies of intracellular transport of dna - devices employed relatively small nanoassemblies ≤ 20 - nm . [SEP]
[CLS] this size may pose concerns of penetration through certain tissues in the body , e . g . extravasation into perivascular spaces through endothelial fenestrae or caveoli . [SEP]
[CLS] this could impair sitespecificity of action and restrict potential applications . [SEP]
[CLS] also , this size is well below that of endocytic vesicles that form on the plasmalemma , e . g . caveoli are ~ 50 - 80 - nm , clathrin pits are ~ 100 - 150 - nm , and macropinosomes or phagasomes are ≥ 1 - μm . [SEP]
[CLS] hence , such dnadevices may enter these compartments in a passive manner as cells B-material continuously display endocytic activity by concomitant means . [SEP]
[CLS] this was the case for spherical - dna carriers and dna cages of such small size , which entered cells B-material via non - specific scavenger receptors and / or non - selective macropinocytosis , making selective intracellular delivery difficult . [SEP]
[CLS] this remained the case even after targeting selected cell B-material - I-material surface I-material receptors I-material , such as the example of anti - her2 conjugated spherical - dna devices . [SEP]
[CLS] although conjugation of this antibody B-material to dna strands did not affect its affinity , this was reduced 10 - fold after conjugation on the spherical B-nanoparticle nanoparticle I-nanoparticle , so that these particles displayed only 3 - 8 - fold increased cell B-event binding as compared to non - targeted counterparts when tested at physiological temperature . [SEP]
[CLS] as a result , uptake was largely mediated by scavenger receptors instead of her2 , resulting in no differences in uptake of targeted vs . untargeted particles by 24 h . [SEP]
[CLS] spherical - dna [SEP]
[CLS] particles and dnasomes of larger size ( ~ 80 - 120 - nm ) were speculated to access into cells B-material via scavenger receptors or lipid - raft - like caveolae domains , respectively , although this may be due to the fact that such particles were not targeted to any particular marker . [SEP]
[CLS] hence , size and targeting valency are key elements to optimize the potential of dnadevices designed to facilitate intracellular access . [SEP]
[CLS] these limitations were overcome in this study by employing a model with larger size ( ~ 200 - nm ) and higher valency ( ≥20 ) , which resulted in markedly greater selectivity : ~ 190 - fold enhanced cellular binding for anti - icam nds over untargeted counterparts and efficient ( 82 % ) uptake by a selected endocytic mechanism ( e . g . cam - mediated endocytosis B-event for this formulation ) . [SEP]
[CLS] given this , dna - devices such as those shown here are preferable to achieve selective drug delivery while previous configurations may show greater performance where non - selective targeting is sufficient ( e . g . enhanced permeability retention in tumors B-material , etc . ) . [SEP]
[CLS] dna - devices of size viable for endocytosis B-event and sufficient targeting valency can provide : ( a ) specific binging to selected cell - surface markers , ( b ) efficient endocytosis B-event into cells B-material via the associated pathway , ( c ) cytosolic delivery of a broad range of compounds ( drugs , sugars , proteins B-material , nucleic B-material acids I-material ) , ( d ) subsequent access to other intracellular compartments ( e . g . the nucleus ) , and ( e ) functional intracellular effects , ( f ) without apparent cytotoxicity B-property . [SEP]
[CLS] the properties and translational implications of the strategy presented here add significantly to the knowledge and potential applications of dna - devices . [SEP]
[CLS] modulable physicochemical properties , versatility of assembly , potential for manipulation by chemical , enzymatic , and physical means , and full degradability in the physiological environment , make dna an interesting material B-material for this purpose . [SEP]
[CLS] furthermore , dna - devices can be designed to delay degradation by dnases and recognition by the immune system due to steric hindrance . [SEP]
[CLS] although in vivo performance and safety remain to be investigated and optimized , this study shows that dna - devices hold potential to bypass cellular membranous barriers B-property for delivery of a broad spectrum of compounds intracellularly , further shifting the now questionable paradigm of dna being unable to enter cells B-material . [SEP]
[CLS] antibodies B-material recognized human icam - 1 ( atcc ; manassas , va ) , pecam - 1 ( a gift from centocor , malvern , pa ) , transferrin receptor B-material ( tfr ; emd millipore , billerica , ma ) , or mannose - 6 - phosphate receptor B-material ( m6pr ; abcam , cambridge , ma ) . [SEP]
[CLS] non - specific iggs were from jackson immunoresearch ( pike west grove , pa ) . [SEP]
[CLS] biotin - phalloidin was from molecular probes ( eugene , or ) . [SEP]
[CLS] rhodamine - albumin - nls and catalase were from sigma aldrich ( st . louis , mo ) . [SEP]
[CLS] plasmid pegfp - rhoa [SEP]
[CLS] experimental section antibodies B-material and reagentswas from addgene ( cambridge , ma ) . [SEP]
[CLS] live / dead ® cell B-property viability I-property kit [SEP]
[CLS] was from live technologies ( grand island , ny ) . [SEP]
[CLS] liposomes B-nanoparticle were from encapsula nano sciences ( brentwood , tn ) . [SEP]
[CLS] ultraamp [UNK] signal amplifiers used as nds were from genisphere ( hatfield , pa ) and polystyrene nps B-nanoparticle from polysciences ( warrington , pa ) . [SEP]
[CLS] other reagents were from sigma - aldrich ( st . louis , mo ) . [SEP]
[CLS] model polymer B-material nds or nps B-nanoparticle were prepared by incubating B-technique generation 4 ultraamp [UNK] dna dendrimers B-nanoparticle ( displaying ~ 20 anti - mouse igg molecules ) or 100 - nm fitc - polystyrene particles , respectively , with anti - icam , anti - pecam , anti - tfr , or anti - m6pr , or igg . [SEP]
[CLS] this allows antibody B-material binding to secondary igg on nds ( figure 1a ) or antibody B-material adsorption on the surface of polystyrene nps B-nanoparticle . [SEP]
[CLS] uncoated antibodies B-material were removed from np B-nanoparticle preparations by centrifugation and coated counterparts were resuspended in phosphatebuffered saline ( pbs ) with 1 % bovine serum albumin ( bsa ) , and sonicated to avoid aggregation . [SEP]
[CLS] size and polydispersity were measured by dls ( malvern zetasizer , worcestershire , uk ) . [SEP]
[CLS] as a note , polystyrene particles were selected as model polymer B-material carriers since after coating B-material with antibodies B-material , they have been shown to provide similar targeting , endocytosis B-event , and in vivo biodistribution as biodegradable B-property poly ( lactic - co - glycolic acid ) nps B-nanoparticle , allowing for comparison with historical data . [SEP]
[CLS] cell B-material culture human umbilical vein endothelial cells B-material ( huvecs ; lonza , walkersville , md ) , mesothelioma ren cells B-material , skin fibroblasts , or epithelial colorectal adenocarcinoma caco - 2 cells B-material ( american type culture collection , manassas , va ) were seeded on coverslips in 24well plates and treated with tnf - α 16 h prior to experiments . [SEP]
[CLS] cells B-material were cultured with antibiotics at 37°c , 5 % co 2 , and 95 % relative humidity in : ( huvecs ) m199 supplemented with 15 % fetal bovine serum ( fbs ) , 15 μg / ml endothelial cell B-material growth supplement , 2 mm lglutamine , and 100 μg / ml heparin ; ( ren ) rpmi 1640 containing 10 % fbs , and 2 mm lglutamine ; or ( fibroblasts and caco - 2 ) dmem supplemented with 10 % fbs and 4 . 50 g / l glucose ( gibcobrl , grand island , ny ) . [SEP]
[CLS] in all cases , incubation B-technique of nds or nps B-nanoparticle with cells B-material was conducted in the presence of complete cell B-material medium ( containing 10 - 20 % serum ) supplemented with 1 % bsa . [SEP]
[CLS] cells B-material were incubated B-technique for the indicated times with nds or fitc - labeled nps B-nanoparticle coated with non - specific igg or with targeting antibodies B-material at 4°c or 37°c . [SEP]
[CLS] for inhibition experiments , cells B-material were incubated B-technique with anti - icam nds in the presence of anti - icam in the medium or with endocytic inhibitors , including 50 μm mdc ( an inhibitor of clathrin coated pits ) , 1 μg / ml filipin ( an inhibitor of caveolar pathways ) , 3 mm amiloride ( an inhibitor of icam - 1 pathway and macropinocytosis ) , or 0 . 5 μm wortmannin ( an inhibitor of macropinocytosis ) . [SEP]
[CLS] non - bound carriers were washed and cells B-material were fixed with 2 % paraformaldehyde . [SEP]
[CLS] to quantify binding , fitc - labeled nps B-nanoparticle were directly imaged by fluorescence B-technique microscopy I-technique , while nd samples were permeabilized with 0 . 2 % triton x - 100 and incubated B-technique with fitc ( green ) - labeled secondary antibodies B-material to stain all cell - associated nds ( see microscopy B-technique settings below ) . [SEP]
[CLS] for internalization , nds coated with alexa fluor 488 ( green ) - labeled antibody or antibodycoated fitc ( green ) - labeled nps B-nanoparticle were used . [SEP]
[CLS] after cell B-material fixation ( without permeabilization ) , samples were incubated B-technique with texas - red secondary antibodies B-material . [SEP]
[CLS] this allows staining of the antibody B-material coat B-material of carriers located on the cell B-material surface in texas - red . [SEP]
[CLS] samples were analyzed by fluorescence B-technique microscopy I-technique to quantify total carriers ( which appear green ) and surfacebound carriers ( which appear yellow given co - localization of red + green ) , from which internalized carriers were calculated . [SEP]
[CLS] cell B-material borders were identified by phase - contrast . [SEP]
[CLS] samples were analyzed using a 60x planapo objective and olympus ix81 microscope ( olympus inc . , center valley , pa ) with fluorescence B-property filters ( semrock , rochester , ny ) for texas - red ( ex . bp360 - 370 nm , em . ba590 - 800 + nm ) and fitc ( ex . bp460 - 490 nm , em . ba515 - 550 nm ) . [SEP]
[CLS] images were taken with orca - er camera ( hamamatsu , bridgewater , nj ) and analyzed using image - pro 6 . 3 ( media cybernetics inc . , bethesda , md ) . [SEP]
[CLS] to minimize potential artifacts due to slightly different focal planes , nd vs . np B-nanoparticle intensities , fluorescent B-property spots containing more than one carrier , etc . , all pictures were taken at the nucleus midsection , the number ( not intensity ) of spots whose fluorescence B-property surpassed a threshold background was quantified , and finally then the number of carriers within that spot was based on the intensity expected for a nd or np B-nanoparticle and the 2 - d area to be occupied by a 200 - nm object . [SEP]
[CLS] for co - internalization of carrier and cargo within common endocytic vesicles , cells B-material were incubated B-technique at 37°c with anti - icam nds or anti - icam nps B-nanoparticle along with the agents indicated below ( referred to as pulse ) . [SEP]
[CLS] cells B-material were then washed to stop further binding and uptake , and incubated B-technique at 37°c with cell B-material medium to enable intracellular trafficking of the internalized agents ( referred to as chase ) . [SEP]
[CLS] cargoes were : ( a ) 2 mg / ml texas - red dextran B-material ( 15 min pulse , 1 h or 5 h chase ) , ( b ) biotin - phalloidin ( 30 min pulse , 5 h chase ) , ( c ) rhodaminealbumin - nls ( 30 min pulse , 5 h chase ) , ( d ) plasmid pegfp - rhoa ( 5 h pulse , 48 h chase ) , or ( e ) catalase ( 3 h continuous incubation B-technique ) . [SEP]
[CLS] for dextran B-material , biotin - phalloidin , albumin - nls , and egfp - rhoa plasmid , cells B-material were fixed and visualized by fluorescence B-technique microscopy I-technique ( using additionally texas - red streptavidin for biotin - phalloidin ; note that due to presence of biotin in the nucleus , texas - red streptavidin also stains the nucleus ) . [SEP]
[CLS] the number of dextranpositive objects ≥ 100 - nm and total pixel area occupied by the signal were quantified . [SEP]
[CLS] phalloidin staining of f - actin or nuclear vs . endocytic distribution of albumin - nls were qualitatively assessed . [SEP]
[CLS] for catalase , after 3 h incubation B-technique , cells B-material were challenged with 50 μm h 2 o 2 ( 16 h ) , washed , and viability was tested using the live / dead ® kit and fluorescence B-technique microscopy I-technique to quantify calcein - positive ( live , green ) vs . ethidium homodimer - 1 - positive ( dead , red ) cells B-material . [SEP]
[CLS] cells B-material were incubated B-technique at 37°c for 5 h with anti - icam nds or anti - icam nps B-nanoparticle , then washed . [SEP]
[CLS] viability was estimated 48 h later , as described above . [SEP]
[CLS] liposomes B-nanoparticle suspended in pbs at ph 7 . 4 or 4 . 5 were incubated B-technique for 30 min at room temperature in the absence or presence of untargeted nds . [SEP]
[CLS] triton x - 100 ( 2 % ) was used as a binding and endocytosis B-event of targeted nucleodendrimers . [SEP]
[CLS] ( a ) nucleodendrimers consist of dna modules containing a central dsdna region ( waist ) with four ssdna ends ( arms ) , where modules assemble by complementarity into layers surrounding a central core B-material . [SEP]
[CLS] outer arms are functionalized with mouse igg secondary antibody B-material , to which a targeting antibody B-material can be coupled by affinity . [SEP]
[CLS] intracellular distribution of targeted nucleodendrimers . [SEP]
[CLS] fluorescence B-technique microscopy I-technique ( top ) and quantification ( bottom ) of huvecs incubated B-technique at 37°c for either 1 h or 3 h with anti - icam nds or anti - icam nps B-nanoparticle . [SEP]
[CLS] both formulations are labeled with a green fluorophore ( alexa fluor 488 - anti - icam for nds or fitc - nps B-nanoparticle , respectively ) . [SEP]
[CLS] surface - bound carriers are then labeled using a texas - red - conjugated secondary antibody B-material , hence they appear yellow ( red + green ) , while internalized counterparts are green . [SEP]
[CLS] perinuclear region refers to the area comprised ≤ 2 μm around the nucleus . [SEP]
[CLS] scale bar = 10 - μm . [SEP]
[CLS] * compares nds vs . nps B-nanoparticle . [SEP]
[CLS] three symbols represent p < 0 . 001 . [SEP]
[CLS] for each formulation , no significant difference was found between 1 h and 3 h . [SEP]
[CLS] effect of nucleodendrimers on membranous vesicles . [SEP]
[CLS] percent degradation of liposomes B-nanoparticle ( % lip . degr . ) quantified by measuring their optical density ( od ; 595 nm ) in a ph 4 . 5 solution , after 30 min incubation B-technique at room temperature with nds or with 2 % triton x - 100 ( tx100 ) used as a positive control . [SEP]
[CLS] ( b ) fluorescence B-technique microscopy I-technique ( top ) and quantification ( bottom ) of huvecs incubated B-technique for 1 h with alexa fluor 488 - ( green ) - labeled anti - icam nds or igg nds , followed by staining of surface - bound nds with texas - redconjugated secondary antibody B-material . [SEP]
[CLS] surface - bound nds appear yellow ( red + green ) and internalized counterparts are green . [SEP]
[CLS] inset = phase - contrast verifying presence of cells B-material . [SEP]
[CLS] scale bar = 10 - μm . [SEP]
[CLS] * compares 4°c vs . 37°c . [SEP]
[CLS] # compares igg nds vs . anti - icam nds . [SEP]
[CLS] dashed lines = cell B-material borders . [SEP]
[CLS] ( b ) endocytosis B-event of anti - icam nds ( 1 h - 37°c ) in the presence of amiloride ( aml ) , filipin ( fil ) , monodansylcadaverine ( mdc ) , or wortmannin ( wrt ) . [SEP]
[CLS] * compares inhibitors to control cells B-material . [SEP]
[CLS] ( a - b ) two or three symbols represent p < 0 . 01 or p < 0 . 001 , respectively . [SEP]
[CLS] subcellular distribution of a fluid - phase marker mediated by targeted nucleodendrimers . [SEP]
[CLS] fluorescence B-technique microscopy I-technique ( top ) of huvecs co - incubated B-technique at 37°c with anti - icam nds ( full panels ) or anti - icam nps B-nanoparticle ( insets ) in the presence of texas - red dextran B-material , followed by fixation and nuclear staining with dapi ( blue ) . [SEP]
[CLS] scale bar = 10 - μm . [SEP]
[CLS] dashed lines = cell B-material borders . [SEP]
[CLS] the number of texas red - positive endocytic vesicles and total area occupied by fluorescence B-property pixels were quantified ( bottom ) . [SEP]
[CLS] δ = fold change between 3 h and 1 h , where * p < 0 . 05 . [SEP]
[CLS] 5 . intracellular delivery of various compounds by targeted nucleodendrimers . [SEP]
[CLS] ( a - b ) fluorescence B-technique microscopy I-technique of huvecs co - incubated B-technique at 37°c in a pulse - chase mode ( see methods section ) with anti - icam nds or anti - icam nps B-nanoparticle along with ( a ) biotin - phalloidin or ( b ) rhodamine - albumin nuclear localization sequence chimera ( albumin - nls ) . [SEP]
[CLS] biotinphalloidin was visualized with texas - red streptavidin ( note : cell B-material nuclei naturally contain biotin ) . [SEP]
[CLS] arrows = distribution to the intended intracellular targets , including f - actin in ( a ) or the nucleus in ( b ) . [SEP]
[CLS] arrowheads = distribution to intracellular off - target sites , such as lack of f - actin staining in ( a ) or perinuclear distribution in ( b ) . [SEP]
[CLS] ( c ) fluorescent B-property protein B-material expression 48 h after co - incubation B-technique of huvecs for 5 h at 37°c with anti - icam nds or anti - icam nps B-nanoparticle and egfp - rhoa - encoding plasmid . [SEP]
[CLS] inset = phase - contrast verifying presence of cells B-material . [SEP]
[CLS] ( a - c ) scale bar = 10 - μm . [SEP]
[CLS] effect of model cargoes delivered intracellularly by targeted nucleodendrimers . [SEP]
[CLS] fluorescence B-technique microscopy I-technique ( top ) and quantification ( bottom ) of huvecs viability after 16 h exposure to 50 μm h 2 o 2 . [SEP]
[CLS] cells B-material had been pre - incubated B-technique with catalase and anti - icam nds or anti - icam nps B-nanoparticle 3 h prior to h 2 o 2 oxidative challenge . [SEP]
[CLS] green = calcein - positive live cells B-material . [SEP]
[CLS] red = ethidium homodimer - 1 - positive dead cells B-material . [SEP]
[CLS] scale bar = 30 - μm . [SEP]
[CLS] * compares nds or nps B-nanoparticle in control condition vs . h 2 o 2 insult . [SEP]
[CLS] # compares nds vs . nps B-nanoparticle . [SEP]
[CLS] two or three symbols represent p < 0 . 01 or p < 0 . 001 , respectively . [SEP]
[CLS] 7 . targeting of nucleodendrimers to various receptors and cell B-material types . [SEP]
[CLS] ( a ) fluorescence B-technique microscopy I-technique of ren cells B-material , skin fibroblasts , caco - 2 cells B-material after 1 h incubation B-technique at 37°c with anti - icam nds . [SEP]
[CLS] ( b ) fluorescence B-technique microscopy I-technique of huvecs after 1 h incubation B-technique at 37°c with nds coupled to antibodies B-material recognizing pecam - 1 , transferrin receptor B-material ( tfr ) , or mannose - 6 - phosphate receptor B-material ( m6pr ) . [SEP]
[CLS] nds were visualized after permeabilization and staining with fitc - secondary antibody B-material . [SEP]
[CLS] scale bar = 10 - μm . [SEP]
[CLS] dashed lines = cell B-material borders . [SEP]
[CLS] 8 . effect of nucleodendrimers on the cell B-property viability I-property . [SEP]
[CLS] fluorescence B-technique microscopy I-technique ( left ) and quantification of the number of huvecs ( middle ) and their viability ( right ) 48 h after incubation B-technique with anti - icam nds for 5 h . green = calcein - positive live cells B-material . [SEP]
[CLS] red = ethidium homodimer - positive dead cells B-material . [SEP]
[CLS] scale bar = 500 - μm . [SEP]
[CLS] recent developments in integrating high selectivity of functional dna , such as dnazyme and aptamers , with efficient dna delivery into cells B-material by gold B-nanoparticle nanoparticles I-nanoparticle or superior near - infrared optical properties of upconversion nanoparticles B-nanoparticle are reviewed . [SEP]
[CLS] their applications in sensing and imaging small organic metabolites , toxins , metal B-material ions B-material , ph , dna , rna , proteins B-material , and pathogens are summarized . [SEP]
[CLS] the advantages and future directions of these functional dna materials are discussed . [SEP]
[CLS] the past decade of biotechnology has witnessed exciting development and integration of biology with nanotechnology and is beginning to enjoy the fruits of this integration as a result of its applications in sensing and imaging . [SEP]
[CLS] nanomaterials B-material possess many unique properties , such as surface plasmon effect for gold B-nanoparticle nanoparticles I-nanoparticle ( aunp B-nanoparticle ) and non - linear optical processes of upconversion nanoparticles B-nanoparticle ( ucnp ) , making them superior choices for signal transductions in sensing and imaging applications . [SEP]
[CLS] however , these nanomaterials B-material do not have any selectivity toward targets of interests , which can be complemented by biomolecules with high selectivity very well . [SEP]
[CLS] therefore a number of nanomaterials B-material have been combined with biomolecules , among which functional dna ( fdna ) has shown the most promise . [SEP]
[CLS] fdnas are short single - stranded dna molecules with enzymatic function ( called dnazymes or deoxyribozymes ) , biorecognition function ( called aptamers ) or both ( called aptazymes ) . [SEP]
[CLS] they are obtained by in vitro selection or systematic evolution of ligands by exponential enrichment ( selex ) . [SEP]
[CLS] one advantage of selex is that , in principle , the process can be tailored to obtain fdnas selective for almost any target of interest , ranging from metal B-material ions B-material , small organic metabolites to large proteins B-material and even whole cells B-material . [SEP]
[CLS] fdnas are also much more stable than other biomolecules and easier to be conjugated to nanomaterials B-material . [SEP]
[CLS] these properties make fdnas an ideal choice to integrate them with nanomaterials B-material for sensing and imaging applications , with the majority of success outside cellular environment , such as environmental monitoring , when sample matrix is not very complex . [SEP]
[CLS] to realize its full potentials , fdna nanomaterials B-material have been developed for applications in living cells B-material . [SEP]
[CLS] this review focuses on these recent achievements by highlighting the impact of fdna - functionalized aunps B-nanoparticle and luminescent B-property ucnps in sensing and imaging in living cells B-material . [SEP]
[CLS] aunps B-nanoparticle are among the most extensively studied nanomaterials B-material because they are stable , and easy to synthesize and functionalize . [SEP]
[CLS] the unique properties , such as distance dependent surface plasmon effects that can result in dramatic color changes with extinction coefficients that are several hundred times higher than the best organic dyes , make them excellent sensing platform , which has been reviewed elsewhere . [SEP]
[CLS] for sensing and imaging in living cells B-material , the aunps B-nanoparticle have been shown to confer greater stability of dnas against degradation and allow delivery of dna into cells B-material . [SEP]
[CLS] the first such demonstration is intracellular atp detection in hela cells B-material using dna aptamerfunctionalized aunps B-nanoparticle ( figure 2a ) . [SEP]
[CLS] the presence of a dense layer of the aptamer on the surface of the aunps B-nanoparticle was shown to enhance aptamer stability inside the cell B-material and provides a mechanism for cellular uptake ( figure 2b ) . [SEP]
[CLS] in addition to organic metabolites , such as atp , metal B-material ions B-material play important roles in cells B-material and yet sensing and imaging agents for metal B-material ions B-material in living cells B-material are quite limited . [SEP]
[CLS] while dnazymes specific a wide variety of metal B-material ions B-material have been obtained and converted to fluorescent B-property , colorimetric , electrochemical sensors and magnetic B-property resonance imaging ( mri ) contrast B-technique agents I-technique outside cells B-material , intracellular metal B-material sensing and imaging was reported only recently . [SEP]
[CLS] in this work , 13 nm aunps B-nanoparticle were functionalized with the uranyl - specific 39e dnazyme , and fluorophore - modified substrate was quenched by both the aunp B-nanoparticle and a molecular quencher ( figure 2c ) . [SEP]
[CLS] the dnazyme - aunp B-nanoparticle nanoprobe B-nanoparticle was shown to cleave the substrate in the presence of uranyl and subsequently resulted in release of a shorter , cy3modified product and thus increase in fluorescence B-property signal . [SEP]
[CLS] it was demonstrated that the dnazyme - aunp B-nanoparticle probe can efficiently enter hela cells B-material and serve as an intracellular metal B-material ion B-material nanosensor B-nanoparticle ( figure 2d ) . [SEP]
[CLS] the dnazyme - aunps B-nanoparticle can not only detect metal B-material ions B-material , but also target mrna in cells B-material . [SEP]
[CLS] the 10 - 23 dnazyme functionalized aunps B-nanoparticle is able to specifically target and cleave cancerassociated mrna in breast cancer cells B-material . [SEP]
[CLS] aunps B-nanoparticle were shown to protect the dnazyme from hydrolytic cleavage by dnaases and were able to reduce gdf15 gene expression by ~ 60 % . [SEP]
[CLS] in this work , selective targeting of the desired mrna was achieved by changing the sequence of the 10 - 23 dnazyme binding arms to target a specific rna sequence . [SEP]
[CLS] dnazymes have the advantage of being more stable in comparison to short interfering rnas ( sirna ) . [SEP]
[CLS] furthermore , dnazyme - based gene regulation mechanism is independent of cellular machineries like rna - induced silencing complex ( risc ) . [SEP]
[CLS] dual functionalization of aunp B-nanoparticle with both dnazyme and sirna is of special interest specially in targeting genes that regulate risc function . [SEP]
[CLS] in addition , detection of cancer - related genes and dna biomarkers B-property has also been demonstrated using a dna - aunp B-nanoparticle system . [SEP]
[CLS] aunps B-nanoparticle functionalized with hairpin dna in a molecular beacon ( mb ) format was used for intracellular detection of a cancer associated gene , signal transducer and activator of transcription B-event 5b ( stat5b ) , expression level in mcf - 7 cancer cells B-material . [SEP]
[CLS] similarly , expression of tyrosinase , a melanoma - associated gene , was detected in melanoma cells B-material using hairpin dnafunctionalized aunps B-nanoparticle . [SEP]
[CLS] it was shown that dna functionalized aunps B-nanoparticle could be used in combination with specific antibody B-material to target desired cells B-material . [SEP]
[CLS] this system showed cell selective uptake while conferring significant gene targeting and knockdown . [SEP]
[CLS] in order to prevent false positive results to improve the reliability of detection , a multicolor fluorescent B-property nanoprobe B-nanoparticle has been developed to detect cancer . [SEP]
[CLS] aunps B-nanoparticle were functionalized with recognition dna sequences for three specific tumor - related mrna transcripts B-event . [SEP]
[CLS] each recognition sequence was hybridized to a fluorophore - functionalized complement dna strand , whose fluorescence B-property was quenched by the aunp B-nanoparticle . [SEP]
[CLS] in the presence of the target mrna , release of the reporter caused fluorescence B-property signal to increase . [SEP]
[CLS] this platform was used to simultaneously detect three different tumor - related mrnas in living cells B-material . [SEP]
[CLS] similarly , aunps B-nanoparticle functionalized with two recognition dna sequences , one as internal control ( targeting actin mrna ) and the other one for designated target mrna , was shown to improve precision of determining mrna levels in individual cells B-material and correct cell - to - cell variations . [SEP]
[CLS] aunps B-nanoparticle functionalized with both cancer - targeting and drug - carrier dnas were also prepared , to improve cancer cell B-material imaging and therapy . [SEP]
[CLS] in this design , the cancertargeting dna was modified with folic B-material acid I-material and the drug - carrier dna was a molecular beacon ( mb ) loaded with doxorubicin B-material . [SEP]
[CLS] the mb was designed to release the drug only in presence of tumor B-material mrna . [SEP]
[CLS] intracellular sensing based on fdna and aunps B-nanoparticle is not limited to small molecules or biomarkers B-property . [SEP]
[CLS] recently , core - satellite assembly of two different sizes of aunps B-nanoparticle prepared by an i - motif dna linker was used to detect ph changes in the endocytic pathway ( figure 3a ) . [SEP]
[CLS] the i - motif consists of cytosine - rich single stranded dna that adopts a quadruplex structure under acidic conditions ( ph ~ 5 . 0 ) . [SEP]
[CLS] in this study 14 nm satellite aunps B-nanoparticle were functionalized with i - motif ssdna . [SEP]
[CLS] satellite aunps B-nanoparticle were assembled on the 50 nm core B-material aunp B-nanoparticle through hybridization with i - motif complementary dna on the core B-material particle to form a core - satellite nanostructure ( csns ) . [SEP]
[CLS] raw 264 . 7 macrophage cells B-material were used to study the well - known drop of ph in the endocytic pathway from 5 . 9 - 6 . 2 in early endosomes to 4 . 7 - 5 . 5 in lysosomes ( figure 3b ) . [SEP]
[CLS] bright yellow csnss were taken up efficiently by macrophages through endocytosis B-event . [SEP]
[CLS] due to the ph drop and i - motif formation that led to disassembly of csnss , the yellow scattering signal became green ( figure 3c ) . [SEP]
[CLS] in a separate study , cellular imaging and endocytic trafficking of proteins B-material were demonstrated using nucleolin and prion aptamers in combination with aunps B-nanoparticle . [SEP]
[CLS] fdna and aunps B-nanoparticle have been also applied for cancer cell B-material targeting and imaging in combination of other nanomaterials B-material such as iron B-nanoparticle oxide I-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] in this study goldcoated iron B-material oxide I-material nanoroses functionalized with sgc8 aptamers were shown to specifically recognize ccrf - cem leukemia cells B-material and enter them through endocytosis B-event process . [SEP]
[CLS] such a combination of gold B-material and iron B-material oxide I-material nanomaterials B-material with fdna provided a multifunctional theranostic tool for mri ( due to iron B-material oxide I-material inner core B-material ) and photothermal therapy ( due the gold B-material shell B-material ) . [SEP]
[CLS] luminescent B-property upconversion nanoparticles B-nanoparticle ( ucnps ) have distinctive properties that make them ideal nanoprobe B-nanoparticle for targeted imaging and selective sensing . [SEP]
[CLS] unlike most other optical probes , ucnps can convert near - infrared ( nir ) excitation light into shorter wavelength visible luminescence B-property . [SEP]
[CLS] this property , in addition to their high photostability and low cytotoxicity B-property , make them suitable for deep tissue bioimaging . [SEP]
[CLS] a major drawback of ucnps is lack of general methods to make them water soluble B-property and functionalizable . [SEP]
[CLS] to overcome this issue , several approaches such as silica coating B-material , oxidation of capped ligands , and ligand - free treatments have been reported . [SEP]
[CLS] recently , our lab has developed a simple and general method to engineer uncps whose surface has been coated with a monolayer of pegylated phospholipids . [SEP]
[CLS] such ucnps have been shown to be water soluble and biocompatible B-property and could be functionalized by a number of targeting molecules for a wide range of applications including selective sensing or imaging . [SEP]
[CLS] as a proof of concept , pegylated phospholipids conjugated with folate were used to functionalize ucnps , which were then tested successfully for imaging cancer cells B-material with high specificity . [SEP]
[CLS] to expand the targeting ability further , our group developed a one - step strategy to prepare uniform dna - modified ucnps ( figure 4a ) . [SEP]
[CLS] it was shown that dna molecules remained functional on the surface of ucnps and were able to assemble into hybrid structures such as aunp B-nanoparticle - ucnp nanostructures . [SEP]
[CLS] more importantly , dna aptamerfunctionalized ucnps were able to specifically target cancer cells B-material and cross the cell membrane with excellent internalization efficiency without the use of any transfection agents ( figure 4b ) . [SEP]
[CLS] therefore dna - ucnps are excellent choices for dna delivery and targeted imaging of live cells B-material ( figure 4c owing to the beneficial properties of ucnps , many groups have utilized them in a variety of biomolecule sensing platforms . [SEP]
[CLS] ucnps in a fret sensor platform have been combined with graphene oxide B-material ( go ) as an acceptor to detect ssdna in human serum . [SEP]
[CLS] similarly , fluorescent B-property silver B-material clusters , which have upconversion properties , have been used in dna biosensing and nucleobase recognition . [SEP]
[CLS] multicolor ucnps functionalized with aptamers have been reported for multiplexed and simultaneous sensing of mycotoxins . [SEP]
[CLS] in this study , functionalized ucnps with ochratoxin a ( ota ) - aptamers and fumonisin b1 ( fb1 ) - aptamers were quenched by go due to the interaction of go with nucleobases of the aptamers . [SEP]
[CLS] in the presence of ota and fb1 , and upon release of go , the fluorescence B-property intensity of ucnps were shown to have a concentration - dependent increase . [SEP]
[CLS] the limits of detection ( lod ) were 0 . 02 ng / ml for ota and 0 . 1 ng / ml for fb1 . [SEP]
[CLS] in other similar examples , atp , enterotoxin b ( eb ) and , thrombin nanosensors B-nanoparticle have been reported using ucnps ( as fret donor ) functionalized with an atp , eb or thrombin aptamer in combination with either go , black hole quencher - 3 or carbon B-material nanoparticles B-nanoparticle ( as a fret acceptor ) , respectively . [SEP]
[CLS] metal B-material ion B-material detection has been also demonstrated using ucnps and gold B-material nanorods B-nanoparticle in combination with mercury ( ii ) aptamer . [SEP]
[CLS] this system was tested for detecting mercury B-material in human serum . [SEP]
[CLS] while current lods may be sufficient to detect certain targets such as small organic molecules or metal B-material ions B-material , they may not be sufficient for detection of other targets , such as pathogens . [SEP]
[CLS] to address this issue , aptamer - functionalized ucnps were combined with dna aptamer - modified magnetic B-nanoparticle nanoparticles I-nanoparticle in order to concentrate and detect bacterial pathogens . [SEP]
[CLS] two different types of ucnps with different colors were functionalized with aptamers for salmonella typhimurium or staphylococcus aureus and were used to selectively capture and detect these pathogens , with a lod of < 10 cfu ml −1 . [SEP]
[CLS] nanomaterials B-material have brought a new and powerful paradigm in biosensing . [SEP]
[CLS] along with advances in the field of nanomaterials B-material , the combination of novel nanomaterials B-material with functional nucleic B-material acids I-material as biological recognition and targeting elements has enabled functionalized nanomaterials B-material to be used in life science research . [SEP]
[CLS] despite the advances recently made with application of functional nanomaterials B-material in biosensing , there are still many challenges that remain to be addressed . [SEP]
[CLS] first , more reliable and efficient molecular and cellular markers need to be made available . [SEP]
[CLS] for example , prostate - specific antigen ( psa ) , a well known protein - based prostate cancer maker , is a poor disease marker with high rates of false positive and false negatives . [SEP]
[CLS] both knowledge of reliable and effective biomarkers B-property and selective and specific recognition elements for those biomarkers B-property are required for successful applications in a sensor platform . [SEP]
[CLS] in addition , despite tremendous progress in detecting nucleic B-material acids I-material and proteins B-material in vivo , detection of small molecules and metal B-material ions B-material as a disease marker or biochemical pathway cofactors has lagged behind . [SEP]
[CLS] by utilizing selex or in vitro selection , it is possible in principle to select functional nucleic B-material acids I-material for any target , whether aptamer for small molecular target or dnazyme for metal B-material ion B-material cofactor . [SEP]
[CLS] therefore , selection for more aptamers and dnazymes needs to be carried out in order to provide efficient recognition elements for different biosensing applications , especially for small molecules and metal B-material ions B-material . [SEP]
[CLS] furthermore , new nanomaterials B-material with improved biocompatibility B-property and better optical properties and lower cytotoxicity B-property are expected to advance their usage in intracellular and biological sensing . [SEP]
[CLS] utilizing two or more nanomaterials B-material in a hybrid format can produce better nanocomposite materials with desired properties , such as multiple functionalities and controlled spatial positioning . [SEP]
[CLS] to utilize the beneficial properties of different nanomaterials B-material , better strategies need to be developed for functionalization of nanomaterials B-material with recognition elements , such as dna aptamers , to generate effective biosensing platforms . [SEP]
[CLS] overcoming these challenges will allow functional dna nanomaterials B-material to be effective sensors and imaging agents not only in living cells B-material , but also in animals and human for better medical diagnostics and personal medicine . [SEP]
[CLS] [ 48 ] [SEP]
[CLS] similarly , ucnps functionalization with dna was demonstrated using click chemistry [ 49 ] . [SEP]
[CLS] it was shown that dna remained functional after attachment to ucnps and also enhanced water B-material solubility B-property of the nanoparticles B-nanoparticle . [SEP]
[CLS] confocal microscopy B-technique was used to demonstrate the ability of dna functionalized ucnps for live cell imaging [ 49 ] . [SEP]
[CLS] 1 . general representation of functional dna based nanomaterials B-material and their application in intracellular sensing and imaging . [SEP]
[CLS] figure 2 . a ) schematic representation of intracellular atp probe based on aptamer nano - flare system ; b ) fluorescence B-technique microscopy I-technique images of hela cells B-material incubated B-technique with aptamer nano - flares ( top ) and control particles ( bottom ) . [SEP]
[CLS] adapted from [ 21 * * ] ; c ) schematic representation of the first intracellular dnazyme based metal B-material ion B-material probe for detecting uranyl ion B-material in living cells B-material ; d ) fluorescence B-technique microscopy I-technique images of hela cell B-material treated with ( top ) or without ( bottom ) uranyl . [SEP]
[CLS] adopted from [ 29 * * ] . [SEP]
[CLS] figure 3 . a ) schematic representation of the formation and ph - induced disassembly of the coresatellite nanostructures ( csns ) ; b ) proposed endocytosis B-event mechanism for i - motif based ph detection ; c ) overlaid dark field and fluorescence B-property images of raw264 . 7 cells B-material incubated B-technique with i - motif and control csns after 15 min incubation B-technique and 30 min post - incubation B-technique . [SEP]
[CLS] framed areas are magnified in insets . [SEP]
[CLS] adopted from [ 37 * * ] . [SEP]
[CLS] figure 4 . a ) schematic illustration of functionalizing ucnps with cancer specific dna aptamer for cancer cell B-material targeting ; b ) fluorescence B-technique microscopy I-technique images of mcf - 7 breast cancer cells B-material treated with aptamer modified ucnps ( apt - ucnps , top ) and random dna modified ucnps ( rnd - ucnps , bottom ) . [SEP]
[CLS] adapted from [ 48 * * ] ; c ) schematic representation of the " click " - conjugation of an alkyne - modified oligonucleotide ( cu ) to an azidomodified ucnp ; d ) bright ucnp dna as " nano - sized " lamp for live cell B-material imaging in the near - ir range . [SEP]
[CLS] adapted from [ 49 * ] [SEP]
[CLS] the use of nanoparticles B-nanoparticle ( nps B-nanoparticle ) in biology and medicine requires a molecular - level understanding of how nps B-nanoparticle interact with cells B-material in a physiological environment . [SEP]
[CLS] a critical difference between well - controlled in vitro experiments and in vivo applications is the presence of a complex mixture of extracellular proteins B-material . [SEP]
[CLS] it has been established that extracellular serum proteins B-material present in blood will adsorb onto the surface of nps B-nanoparticle , forming a " protein B-material corona I-material " . [SEP]
[CLS] our goal was to understand how this protein B-material layer affected cellular - level events , including np B-nanoparticle binding , internalization , and transport . [SEP]
[CLS] a combination of microscopy B-technique , which provides spatial resolution , and spectroscopy B-technique , which provides molecular information , is necessary to probe protein−np−cell interactions . [SEP]
[CLS] initial experiments used a model system composed of polystyrene nps B-nanoparticle functionalized with either amine B-material or carboxylate B-material groups I-material to provide a cationic B-material or anionic surface , respectively . [SEP]
[CLS] serum proteins B-material adsorb onto the surface of both cationic B-material and anionic nps B-nanoparticle , forming a net anionic protein−np complex . [SEP]
[CLS] although these protein−np complexes have similar diameters and effective surface charges , they show the exact opposite behavior in terms of cellular binding . [SEP]
[CLS] in the presence of bovine serum albumin ( bsa ) , the cellular binding of bsa−np complexes formed from cationic B-material nps B-nanoparticle is enhanced , whereas the cellular binding of bsa−np complexes formed from anionic nps B-nanoparticle is inhibited . [SEP]
[CLS] these trends are independent of np B-nanoparticle diameter or cell B-material type . [SEP]
[CLS] similar results were obtained for anionic quantum B-nanoparticle dots I-nanoparticle and colloidal gold B-material nanospheres B-nanoparticle . [SEP]
[CLS] using competition assays , we determined that bsa−np complexes formed from anionic nps B-nanoparticle bind to albumin receptors on the cell B-material surface . [SEP]
[CLS] bsa−np complexes formed from cationic B-material nps B-nanoparticle are redirected to scavenger receptors . [SEP]
[CLS] the observation that similar nps B-nanoparticle with identical protein B-material corona I-material compositions bind to different cellular receptors suggested that a difference in the structure of the adsorbed protein B-material may be responsible for the differences in cellular binding of the protein−np complexes . [SEP]
[CLS] circular dichroism spectroscopy B-technique , isothermal titration calorimetry , and fluorescence B-technique spectroscopy I-technique show that the structure of bsa is altered following incubation B-technique with cationic B-material nps B-nanoparticle , but not anionic nps B-nanoparticle . [SEP]
[CLS] singleparticle - tracking fluorescence B-technique microscopy I-technique was used to follow the cellular internalization and transport of protein−np complexes . [SEP]
[CLS] the single particle - tracking experiments show that the protein B-material corona I-material remains bound to the np B-nanoparticle throughout endocytic uptake and transport . [SEP]
[CLS] the interaction of protein−np complexes with cells B-material is a challenging question , as the adsorbed protein B-material corona I-material controls the interaction of the np B-nanoparticle with the cell B-material ; however , the np B-nanoparticle itself alters the structure of the adsorbed protein B-material . [SEP]
[CLS] a combination of microscopy B-technique and spectroscopy B-technique is necessary to understand this complex interaction , enabling the rational design of nps B-nanoparticle for biological and medical applications . [SEP]
[CLS] nanoparticles B-nanoparticle ( nps B-nanoparticle ) are increasingly important for biological applications ranging from cellular imaging to drug delivery . [SEP]
[CLS] in these applications , nps B-nanoparticle encounter a complex mixture of cells B-material and extracellular proteins B-material . [SEP]
[CLS] for example , nps B-nanoparticle injected into the bloodstream are exposed to red and white blood cells B-material , clotting factors , and serum proteins B-material . [SEP]
[CLS] similarly , nps B-nanoparticle used for cellular experiments are exposed to the serum proteins B-material used as a nutrient source for cultured cells B-material . [SEP]
[CLS] serum consists of hundreds of distinct proteins B-material isolated from blood plasma following the removal of clotting factors . [SEP]
[CLS] these extracellular serum proteins B-material adsorb onto the np B-nanoparticle surface , forming a protein B-material " corona I-material " ( figure 1 ) . [SEP]
[CLS] poly ( ethylene glycol ) ( peg ) can reduce the adsorption of serum proteins B-material on nps B-nanoparticle , but complete inhibition of corona formation remains a challenge . [SEP]
[CLS] understanding the protein B-material corona I-material is crucial for understanding how nps B-nanoparticle interact with cells B-material , as the corona B-material proteins I-material control the specific cellular receptors used by the protein−np complex , the cellular internalization pathway , and even the immune response [SEP]
[CLS] ■ adsorption of proteins B-material on np B-nanoparticle surfaces : protein B-material corona I-material a I-material protein B-material corona I-material has been observed on a diverse range of nps B-nanoparticle , including polymeric nps B-nanoparticle , silica nps B-nanoparticle , quantum B-nanoparticle dots I-nanoparticle , [SEP]
[CLS] iron [SEP]
[CLS] oxide B-material nps B-nanoparticle , 38−40 silver B-material nanoclusters , [SEP]
[CLS] silver B-material nps B-nanoparticle , [SEP]
[CLS] gold B-material nanorods B-nanoparticle , [SEP]
[CLS] and gold B-material nps B-nanoparticle [SEP]
[CLS] for most nps B-nanoparticle , the corona is dominated by albumin , the most abundant protein B-material in serum ( 55 % ) . [SEP]
[CLS] 12−14 however , lowerabundance proteins B-material , such as immunoglobulins B-material , apolipoproteins , and fibrinogen , are also found in the corona , in some cases at higher concentrations than albumin despite their relatively low concentrations in plasma . [SEP]
[CLS] an " adsorbome " has been identified consisting of 125 plasma proteins B-material that have been detected on np B-nanoparticle surfaces . [SEP]
[CLS] the composition of corona B-material proteins I-material is dynamic ( figure 1 ) . [SEP]
[CLS] the " soft corona " that forms initially reflects the relative abundance of individual serum proteins B-material . [SEP]
[CLS] over time , weakly bound , low - affinity proteins B-material are displaced by high - affinity , tightly bound proteins B-material that comprise the " hard corona " . [SEP]
[CLS] within the payne lab , we have observed that albumin is the most abundant protein B-material adsorbed on polystyrene nps B-nanoparticle , semiconductor quantum B-nanoparticle dots I-nanoparticle , and colloidal gold B-material nps B-nanoparticle following exposure to serum proteins B-material . [SEP]
[CLS] we isolate the corona B-material proteins I-material by repeated centrifugation and resuspension in water B-material ( figure 2 ) using a method adapted from dawson et al . that is optimized for each type of np B-nanoparticle . [SEP]
[CLS] after each centrifugation step , the supernatant is loaded onto a polyacrylamide B-technique gel I-technique for electrophoresis B-technique . protein B-material in the supernatant is detected with a coomassie - like protein B-material stain . [SEP]
[CLS] after protein B-material is no longer detected in the supernatant , sodium B-material dodecyl sulfate ( sds ) , a detergent , is added to the protein−np pellet to solubilize any remaining protein B-material adsorbed on the np B-nanoparticle . [SEP]
[CLS] this mixture , which contains the hard corona B-material proteins I-material , is then loaded onto the gel . [SEP]
[CLS] within the mixture of fetal bovine serum ( fbs ) proteins B-material , the presence of albumin is indicated by a protein B-material band at 66 kda , the molecular weight of bovine serum albumin ( bsa ) . [SEP]
[CLS] we and others have observed that serum proteins B-material adsorb onto the surface of both cationic B-material and anionic nps B-nanoparticle . [SEP]
[CLS] our initial experiments used cationic B-material , aminemodified , polystyrene nps B-nanoparticle and anionic , carboxylate B-material - modified , polystyrene nps B-nanoparticle ( 40−200 nm , fluospheres , invitrogen ) as model nps B-nanoparticle with the same composition but opposite charge . [SEP]
[CLS] these nps B-nanoparticle are embedded with a yellow−green fluorophore for fluorescence B-technique microscopy I-technique and flow B-technique cytometry I-technique . [SEP]
[CLS] the diameter and effective surface charge of the nps B-nanoparticle was characterized within our lab ( nano - zs , malvern instruments ) . [SEP]
[CLS] fbs was used as a representative mixture of serum proteins B-material . [SEP]
[CLS] formation of a protein B-material corona I-material was confirmed by zeta B-property potential I-property measurements and gel B-technique electrophoresis I-technique . [SEP]
[CLS] importantly , after serum proteins B-material adsorb onto the surface , the cationic B-material and anionic nps B-nanoparticle are indistinguishable . [SEP]
[CLS] both are anionic ( figure 3a ) , and the main protein B-material adsorbed on the surface is bsa ( figure 3b ) . [SEP]
[CLS] the initially cationic B-material nps B-nanoparticle ( zeta B-property potential I-property = + 20 mv ) become anionic ( −19 mv ) following corona formation . [SEP]
[CLS] the anionic nps B-nanoparticle ( −31 mv ) show a slight increase in zeta B-property potential I-property ( −27 mv ) , reflecting the charge of the adsorbed albumin . [SEP]
[CLS] these results are in good agreement with previous work showing that although proteins B-material present in serum possess a net negative formation of a protein B-material corona I-material on 200 nm amine - modified polystyrene nps B-nanoparticle confirmed with sds - page . [SEP]
[CLS] nps B-nanoparticle ( 15 pm ) were incubated B-technique with fbs ( 10 % v / v ) for 10 min at 4 °c . [SEP]
[CLS] wash steps , consisting of repeated centrifugation ( 16 000g , 10 min ) , removal of supernatant , and resuspension in water B-material , were used to removed unbound proteins B-material from the protein−np complexes . [SEP]
[CLS] after each wash step , the supernatant ( s ) was loaded onto the gel . [SEP]
[CLS] s1 was diluted to 10 % v / v due to the high protein B-material concentration . [SEP]
[CLS] after five wash steps ( s5 ) , protein B-material is no longer visible in the supernatant . [SEP]
[CLS] sds was used to remove the protein B-material from the np B-nanoparticle surface ( np B-nanoparticle + sds ) . [SEP]
[CLS] as a control , incubation B-technique in water B-material does not remove the protein B-material corona I-material ( np B-nanoparticle + h 2 o ) . [SEP]
[CLS] fbs was run for comparison . [SEP]
[CLS] adapted from ref 24 . [SEP]
[CLS] copyright 2012 american chemical society . [SEP]
[CLS] charge , regions of positive and negative charge allow proteins B-material to form complexes with both cationic B-material and anionic nps B-nanoparticle , nanorods B-nanoparticle , and planar surfaces . [SEP]
[CLS] for example , serum albumin is net negatively charged at physiological ph with an isoelectric point at ph 4 . 7 , but it contains 60 positively charged lysine B-material groups . [SEP]
[CLS] ■ cellular binding of protein−np complexes although the complexes formed from cationic B-material and anionic polystyrene nps B-nanoparticle are indistinguishable in terms of charge and protein B-material corona I-material following incubation B-technique with fbs ( figure 3 ) , they have opposite trends in cellular binding ( figure 4 ) . [SEP]
[CLS] fluorescence B-technique microscopy I-technique shows that in the presence of 10 % fbs , the concentration of fbs typically used to culture cells B-material , the cellular binding of cationic B-material nps B-nanoparticle is increased . [SEP]
[CLS] in comparison , the cellular binding of anionic nps B-nanoparticle is decreased in the presence of fbs . [SEP]
[CLS] in both cases , it should be noted that the nps B-nanoparticle form a protein−np complex immediately following exposure to fbs . [SEP]
[CLS] these trends were observed for multiple np B-nanoparticle diameters ( 40− 200 nm ) and multiple cell B-material types ( monkey kidney epithelial cells B-material ( bs - c - 1 ) , human cervical cancer cells B-material ( hela ) , and chinese hamster ovary ( cho ) cells B-material ) . [SEP]
[CLS] using fbs , it is possible that a low - abundance protein B-material not visible in the gel is responsible for this difference in np B-nanoparticle binding . [SEP]
[CLS] for example , one protein B-material adsorbs B-property onto cationic B-material nps B-nanoparticle and enhances binding and a different protein B-material present in the mixture of fbs proteins B-material adsorbs B-property on anionic nps B-nanoparticle and inhibits binding . [SEP]
[CLS] to test this possibility , cellular binding experiments were repeated using only bsa ( ≥98 % purity , fisher ) . [SEP]
[CLS] this removes the possibility that a low - abundance protein B-material is responsible for the observed binding trends . [SEP]
[CLS] results with bsa were identical to those with fbs ( figure 5 ) . [SEP]
[CLS] competition assays were used to identify the cell B-material surface I-material receptor I-material used by the bsa−np complexes ( figure 6 ) . [SEP]
[CLS] cellular binding of complexes formed from anionic nps B-nanoparticle is inhibited by free bsa ( figure 5a ) , suggesting that competition for the bsa receptor B-material is responsible for the cellular binding and internalization of albumin . [SEP]
[CLS] this was tested using flow B-technique cytometry I-technique . [SEP]
[CLS] flow B-technique cytometry I-technique measures fluorescence B-property intensity per cell B-material in a highthroughput flow system . [SEP]
[CLS] although flow B-technique cytometry I-technique lacks spatial resolution , it has the advantage of measuring [UNK] 000 cells B-material / min . [SEP]
[CLS] using flow B-technique cytometry I-technique , we observed that increasing concentrations of bsa led to decreased binding of 93 nm carboxylate - modified polystyrene nps B-nanoparticle ( figure 6a ) . [SEP]
[CLS] at a bsa concentration of 10 mg • ml −1 , similar to the concentration of protein B-material used in cell B-material culture , np B-nanoparticle binding was reduced to 32 % in comparison to a normalized value of 100 % in the absence of bsa [SEP]
[CLS] this shows that bsa−nps formed from anionic polystyrene nps B-nanoparticle compete with free bsa for cellular receptors . [SEP]
[CLS] in comparison , bsa−nps formed from cationic B-material polystyrene nps B-nanoparticle show increased binding in the presence of free bsa ( figure 5b ) , indicating that a different cellular receptor B-material is used by these complexes . [SEP]
[CLS] a possible class of receptors for the bsa− nps B-nanoparticle formed from cationic B-material nps B-nanoparticle are scavenger receptors . [SEP]
[CLS] these cell B-material surface I-material receptors I-material bind disrupted albumin and have been identified previously in the cellular binding of oligonucleotidefunctionalized gold B-material nps B-nanoparticle . [SEP]
[CLS] to determine if scavenger receptors are the cellular binding site of the bsa−nps formed from cationic B-material nps B-nanoparticle , we used polyinosinic acid as a competitor . [SEP]
[CLS] this polyanionic molecule is a competitor for scavenger receptors . [SEP]
[CLS] if bsa−nps bind to scavenger receptors , then we expect the addition of polyinosinic acid to compete with the bsa−nps for binding sites on the cell B-material surface , thereby inhibiting the cellular binding of the nps B-nanoparticle . [SEP]
[CLS] the approach is identical to that used for the anionic nps B-nanoparticle , with polyinosinic acid rather than free bsa used as a competitor . [SEP]
[CLS] flow B-technique cytometry I-technique shows a decrease in cellular binding of 87 nm amine - modified polystyrene nps B-nanoparticle , with 100 % binding ( normalized ) decreased to 25 % in the presence of 2 . 5 mg • ml −1 polyinosinic acid ( figure 6b ) . [SEP]
[CLS] a control experiment with polyadenylic acid ( 2 . 5 mg • ml −1 ) , a similar molecule that does not compete for scavenger receptors , showed no significant competition with the 87 nm nps B-nanoparticle . [SEP]
[CLS] the fluorescence B-technique microscopy I-technique images and flow B-technique cytometry I-technique experiments show that the same protein B-material ( bsa ) adsorbed on two different nps B-nanoparticle ( cationic B-material and anionic polystyrene ) leads to binding of these protein−np complexes to two different cellular receptors , scavenger receptors or native albumin receptors . [SEP]
[CLS] differences in np−cell B-nanoparticle interactions have also been observed for cationic B-material and anionic polymer - modified gold B-material nps B-nanoparticle ( 10−16 nm ) , which show different rates of cellular uptake despite the formation of identical protein B-material coronas I-material . [SEP]
[CLS] we proposed that a difference in protein B-material structure following adsorption on the polystyrene np B-nanoparticle surface leads to this difference in cellular binding . [SEP]
[CLS] ■ secondary structure of corona B-material proteins I-material determines the cell B-material surface I-material receptor I-material circular dichroism ( cd ) spectroscopy B-technique was used to probe the structure of bsa following exposure to cationic B-material and anionic polystyrene nps B-nanoparticle . [SEP]
[CLS] cd spectroscopy B-technique utilizes a difference in the absorption of left and right circularly polarized light to probe protein B-material secondary structure . [SEP]
[CLS] cd spectroscopy B-technique showed that exposure to anionic nps B-nanoparticle did not perturb the secondary structure of bsa ( figure 7a ) . [SEP]
[CLS] isolated bsa has 65 % α - helix structure , calculated at 208 nm . [SEP]
[CLS] incubation B-technique of 60 and 200 nm anionic nps B-nanoparticle with bsa resulted in minimal changes to the percent α - helicity , 71 and 63 % , respectively . [SEP]
[CLS] in comparison , incubation B-technique of bsa with 58 and 200 nm cationic B-material nps B-nanoparticle led to a substantial change in α - helicity , 48 and 37 % , respectively . [SEP]
[CLS] differences in protein−np interactions for anionic and cationic B-material polystyrene nps B-nanoparticle are also observed in the thermodynamics of protein B-material adsorption on the np B-nanoparticle surface . [SEP]
[CLS] both isothermal titration calorimetry ( itc ) and fluorescence B-technique spectroscopy I-technique measure a greater equilibrium association constant for the adsorption of bsa on anionic nps B-nanoparticle ( table 1 ) . [SEP]
[CLS] isothermal titration calorimetry also showed a greater number of bsa molecules adsorbed on the anionic nps B-nanoparticle , with 230 % coverage on anionic 60 nm nps B-nanoparticle and 8 % coverage on cationic B-material 58 nm nps B-nanoparticle . [SEP]
[CLS] this value for cationic B-material nps B-nanoparticle is likely an underestimate because it assumes an end - on model that may not be appropriate for a denatured protein B-material and ignores aggregation that occurs for the cationic B-material nps B-nanoparticle under the buffer conditions necessary for calorimetry . [SEP]
[CLS] like itc , fluorescence B-technique spectroscopy I-technique showed a greater equilibrium association constant for the adsorption of bsa on anionic nps B-nanoparticle ( 1 . 8 ± 0 . 1 × 10 9 m −1 ) compared to that of cationic B-material nps B-nanoparticle ( 7 . 7 ± 0 . 1 × 10 8 m −1 ) ( figure 7b ) . [SEP]
[CLS] taken together , these results suggest that disrupted bsa on the surface of cationic B-material polystyrene nps B-nanoparticle causes the bsa−np complexes to bind to scavenger receptors . [SEP]
[CLS] it is also possible that adsorption of bsa on the np B-nanoparticle surface could expose new peptide B-material sequences . [SEP]
[CLS] these epitopes could then direct the protein−np complex to alternative receptors . [SEP]
[CLS] however , as we observe bsa denaturation with cd spectroscopy B-technique and binding to a scavenger receptor B-material known to bind disrupted bsa , it is likely that protein B-material disruption , rather than altered epitope exposure , is the main reason for the binding of bsa−np complexes formed from cationic B-material nps B-nanoparticle to scavenger receptors . [SEP]
[CLS] protein B-material adsorption on planar surfaces is known to alter structure and lead to partial denaturation . [SEP]
[CLS] 52 , 59−61 a similar disruption of protein B-material structure has been observed previously for nps B-nanoparticle . [SEP]
[CLS] in the case of albumin , disruption of secondary structure has been observed following adsorption to silver B-material nps B-nanoparticle , zinc B-material oxide I-material nps B-nanoparticle , gold B-material nps B-nanoparticle , and gold B-material nanorods B-nanoparticle . [SEP]
[CLS] structural changes have also been observed for lower abundance plasma proteins B-material including fibrinogen , 27 , 46 , 71 lysozyme , 72 cytochrome c , and chymotrypsin . [SEP]
[CLS] ■ corona B-material proteins I-material remain bound during np B-nanoparticle [SEP]
[CLS] the protein B-material corona I-material ultimately determines the cell B-material surface I-material receptors I-material used by the protein−np complex , as described above , and the subsequent cellular internalization of the np B-nanoparticle . [SEP]
[CLS] to monitor serum proteins B-material and nps B-nanoparticle during cellular internalization , we carried out two - color fluorescence B-technique microscopy I-technique single particle tracking experiments using fluorescently B-property labeled cationic B-material polystyrene nps B-nanoparticle ( green ) and serum proteins B-material ( red ) ( figure 8 ) . [SEP]
[CLS] serum proteins B-material and nps B-nanoparticle bind to the cell B-material as a single complex and remain bound for at least 18 h . [SEP]
[CLS] incubating B-technique cells B-material at 4 °c allows protein−np binding but inhibits internalization . [SEP]
[CLS] after warming the cells B-material to 37 °c , it is possible to track the internalization of the serum proteins B-material and nps B-nanoparticle simultaneously . [SEP]
[CLS] we find that bsa−nps are internalized as a single complex and remain colocalized as they are transported through the cell B-material . [SEP]
[CLS] transport is microtubule - dependent , indicative of endosomes or lysosomes undergoing active transport . [SEP]
[CLS] these experiments have two important implications . [SEP]
[CLS] first , corona B-material proteins I-material determine the cellular transport of nps B-nanoparticle , as they are not displaced during np B-nanoparticle interactions with cells B-material . [SEP]
[CLS] binding to two different cell B-material surface I-material receptors I-material suggests that the bsa−np complexes formed from cationic B-material and anionic polystyrene nps B-nanoparticle may use different endocytic pathways , with different rates , to reach the lysosomes . [SEP]
[CLS] second , proteins B-material remain bound as the np B-nanoparticle is internalized and transported through the cell B-material . [SEP]
[CLS] the use of nps B-nanoparticle in biology and medicine requires understanding the interactions among nps B-nanoparticle , proteins B-material , and cells B-material . [SEP]
[CLS] our experiments show that serum proteins B-material adsorb onto the surface of both cationic B-material and anionic nps B-nanoparticle . [SEP]
[CLS] protein B-material structure can be altered by adsorption on a surface , 52 , 59−61 including np B-nanoparticle surfaces . [SEP]
[CLS] for cationic B-material polystyrene nps B-nanoparticle , a change in the secondary structure of bsa redirects the protein−np complex to scavenger receptors . [SEP]
[CLS] in comparison , bsa adsorbed on anionic polystyrene nps B-nanoparticle retains its native structure , resulting in binding of bsa−nps to albumin receptors . [SEP]
[CLS] in the case of anionic nps B-nanoparticle , a similar trend was observed for carboxylatemodified quantum B-nanoparticle dots I-nanoparticle and citrate - modified colloidal gold B-material nps B-nanoparticle , despite the differences in np B-nanoparticle diameter , material B-material , and surface modification . [SEP]
[CLS] the protein B-material and np B-nanoparticle remain complexed during cellular internalization and transport . [SEP]
[CLS] these experiments illustrate the importance of serum protein B-material structure , not just composition , for the cellular binding , internalization , and transport of nps B-nanoparticle ( figure 9 ) . [SEP]
[CLS] moving forward , additional research is necessary to understand how the structure of other serum proteins B-material is affected by adsorption on nps B-nanoparticle , as each protein B-material will vary . [SEP]
[CLS] the number of experiments required to investigate each serum protein B-material and np B-nanoparticle of interest is intractable , making simulations necessary . [SEP]
[CLS] coarsegrained molecular dynamics ( md ) simulations have recently examined the structure of corona B-material proteins I-material . [SEP]
[CLS] a comparison of protein B-material structure , cd spectra , and md simulations can be used to predict how a specific protein B-material will be affected by adsorption on a np B-nanoparticle surface . [SEP]
[CLS] in addition to computational approaches , xray spectroscopy B-technique and small - angle neutron scattering will provide new and complementary molecular information . [SEP]
[CLS] our results have important implications for the design of nps B-nanoparticle to target specific populations of cells B-material or subcellular locations , a central goal for nanomedicine . [SEP]
[CLS] corona B-material proteins I-material have dedicated cell B-material surface I-material receptors I-material that can be used for the binding and internalization of protein−np complexes . [SEP]
[CLS] for successful targeting , the targeting B-material ligand I-material must have a greater affinity for its receptor B-material than the nonspecifically adsorbed serum proteins B-material have for their receptors . [SEP]
[CLS] it is likely that competition between targeting B-material ligands I-material and nonspecifically adsorbed serum proteins B-material is responsible for the challenges associated with in vivo np B-nanoparticle targeting . [SEP]
[CLS] for example , transferrin - functionalized silica nps B-nanoparticle bind to native transferrin receptors in vitro , but their targeting capabilities are masked by the adsorption of serum proteins B-material . [SEP]
[CLS] this highlights the importance of fundamental , molecular - level research to inform translational applications such as rationally designed nps B-nanoparticle for drug and gene delivery . [SEP]
[CLS] schematic of protein B-material corona I-material formation on a nanoparticle B-nanoparticle ( np B-nanoparticle ) surface . [SEP]
[CLS] protein B-material adsorption is a kinetic ( k ) and thermodynamic ( k ) function of both the individual proteins B-material and np B-nanoparticle properties such as surface modification , composition , and diameter . [SEP]
[CLS] initially , highabundance and / or high - mobility B-property proteins B-material bind to the nanoparticle B-nanoparticle surface . [SEP]
[CLS] over time , these proteins B-material are replaced by lower - mobility B-property proteins B-material with a higher binding affinity . [SEP]
[CLS] serum proteins B-material commonly observed in np B-nanoparticle coronas are shown as a representative corona : serum albumin , immunoglobulin B-material g1 I-material ( igg1 ) , alpha - 2 macroglobulin ( a2m ) , and apolipoprotein a - 1 ( apoa1 ) . [SEP]
[CLS] modified with permission from ref 19 . [SEP]
[CLS] copyright 2013 john wiley and sons . [SEP]
[CLS] formation of a protein B-material corona I-material on 200 nm amine - modified polystyrene nps B-nanoparticle confirmed with sds - page . [SEP]
[CLS] nps B-nanoparticle ( 15 pm ) were incubated B-technique with fbs ( 10 % v / v ) for 10 min at 4 °c . [SEP]
[CLS] wash steps , consisting of repeated centrifugation ( 16 000g , 10 min ) , removal of supernatant , and resuspension in water B-material , were used to removed unbound proteins B-material from the protein−np complexes . [SEP]
[CLS] after each wash step , the supernatant ( s ) was loaded onto the gel . [SEP]
[CLS] s1 was diluted to 10 % v / v due to the high protein B-material concentration . [SEP]
[CLS] after five wash steps ( s5 ) , protein B-material is no longer visible in the supernatant . [SEP]
[CLS] sds was used to remove the protein B-material from the np B-nanoparticle surface ( np B-nanoparticle + sds ) . [SEP]
[CLS] as a control , incubation B-technique in water B-material does not remove the protein B-material corona I-material ( np B-nanoparticle + h 2 o ) . [SEP]
[CLS] fbs was run for comparison . [SEP]
[CLS] adapted from ref 24 . [SEP]
[CLS] copyright 2012 american chemical society [SEP]
[CLS] cationic B-material and anionic nps B-nanoparticle form similar protein−np complexes . [SEP]
[CLS] ( a ) zeta B-property potential I-property of 200 nm polystyrene nps B-nanoparticle in water B-material and after incubation B-technique with minimum essential medium ( mem ) supplemented with 10 % fbs . [SEP]
[CLS] a series of five washes consisting of centrifugation ( 16 000g , 10 min ) and resuspension was used to remove unbound protein B-material . [SEP]
[CLS] adapted from ref 24 . [SEP]
[CLS] copyright 2012 american chemical society . [SEP]
[CLS] ( b ) gel B-technique electrophoresis I-technique of the washed 200 nm protein−np complexes . [SEP]
[CLS] sds was used to remove the protein B-material corona I-material from the nps B-nanoparticle . [SEP]
[CLS] bsa ( 66 kda ) was run for comparison . [SEP]
[CLS] fluorescence B-technique microscopy I-technique images show cellular binding of cationic B-material and anionic polystyrene nps B-nanoparticle ( green ) in mem and mem supplemented with fbs ( mem + 10 % fbs ) to monkey kidney epithelial cells B-material ( bs - c - 1 ) . [SEP]
[CLS] binding experiments were carried out at 4 °c to allow cellular binding but not internalization . [SEP]
[CLS] 75−77 nuclei are stained with dapi ( blue ) . [SEP]
[CLS] adapted from ref 24 . [SEP]
[CLS] copyright 2012 american chemical society [SEP]
[CLS] fluorescence B-technique microscopy I-technique images show cellular binding of cationic B-material and anionic polystyrene nps B-nanoparticle ( green ) in mem and mem supplemented with bsa ( mem + 10 mg • ml −1 bsa ) to monkey kidney epithelial cells B-material ( bs - c - 1 ) . [SEP]
[CLS] this concentration of bsa is approximately equal to the total protein B-material present in mem supplemented with 10 % fbs , shown in figure 4 . [SEP]
[CLS] binding experiments were carried out at 4 °c . [SEP]
[CLS] nuclei are stained with dapi ( blue ) . [SEP]
[CLS] ( a ) 93 nm carboxylate B-material - modified nps B-nanoparticle . [SEP]
[CLS] ( b ) 87 nm amine - modified nps B-nanoparticle . [SEP]
[CLS] adapted from ref 55 . [SEP]
[CLS] copyright 2014 american chemical society [SEP]
[CLS] identification of cell B-material surface I-material receptors I-material using cellular binding competition assays measured with flow B-technique cytometry I-technique . [SEP]
[CLS] ( a ) cellular binding of 93 nm anionic , carboxylate - modified polystyrene nps B-nanoparticle in mem with increasing concentrations of bsa . [SEP]
[CLS] ( b ) cellular binding of 87 nm cationic B-material , aminemodified polystyrene nps B-nanoparticle in mem supplemented with 10 mg • ml −1 bsa with increasing concentrations of polyinosinic acid , a competitor for scavenger receptors . [SEP]
[CLS] 22 , 56−58 adapted from ref 55 [SEP]
[CLS] copyright 2014 american chemical society [SEP]
[CLS] 7 . molecular properties of bsa after exposure to anionic and cationic B-material polystyrene nps B-nanoparticle . [SEP]
[CLS] ( a ) cd spectra of bsa in the presence of 60 nm carboxylate - modified nps B-nanoparticle ( red ) , in the presence of 58 nm amine - modified nps B-nanoparticle ( blue ) , and in the absence of nps B-nanoparticle ( black ) . [SEP]
[CLS] representative spectra are the average of 10 consecutive scans , smoothed with a savitzy−golay least - squares fit . [SEP]
[CLS] standard deviation from the 10 scans is shown by the shaded region of each line . [SEP]
[CLS] ( b ) stern−volmer plot of bsa quenching in the presence of 60 nm carboxylate - modified nps B-nanoparticle ( red ) and 58 nm amine - modified nps B-nanoparticle ( blue ) . [SEP]
[CLS] solid lines correspond to an exponential fit of the raw fluorescence B-property data . [SEP]
[CLS] dashed lines are a linear fit of the initial slope used to calculate an effective equilibrium constant . [SEP]
[CLS] error bars show the standard deviation from three experiments . [SEP]
[CLS] adapted from ref 55 . [SEP]
[CLS] copyright 2014 american chemical society [SEP]
[CLS] thermodynamic parameters of bsa adsorption on anionic and cationic B-material polystyrene nps B-nanoparticle measured with itc a np B-nanoparticle surface B-nanoparticle group k a ( 10 5 m −1 ) δh ( 10 4 kj • mol −1 ) mean and standard deviation from n = 3 ( cooh ) or n = 4 ( nh 2 ) measurements . [SEP]
[CLS] reprinted from ref 55 . [SEP]
[CLS] copyright 2014 american chemical society [SEP]
[CLS] 8 . cellular binding and internalization of protein−np complexes . [SEP]
[CLS] ( a ) fluorescence B-technique microscopy I-technique image shows bsa−np complexes formed from 87 nm amine - modified nps B-nanoparticle bound to bs - c - 1 cells B-material at 4 °c . [SEP]
[CLS] np B-nanoparticle fluorescence B-property appears green , protein B-material fluorescence B-property is red , and protein−np complexes are yellow as a result of colocalization . [SEP]
[CLS] nuclei are stained with dapi ( blue ) . [SEP]
[CLS] adapted from ref 23 by permission of the royal society of chemistry . [SEP]
[CLS] ( b ) single - particle trajectories of bsa and 87 nm amine - modified nps B-nanoparticle during and after internalization into bs - c - 1 cells B-material at 37 °c . [SEP]
[CLS] stars indicate the start of the trajectory . [SEP]
[CLS] adapted from ref 25 by permission of the royal society of chemistry . [SEP]
[CLS] mail : christine . payne @ chemistry . gatech . edu . [SEP]
[CLS] phone : ( 404 ) 385 - 3125 . fax : ( 404 ) 385 - 6057 . [SEP]
[CLS] present address § department of biomedical engineering , emory university , 1760 haygood drive , atlanta , georgia 30322 , united states . [SEP]
[CLS] notes the authors declare no competing financial interest . [SEP]
[CLS] biographies candace c . fleischer received b . s . ( 2005 ) and m . s . ( 2007 ) degrees in chemistry from western washington university . [SEP]
[CLS] her m . s . thesis was completed under the mentorship of professor steven r . emory . [SEP]
[CLS] she spent three years teaching prior to beginning the ph . d . program at georgia tech . [SEP]
[CLS] she earned a ph . d . in chemistry in 2014 under the mentorship of professor christine k . payne and is currently a postdoctoral researcher in the lab of professor xiaoping hu in the department of biomedical engineering at emory university . [SEP]
[CLS] christine k . payne received a b . s . ( 1998 ) in chemistry from the university of chicago . [SEP]
[CLS] she obtained a ph . d . ( 2003 ) in chemistry from the university of california , berkeley , under the mentorship of professor charles b . harris . [SEP]
[CLS] she spent three years in the department of chemistry and chemical biology at harvard university as a nih nrsa postdoctoral fellow with professor xiaowei zhuang . [SEP]
[CLS] she joined the faculty of the school of chemistry and biochemistry at georgia tech in 2007 . [SEP]
[CLS] her interests include nanoparticle−cell interactions , conducting polymer−cell interactions , cellular biophysics , and fluorescence B-technique microscopy I-technique . [SEP]
[CLS] acknowledgmentswe acknowledge the nih director ' s new innovator award ( 1dp2od006470 , c . k . p . ) and a u . s . doed molecular biophysics and biotechnology gaann fellowship ( p200a120190 , c . c . f . ) . [SEP]
[CLS] ■ references ( 1 ) de , m . ; ghosh , p . s . ; rotello , v . m . applications of nanoparticles B-nanoparticle in biology . [SEP]
[CLS] adv . mater . 2008 , 20 , 4225−4241 [SEP]
[CLS] schematic illustrating the importance of the structure of corona B-material proteins I-material on np−cell interactions . [SEP]
[CLS] bsa adsorbed on anionic polystyrene nps B-nanoparticle retains its native structure , allowing the bsa−np complexes to bind to native albumin receptors . [SEP]
[CLS] in comparison , bsa adsorbed on cationic B-material polystyrene nps B-nanoparticle is disrupted , causing the bsa− np B-nanoparticle complexes to bind to scavenger receptors . [SEP]
[CLS] corona B-material proteins I-material remain bound to the np B-nanoparticle throughout cellular internalization and transport . [SEP]
[CLS] resistance to the chemotherapeutic agent cisplatin B-material is a major limitation for the successful treatment of many cancers . [SEP]
[CLS] development of novel strategies to overcome intrinsic and acquired resistance to chemotherapy is of critical importance to effective treatment of ovarian cancer and other types of cancers . [SEP]
[CLS] we have sought to re - sensitize resistant ovarian cancer cells B-material to chemotherapy by co - delivering chemotherapeutics and pooled sirnas targeting multi - drug resistance ( mdr ) genes using self - assembled nanoscale coordination polymers B-material ( ncps ) . [SEP]
[CLS] in this work , ncp - 1 particles with trigger release properties were first constructed by linking cisplatin B-material prodrug - based bisphosphonate bridging ligands with zn 2 + metal B-material - connecting points and then coated with a cationic B-material lipid B-material layer , followed by the adsorption of pooled sirnas targeting three mdr genes including survivin , bcl - 2 , and p - glycoprotein via electrostatic interactions . [SEP]
[CLS] the resulting ncp - 1 / sirna particles promoted cellular uptake of cisplatin B-material and sirna and enabled efficient endosomal escape in cisplatin - resistant ovarian cancer cells B-material . [SEP]
[CLS] by down - regulating the expression of mdr genes , ncp - 1 / sirnas enhanced the chemotherapeutic efficacy as indicated by cell B-technique viability I-technique assay I-technique , dna ladder , and flow B-technique cytometry I-technique . [SEP]
[CLS] local administration of ncp - 1 / sirnas effectively reduced tumor B-material sizes of cisplatin - resistant skov - 3 subcutaneous xenografts . [SEP]
[CLS] this work shows that the ncp - 1 / sirna platform holds great promise in enhancing chemotherapeutic efficacy for the effective treatment of drug - resistant cancers . [SEP]
[CLS] ovarian cancer is the fifth most prevalent cancer among women in the united states , with a lifetime risk of 1 . 4 % - 1 . 8 % . [SEP]
[CLS] development of multi - drug resistance ( mdr ) in ovarian cancer cells B-material is one of the major obstacles for effective chemotherapy . [SEP]
[CLS] after repeated treatment with anticancer B-property chemotherapeutic agents , tumor B-material cells B-material develop strategies to increase their resistance to chemotherapy to avoid programmed death by activating antiapoptotic pathways . [SEP]
[CLS] although a combination of chemotherapeutic agents are often used to prevent drug resistance in cancer patients , the ability of the cancer cells B-material to adapt and develop one or more drug resistance pathways ultimately leads to failure in the treatment of ovarian and other cancers . [SEP]
[CLS] in ovarian cancer , the majority of patients ( 50 - 75 % ) have recurrent and incurable cancer after their successful initial treatment with platinum B-material drug or paclitaxel B-material . [SEP]
[CLS] the development of therapeutic strategies to overcome drug resistance will have a great impact on the treatment of ovarian cancer . [SEP]
[CLS] small interfering rna ( sirna ) has the ability to disrupt cellular mdr pathways by silencing relevant gene expressions , opening the door for re - sensitizing the cancer cells B-material which have acquired resistance to anticancer B-property drugs . [SEP]
[CLS] however , drug resistance often involves multiple and dynamically acquired mdr mechanisms as a result of the overexpression of drug efflux pumps ( e . g . , p - glycoprotein , p - gp or mdr1 ) ; multidrug resistance protein B-material , mrp , anti - apoptotic proteins B-material ( e . g . , bcl - 2 , survivin ) , oncogenes ( e . g . , c - myc ) , and regulators of drug metabolism ( e . g . , pregnane x receptor B-material , pxr ) . [SEP]
[CLS] p - gp is overexpressed in the malignant tissues and becomes an attractive target to overcome mdr . [SEP]
[CLS] bcl - 2 is responsible for the activation of cellular anti - apoptotic defense . [SEP]
[CLS] survivin has a functional role in caspase inhibition to lead to negative regulation of apoptosis B-event , and is upregulated in most human tumors B-material , making it a potential target for cancer treatment . [SEP]
[CLS] we hypothesized that delivering pooled sirnas , including sip - gp , sibcl - 2 , and sisurvivin , targeting different molecular signaling pathways would be an effective approach to overcoming drug resistance in cancers . [SEP]
[CLS] nanoparticulate delivery systems have been shown to improve therapeutic efficacy of anticancer B-property chemotherapeutics by enhancing drug delivery to tumors B-material and thereby reducing general toxicity B-property through the enhanced permeability and retention ( epr ) effect . [SEP]
[CLS] however , free nucleic B-material acid I-material drugs , such as sirnas , cannot be used for cancer treatment in vivo due to their ready degradation by nucleases . [SEP]
[CLS] nanoparticles B-nanoparticle have been shown to provide protection to sirnas and mediate efficient gene silencing in cancer cells B-material . [SEP]
[CLS] codelivery of anticancer B-property chemotherapeutics and sirnas targeting mdr genes in the same nanoparticle B-nanoparticle can drastically enhance therapeutic efficacy by overcoming drug resistance in cancer cells B-material . [SEP]
[CLS] several nanoparticle B-nanoparticle systems , including liposomes B-nanoparticle , carbon B-nanoparticle nanotubes I-nanoparticle , and polymer B-material micelles B-material , have recently been used to co - deliver sirna and cisplatin B-material to cancer cells B-material both in vitro and in vivo in order to enhance therapeutic responses . [SEP]
[CLS] herein we report a novel self - assembled nanoscale coordination polymer B-material ( ncp ) system for the co - delivery of cisplatin B-material and pooled sirnas to cisplatin - resistant ovarian cancer cells B-material ( es - 2 , ovcar - 3 , skov - 3 , and a2780 / cddp cells B-material ) to overcome drug resistance by resensitizing the cells B-material to cisplatin B-material treatment . [SEP]
[CLS] ncps are self - assembled from metal B-material ions B-material and organic bridging ligands , which have numerous advantages over conventional drug delivery systems such as high drug loadings , tunable compositions , shapes , and sizes , and readily modifiable surface . [SEP]
[CLS] our group has developed a series of ncps as potential delivery systems for chemotherapeutics . [SEP]
[CLS] in particular , we recently reported the self - assembly of zinc B-material bisphosphonate ncps that contain 48 wt % cisplatin B-material prodrug by linking the cisplatin B-material prodrug cis , cis , trans - [ pt ( nh3 ) 2 cl 2 ( oconhp ( o ) ( oh ) 2 ) 2 ] with zn 2 + ions B-material . [SEP]
[CLS] upon pegylation with the 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ( dopc ) / cholesterol / 1 , 2distearoyl - sn - glycero - 3 - phosphoethanolamine - n - [ amino ( polyethylene glycol ) - 2000 ] ( dspe - peg2k ) coating B-material , these ncps showed much enhanced stability in the blood circulation and possessed superior anticancer B-property efficacy in multiple non - resistant murine tumor B-material xenograft models compared to free drugs . [SEP]
[CLS] in this study , we used cationic B-material lipid B-material 1 , 2 - dioleoyl - 3 - trimethylammonium - propane ( dotap ) instead of dopc to coat B-material ncp - 1 to yield positively charged particles which strongly bind to negatively charged sirnas via electrostatic interactions to afford sirna - encapsulated ncp - 1 particles ( ncp - 1 / sirnas ) ( scheme 1 ) . [SEP]
[CLS] we examined sirna protection , sirna release profiles , and cell B-material uptake and mdr gene silencing efficiency of ncp - 1 / sirnas in ovarian cancer cells B-material . [SEP]
[CLS] we evaluated the in vitro anticancer B-property effect of ncp - 1 / sirnas by cell B-technique viability I-technique assay I-technique , dna ladder , annexin v staining , and flow B-technique cytometry I-technique in cisplatin - resistant ovarian cancer cells B-material . [SEP]
[CLS] finally , we demonstrated the significantly enhanced chemotherapeutic efficacy of ncp - 1 / sirnas in skov - 3 tumor bearing mice . [SEP]
[CLS] all of the starting materials were purchased from sigma - aldrich and fisher ( usa ) , unless otherwise noted , and used without further purification . [SEP]
[CLS] 1 , 2 - dioleoyl - sn - glycero - 3 - phosphate ( dopa ) , 1 , 2 - dioleoyl - 3 - trimethylammonium - propane ( dotap ) , cholesterol , and 1 , 2distearoyl - sn - glycero - 3 - phosphoethanolamine - n - [ amino ( polyethylene glycol ) 2000 ] ( dspe - peg2k ) were purchased from avanti polar lipids B-material ( usa ) . [SEP]
[CLS] the sirna duplexes were supplied by dharmacon ( usa ) and dissolved in diethylpyrocarbonate ( depc ) - treated water B-material before use . [SEP]
[CLS] survivin sirna ( sisurvivin ) , bcl - 2 sirna ( sibcl - 2 ) , and p - gp sirna ( sip - gp ) contained the anti - sense sequences of 5 ' - ggaccaccgcaucucuacadtdt - 3 ' , 5 ' - uucggcauuaggccuuccgdtdg - 3 ' , and 5 ' - agcttataatggatgtact - 3 ' , respectively . [SEP]
[CLS] tamra - labeled survivin sirna was bought from dharmacon ( usa ) and used for quantification and confocal B-technique laser I-technique scanning I-technique microscopy I-technique ( clsm ) . [SEP]
[CLS] three kinds of human ovarian cancer cells B-material , es - 2 , ovcar - 3 , and skov - 3 cells B-material and murine macrophage raw 264 . 7 cells B-material [SEP]
[CLS] were from the american type culture collection ( rockville , md , usa ) . [SEP]
[CLS] es - 2 and skov - 3 cells B-material were cultured in mccoy ' s 5a medium containing 10 % fetal bovine serum ( fbs ) . [SEP]
[CLS] ovcar - 3 cells B-material were cultured in rpmi - 1640 medium ( gibco , grand island , ny , usa ) containing 10 % fbs . [SEP]
[CLS] raw 264 . 7 cells B-material were cultured in dmem medium containing 10 % fbs . [SEP]
[CLS] cisplatin B-material - sensitive and - resistant human ovarian cancer cells B-material a2780 and a2780 / cddp were obtained from developmental therapeutics core B-material , northwestern university and were cultured in rpmi 1640 containing 10 % fbs . [SEP]
[CLS] athymic female nude mice ( 8 weeks , 20 - 25 g ) were provided by harlan laboratories , inc . ( usa ) . [SEP]
[CLS] the study protocol was reviewed and approved by the institutional animal care and use committee ( iacuc ) at the university of chicago . [SEP]
[CLS] ncp - 1 nanoparticles B-nanoparticle were prepared according to our previous report with modifications . [SEP]
[CLS] briefly , the cisplatin B-material prodrug , cis , cis , trans - [ pt ( nh 3 ) 2 cl 2 ( oconhp ( o ) ( oh ) 2 ) 2 ] , was synthesized following our previous procedures . [SEP]
[CLS] two hundred microliters of 25 mg / ml cisplatin B-material prodrug solution and 0 . 2 ml of 100 mg / ml zn ( no 3 ) 2 aqueous solution were added to 5 ml of 0 . 3 m triton x - 100 / 1 . 5 m 1 - hexanol in cyclohexane mixture , respectively , to form w = 7 . 4 microemulsions . [SEP]
[CLS] two hundred microliter of dopa ( 200 mg / ml in chcl 3 ) was added to the cisplatin B-material prodrug microemulsion and the stirring was continued for 15 min until a clear solution formed . [SEP]
[CLS] the two microemulsions were combined , and the resultant 10 ml of microemulsion was stirred for 30 additional minutes to yield dopa - coated ncp particles , which were then washed with cyclohexane and ethanol to remove excess dopa , and dispersed in thf . [SEP]
[CLS] the cationic B-material lipid coated ncp - 1 was prepared by adding a thf solution of dotap , cholesterol ( molar ratio of dotap / cholesterol = 2 : 1 ) , 20 mol % dspe - peg2k , and dopacoated ncp to 30 % ( v / v ) ethanol / water B-material at 50 °c . [SEP]
[CLS] thf and ethanol were completely evaporated and the ncp - 1 solution was allowed to cool down to room temperature . [SEP]
[CLS] the ncp - 1 was centrifuged at 13000 rpm for 30 min followed by the removal of the supernatant and re - suspending the nanoparticle B-nanoparticle precipitate in depc - treated water B-material . [SEP]
[CLS] the control nanoparticles B-nanoparticle ( zn control ) were prepared with the same method except that sodium B-material pyrophosphate decahydrate was used instead of cisplatin B-material prodrug to form the ncps . [SEP]
[CLS] sisurvivin , sibcl - 2 , and sip - gp were dissolved in depc - treated water B-material at weight ratio of 1 : 1 : 1 to achieve 2 mg / ml pooled sirna solution . [SEP]
[CLS] cationic B-material lipid coated ncp - 1 ( 2 mg / ml ) was mixed with sirna solution ( 2 mg / ml ) at weight ratio of cisplatin B-material : sirna = 4 : 1 , and the mixture was stirred at 800 rpm and at room temperature for 30 min to allow the adsorption of negatively charged sirna onto positively charged ncp - 1 surface . [SEP]
[CLS] inductively coupled plasma - mass spectrometry ( icp - ms , agilent technologies , usa ) was utilized to analyze the pt concentration of ncp to calculate the cisplatin B-material loading B-property efficiency I-property . [SEP]
[CLS] the particle size and zeta B-property potential I-property of ncp - 1 and ncp - 1 / sirnas in phosphate buffered solution ( pbs ) were determined with zetasizer ( nano zs , malvern , uk ) . [SEP]
[CLS] transmission electron micrsocopy ( tem , jem 100cx - ii , joel , japan ) was used to observe the morphology of ncp - 1 and ncp - 1 / sirnas . [SEP]
[CLS] the in vitro stability of ncp - 1 / sirnas in terms of particle size and polydispersity index ( pdi ) was evaluated in phosphate buffer saline ( pbs ) supplemented with 5 mg / ml bovin serum albumin ( bsa ) . [SEP]
[CLS] ncp - 1 / sirna ( 1 mg / ml ) was incubated B-technique with pbs containing 5 mg / ml bsa at 37 °c for 12 h , and the particle size and pdi were monitored during the 12 - h incubation B-technique by dls . [SEP]
[CLS] the association of sirna with ncp - 1 was first determined with gel retardation assay on 4 % ( w / v ) agarose B-technique gel I-technique electrophoresis I-technique containing 0 . 25 µg / ml of eb . [SEP]
[CLS] we also quantitatively determined the encapsulation B-property efficiency I-property ( ee ) of sirna onto ncp - 1 by fluorimetry . [SEP]
[CLS] tamra - labeled sirna was encapsulated into ncp - 1 and the nanoparticle B-nanoparticle suspension was centrifuged at 13 , 000 rpm for 30 min . [SEP]
[CLS] the amount of free tamra - sirna in the supernatant was determined with fluorimetry based on the standard curve ( tamra , λ ex = 565 nm , λ em = 580 nm ) . [SEP]
[CLS] ee was calculated from the following equation : [SEP]
[CLS] where w 0 and w 1 stand for the content of total sirna and free sirna in the supernatant , respectively . [SEP]
[CLS] ncp - 1 / sirnas containing 1 µg of sirna was mixed with equal volume of fbs . [SEP]
[CLS] after incubation B-technique at 37 °c for a pre - determined time , the mixture was heated at 80 °c for 5 min to inactivate the nucleases and disrupt the ncp - 1 structure . [SEP]
[CLS] thus , the sirna was dissociated from ncp - 1 / sirna , and its integrity was subsequently evaluated on 4 % ( w / v ) agarose B-technique gel I-technique electrophoresis I-technique . [SEP]
[CLS] a free sirna solution containing 1 µg of sirna served as control . [SEP]
[CLS] image j was used to quantify the intensity of each sirna migration band in order to give the quantitative sirna degradation curve . [SEP]
[CLS] the intensity of the band representing sirna control ( containing 1 µg of sirna ) served as 100 % intensity . [SEP]
[CLS] as for the evaluation of sirna release profiles from ncp - 1 / sirnas , nanoparticles B-nanoparticle containing 1 µg of tamra - sirna were incubated B-technique with 1 ml of pbs at 37 °c with shaking . [SEP]
[CLS] at pre - determined time intervals , the suspension was centrifuged at 13 , 000 rpm for 10 min and 0 . 5 ml of the supernatant was quantified for the tamra - sirna content by fluorimetry . [SEP]
[CLS] an equal volume of the release medium was added , and the precipitate was resuspended before further incubation B-technique . [SEP]
[CLS] the release profiles of cisplatin B-material from the lipid coated ncp - 1 and ncp - 1 / sirnas were investigated . [SEP]
[CLS] the release profiles were performed in 400 ml of 6 . 7 mm pbs buffer at 37°c . [SEP]
[CLS] the ncp - 1 ( 3 mg ) was suspended in 5 ml of 6 . 7 mm pbs buffer in a 10 , 000 mwco pleated dialysis bag . [SEP]
[CLS] the dialysis bag containing nanoparticle B-nanoparticle suspension was then put in the beaker followed by the addition of 400 ml of 5 mm pbs buffer , and the system was incubated B-technique at 37 °c under stirring . [SEP]
[CLS] periodically , 1 ml aliquots of the solution were removed , and 1 ml of fresh buffer solution was added to the beaker . [SEP]
[CLS] the removed aliquots were collected and analyzed by icp - ms for pt content . [SEP]
[CLS] for the experiments carried out under reducing environments , 6 . 7 mm pbs supplemented with 5mm cysteine B-material was used for the release profiles . [SEP]
[CLS] three kinds of ovarian cancer cell B-material lines including es - 2 , ovcar - 3 , and skov - 3 cells B-material were seeded on a 24 - well plate at 1×10 5 cells B-material per well and cultured for 24 h . tamra - sirnacontaining ncp - 1 / sirna and free tamra - sirna solution ( 2 mg / ml ) were added ( 0 . 4 µg sirna / well ) . [SEP]
[CLS] following a 4 - h incubation B-technique , cells B-material were washed with pbs three times and then lysed with 0 . 5 % ( w / v ) sodium B-material dodecyl sulfate ( sds , ph 8 . 0 ) . [SEP]
[CLS] the lysate was quantified for tamra - sirna by fluorimetry and protein B-material content by the bca kit ( promega , usa ) . [SEP]
[CLS] uptake level was expressed as the amount of tamra - sirna associated with 1 mg of cellular protein B-material . [SEP]
[CLS] in order to determine the amount of pt internalized by the skov - 3 cells B-material , the cells were collected and centrifuged at 3000 rpm 5 min after incubating B-technique with free cisplatin B-material , ncp - 1 , and ncp - 1 / sirnas for 4 h or 24 h , respectively . [SEP]
[CLS] the empty centrifuge tubes were first weighed and their weights were recorded as w 1 . [SEP]
[CLS] the cell B-material suspension was added to the tubes followed by centrifugation . [SEP]
[CLS] the supernatant was discarded and the cell B-material pellet in the tubes was completely dried . [SEP]
[CLS] then the tubes containing cell B-material pellet were weighed again and their weights were recorded as w 2 . [SEP]
[CLS] the weight of the cell B-material pellet was obtained by subtracting w 1 from w 2 . [SEP]
[CLS] the cell B-material pellets were then digested in concentrated nitric acid for 24 h . [SEP]
[CLS] the amount of internalized pt was determined by icp - ms . [SEP]
[CLS] a time dependent study of endosomal escape was performed by incubating B-technique ncp - 1 / sirnas with skov - 3 cells B-material for 10 , 30 , 60 , 90 , and 120 min followed by lysotracker green ( 100 nm ) and dapi ( 10 µg / ml ) staining . [SEP]
[CLS] the time - dependent co - localization of ncp - 1 / sirnas and endosome / lysosome was observed under clsm ( olympus fv1000 , japan ) , and the colocalization efficiency was quantitatively determined using image j ( co - localization threshold ) based on the clsm images . [SEP]
[CLS] es - 2 , ovcar - 3 , and skov - 3 cells B-material were seeded at 2×10 5 cells B-material per well in 24 - well plates and further cultured for 24 h . [SEP]
[CLS] the culture media were replaced by 1 ml of pre - warmed and fresh culture media containing 10 % fetal bovine serum ( fbs ) prior to the experiment . [SEP]
[CLS] ncp - 1 / sirnas , ncp - 1 / sibcl - 2 , ncp - 1 / sip - gp , ncp - 1 / sisurvivin , zn control / sirnas , and ncp - 1 were added to the cells B-material at a sirna dose of 30 nm , corresponding to the cisplatin B-material dose of 1 . 6 µg per well . [SEP]
[CLS] following incubation B-technique for 4 h , the culture media were replaced by pre - warmed and fresh culture media containing 10 % fbs , and additional 20 h of incubation B-technique was allowed . [SEP]
[CLS] the supernatant of the culture media was collected for the determination of extracellular survivin and p - gp production by elisa ( r & d systems , usa ; mybiosource , usa ) following manufacturer ' s instructions . [SEP]
[CLS] the cells B-material were lysed , and the bcl - 2 amount in the lysate was quantified by elisa ( r & d systems , usa ) . [SEP]
[CLS] dose - dependent in vitro transfection was carried out on skov - 3 cells B-material , and the gene silencing efficiency mediated by ncp - 1 / sirnas was compared with commercialized transfection reagent lipofectamine rnaimax ( life technology , usa ) . [SEP]
[CLS] in addition , rna was isolated from the transfected skov - 3 cells B-material according to the trizol reagent protocol ( invitrogen , usa ) , and cdna was synthesized from 500 ng of total rna using primescript ® rt reagent kit ( takara biotechnology co . ltd ) according to the manufacturer ' s instructions . [SEP]
[CLS] synthesized cdna , forward and reverse primers , and the sybr premix ex [UNK] ( takara biotech . co . , ltd . ) were run on the cfx96 real - time pcr detection system ( bio - rad , usa ) for the evaluation of cellular bcl - 2 , survivin , and pgp mrna levels . [SEP]
[CLS] sequences of the primers used were designed with primer bank ( supporting information table s1 ) . [SEP]
[CLS] β - actin was used as an internal loading control . [SEP]
[CLS] es - 2 , ovcar - 3 , and skov - 3 cells B-material were seeded at 5000 cells B-material per well while a2780 and a2780 / cddp cells B-material were seeded at 2500 cells B-material per well in 96 - well plates and further cultured for 24 h . [SEP]
[CLS] the culture media were replaced by 100 µl of fresh culture media containing 10 % fbs . [SEP]
[CLS] cisplatin B-material solution , ncp - 1 , ncp - 1 / sirnas , ncp - 1 / sibcl - 2 , ncp - 1 / sip - gp , ncp - 1 / sisurvivin , and zn control / sirnas were added to the cells B-material at different cisplatin B-material or sirna dose . [SEP]
[CLS] following incubation B-technique for 72 h , the cell B-property viability I-property was determined by ( 3 - ( 4 , 5dimethylthiazol - 2 - yl ) - 5 - ( 3 - carboxymethoxyphenyl ) - 2 - ( 4 - sulfophenyl ) - 2h - tetrazolium ) ( mts ) assay ( promega , usa ) according to manufacture instructions . [SEP]
[CLS] the concentrations of cisplatin B-material and sirna required to inhibit cell B-material growth by 50 % ( ic 50 values ) were calculated . [SEP]
[CLS] 2 . 9 . [SEP]
[CLS] apoptosis B-event [SEP] B-event
[CLS] 2 . 9 . 1 [SEP]
[CLS] dna ladder - es - 2 , ovcar - 3 , and skov - 3 cells B-material were seeded at 1×10 6 cells B-material per well in 6 - well plates and further cultured for 24 h . [SEP]
[CLS] the culture media were replaced by 2 ml of fresh culture media containing 10 % fbs . [SEP]
[CLS] ncp - 1 and ncp - 1 / sirnas were added to the cells B-material at a cisplatin B-material concentration of ic 80 . [SEP]
[CLS] following incubation B-technique for 24 h , total dna of cancer cells B-material was extracted using dna ladder isolation kit ( invitrogen , usa ) according to the manufacture instructions and examined for dna fragmentation on a 2 % ( w / v ) agarose B-technique gel I-technique electrophoresis I-technique at 35 v for 5 h . [SEP]
[CLS] 2 . 9 . 2 annexin v staining - coverslips putting in the 6 - well plates were seeded with es - 2 , ovcar - 3 , and skov - 3 cells B-material at the density of 1×10 6 cells B-material per well . [SEP]
[CLS] the cells B-material were incubated B-technique at 37°c and 5 % co 2 for 24 h prior to nanoparticle B-nanoparticle treatment . [SEP]
[CLS] tamra - sirna loaded ncp - 1 / sirnas were incubated B-technique with cells B-material at 37°c and 5 % co 2 for 24 h . [SEP]
[CLS] then , the cells B-material were washed with pbs , fixed with iced 4 % paraformaldehyde , and stained with 10 µg / ml of dapi and alexa fluor 488 conjugated annexin v ( invitrogen , usa ) according to the manufacturer ' s instructions . [SEP]
[CLS] the cells B-material were observed using confocal B-technique laser I-technique scanning I-technique microscopy I-technique ( clsm , zeiss lsm710 , german ) at excitation wavelength of 405 nm , 488 nm , and 546 nm to visualize nuclei ( blue fluorescence B-property ) , cell B-material apoptosis B-event ( green fluorescence ) and nanoparticle B-nanoparticle internalization ( red fluorescence B-property ) , respectively . [SEP]
[CLS] 2 . 9 . 3 . [SEP]
[CLS] flow B-technique cytometry I-technique - skov - 3 cells B-material were seeded at 1×10 6 cells B-material per well in 6 - well plates and further cultured for 24 h . [SEP]
[CLS] the culture media were replaced by 2 ml of fresh culture media containing 10 % fbs . [SEP]
[CLS] free cisplatin B-material solution , zn control , zn control / sirnas , ncp - 1 , ncp - 1 / sirnas , ncp - 1 / sibcl - 2 , ncp - 1 / sip - gp , and ncp - 1 / sisurvivin were added to the cells B-material , respectively , at cisplatin B-material concentration of 5 µm or equivalent nanoparticle B-nanoparticle concentration of 20 µg / ml . [SEP]
[CLS] cells B-material incubated B-technique with saline served as control . [SEP]
[CLS] following incubation B-technique for 24 h , the floating and adherent cells B-material were collected by cell B-material scraper and stained with alexa fluor 488 annexin v / dead cell B-material apoptosis B-event kit with alexa fluor 488 annexin v and pi ( invitrogen , usa ) according to the manufacturer ' s instructions . [SEP]
[CLS] the apoptosis B-event was examined on a flow cytometer ( lsrii blue , bd , usa ) . [SEP]
[CLS] skov - 3 cells B-material or raw 264 . 7 cells B-material were seeded at 2×10 5 cells B-material per well in 24 - well plates and further cultured for 24 h . [SEP]
[CLS] the culture media were replaced by 1 ml of fresh culture media containing 10 % fbs prior to the experiment . [SEP]
[CLS] ncp - 1 and ncp - 1 / sirnas were added to the cells B-material at a sirna dose of 0 . 4 µg ( 30 nm ) per well , corresponding to the cisplatin B-material dose of 1 . 6 µg per well . [SEP]
[CLS] following incubation B-technique for 72 h , the supernatant of the culture media was collected for the determination of tnf - α , il - 6 , and ifn - γ by elisa ( r & d systems , usa ) following manufacturer ' s instructions . [SEP]
[CLS] cells B-material treated with saline served as controls . [SEP]
[CLS] tumor bearing mice were established by subcutaneous inoculation of skov - 3 cell B-material suspension ( 5×10 6 cells B-material per mouse ) into the right flank region of 8 - week athymic female nude mice . [SEP]
[CLS] after the tumor B-material volume reached approximately 100 mm 3 , the mice were randomly divided into 5 groups ( n = 6 ) and intratumorally injected with pbs , free cisplatin B-material plus free pooled sirna solution , ncp - 1 , zn control / sirnas , and ncp - 1 / sirnas at equivalent cisplatin B-material dose of 1 mg / kg and sirna dose of 0 . 25 mg / kg once every week ( total three injections ) . [SEP]
[CLS] tumor B-material volumes and body weights were monitored three times every week . [SEP]
[CLS] tumor B-material volumes were calculated as follows : ( width 2 × length ) / 2 . [SEP]
[CLS] finally , all mice were sacrificed 19 days after last injection , and the excised tumors B-material were weighed . [SEP]
[CLS] the mrna expression levels and protein B-material productions of bcl - 2 , p - gp , and survivin in the tumor B-material were evaluated by realtime - pcr and elisa , respectively . [SEP]
[CLS] one hundred microgram of tumor B-material was homogenized with radioimmunoprecipitation assay buffer ( ripa buffer ) and then centrifugated at 12 , 000 rpm for 15 min at 4 °c . [SEP]
[CLS] the amounts of bcl - 2 , p - gp , and survivin in the supernatant were measured by elisa and normalized with total protein B-material content determined using the bca kit . [SEP]
[CLS] another 100 µg of tumor B-material was homogenized in liquid nitrogen B-material , and the rna in the tumor B-material tissues was extracted with the trizol reagent and the intracellular bcl - 2 , survivin , and p - gp mrna levels were thereafter monitored by realtime - pcr . [SEP]
[CLS] tdt - mediated dutp nick end labeling ( tunel ) reaction was performed on 5 - µm frozen tumor B-material sections using dna fragmentation detection kit ( life technology , usa ) according to the manufacturer ' s instructions and observed clsm . [SEP]
[CLS] dna fragment in apoptotic cells B-material was stained with fluorescein - conjugated deoxynucleotides ( green ) and the nuclei were stained with dapi ( 10 µg / ml ) . [SEP]
[CLS] the percentage of apoptotic cells B-material was determined by the number ratio of tunel - positive cells B-material / total cells by image j . [SEP]
[CLS] blood was collected at the endpoint of in vivo antitumor efficacy experiment , and the serum was separated . [SEP]
[CLS] the serum concentrations of tnf - α , ifn - γ , and il - 6 were detected by elisa ( r & d systems , usa ) to evaluate the immunogenic B-property response evoked by ncp - 1 / sirnas . [SEP]
[CLS] the plasma concentration of ige was determined by elisa ( r & d systems , usa ) to check the induction of hypersensitivity by ncp - 1 / sirnas . [SEP]
[CLS] liver , lungs , spleen , and kidneys were also excised after the mice were sacrificed , and then fixed with formalin . [SEP]
[CLS] paraffin - embedded 5 µm tissue sections were stained with hematoxylin and erosin ( h & e ) and observed for toxicity B-property with light microscopy B-technique . [SEP]
[CLS] ncp particles containing a cisplatin B-material prodrug , cis , cis , trans - [ pt ( nh 3 ) 2 cl 2 ( oconhp ( o ) ( oh ) 2 ) 2 ] , were synthesized according to our previous report , and further coated with dotap , cholesterol ( molar ratio of dotap / cholesterol = 2 : 1 ) , and 20 mol % dspe - peg2k to afford cationic B-material ncp - 1 . [SEP]
[CLS] transmission B-technique electron I-technique microscopy I-technique ( tem ) images indicated the formation of spherical particles of ncp - 1 coated with cationic B-material lipid B-material dotap ( figure 1a ) . [SEP]
[CLS] dynamic B-technique light I-technique scattering I-technique ( dls ) measurements showed that the diameter , polydispersity index ( pdi ) , and surface charge of ncp - 1 were 134 . 2 ± 3 . 4 nm , 0 . 076 ± 0 . 013 , and 16 . 3 ± 2 . 6 mv , respectively . [SEP]
[CLS] the cisplatin B-material loading was determined to be 25 . 8±2 . 2 wt . % by inductively coupled plasma - mass spectrometry ( icp - ms ) . [SEP]
[CLS] after complete removal of extra lipids B-material and free liposomes B-nanoparticle by centrifugation , pooled sirnas ( sisurvivin , sibcl - 2 , and sip - gp ) were directly adsorbed onto the surface of ncp - 1 through electrostatic interactions . [SEP]
[CLS] sisurvivin , sibcl - 2 , and sip - gp contained the anti - sense sequences of 5 ' - ggaccaccgcaucucuacadtdt - 3 ' , 5 ' - uucggcauua - ggccuuccgdtdg - 3 ' , and 5 ' - agcttataatggatgtact - 3 ' , respectively . [SEP]
[CLS] transmission B-technique electron I-technique microscopy I-technique ( tem ) images showed the presence of monodisperse spherical particles of ~ 30 nm in diameter for ncp - 1 / sirnas ( figure 1b ) . [SEP]
[CLS] particle size , pdi , and surface charge of ncp - 1 / sirnas were determined to be 156 . 3 ± 6 . 7 nm , 0 . 087 ± 0 . 021 , and −3 . 1 ± 0 . 5 mv , respectively , by dls . [SEP]
[CLS] the bigger particle size given by dls compared to tem is attributed to the lipid B-material layer and sirnas on the particle surface as well as the hydrogel nature of the ncp - 1 core B-material . [SEP]
[CLS] the slightly increased particle size and negative charge of ncp - 1 / sirnas are consistent with the successful sirna loading . [SEP]
[CLS] zn control particles loaded with sirna was synthesized under similar conditions , and their size , pdi , and surface charge were 144 . 2 ± 2 . 4 nm , 0 . 102 ± 0 . 022 , and −2 . 9 ± 0 . 4 mv , respectively . [SEP]
[CLS] ncp - 1 / sirnas also exhibited enhanced stability as manifested by constant particle size and pdi in serum - containing phosphate buffer saline ( pbs ) over a period of 12 h ( figure s1 , si ) . [SEP]
[CLS] gel B-technique electrophoresis I-technique indicated complete capture of sirnas by ncp - 1 at a cisplatin B-material / sirna weight ratio of 4 ( figure s2 , si ) . [SEP]
[CLS] we also quantitatively examined the sirna encapsulation B-property efficiency I-property ( ee ) by fluorimetry . [SEP]
[CLS] fluorescence - labeled sirnas was used to form ncp - 1 / sirnas , and the ee was determined to be 91 . 2 ± 4 . 9 % . [SEP]
[CLS] successful sirna delivery requires both sufficient protection of the cargo from nuclease degradation and its efficient release to the cell B-material cytoplasm as a large number of intact sirna molecules are required for mrna recognition and rna interference ( rnai ) in the cytoplasm . [SEP]
[CLS] ncp - 1 / sirnas efficiently protected sirnas from nuclease degradation : upon incubation B-technique with serum for up to 4 h migration bands of ncp - 1 / sirnas were clearly observed ( figure 1c ) . [SEP]
[CLS] in contrast , the migration bands of free sirna solution quickly faded with time due to the degradation of sirnas . [SEP]
[CLS] the rate of sirna degradation was estimated by measuring the intensity of sirna bands with image j . after 2 h of incubation B-technique , less than 20 % the free sirna remained intact while more than 80 % of sirna was detected for ncp - 1 / sirna ( figure s3 , si ) . [SEP]
[CLS] after 4 h of incubation B-technique , ~ 12 . 5 % of the free sirna remained in solution while 56 . 2 % of the sirna loaded onto ncp - 1 was detected ( figure s3 , si ) . [SEP]
[CLS] upon loading onto the ncp - 1 surface , sirnas are likely lying flat to increase electrostatic interactions with dotap molecules and are shielded by dspe - peg2k molecules from nuclease binding and degradation . [SEP]
[CLS] on the other hand , ncp - 1 / sirnas effectively released sirnas in pbs with a complete sirna release after 24 h ( figure s4 , si ) , suggesting that sufficient amount of sirna could be released into the cytoplasm once ncp - 1 / sirnas were internalized by cells B-material . [SEP]
[CLS] the gradual release of sirnas from the ncp - 1 surface is believed to account for the slow degradation of sirnas after incubation B-technique with serum over time . [SEP]
[CLS] at the same time , the cisplatin B-material could be released from the ncp - 1 upon entering the cells B-material triggered by the higher intracellular cysteine B-material and glutathione concentrations ( figure s5 , si ) . [SEP]
[CLS] after sirna loading , the cisplatin B-material release was slightly retarded ( figure s5 , si ) , which might be due to the fact that the sirna layer further retarded the penetration of cysteine B-material through the lipid B-material bilayer I-material . [SEP]
[CLS] we evaluated the ability of ncp - 1 / sirnas to deliver cisplatin B-material and sirnas to different ovarian cancer cells B-material . [SEP]
[CLS] compared to the free sirna solution , sirna uptake of ncp - 1 / sirnas was significantly enhanced ( figure 2a ) , indicating that ncp - 1 / sirnas could assist in the sirna internalization . [SEP]
[CLS] the sirna uptake was also directly observed using confocal B-technique laser I-technique scanning I-technique microscopy I-technique ( clsm ) . [SEP]
[CLS] large amounts of sirna ( red fluorescence B-property ) were located in the cytoplasm of all three ovarian cancer cell B-material lines ( figure s6 , si ) . [SEP]
[CLS] we believe that cationic B-material dotap and optimal particle size of ncp - 1 / sirnas were beneficial to sirna uptake . [SEP]
[CLS] the cisplatin B-material internalization was also promoted by the ncp - 1 / sirnas ( figure 2b ) , which might be ascribed to the decreased nanoparticle B-nanoparticle / drug efflux pump by down - regulating the p - gp expression . [SEP]
[CLS] besides high sirna uptake levels , successful endo / lysosomal escape is also required for efficient sirna - mediated gene silencing . [SEP]
[CLS] we have used clsm to study the sirna endosomal escape process . [SEP]
[CLS] as shown in fig 2d , the extent of co - localization between the sirna fluorescence B-property and that of lysotracker rapidly decreased over time . [SEP]
[CLS] after a 2 - h incubation B-technique , the majority of sirna ( ~ 70 % ) encapsulated in the ncp - 1 / sirnas has escaped from endo / lysosome compartments , as demonstrated by the steady decrease of colocalization between red fluorescence B-property from the sirna and green fluorescence from lysotracker green ( that tracks endo / lysosome membranes ) over the 2 - h period ( figure 2c & d , figure s7 & s8 , si ) . [SEP]
[CLS] the enhanced sirna release from endo / lysosomes by ncp - 1 / sirnas is likely mediated by the ion B-material - pair formation between the positively charged groups of dotap and the negatively charged groups of endosome membrane . [SEP]
[CLS] clustered ion B-material - pairs are known to de - stabilize both the endosome membrane and the cationic B-material lipid coated vector . [SEP]
[CLS] upon overcoming the several barriers B-property to gene transfection , including sirna encapsulation , protection , release , cell B-material internalization , and endosomal escape , ncp - 1 / sirnas evoked potent gene silencing in terms of mrna expression and protein B-material production in ovarian cancer cells B-material ( figure s9 , si and figure 3 ) as determined by realtime - pcr and enzymelinked immunosorbent assays ( elisa ) , respectively . [SEP]
[CLS] we compared the gene knockdown efficiency of ncp - 1 / pooled sirnas to those of ncp - 1 / individual sirna in order to determine if pooled sirnas work synergistically to downregulate the expression of relevant proteins B-material responsible for drug resistance . [SEP]
[CLS] pooled sirnas contain one - third of each individual sirna in the ncp - 1 / sirnas , with the same total sirna dose as that of ncp - 1 / individual sirna . [SEP]
[CLS] the specific gene knockdown efficiency mediated by ncp - 1 loaded pooled sirnas and individual sirnas as essentially identical , suggesting that pooled sirnas likely elicit synergistic effects in silencing multiple genes . [SEP]
[CLS] in addition , zn control / sirnas were also capable of down - regulating gene expression ( figure 3 ) . [SEP]
[CLS] the slightly decreased survivin and bcl - 2 expression levels in ncp - 1 might be attributed to the cytotoxicity B-property induced by cisplatin B-material incorporated in the nanoparticles B-nanoparticle that influenced the expression levels of tumor B-material growth relevant genes including survivin and bcl - 2 . [SEP]
[CLS] the gene transfection B-property efficiency I-property mediated by ncp - 1 / sirnas was also compared with commercialized lipofecatmine rnaimax ( lipo ) . [SEP]
[CLS] as shown in figure 3d - f , the gene silencing efficiency of ncp - 1 / sirnas was comparable to lipo at the recommended sirna dose of 3 nm . when the sirna dose was reduced to 0 . 75 nm , ncp - 1 / sirnas was significantly more potent than lipo . [SEP]
[CLS] additionally , ncp - 1 / sirnas showed prolonged gene silencing with transfection B-property efficiency I-property of up to 50 % in 3 days , suggesting their preferable stability and high potency ( figure s10 , si ) . [SEP]
[CLS] to further examine whether the efficient and simultaneous suppression of survivin , bcl - 2 , and p - gp can effectively increase the chemotherapy efficacy of cisplatin B-material , the cytotoxicity B-property of free cisplatin B-material , ncp - 1 , zn control / sirnas , and ncp - 1 / sirnas was assessed . [SEP]
[CLS] by the codelivery of cisplatin B-material and pooled sirnas , all four cisplatin - resistant ovarian cancer cell B-material lines could be re - sensitized by ncp - 1 / sirnas , as evidenced by the drastically decreased cisplatin B-material ic 50 ( the dose of a drug required for 50 % inhibition ) values compared to either free cisplatin B-material or ncp - 1 ( table 1 , figure s11 - s13 & s15 , si ) . [SEP]
[CLS] in es - 2 , ovcar - 3 , skov - 3 , and a2780 / cddp cells B-material , the cisplatin B-material ic 50 of ncp - 1 / sirnas showed a 102 - , 7 - , 140 - , and 16 - fold decrease compared to ncp - 1 , respectively . [SEP]
[CLS] ncp - 1 / individual sirna treatment was only slightly more potent than ncp - 1 with the exception of ncp - 1 / sisurvivin on skov - 3 cells B-material ( with a 21 - fold decrease in ic 50 when compared to ncp - 1 ) ; the ic 50 values for ncp - 1 / individual sirna samples were only up to 2 . 6 times lower than that of ncp - 1 . [SEP]
[CLS] even the ic 50 of ncp - 1 / sisurvivin on skov - 3 cells B-material is 6 . 5 times higher than than of ncp - 1 / sirnas . [SEP]
[CLS] these results indicate that ncp - 1 / sirnas are much more potent than ncp - 1 / individual sirna , consistent with the more effective gene knockdown as discussed earlier . [SEP]
[CLS] in cisplatin - sensitive a2780 cell B-material , free cisplatin B-material , ncp - 1 , and ncp - 1 / sirnas evoked similar cytotoxicity B-property ( table 1 , figure s14 , si ) . [SEP]
[CLS] therefore , we believe that delivering pooled sirnas to silence the expression of multiple mdr genes simultaneously is more effective at overcoming drug resistance than targeting a single specific gene . [SEP]
[CLS] importantly , ncp - 1 / sirnas were able to enhance the anticancer B-property efficacy in different types of ovarian cancer cells B-material : es - 2 is originated from clear cell ovarian carcinoma ; ovcar - 3 is from serous adenocarcinoma ; skov - 3 , a2780 , and a2780 / cddp are from adenocarcinoma . [SEP]
[CLS] we also evaluated the cytotoxicity B-property of zn control / sirnas at the sirna doses corresponding to cisplatin B-material ic 50 values . [SEP]
[CLS] no obvious differences were observed between the cell B-property viability I-property of zn control / sirnas and control ( 88 . 3±2 . 1 % , 89 . 4±3 . 1 % , 94 . 2±5 . 6 % , 102 . 9±4 . 5 % , and 89 . 4±10 . 2 % for es - 2 , ovcar - 3 , skov - 3 , a2780 , and a2780 / cddp , respectively ) , indicating that the drastically elevated anticancer B-property efficacy of ncp - 1 / sirnas results from the synergy between gene regulation by pooled sirnas and chemotherapeutic effects of cisplatin B-material . [SEP]
[CLS] the cell B-material apoptosis B-event induced by ncp - 1 / sirnas was analyzed by annexin v conjugate staining , dna ladder , and flow B-technique cytometry I-technique . [SEP]
[CLS] as shown in figure 4a , ovarian cancer cells B-material were stained with annexin v - fitc conjugate after incubating B-technique with ncp - 1 / sirnas for 24 h , indicating that the nanoparticles B-nanoparticle successfully induced cancer cell B-material apoptosis B-event . [SEP]
[CLS] the presence of the characteristic dna ladder in the ncp - 1 / sirnas rather than ncp - 1 indicated that codelivery of cisplatin B-material and pooled sirna successfully induce cell B-material apoptosis B-event in cisplatinresistant cells B-material by silencing the mdr gene expression ( figure 4b ) . [SEP]
[CLS] as shown in figure s16 ( si ) , no apoptosis B-event was observed in the zn control group after a 24 - h incubation B-technique by flow B-technique cytometry I-technique . [SEP]
[CLS] healthy cell B-material percentages of ncp - 1 , ncp - 1 / sibcl - 2 , ncp - 1 / sip - gp , ncp - 1 / sisurvivin , ncp - 1 / sirnas , zn control / sirnas , and free cisplatin B-material solution were determined to be 79 . 8 % , 49 . 1 % , 51 . 9 % , 39 . 4 % , 25 . 2 % , 94 . 8 % , and 67 . 0 % , respectively , indicating that ncp - 1 / sirnas was the most potent in inducing apoptosis B-event ( figure 4c , figure s16 , table s2 , si ) . [SEP]
[CLS] additionally , ncp - 1 / sirnas evoked no immunogenic B-property response in raw 264 . 7 macrophage and skov - 3 cells B-material ( figure s17 , si ) , indicating the lack of immuno - toxicity B-property of ncp - 1 / sirnas . [SEP]
[CLS] local delivery of chemotherapeutic drugs is an efficient strategy for achieving high drug doses at the target site while minimizing systemic exposure . [SEP]
[CLS] encouraged by significantly enhanced in vitro anticancer B-property efficacy , we evaluated the in vivo antitumor effect of ncp - 1 / sirnas in cisplatin - resistant skov - 3 subcutaneous xenografts . [SEP]
[CLS] as depicted in figure 5 , no antitumor efficacy was observed for free cisplatin B-material ( 1 mg / kg dose ) plus free pooled sirnas ( 0 . 25 mg / kg dose ) , ncp - 1 ( 1 mg / kg dose ) , and zn control / sirnas ( 0 . 25 mg / kg dose ) , of which the p values were 0 . 8311 , 0 . 1502 , 0 . 8594 compared to control by two - tail t - test , respectively . [SEP]
[CLS] in contrast , ncp - 1 / sirnas at the same cisplatin B-material and / or sirna doses exhibited remarkable tumor B-material regression ; the average tumor B-material size decreased from 97±6 to 38±20 mm 3 ( p = 0 . 0010 vs . control ) . [SEP]
[CLS] compared to the control group , the mrna expression levels of bcl - 2 , survivin , and p - gp in the tumors B-material of mice receiving local injection of ncp - 1 / sirnas were significantly reduced by 58 . 8 % , 51 . 7 % , and 48 . 7 % , respectively ( figure s18 ) . [SEP]
[CLS] accordingly , the bcl - 2 , p - gp , and survivin protein B-material production of tumors B-material treated with ncp - 1 / sirnas were down - regulated by 74 % , 48 % , and 84 % , respectively , in comparison to the control ( figure 5d ) . [SEP]
[CLS] the significant knockdown of bcl - 2 , p - gp , and survivin in the tumor B-material site presumably sensitized the tumor B-material cells B-material towards cisplatin B-material treatment , leading to the much enhanced antitumor effect by the co - delivery of cisplatin B-material and sirnas . [SEP]
[CLS] the tunel assay showed that the fluorescence B-property intensity of dna fragmentation and the relative percentage of apoptotic cells B-material in the ncp - 1 / sirnas group were higher than those in the other groups , indicating their superior anticancer B-property efficacy ( figure 5c & d ) . [SEP]
[CLS] no significant body weight loss or immunogenic B-property response was observed after repeated treatment with ncp - 1 / sirnas ( figure s19 - s20 , si ) . [SEP]
[CLS] we also performed histological analysis of the kidneys , livers , spleens , and lungs of mice treated with ncp - 1 / sirnas . [SEP]
[CLS] no cast formation , swelling , desquamation , tubular damage or microvillus disappearance was noted in the kidney , indicating that ncp - 1 / sirnas would not induce nephrotoxicity which is a major concern of cisplatin B-material toxicity B-property ( figure s21 ) . [SEP]
[CLS] in addition , ncp - 1 / sirnas treatment did not induce haemorrhages or dystrophy of hepatocytes in liver , increase the number of lymphoid follicles in spleen , or cause inflammatory cell B-material infiltration in lungs ( figure s21 ) . [SEP]
[CLS] these results suggested the safety of ncp - 1 / sirnas when applied in vivo . [SEP]
[CLS] we have reported the first effort of utilizing self - assembled nanoscale coordination polymers B-material as an efficient vehicle B-material to simultaneously deliver cisplatin B-material and pooled sirnas to cisplatin - resistant ovarian cancer cells B-material . [SEP]
[CLS] our results demonstrated that ncp - 1 / sirnas could mediate effective gene silencing in cisplatin - resistant ovarian cancer cells B-material to overcome mdr and re - sensitize the cells B-material to cisplatin B-material treatment . [SEP]
[CLS] consequently , the co - delivery of cisplatin B-material and pooled sirnas drastically enhanced the in vivo chemotherapeutic effects in cisplatinresistant skov - 3 ovarian cancer mouse model . [SEP]
[CLS] the versatility of the ncp delivery platform should allow its further optimization for potential clinical translation to treat late stage , resistant cancers . [SEP]
[CLS] schematic showing the compositions of ncp - 1 / sirnas . [SEP]
[CLS] upon entering cancer cells B-material , the intracellular reducing environment will trigger the cisplatin B-material release via reductive degradation of ncp - 1 . [SEP]
[CLS] cisplatin B-material ic 50 ( µm ) in es - 2 , ovcar - 3 , skov - 3 , a2780 and a2780 / cddp cells B-material after a 72 - h incubation B-technique . [SEP]
[CLS] 1 . tem images of ncp - 1 coated with dotap ( a ) and ncp - 1 / sirnas ( b ) showing the spherical , mono - dispersed , and well - defined morphology . [SEP]
[CLS] ( c ) enhanced serum stability of sirnas that were loaded into ncp - 1 as evaluated by electrophoresis B-technique . [SEP]
[CLS] 2 . ( a ) cellular uptake amount of sirna in ovarian cancer cells B-material after incubating B-technique for 4 h ( n = 3 ) . [SEP]
[CLS] ( b ) cellular uptake amount of pt in skov - 3 cells B-material after incubating B-technique for 4 h and 24 h ( n = 3 ) . [SEP]
[CLS] ( c ) time - dependent endosomal escape of ncp - 1 / sirnas ( tamra - labeled , red fluorescence B-property ) in skov - 3 cells B-material . [SEP]
[CLS] endosome / lysosome and nuclei were stained with lysotracker green and dapi , respectively . [SEP]
[CLS] bar represented 20 µm . [SEP]
[CLS] ( d ) percent co - localization of sirna and endosome / lysosome quantified by image j based on ( c ) . [SEP]
[CLS] survivin ( a ) , bcl - 2 ( b ) , and p - gp ( c ) relative expression levels in ovarian cancer cells B-material transfected with ncp - 1 , ncp - 1 / sirnas , ncp - 1 / sisurvivin , ncp - 1 / sibcl - 2 , ncp - 1 / sip - gp , and zn control / sirnas at an sirna concentration of 30 nm ( n = 3 ) . [SEP]
[CLS] ( d ) - ( f ) dose - dependent transfection B-property efficiency I-property mediated by ncp - 1 / sirnas and lipofecatamine rnaimax / sirnas ( lipo / sirnas ) in skov - 3 cells B-material ( n = 3 ) . [SEP]
[CLS] ( a ) clsm images showing cell B-material apoptosis B-event and sirna internalization in es - 2 , ovcar - 3 , and skov - 3 cells B-material after incubation B-technique with ncp - 1 / sirnas for 24 h . the sirna was labeled with tamra ( red fluorescence B-property ) . the cells B-material were stained with annexin v fitc conjugate and the nuclei were stained with dapi . [SEP]
[CLS] bar represented 20 µm . [SEP]
[CLS] ( b ) analysis of dna ladder on 2 % ( w / v ) agarose gel at 35 v for 5 h after dna extraction from the ovarian cancers treated with ncp - 1 or ncp - 1 / sirna . [SEP]
[CLS] m : dna marker ; 1 : control ; 2 : ncp - 1 ; 3 : ncp - 1 / sirnas . [SEP]
[CLS] ( c ) annexin v / pi analysis of skov - 3 cells B-material after the incubation B-technique with saline [SEP]
[CLS] 5 . in vivo anticancer B-property effects of ncp - 1 / sirnas on skov - 3 subcutaneous xenograft murine models . [SEP]
[CLS] ( a ) tumor B-material growth curves of the mice receiving intratumoral injections ( n = 6 ) . [SEP]
[CLS] ( b ) photograph of the excised tumors B-material on day 33 . [SEP]
[CLS] 1 : control ; 2 : free cisplatin B-material + free sirnas ; 3 : ncp - 1 ; 4 : zn control / sirnas ; 5 : ncp - 1 / sirnas . [SEP]
[CLS] ( c ) weights of the excised tumors B-material on day 33 . [SEP]
[CLS] indicated values were mean ± sd ( n = 6 ) . [SEP]
[CLS] the p values of free cisplatin B-material plus free sirnas , ncp - 1 , zn control / sirnas , and ncp - 1 / sirnas compared to control were 0 . 9159 , 0 . 0538 , 0 . 6493 , and 0 . 0001 , respectively , by two - tail t - test . [SEP]
[CLS] ( d ) the protein B-material expression levels of [SEP]
[CLS] author manuscript ; available in pmc 2016 january 01 . [SEP]
[CLS] in this review , we summarize recent progresses in the application of synchrotron - based spectroscopic techniques for nucleic B-material acid I-material research that takes advantage of high - flux and highbrilliance electromagnetic radiation from synchrotron sources . [SEP]
[CLS] the first section of the review focuses on the characterization of the structure and folding processes of nucleic B-material acids I-material using different types of synchrotron - based spectroscopies B-technique , such as x - ray absorption B-technique spectroscopy I-technique , x - ray emission B-technique spectroscopy I-technique , x - ray photoelectron spectroscopy B-technique , synchrotron radiation circular dichroism , x - ray footprinting and small - angle x - ray scattering . [SEP]
[CLS] in the second section , the characterization of nucleic acid - based nanostructures , nucleic acid - functionalized nanomaterials B-material and nucleic acid - lipid B-material interactions using these spectroscopic techniques is summarized . [SEP]
[CLS] insights gained from these studies are described and future directions of this field are also discussed . [SEP]
[CLS] the history of synchrotrons can be traced back to 1873 , when james clerk maxwell came up with the theory of electromagnetism that changed our understanding of light . [SEP]
[CLS] the main form of light generated by synchrotrons , x - ray , was discovered by wilhelm rontgen in 1895 when he found that running a high - voltage discharge tube , enclosed in thick black cardboard , could cause the plate covered with barium platinocyanide to glow , even if it was two meters away from the discharge tube . [SEP]
[CLS] since the discovery of this radiation , x - ray has enjoyed its fast development in physical , medical , and biological research . [SEP]
[CLS] to expand the field of applications even further to include areas such as high energy physics , scientists and engineers around the world have designed and realized many ways to accelerate particles , including synchrotron radiation facilities that provide researchers with extremely high - flux electromagnetic radiation , at broad energy ranges from the infrared through the ultraviolet and into the x - ray region . [SEP]
[CLS] synchrotron light sources have enabled tremendous breakthroughs in physics , chemistry and biology , especially structural biology . [SEP]
[CLS] thousands of biomolecule structures are deposited in the protein B-material data bank every year , and more than 80 % of them have been determined with the use of synchrotron - based methods . [SEP]
[CLS] a major class of biomolecules is nucleic B-material acids I-material , which play central roles in the storage and transfer of genetic information . [SEP]
[CLS] even though nucleic B-material acids I-material were first discovered in 1869 by johannes friedrich miescher , the structure of nucleic B-material acids I-material remained a mystery until x - ray diffractograms of dna crystals were recorded by rosalind franklin and the double - helix model was proposed by watson and crick in 1953 . [SEP]
[CLS] the characterization of the double helical structure of dna paved the way for explaining how genetic information is stored and then copied to the next generation . [SEP]
[CLS] with the discoveries of various types of rna molecules with distinct functions , such as messenger rna , transfer rna , non - coding rna , small interfering rna , ribozyme and ribosome , it was revealed that rna molecules can not only serve as a template for protein B-material synthesis , but also form complex three - dimensional ( 3d ) structures for a wide range of functions , including protein B-material synthesis , enzymatic reactions and gene regulation . [SEP]
[CLS] high - resolution structures of these rna molecules play a key role in furthering our understanding of the structural features and mechanisms behind these functions . [SEP]
[CLS] in addition to the dna and rna molecules discovered in nature , systematic evolution of ligands by exponential enrichment ( selex ) , or in vitro selection , has been used to obtain rna or dna molecules in test tubes that can either bind numerous molecules selectively ( called aptamers ) , or can catalyze specific reactions ( called ribozymes for catalytic rna or deoxyribozymes or dnazymes for catalytic dna ) . [SEP]
[CLS] many of these aptamers , ribozymes and dnazymes have been transformed into sensors based on either fluorescence B-property , colorimetry or electrochemistry . [SEP]
[CLS] more recently , due to the intrinsic programmability of dna caused by precise base - pairing with complementary strands , dna molecules are emerging as promising candidates to be used in various areas in nanotechnology , such as dna - directed self - assembly of colloidal nanoparticles B-nanoparticle , dna origami , microchips and dna - based computation . [SEP]
[CLS] given the wide variety of dna and rna molecules and their diverse functions , it is important to characterize their structures in order to understand them . [SEP]
[CLS] it is not surprising that synchrotron - based techniques , such as x - ray spectroscopy B-technique , x - ray footprinting , and small - angle x - ray scattering play key roles in achieving this goal ( figure 1 ) . [SEP]
[CLS] herein , we summarize the applications of synchrotron - based spectroscopic techniques used to characterize nucleic B-material acids I-material . [SEP]
[CLS] instead of giving a comprehensive review on numerous highresolution crystal structures of nucleic B-material acids I-material obtained using a synchrotron light source , we emphasize the different types of synchrotron - based spectroscopic studies which helped to elucidate the properties of nucleic B-material acids I-material , including electronic structures , folding pathways , overall 3d nanostructures , and crystal lattices in the dna - directed self - assembly of nanoparticles B-nanoparticle . [SEP]
[CLS] synchrotron facilities can provide electromagnetic radiation ranging from infrared to x - ray with high brightness , high collimation and wide tunability . [SEP]
[CLS] these properties make synchrotron radiation an ideal light source for a number of spectroscopic applications . [SEP]
[CLS] among different synchrotron - based techniques , x - ray absorption B-technique spectroscopy I-technique ( xas ) , x - ray emission B-technique spectroscopy I-technique ( xes ) , x - ray photoelectron spectroscopy B-technique ( xps ) and synchrotron radiation circular dichroism ( srcd ) are widely used in characterizing conformations and electronic structures of nucleic B-material acids I-material . [SEP]
[CLS] the fundamental principle of xas is based on the ability of x - ray to excite core B-material electrons from an atom B-material ( figure 2 ) . [SEP]
[CLS] x - rays are often described in terms of the energy they carry , which can range from less than 1 kev to greater than 100 kev . [SEP]
[CLS] the x - ray with energies above 10 kev is often referred to as the hard x - ray , while the lower energy x - ray is referred to as the soft x - ray . [SEP]
[CLS] when the absorption coefficient of a given element is measured over a range of excitation energies , a sharp increase in the absorption coefficient at a certain energy level can be observed . [SEP]
[CLS] such an absorption increase is referred to as the absorption edge , which occurs when the core B-material electron absorbs energy equal to or greater than its binding energy . [SEP]
[CLS] the naming of the edges depends on which shell B-material the core B-material electron is ejected from , with the principal quantum numbers n = 1 , 2 , and 3 corresponding to the k - , l - , and m - edges , respectively . [SEP]
[CLS] from the edge to about 50 ev above the edge is the x - ray absorption near - edge structure ( xanes ) region , while an extended x - ray absorption fine structure ( exafs ) region extends to approximately 1 kev above the edge ( figure 1 ) [SEP]
[CLS] near edge x - ray absorption fine structure ( nexafs ) deals with analysis of soft x - ray 1s xanes spectra and is more relevant for nucleic B-material acid I-material research . [SEP]
[CLS] analyses of pre - edge , edge , xanes and exafs regions reveal information about the electronic structures and the local metal B-material coordination information of the samples . [SEP]
[CLS] as the x - ray excitation creates a hole in the core B-material , an outer - shell B-material electron can relax and emit a photon . [SEP]
[CLS] xes analyzes the energy and intensity of emitted photons generated during electron rearrangement , thereby revealing the electronic structures of the samples . [SEP]
[CLS] xps , on the other hand , studies energy distribution of electrons emitted from x - rayirradiated compounds . [SEP]
[CLS] from this measurement , element composition , chemical formula , and the electronic state of materials can be deduced . [SEP]
[CLS] dna molecules have been used in constructing various nano - devices as molecular wires , and therefore their electronic properties are essential for the design and optimization of these devices . [SEP]
[CLS] in nature , dna contains four nucleobases , adenine ( a ) , guanine ( g ) , cytosine ( c ) , and thymine ( t ) . [SEP]
[CLS] to gain a better understanding of the electronic properties of dna molecules , the chemical structures of nucleobases have been extensively characterized using xas and xes . [SEP]
[CLS] by determining the transitions from the inner - shell atomic orbitals to virtual molecular orbitals , information about the electronic structures of a particular element of interest in a nucleobase can be readily obtained . [SEP]
[CLS] as nucleobases are composed of light atoms B-material , mainly carbon B-material , nitrogen B-material and oxygen B-material , with low electron binding energies ( 284 ev for carbon B-material , 410 ev for nitrogen B-material and 543 ev for oxygen B-material ) , soft x - ray is often used to characterize nucleobases . [SEP]
[CLS] the nitrogen B-material atom I-material , as the only element that appears exclusively in nucleobases but not in the ribose or the phosphate backbone , provides the clearest fingerprint of dna , and therefore caught more attention in the early work of nucleobase characterization . [SEP]
[CLS] kirtley et al . used xanes to study the electronic environment of nitrogen B-material in different nucleobases , nucleotides , polynucleotides B-material and calf thymus dna . [SEP]
[CLS] in their study , they changed the chemical environment around nitrogen B-material atoms I-material by introducing oxygen B-material or halogen B-material substitutions on the carbon B-material atoms I-material of the aromatic ring to modify the ring . [SEP]
[CLS] spectral perturbations were observed in these cases , revealing that the fine structures shown in the photoabsorption spectrum of dna near the nitrogen B-material k - edge depended highly on the chemical structure of the nitrogen B-material atom I-material . [SEP]
[CLS] moewes et al . , on the other hand , used carbon B-material kedge xas and xes to study electronic structures of b - dna and its four nucleobases . [SEP]
[CLS] in addition , they performed a systematic study to investigate how different buffer conditions might affect the electronic structure of b - dna using xas and xes . [SEP]
[CLS] on the other hand , macnaughton et al . carried out a more comprehensive study on the four nucleobases by obtaining xas and xes spectra for all three elements ( c , n , and o ) . [SEP]
[CLS] combined with theoretical calculations , they were able to identify the main energy transitions of these nucleobases in the xas and xes spectra . [SEP]
[CLS] by comparing the xes and xas experimental data on a common energy axis , the energy gap between highest occupied molecular orbital ( homo ) and lowest unoccupied molecular orbital ( lumo ) was determined for all four bases . [SEP]
[CLS] furthermore , zubavichus et al . reported a detailed analysis of high - quality nexafs spectra of five nucleotide bases , including three pyrimidine ( cytosine , uracil , and thymine ) and two purine ( adenine and guanine ) for carbon B-material , nitrogen B-material and oxygen B-material k - edges . [SEP]
[CLS] all these studies have provided electronic structures of the nucleobases and reference data that can be useful for the analysis of xas or xes spectra of dna molecules with different sequences as discussed in the next section . [SEP]
[CLS] although the dna molecules use nucleotides as their basic building blocks , interactions between nucleobases , such as hydrogen B-material bonding , make their electronic structures different from those of an individual nucleotide . [SEP]
[CLS] therefore , it is not surprising that the spectra of dsdna exhibited significant differences from the sum of individual spectrum of the nucleotides , supporting the hypothesis that the interactions affect the electronic structure of the nucleotides . [SEP]
[CLS] by studying poly ( dg ) • poly ( dc ) and poly ( da ) • poly ( dt ) dna duplexes separately , hua et al . proposed that the spectral properties of dsdna of mixed bases could be expressed as linear combinations of those of dg • dc and da • dt base pairs , suggesting that hydrogen B-material bonding , rather than stacking between base pairs , affected the electronic structure . [SEP]
[CLS] the electronic structures of the dna molecules were found to be sensitive to its environment , since xas and xes spectra as well as the homo - lumo gap were found to change in different buffers . [SEP]
[CLS] several studies suggested that the homo - lumo gap of a dna duplex is closely related to charge hopping phenomenon . [SEP]
[CLS] guo et al . and kummer et al . determined that the band gap of a dna duplex was 2 . 6 ev and was associated with a transition between the π and π * orbitals . [SEP]
[CLS] circular dichroism ( cd ) is a commonly used technique for studying the secondary structure of nucleic B-material acids I-material . [SEP]
[CLS] in order to obtain more information about the nucleic B-material acids I-material , the wavelength of the cd has been extended to the vacuum ultraviolet ( vuv ) region in order to achieve stronger electron coupling between bases . [SEP]
[CLS] although advanced experimental setups were able to record cd spectra in the wavelength between 170 and 200 nm , low photon fluxes were often a limitation due to strong absorption by air , solvent , buffer , salts B-material and cuvette . [SEP]
[CLS] to overcome this limitation , synchrotron radiation was used to provide high vuv flux , making it possible to obtain srcd data with high signal to noise ratios . [SEP]
[CLS] nielsen , holm and coworkers carried out systematic srcd spectral studies on various types of dna , including single - stranded dna ( ssdna ) , dsdna with different combinations of nucleobases , adenine - thymine triplex , i - motifs and g - quadruplexes . [SEP]
[CLS] g - quadruplexes have been known to adopt different topologies when their structural elements are different . [SEP]
[CLS] 3a illustrates four common quadruplex folding motifs formed by different dna sequences . [SEP]
[CLS] homologous guanine oligonucleotides d ( g ) n are known to form quadruplexes with four parallel strands ( figure 3 ( a ) , a ( 4 ) ) , while d ( g4t4g4 ) yields a bimolecular antiparallel g - quadruplex with diagonal loops ( figure 3 ( a ) , b ( 2 ) ) . [SEP]
[CLS] on the other hand , d ( tagggutagggt ) results in a bimolecular parallel g - quadruplex with double - chainreversal loops ( figure 3 ( a ) , c ( 2 ) ) , and d ( ggttggtgtggttgg ) adopts a self - folded chair - type structure ( figure 3 ( a ) , d ( 1 ) ) . [SEP]
[CLS] by examining the g - quadruplexes with distinct topologies , holm et al . found that the srcd signal from a g - quadruplex was proportional to its length , indicating that the cd signal originated from the g quartets . [SEP]
[CLS] as gquadruplexes with different conformations ( anti or syn ) or handedness displayed similar srcd signals , holm et al . proposed that the srcd signal was not related to the topology of the g - quadruplex . [SEP]
[CLS] in another study , gao et al . used srcd to monitor structural changes of dna molecules wrapped around singled - B-nanoparticle walled I-nanoparticle carbon I-nanoparticle nanotube I-nanoparticle ( swcnt ) upon hg 2 + binding ( figure 4 ) . [SEP]
[CLS] the dna molecules have been known to exhibit strong induced circular dichroism ( icd ) signals when they are wrapped around swcnts , due to the coupling between the transition dipole moments of the optically chiral swcnts and the transition dipole moments of dna . [SEP]
[CLS] hg 2 + - induced extending of the dna caused partial detachment of the dna from the swcnts , resulting in a decrease of the icd signal . [SEP]
[CLS] by taking advantage of this property , they have transformed the dna - swcnt system into a sensor for hg 2 + with a nanomolar detection limit . [SEP]
[CLS] immobilization of nucleic B-material acids I-material on solid surfaces is a fast - growing research area that has given birth to numerous cutting - edge biotechnological applications , such as dna microarrays and biosensors for medical diagnosis and pathogen detection [SEP]
[CLS] to retain the full function after immobilization , nucleic B-material acids I-material must be oriented with proper densities and correct conformations on the surface . [SEP]
[CLS] in order to characterize the structures of nucleic B-material acids I-material at these surfaces , x - ray absorption B-technique spectroscopy I-technique ( xas ) , x - ray photoemission spectroscopy B-technique ( xps ) , reflection absorption B-technique infrared I-technique spectroscopy I-technique ( rairs ) and x - ray diffraction ( xrd ) have been widely applied to provide structural information . [SEP]
[CLS] fujii et al . studied evaporated thin films of nucleobases on au - coated si surfaces and calculated the angles of different bases with respect to the flat surface using nexafs . [SEP]
[CLS] they found that purines were orientated to the surface , with an angle of 15 ± 6° for adenine and 38 ± 1° for guanine . [SEP]
[CLS] uracil had an orientation of 16 ± 4° , but thymine and cytosine were randomly orientated with respect to the surface . [SEP]
[CLS] nexafs and xps have also been used for studying dna bases on cu ( 110 ) , si ( 111 ) and au ( 111 ) surfaces . [SEP]
[CLS] furukawa et al . compared the orientation of purine nucleobases ( g and a ) on cu ( 110 ) surfaces , and found that adenine adsorbates B-property laid almost flat while guanine adsorbates B-property were tilted up on the surface . [SEP]
[CLS] seifert et al . determined the average tilt angles of three nucleobases ( a , c and g ) deposited on hydrogen B-material passivated si ( 111 ) surfaces by nexafs spectra of the carbon B-material k - edge . [SEP]
[CLS] their results revealed that adenine and guanine both laid almost flat on the substrate surface , while cytosine adopted a more upright orientation . [SEP]
[CLS] the adsorption geometry of thymine on au ( 111 ) and cu ( 110 ) surfaces was characterized by plekan et al . using xps and nexafs . [SEP]
[CLS] these spectroscopic results suggested that thymine oriented almost parallel to the au ( 111 ) surface , while it tilted at a steep angle on the cu ( 110 ) surface . [SEP]
[CLS] since the early work of tarlov and co - workers on dna immobilization onto gold B-material surfaces using thiol - terminated ssdna ( thiol - ssdna ) , many spectroscopic characterizations have been carried out in order to understand the surface structures of bound dna . [SEP]
[CLS] surface coverage of the dna molecules has been demonstrated to be an important factor in maximizing hybridization efficiency . [SEP]
[CLS] hybridization of complementary dna to densely packed hs - ssdna on a gold B-material surface was found to be impeded due to steric and electrostatic factors . [SEP]
[CLS] to maximize the hybridization , spacer B-material molecules were often introduced into the system to vary the coverage of ssdna on a materials surface . [SEP]
[CLS] lee et al . studied kinetics of 11 - mercapto - 1 - undecanol ( mcu ) displacing hs - ssdna on a gold B-material surface with nexafs , and proposed that the thiol - ssdna oligomers reoriented toward a more upright position upon mcu incorporation . [SEP]
[CLS] as shown in figure 5 , during short - term backfill of mcu , the vacant surface sites surrounding the loosely packed hs - ssdna were occupied by the mcu immobilization . [SEP]
[CLS] upon extending the mcu backfill time , the dna molecules were gradually replaced and non - specific interactions between the nucleobase nitrogen B-material and gold B-material were also weakened , leading to the overall change of ssdna orientation toward a more upright position . [SEP]
[CLS] the nexafs spectra have also been measured to reveal the orientation and electronic structures of dna or peptide B-material nucleic B-material acid I-material ( pna ) molecules on gold B-material , pyrite and inas surfaces . [SEP]
[CLS] structures of metal B-material - nucleic B-material acid I-material complexes - xas has served as an importanttool to provide structural information of metal B-material - binding sites in metal B-material - nucleic B-material acid I-material complexes . [SEP]
[CLS] the interactions between metal B-material ions B-material and nucleic B-material acids I-material involves mostly nitrogen B-material and oxygen B-material atoms I-material . [SEP]
[CLS] due to the inability of exafs to differentiate between oxygen B-material and nitrogen B-material , it is challenging to pinpoint coordinating residues . [SEP]
[CLS] however , the exafs can still provide valuable structural information about metal B-material - ligand distances , as well as coordination numbers . [SEP]
[CLS] as the most abundant transition metal B-material ion B-material in cells B-material , iron B-material serves as the metal B-material cofactor for many enzymes that catalyze redox reactions . [SEP]
[CLS] however , when in excess , iron B-material is believed to generate oxidative stress that can induce damage to cell B-material structures and nucleic B-material acids I-material by converting hydrogen B-material peroxide ( h 2 o 2 ) into highly reactive hydroxyl radicals ( • oh ) through the fenton reaction . [SEP]
[CLS] in mammalian cells B-material , fe ( ii ) has been found to form complex with dna and generate • oh from h 2 o 2 . [SEP]
[CLS] the • oh can then attack nearby residues and cause oxidative damage to dna . [SEP]
[CLS] to understand the coordination of the iron B-material - dna complex and its implication in mutagenesis , bertoncini et al . studied fe - dna interaction using xanes and exafs , and found that only oxygen B-material coordinated with fe ( iii ) while nitrogen B-material and oxygen B-material could bind to fe ( ii ) [SEP]
[CLS] in addition to metal - dna complexes , metal - binding sites in rna molecules have also been identified and their specific catalytic functions have been characterized . [SEP]
[CLS] ribonuclease p ( rnase p ) is a type of ribonuclease that cleaves rna . [SEP]
[CLS] unlike other protein - based rnases , rnase p is unique since it is a ribozyme that uses rna to perform catalytic reactions in the same way as protein - based enzymes . [SEP]
[CLS] rnase p cleaves off a precursor sequence of rna on trna , and this reaction has been found to be metal B-material - ion dependent . [SEP]
[CLS] koutmou et al . combined nmr and xas spectroscopy B-technique to identify and characterize inner - sphere metal - binding sites in a stem - loop rna that served as a model for the most highly conserved p4 helix of rnase p . [SEP]
[CLS] exafs spectra revealed inner - sphere binding of zn 2 + to one or more of the bases of p4 helix with six - coordinate geometry . [SEP]
[CLS] together with nmr characterization , they identified the localization of the metal - binding residues in the active site of p4 helix . [SEP]
[CLS] such an approach can also be used as a general method for characterizing inner - sphere metal - binding sites in other nucleic B-material acids I-material . [SEP]
[CLS] cisplatin B-material , as well as many other related platinum B-material ( pt ) complexes , is known to be a potent anticancer B-property drug through its interactions with dna . [SEP]
[CLS] to understand the formation of the drug - dna complex , the interaction between the platinum B-material complexes and dna has been studied using exafs as well as x - ray crystallography and nmr [SEP]
[CLS] teo et al . used exafs to reveal the interaction between pt and dna . [SEP]
[CLS] their findings suggested that four pt - n ( or o ) bonds at 2 . 03 a were present in the complex in presumably square planar geometry . [SEP]
[CLS] hitchcock et al . reported exafs study of a dna complex with a pt dimer , revealing that the likely binding sites of the pt dimer were guanine bases . [SEP]
[CLS] binding of the pt dimer to these sites interfered with the hydrogen B-material bonding between dna strands and impeded the replication process . [SEP]
[CLS] kobayashi et al . showed a complex made of dna and chloroterpyridine platinum B-material ( pttc ) could potentially behave as a radiosensitizer for radiotherapy , since it could bind to plasmid dna and resulted in an increased yield of strand breaks under x - ray irradiation . [SEP]
[CLS] besides cisplatin B-material , bleomycin B-material , an antitumor antibiotic used in cancer chemotherapy , has been characterized in its complex form with fe ( ii ) using xas and magnetic B-property circular dichroism ( mcd ) by solomon and co - workers . [SEP]
[CLS] their results suggest that the general interaction of the fe ( ii ) - blm complex with dna alters the ligand field of blm , leading to a reaction with o 2 to cleave dna . [SEP]
[CLS] in addition to naturally occurring dna and rna molecules , many of the in vitro selected functional nucleic B-material acids I-material , such as ribozymes , dnazymes and aptamers , can also use metal B-material ions B-material as their cofactors . [SEP]
[CLS] however , structural characterization , especially x - ray crystallography of functional nucleic B-material acids I-material , is very challenging in many cases , probably due to more dynamic nature of these functional nucleic B-material acids I-material than those of proteins B-material . [SEP]
[CLS] therefore , only a limited number of crystals or nmr structures of functional nucleic B-material acids I-material have been reported . [SEP]
[CLS] as an alternative approach , xas has shown to be a versatile technique to probe the structure of metal B-material sites in these functional nucleic B-material acids I-material in solution . [SEP]
[CLS] thrombin - binding aptamer ( tba ) is a 15 - mer dna oligonucleotide that binds thrombin and inhibits thrombin activity . [SEP]
[CLS] it folds into a unimolecular quadruplex in the presence of k + . [SEP]
[CLS] it has been revealed that tba can fold in the presence of pb 2 + , with its confirmation similar to that obtained in the presence of k + . [SEP]
[CLS] smirnov et al . utilized exafs to study the dna - metal binding site in tba . [SEP]
[CLS] their results indicated that the pb 2 + was located in the region between the two quartets in tba , which was consistent with the structure model in which pb 2 + was between two layers of g - quadruplex , and coordinated by eight guanine oxygen B-material atoms I-material . [SEP]
[CLS] mercury B-material is a toxic B-property heavy metal B-material ion B-material in water B-material and soil . [SEP]
[CLS] double - stranded dna molecules with consecutive t - t mispairs have been shown to display a strong binding affinity to mercury B-material . [SEP]
[CLS] based on this property , dna - based mercury B-material sensors have been developed . [SEP]
[CLS] ravel , lu and coworkers performed exafs study on the hg ( ii ) - binding to the t - t mispairs . [SEP]
[CLS] they observed a significant difference in exafs spectra of mercury B-material in the presence and in the absence of dna ( figure 6 ) . [SEP]
[CLS] further fitting with ifeffit showed mercury B-material directly bound to a six - membered ring at 2 . 04 a , likely to be a thymine pyrimidine ring . [SEP]
[CLS] the data is consistent with a thymine - mercury - thymine model based on nmr studies . [SEP]
[CLS] small - angle x - ray scattering ( saxs ) is a technique that can be used to probe the structures and interactions of biomolecules in solution at a low resolution ( 1 - 2 nm ) . [SEP]
[CLS] the main principle of saxs was developed by andre guinier in the 1930s , following his studies of metallic alloys . [SEP]
[CLS] saxs was found to provide not just information on the sizes and shapes of particles but also information on the internal structures of disordered and partially disordered systems . [SEP]
[CLS] the method was adopted for characterizing biomolecules in solution in the 1960s . [SEP]
[CLS] since most biomolecules , like proteins B-material and nucleic B-material acids I-material , have sizes larger than the wavelength of x - rays , they can give a relatively good saxs scattering pattern . [SEP]
[CLS] information about the size , shape , compactness and molecular weight of molecules in solution can be readily obtained using this method . [SEP]
[CLS] therefore , it is a powerful probe for examining protein B-material and nucleic B-material acid I-material conformations . [SEP]
[CLS] compared with crystallization and nmr spectroscopy B-technique , saxs does not require high - quality crystals as in crystallography , nor does it have strict limits on the molecular weight of the sample as in nmr spectroscopy B-technique . [SEP]
[CLS] saxs can also tolerate a variety of conditions for measurement , ranging from physiological to highly denaturing conditions . [SEP]
[CLS] breakthrough in saxs data analysis methods , such as those reported by svergun and stuhrmann in the 1990s , had a significant impact on the use of saxs for scattering studies of wide range of molecules in solution . [SEP]
[CLS] their procedures allowed extraction of meaningful three - dimensional details from one - dimensional scattering data , resulting in reliable ab initio shape and domain structure determination for the first time . [SEP]
[CLS] meanwhile , the increasing availability of third - generation synchrotron sources , accompanied by improvement in time resolutions down to the sub - milliseconds , has largely increased the users of saxs and created excellent opportunities for a variety of biological applications in the past decade . [SEP]
[CLS] here , we focus on the recent development and applications of saxs in structural studies of nucleic B-material acids I-material . [SEP]
[CLS] the discovery by cech and altman in the early 1980s that rna can act as an enzyme has led to considerable interest in elucidating the structure and function of rna molecules . [SEP]
[CLS] in addition , there is also an emerging awareness of the extensive involvement of rna machineries in gene - control processes . [SEP]
[CLS] numerous riboswitches with complex folded domains have been identified in the non - coding region of mrnas in prokaryotes . [SEP]
[CLS] these riboswitches change their structures upon binding of specific metabolites , and influence transcription B-event or translation at different levels , and thereby controlling many biological processes . [SEP]
[CLS] the complex tertiary structures of ribozymes and riboswitches , and folding process of their tertiary structures have been extensively studied during the past two decades . [SEP]
[CLS] to build the relationship between structure and function of these special rna molecules , x - ray crystallography has served as a powerful tool for obtaining structural information with atomic resolution . [SEP]
[CLS] however , to probe the intermediates B-property in the folding pathways , unfolded or partially folded conformations commonly coexist in the system , resulting in a complex system where x - ray crystallography cannot be readily applied . [SEP]
[CLS] instead , saxs has been shown to be one of the major tools for obtaining global information on the size and shape of folding intermediates B-property of rna molecules in solution , since it provides quantitative characterization of mixtures by measuring the radius of gyration of molecules with 1 - 3 nm resolution . [SEP]
[CLS] moreover , time - resolved experiments carried out with rapid mixing methods to trigger folding can provide unique information about the structures of transient intermediates B-property populated during the folding process . [SEP]
[CLS] one fundamental question in rna folding is the nature of the rate - limiting step . [SEP]
[CLS] the early effort in understanding how ribozymes fold dates back to 2000s . [SEP]
[CLS] russell et al . reported an initial study using saxs to monitor the changes in the overall size and shape of the intermediates B-property as tetrahymena group i ribozyme folds ( figure 7 ( a ) ) . [SEP]
[CLS] by measuring the radius of gyration using saxs , they found that the native ribozyme , formed in the presence of mg 2 + , adopted a more compact and globular conformation than the unfolded ribozyme . [SEP]
[CLS] moreover , time - resolved measurements suggested that the ribozyme collapsed into a compact intermediate B-property within a few milliseconds after addition of mg 2 + , with a rate constant at least 20 - fold faster than the overall rate constant for folding ( figure 7 ( b ) ) . [SEP]
[CLS] this fast compaction was found to last on the order of 100 ms , before slower rearrangements of misfolded intermediates B-property took place . [SEP]
[CLS] the fully functional state eventually formed on the timescale of about 100 s . their results indicate that there is a kinetic trap involved in the folding process , and rearrangement of misfolded structures is the rate - limiting step . [SEP]
[CLS] in a subsequent study of the ribozyme folding process , das et al . designed a quintuple mutant of the tetrahymena ribozyme to destabilize the tertiary contacts within the folded ribozyme . [SEP]
[CLS] time - resolved saxs measurements showed that collapse to the compact intermediate B-property upon the addition of salt B-material still occurred , even in the absence of the specific tertiary interactions . these results suggested that the initial compaction was a result of nonspecific shielding of the coulomb forces by the added counter ions . however , the tertiary hydrogen B-material bond contacts were found to be important in the subsequent compaction on the timescale of 100 ms . [SEP]
[CLS] similarly , the compaction process of yeast trna phe , the catalytic domain of the bacillus subtilis rnase p rna , the candida group i ribozyme ( ca . l - 11 ) and the azoarcus group i ribozyme were investigated using saxs or small - angle neutron scattering ( sans ) by different groups . [SEP]
[CLS] in contrast to the tetrahymena ribozyme , these ribozymes exhibited rapid folding processes from the initial collapse to the native state , suggesting the folding pathways did not involve long - lived kinetic traps . [SEP]
[CLS] to reveal the interplay between core B-material and peripheral elements in ribozyme folding , baird et al . used saxs combined with chemical and nuclease mapping , cd and molecular modeling to investigate the folding intermediates B-property of the specificity domain , or s - domain , of the bacillus subtilis rnase p rna [SEP]
[CLS] the structure of this rna is composed of four tertiary structural modules , including a rigid core B-material , a four - way junction , a gaaa tetraloop - receptor B-material and an unusual motif involving two tertiary interacting loops ( figure 8 ( a ) ) . [SEP]
[CLS] their findings suggested that in the thermodynamic folding pathway , an intermediate B-property containing two of the four native tertiary modules was populated . [SEP]
[CLS] the size and shape of the native rna and the intermediate B-property structures were characterized by saxs measurements . [SEP]
[CLS] the results indicated that the size of the intermediate B-property was bigger and the shape was more extended than those for the native rna . [SEP]
[CLS] the intermediate B-property lacked the core B-material , and folding from the intermediate B-property to the native structure involved the formation of the core B-material as well as significant conformational changes that bring two peripheral helices close to each other in order to form the tetraloopreceptor interaction ( figure 8 ( b ) ) . [SEP]
[CLS] to compare how the charge and size of the cations B-material influenced the collapse transition in different ribozymes , moghaddam et al . used saxs to monitor the changes in the radius of gyration of the azoarcus and tetrahymena ribozymes with different cations B-material [SEP]
[CLS] their findings suggested that both ribozymes underwent collapse transition to native conformations in all of the counter ions B-material they tested ( na + , k + , mg 2 + , ca 2 + , co ( nh 3 ) 6 3 + , ba 2 + , sr 2 + , and spermidine 3 + ) . small , multivalent cations B-material induced the collapse of both ribozymes more efficiently than monovalent ions B-material . [SEP]
[CLS] however , counter ion - induced collapse and formation of tertiary interactions occurred at the same time for the azoarcus ribozyme , while the folding of the tetrahymena ribozyme was composed of two distinct transitions in the presence of increasing concentrations of mg 2 + , which was best explained by a three - state model . [SEP]
[CLS] besides ribozymes , saxs has also been used to study the thermodynamic folding pathways of riboswitches . [SEP]
[CLS] for example , lipfert et al . studied the folding of a glycine - dependent riboswitch ( vci - ii ) from vibrio cholerae using saxs in combination with hydroxyl radical footprinting . [SEP]
[CLS] they proposed a three - state thermodynamic model for energy coupling between magnesium - induced folding and glycine B-material binding . [SEP]
[CLS] under low salt B-material condition without any mg 2 + , the vci - ii riboswitch adopted an extended overall conformation indicating unfolded structures . [SEP]
[CLS] addition of millimolar concentrations of mg 2 + in the absence of glycine B-material resulted in a significant compaction and partial folding . [SEP]
[CLS] addition of glycine B-material in the presence of millimolar mg 2 + lead to further compaction mediated by additional tertiary packing interactions and further binding of mg 2 + . [SEP]
[CLS] using a 3d reconstruction algorithm dammin , they were able to obtain low - resolution 3d structures for all three states derived from saxs measurements ( figure 9a ) . [SEP]
[CLS] with the development of different algorithms for reconstructing 3d density maps from saxs profiles , the overall structures of several rna and dna molecules have also been successfully reconstructed from their saxs data . [SEP]
[CLS] using bead model representations of the macromolecules as an input to calculation , which had been commonly used for reconstructing proteins B-material and their complexes previously , lipfert et al . reported reconstructed low - resolution density maps for three different types of rna molecules , including yeast trna phe , a 24 bp b - dna duplex and the p4 - p6 domain of the tetrahymena group i intron ( figure 9b - d ) . [SEP]
[CLS] the reconstructed structures are in good agreement with actual crystal structures . [SEP]
[CLS] their approach demonstrated that , besides proteins B-material , low - resolution reconstruction methods could also be applied to nucleic B-material acids I-material . [SEP]
[CLS] such a method is potentially applicable to other biomolecules for obtaining structural information when the highresolution structure is not available . [SEP]
[CLS] although saxs can provide reliable data for investigating rna molecules , in practice , predicted shapes or confirmations can be inconsistent with actual structures due to the existence of misfolded rna molecules in a sample . [SEP]
[CLS] to improve saxs data for structural analyses , rambo et al . reported using a size exclusion chromatographic purification as a general approach to reduce the heterogeneity of the samples and therefore largely improve the accuracy of predicted shapes for a variety of rna molecules , including ribosomal subunits , trnas , ribozymes and riboswitches . [SEP]
[CLS] the past decade has witnessed significant improvements in saxs data collection techniques and computational algorithms for analyzing saxs data . [SEP]
[CLS] with continuous development , saxs can be an invaluable tool for providing structural insights into biomolecules in the absence of prior structural information . [SEP]
[CLS] besides forming right - handed double helices as genetic materials , nucleic B-material acids I-material have also been found to be capable of adopting a variety of non - canonical structures in vitro or in vivo for different functions . [SEP]
[CLS] for example , eukaryotic chromosomes are terminated with telomeres containing small , tandemly repeated guanidine - rich dna sequences . [SEP]
[CLS] in vitro biophysical characterization indicates that these g - rich sequences can form four - stranded structures , known as g - quadruplexes , under physiologically relevant conditions . [SEP]
[CLS] structures of g - quadruplexes are usually made of sets of four guanine bases held in plane through hoogsteen hydrogen B-material bonding and then stacked on top of each other . [SEP]
[CLS] the quadruplex structures can be further stabilized by cations B-material , especially k + ions B-material , which are located in the central channel formed between each pair of tetrads . [SEP]
[CLS] recently , a low - resolution structure of a g - quadruplex derived from human telomere repeats , d ( ttaggg ) 4 , was obtained on the basis of saxs data collected by kozak et al . . [SEP]
[CLS] together with crystal structures and nmr structures , the structure and potential conformational changes of g - quadruplex in solution were revealed , providing useful information for elucidating functions of g - quadruplexes in vivo . [SEP]
[CLS] in addition to g - quadruplexes , dna molecules have also been shown to form an i - motif in solution . [SEP]
[CLS] the i - motif is a four - stranded dna structure formed by intramolecular noncanonical base - pair interactions between protonated and unprotonated cytosines under slightly acidic conditions ( e . g . , ph < 6 . 5 ) . [SEP]
[CLS] based on the fact that the i - motif can reversibly undergo a structural change driven by a ph change , both dna nanomachines that can generate motions for multiple cyclings and dna nanodevices B-nanoparticle that can map intracellular ph gradient in real time have been demonstrated . [SEP]
[CLS] detailed structural information of imotif dna in solution at various ph conditions was characterized using saxs technique by jin et al . [SEP]
[CLS] their observations indicated that the i - motif dna molecules adopted multiple confirmations over a wide ph range , providing structural basis for future design of dnabased nanodevices B-nanoparticle . [SEP]
[CLS] moreover , over the past two decades , there have been increasing efforts invested in isolating new types of functional nucleic B-material acids I-material in vitro . [SEP]
[CLS] with a combinatorial method named in vitro selection or systematic evolution of ligands by exponential enrichment ( selex ) , dna or rna molecules that can bind specific targets with high affinity , or possess catalytic activities in the presence of metal B-material ions B-material or small molecules have been selected . numerous kinds of sensors or targeted drug delivery systems have been designed based on these aptamers and dnazymes . [SEP]
[CLS] however , compared to the rapid development of sensor devices , little is known about the three dimensional structures of these functional nucleic B-material acids I-material , and only a limited number of crystal structures of a few aptamers and one misfolded dnazyme are available . [SEP]
[CLS] in a complementary approach to fret studies on dnazymes , saxs combined with other characterization techniques have been shown to be possible to obtain the 3d structural information of aptamers alone or in complex with their targets . using saxs in combination of fluorescence B-technique correlation I-technique spectroscopy I-technique ( fcs ) , werner et al . characterized the structure of an rna aptamer named srb2m , which has a high affinity to a dye called sulforhodamine b . [SEP]
[CLS] the aptamer , as well as the sulforhodamine b - aptamer complex , were found to form dimers predominantly in solution ( figure 10 ) . [SEP]
[CLS] interaction of another dye , named patent blue v ( pbv ) , with the srb2m led to a dissociation of the srb2m dimers into monomers B-material . [SEP]
[CLS] more recently , reinstein et al . used saxs to characterize a cocaine - binding aptamer either in its free form or substrate - bound form . [SEP]
[CLS] their ab initio shape reconstruction structures are also consistent with the structural - switching binding mechanism . [SEP]
[CLS] with these latest examples , continuous progress in obtaining the structural information of these non - canonical nucleic B-material acids I-material is highly desired , since it can not only guide the future design and optimization of sensors , but also provide insights into the mechanisms behind the functions of aptamers and dnazymes . [SEP]
[CLS] in addition to saxs , another method that has been commonly used for probing nucleic B-material acid I-material structures is footprinting . [SEP]
[CLS] the term ' footprinting ' describes a method for studying the sequence - specific binding B-event of I-event proteins I-event to dna . [SEP]
[CLS] by utilizing dnase , a nuclease that can easily digest dna exposed to the solvent but unable to cut dna that is ' protected ' by proteins B-material , scientists are able to see the protective ' footprint ' of the binding protein B-material on the dna sequence . [SEP]
[CLS] since then , footprinting has been used to examine ligand binding or conformational changes by quantifying the solvent accessibility of the backbone of nucleic B-material acid I-material through their sensitivity to enzymatic or chemical cleavage . [SEP]
[CLS] the protected region with less solvent accessibility can be revealed by separating the reaction products using gel B-technique electrophoresis I-technique . [SEP]
[CLS] free linear nucleic B-material acid I-material molecules usually give bands with even intensity , since the probability of each nucleotide being attacked by nuclease is almost the same . [SEP]
[CLS] in contrast , the ' protected ' nucleic B-material acid I-material molecules will show decreased intensity of certain bands , corresponding to the protected region that has less chance to be attacked ( figure 11 ) . [SEP]
[CLS] among different types of reagents used for footprinting assays , such as endonuclease dnase i and hydroxyl radical ( • oh ) , • oh has proven to have significant advantages over other reagents , because it has van der waals surface area and solvent properties similar to those of water B-material molecules . [SEP]
[CLS] therefore , • oh is an ideal radical for probing solvent accessibility with single nucleotide resolution . [SEP]
[CLS] it can react with dna and rna by attacking the phosphodiester backbone , and it is insensitive to base sequence or secondary structure . [SEP]
[CLS] in the early days , • oh was usually generated by fenton reaction using fe - edta , or from homolytic dissociation of peroxynitrous acid . [SEP]
[CLS] however , these two reagents commonly used to generate • oh could not be used for time - resolved footprinting with timescales of less than seconds . [SEP]
[CLS] with the development of synchrotron facilities , researchers have been able to use high - flux synchrotron x - ray beam to generate • oh from water B-material in milliseconds and map the dynamic structures of nucleic B-material acid I-material at subsecond time scale . [SEP]
[CLS] high flux provided by whitelight x - ray beams shortened reaction time for generating • oh tremendously and has led to many breakthroughs in the study of nucleic B-material acid I-material folding process . [SEP]
[CLS] compared with saxs , which is commonly used for obtaining overall shape of nucleic B-material acid I-material , synchrotron - generated hydroxyl radical footprinting provided a unique approach for probing rna conformations with single nucleotide resolution . [SEP]
[CLS] among many large rna molecules , the tetrahymena thermophilia ribozyme is one the best - characterized ribozymes with regard to its structure , folding pathways and enzymatic activity ( figure 12 ( a ) ) . [SEP]
[CLS] it is composed of at least three domains of tertiary structure , " p4 - p6 " , " p1 - p3 " and " p3 + p7 - p9 " . [SEP]
[CLS] it was found that these individual domains can reassemble into active ribozyme when they are mixed together . [SEP]
[CLS] of the three domains , p4 - p6 is the only domain that can fold independently ( figure 12 ( b ) ) . [SEP]
[CLS] in addition to the folding mechanism for individual domains , the complete folding pathway of mg 2 + - dependent tetrahymena ribozyme was characterized by several groups using synchrotron footprinting ( figure 12 ( c ) ) . [SEP]
[CLS] they determined the folding kinetics by monitoring the changes in solvent accessibility of individual site as a function of time . [SEP]
[CLS] the folding process starts with rapid collapse of rna molecule into a partially disordered state , followed by a slow step allowing rna to rearrange into active structures . [SEP]
[CLS] it was revealed that the most stable domain of the tertiary structure formed within 3 seconds , followed by sequential folding of the peripheral helices and the catalytic core B-material on the timescale of minutes . [SEP]
[CLS] the different effects of monovalent and divalent ions B-material on the rates and hierarchy of tetrahymena ribozyme folding has been studied by uchida et al . and shcherbakova et al . [SEP]
[CLS] the tetrahymena ribozyme folds to the same overall structure in the presence of either divalent or monovalent ions B-material . [SEP]
[CLS] however , the rate of folding is much faster and the solvent accessibility of nucleotides is more in monovalent ions B-material than in divalent ions . [SEP]
[CLS] unlike many large rnas such as the aforementioned tetrahymena ribozyme , the azoarcus group i ribozyme is small in size . [SEP]
[CLS] it retains the conserved catalytic core B-material common to all other group i ribozymes , but lacks the peripheral domains that stabilize folding intermediates B-property of the large ribozymes . [SEP]
[CLS] rangan et al . used synchrotron footprinting to probe the assembly and tertiary folding of this ribozyme . [SEP]
[CLS] their findings revealed that tertiary folding occurred much faster for azoarcus ribozyme than other group i ribozymes , and assembly of helices happened before the formation of native tertiary structures . [SEP]
[CLS] a 3d model was constructed based on the analysis , revealing distinct structural features that explained the unusual stability and cooperative folding of azoarcus ribozyme . [SEP]
[CLS] more recently , the use of synchrotron footprinting has been extended to studying noncanonical dna structures , characterizing dynamics of ribosome rna folding and mapping structures of ribosomes and ribozymes inside cells B-material . [SEP]
[CLS] in particular , the development of in vivo footprinting using synchrotron radiation has provided structural biology with a new method for capturing the structural dynamics of nucleic B-material acid I-material in the cellular environment . [SEP]
[CLS] with increasing interest in acquiring and interpreting biomolecular information at physiological conditions , synchrotron footprinting can be a valuable tool that paves the way for a large number of biological and biomaterial applications . [SEP]
[CLS] the discovery of dna double helical structures has laid the cornerstone for molecular biology . [SEP]
[CLS] the intrinsic programmable property of dna to form watson - crick base pairs with complementary strands has also shown to be a foundation for bionanotechnology . [SEP]
[CLS] with a diameter of about 2 nm and a repeat of helical pitch every 3 . 4 - 3 . 6 nm , the stiff helical dna molecule has proven to be a versatile building block for ' bottom - up ' constructions of complex structures with subnanometer precision . [SEP]
[CLS] the concept that using approximately 200 short staple dna strands to direct the folding of a long scaffold strand into desired two - and three - dimensional nanostructures gave birth to the field of dna origami [SEP]
[CLS] since then , there has been an explosive growth in the fabrication of customshaped structures comprised of dna molecules . [SEP]
[CLS] since the early advent of individual dna objects such as a quadrilateral and a cube , numerous artificial dna structures have been constructed , including tubes , 2d and 3d lattices and assemblies . [SEP]
[CLS] moreover , because of the special electronic , magnetic B-property , and photonic properties of programmable assembly of nanomaterials B-material , as well as the potential use of 3d dna crystal lattices in structure biology for protein B-material structure determination , many structural dna and dna origami ' s have been used as platforms to arrange functional nanomaterials B-material such as metals B-material , nanoparticles B-nanoparticle , quantum B-nanoparticle dots I-nanoparticle , carbon B-nanoparticle nanotubes I-nanoparticle and proteins B-material at nanometer scale . [SEP]
[CLS] it has been demonstrated that dna - based nanomaterials B-material can assemble into periodic macroscopic structures in a well - controlled manner , with the precision of such ordered structures difficult to achieve by other means of fabrication . [SEP]
[CLS] the structures of dna assembly and dna - templated assembly of nanoparticles B-nanoparticle are usually characterized by atomic B-technique force I-technique microscopy I-technique ( afm ) , transmission B-technique electron I-technique microscopy I-technique ( tem ) and cryo - electron microscope ( cryo - em ) . [SEP]
[CLS] these instruments are preferred because of their high spatial resolution that is comparable to the size of dna and nanoparticles B-nanoparticle . [SEP]
[CLS] however , despite of their ability to provide high - resolution images of a sample , due to the drying process required during sample preparation and imaging conditions under high vacuum , the morphology of 3d superlattices may be distorted or destroyed as dehydration of the dna would affect the structural integrity of the dna . [SEP]
[CLS] this limitation often resulted in many 3d assemblies of dna - functionalized materials not suitable to be imaged directly using these imaging methods . [SEP]
[CLS] instead , saxs is often used for providing structural information about 3d superlattice structures in solution . [SEP]
[CLS] here we review the recent progress in using saxs for characterizing dna - based and dna - functionalized 3d nanomaterial B-material assemblies . [SEP]
[CLS] anderson et al . reported a covalently closed dna nanocage B-nanoparticle constructed with eight 75 - mer dna strands assembled into a truncated octahedron structure by going through the annealing process followed by dna ligation . [SEP]
[CLS] the 3d structure of the assembled dna cage was characterized by saxs in combination with single - particle image reconstruction based on cryo - tem . [SEP]
[CLS] saxs data showed that the sample contained very well - defined hollow particles , and the inner diameter and overall dimensions of the nanocages B-nanoparticle were predicted with reasonable accuracy . [SEP]
[CLS] the predicted model from saxs data is also in good agreement with cryo - tem analysis ( figure 13 ) . [SEP]
[CLS] in building 3d structures with dna origami , anderson et al . created a dna box by folding six dna origami sheets along a long circular , single - stranded dna . [SEP]
[CLS] the lid was designed with a lock - key system , which could be opened in the presence of externally supplied dna ' keys ' . [SEP]
[CLS] the structure of this dna box was characterized by saxs , cryo - em and afm . [SEP]
[CLS] saxs measurement provided useful information about the native structure of dna origami box in solution without sample fixation . [SEP]
[CLS] the saxs analysis indicated the sample was in well - defined shape and adopted a hollow structure . [SEP]
[CLS] theoretical modeling of the box derived from saxs data gave overall dimensions and wall thickness close to the expected size and was consistent with the size observed by cryo - em . [SEP]
[CLS] because of the unique physical and chemical properties exhibited by nanoarchitectures built from well - organized nanoparticle B-nanoparticle building blocks , assembling nanoparticles B-nanoparticle into designed 2d and 3d structures has attracted a lot of interest in scientific research . [SEP]
[CLS] numerous attempts have been made to precisely control the localization of nanoparticles B-nanoparticle in 3d assemblies , including using small organic molecules , synthetic polymers B-material , proteins B-material and dna - functionalized nanoparticles B-nanoparticle as building blocks for the use in therapeutic , spectroscopic , catalytic and material B-material applications . [SEP]
[CLS] among different strategies , dna has been shown to be the most versatile material B-material for positioning nanoparticles B-nanoparticle in a desired way due to its good programmability and tunability . [SEP]
[CLS] since the initial report on dna - directed self - assembly of gold B-nanoparticle nanoparticles I-nanoparticle by alivisatos and mirkin , a variety of nanostructures based on dna - functionalized nanoparticles B-nanoparticle have been reported . the optical , electrical properties of these nanostructures have been utilized for colorimetric diagnosis , nanophotonic circuits , and high efficiency energy - harvesting devices . [SEP]
[CLS] the saxs , with its ability to provide information on parameters of crystalline lattices and quantitatively determine interparticle distances in periodic structures , served as one of the most widely used techniques for characterizing 3d nano - assemblies . [SEP]
[CLS] in a saxs spectrum , the peak positions and relative heights indicate the structure of the assemblies , while the degree of ordering within the structure is reflected by peak numbers and their widths . [SEP]
[CLS] the electrical properties of gold B-nanoparticle nanoparticle I-nanoparticle assemblies linked by dna were reported by park et al . [SEP]
[CLS] since the electrical properties of gold B-nanoparticle nanoparticle I-nanoparticle assemblies are highly influenced by interparticle distances , the distance between the particles is of great interest to researchers . [SEP]
[CLS] such a property was characterized by saxs in combination with other techniques . [SEP]
[CLS] it was observed that in the solution , the distance between particles increased as the length of the linker dna increased . [SEP]
[CLS] however , for dried aggregates , the interparticle distance was no longer dependent on the length of the linker . [SEP]
[CLS] the electrical properties of dna - protected nanoparticles B-nanoparticle were also quite different from those of gold B-nanoparticle nanoparticle I-nanoparticle films , with the former behaving more as semiconductors , while the latter exhibiting metallic behavior . [SEP]
[CLS] to understand the optical properties of dna - linked gold B-nanoparticle nanoparticle I-nanoparticle aggregates and their relationships with structures at nanometer scale , storhoff et al . characterized the aggregates formed by two types of dna - functionalized gold B-nanoparticle nanoparticles I-nanoparticle linked by dna linkers with three different lengths , 24 , 48 and 72 base pairs . [SEP]
[CLS] they observed that at room temperature , nanoparticle B-nanoparticle assemblies exhibited plasmon frequency changes that had an inverse relationship with the lengths of linkers . twenty - four base linked aggregates showed the largest change , while the frequency shift for the 72 base linked gold B-nanoparticle nanoparticles I-nanoparticle was the smallest . [SEP]
[CLS] however , upon annealing at temperatures near the melting temperature of the dna , the plasmon frequency of longer dna - linked assemblies ( 48 and 72 base pairs ) further red shifted until they were similar to the assemblies formed with the shorter linkers ( 24 base pairs ) . [SEP]
[CLS] this result suggested that under pre - annealing conditions , the 48 and 72 base linked aggregates formed kinetically stable structures , and these structures could be thermally transformed into thermodynamic aggregates upon annealing . [SEP]
[CLS] the saxs characterization showed that the first - order diffraction peak of the long dna - linked aggregates did not shift upon annealing . [SEP]
[CLS] this result suggested that the interparticle distance of these dna - linked aggregates did not change significantly upon annealing . [SEP]
[CLS] instead , the optical properties of the assemblies were proven to be dependent on aggregate size rather than oligonucleotide linker length , upon characterization using sedimentation rate , tem , dynamic B-technique light I-technique scattering I-technique and uv - vis spectroscopy B-technique . [SEP]
[CLS] chi et al . characterized the interparticle distance of a dimer of dna - functionalized gold B-nanoparticle nanoparticles I-nanoparticle using synchrotron - based saxs in combination with molecular simulations . [SEP]
[CLS] their results indicated that the separation distance between the two nanoparticles B-nanoparticle depended on the length of ssdna linker as well as the number of linkers connecting the nanoparticles B-nanoparticle . [SEP]
[CLS] longer ssdna linkers resulted in larger intradimer distances , while multiple linkers between two particles lead to smaller interparticle distances due to off - axis connections by the linker strands ( figure 14 ) . [SEP]
[CLS] with the interparticle structure of 3d assemblies from dna - functionalized nanoparticle B-nanoparticle thoroughly characterized by saxs , [SEP]
[CLS] researchers have been able to interpret how interparticle interactions and dna - nanoparticle B-nanoparticle interactions can affect the 3d structures of these assemblies . [SEP]
[CLS] in an early demonstrations reported by park et al . , the oligonucleotidemodified nanoparticle B-nanoparticle assemblies were found to adopt amorphous structures , with local structures exhibiting scattering patterns that revealed body - centered cubic ( bcc ) , facecentered cubic ( fcc ) or body - centered tetragonal ( bct ) - like structures . [SEP]
[CLS] in the same study , saxs was used to probe the effects of oligonucleotide components on the assembled structures . [SEP]
[CLS] poly - a was found to have higher affinity for gold B-material than poly - t , as poly - t spacer B-material resulted in a significant increase in the interparticle distance . [SEP]
[CLS] these findings provided useful insights into the structural basis for 3d assemblies for better control over their structures . [SEP]
[CLS] in more recent studies , by shortening the length of ' sticky ends ' of dna linkers and allowing weaker dna interactions between particles , mirkin ' s and gang ' s groups independently showed the formation of 3d crystalline assemblies of gold B-nanoparticle nanoparticles I-nanoparticle guided by the interactions between complementary dna molecules conjugated on the surface of particles . [SEP]
[CLS] to probe the internal structures of nanoassemblies into superlattices during heating and cooling processes in real time , both groups used saxs for monitoring the phase behavior of their systems in situ ( figure 15 ) . [SEP]
[CLS] in the binary system reported by gang and coworkers , five sets of dna - capped gold B-nanoparticle nanoparticles I-nanoparticle were prepared . each set contained two kinds of gold B-nanoparticle nanoparticles I-nanoparticle modified with different dna molecules . [SEP]
[CLS] at room temperature , nanoparticles B-nanoparticle assembled into amorphous structures by dna hybridization . [SEP]
[CLS] however , upon heating the assembly to dna melting temperatures ( t m ) and cooling the assembly below t m , two sets of dna - modified nanoparticles B-nanoparticle with longer flexible spacers B-material showed crystalline organization , while systems with shorter dna spacers B-material remained amorphous . [SEP]
[CLS] clear saxs patterns strongly indicated the reversible formation of bcc crystalline structures and remarkable degrees of long - range ordering during heating and cooling cycles ( figure 15 ( b , c ) ) . [SEP]
[CLS] in a parallel study , mirkin ' s group demonstrated that it was possible to use dna for directing the assembly of gold B-nanoparticle nanoparticles I-nanoparticle into two distinct crystalline structures ( figure 15 ( a ) ) . [SEP]
[CLS] by designing a linker sequence that was either self - complementary or non - self - complementary , a singlecomponent system or a binary system could be achieved . [SEP]
[CLS] under thermodynamic control , the single - component system was expected to form a close - packed fcc structure with 12 nearest neighbors to maximize the number of hybridized dna linkages B-property and minimize the energy of the system , while the binary system was predicted to form a non - close - packed bcc structure with 8 nearest neighbors in order to achieve the maximum number of dna hybridization . [SEP]
[CLS] through a careful heating and cooling process , they showed the formation of a fcc crystal using the single - component system . [SEP]
[CLS] more interestingly , due to the competition between the entropic and enthalpic contributions involved in the assembly process at different temperatures , the binary system was found to form bcc structure at room temperature , while it formed fcc structure if it was treated with heating followed by slow cooling . [SEP]
[CLS] moreover , besides dna linkage B-property and temperature , many other variables were also found to participate in affecting the final structure of dna - aunp B-nanoparticle assembly , such as the flexor between the dna linker and aunp B-nanoparticle , the rigidity of hybridized dna linkers and particle size . [SEP]
[CLS] by varying the length of the dna hybridization region in the singlecomponent system , it has been demonstrated that the distance between aunps B-nanoparticle in the fcc crystals can be fine - tuned . [SEP]
[CLS] longer dna connections result in increased nanoparticle B-nanoparticle spacing as well as a decrease in the order of the lattice because the nanoparticles B-nanoparticle are less spatially constrained . [SEP]
[CLS] xiong et al . studied the effect of dna linker length and the number of linkers per particle on the internal structure of dna - aunp B-nanoparticle assembly in a binary system using saxs . [SEP]
[CLS] their findings suggested that linker lengths should be constrained within a limited range for the formation of bcc crystal structure , and the density of linkers on nanoparticles B-nanoparticle controlled the onset of system crystallization . [SEP]
[CLS] since the discovery of dna - directed colloidal crystallization , the mechanism of crystal growth has been extensively studied . [SEP]
[CLS] macfarlane et al . discussed a three - step process for the formation of dna - aunp B-nanoparticle crystals derived from saxs profiles , collected during the growth of crystal systems . [SEP]
[CLS] the whole process is composed of initial dna - aunp B-nanoparticle aggregation , formation of small , well - ordered crystalline domains , followed by small crystal - crystal aggregation , and final rearrangement into large crystal systems with long - range order . [SEP]
[CLS] luo and co - workers comprehensively studied the process of " soft " crystallization using dnacapped nanoparticles B-nanoparticle , and during this process the deformation of soft corona made of dna on the surface of nanoparticles B-nanoparticle was found to occur . [SEP]
[CLS] using saxs to probe the entire crystallization process in real time and in situ , they demonstrated that the soft crystals went through a gradual transition from ' wet crystals ' to ' dry crystals ' accompanied by elastic drying - mediated deformation of dna molecules ( figure 16 ) . [SEP]
[CLS] to fully understand the parameters involved in crystal assembly , macfarlane et al . found general design rules that can address the control over lattice parameters . [SEP]
[CLS] by varying particle size , length of dna linkers , length of spacers B-material and number of sticky end types , nine different kinds of crystal lattices were obtained and characterized by saxs under thermodynamic or kinetic controls . [SEP]
[CLS] this discovery enables researchers to synthesize nanoassemblies in a predicted fashion . [SEP]
[CLS] meanwhile , there is also an increasing interest in using simulations to characterize the process of dna - programmable nanoparticle B-nanoparticle crystallization . [SEP]
[CLS] quantitative prediction of the phase behavior of dna - functionalized colloids has been achieved through computational simulations based on a coarse - grain method . [SEP]
[CLS] more recently , the concept of dna - nanoparticle B-nanoparticle superlattice has been extended to using different types of building blocks , such as anisotropic nanorods B-nanoparticle , rhombic dodecahedra and octahedral nanoparticles B-nanoparticle to synthesize novel crystalline structures . [SEP]
[CLS] novel 2d and 3d structures were observed from anisotropic particle assemblies that could not be achieved by the assembly of symmetric spherical particles . [SEP]
[CLS] spherical nucleic B-material acid I-material nanoparticle B-nanoparticle conjugates have also been used as hollow spacers B-material in the construction of nanoparticle B-nanoparticle superlattices , resulting in new crystalline structures with distinct symmetries that were difficult to obtain previously . [SEP]
[CLS] a general approach to functionalize a wide variety of nanoparticles B-nanoparticle with a dense shell B-material of dna has been reported , largely expanding the building blocks to nanoparticles B-nanoparticle of chemical species besides gold B-material . [SEP]
[CLS] 3d nanoparticle B-nanoparticle assembly with controllable switching of interparticle distances in the superlattices has also been demonstrated by maye et al . and xiong et al . very recently , a topotactic intercalation strategy was reported by mirkin and co - workers to create nanoparticle B-nanoparticle superlattices with greater complexity than simple binary systems . [SEP]
[CLS] this methodology allowed a third nanoparticle B-nanoparticle to be inserted into predetermined sites in the preformed binary lattice , and led to unique crystal structures with much increased complexity . [SEP]
[CLS] the saxs , as a major tool in characterizing 3d structures of dna nanostructures and dna - programmable nanoparticle B-nanoparticle assemblies , has provided us with a comprehensive understanding of the structures constructed , and guides us towards the field of building nanoarchitectures with greater complexity and functionality . [SEP]
[CLS] the structures formed by dna and lipids B-material have attracted significant interests in the past decade after the discovery that lipids B-material can be used as vehicles B-material to deliver dna into mammalian cells B-material . [SEP]
[CLS] as a major type of non - viral vectors , liposomes B-nanoparticle that are formed by closed bilayer membrane B-material shells I-material of I-material lipids I-material are very promising for gene therapy because they are easy to prepare and less immunogenic B-property than viral vectors . [SEP]
[CLS] since the early work of using cationic B-material lipids B-material to form lipid - dna complexes for gene delivery by felgner and co - workers , tremendous efforts have been devoted to the search for new formulations of liposomes B-nanoparticle in order to increase the level of gene transfer efficiency . [SEP]
[CLS] meanwhile , targeted delivery has been proposed by conjugating lipids B-material with different targeting groups , such as small molecules , peptides B-material , antibodies B-material and aptamers . [SEP]
[CLS] to understand how the structures of lipid - dna complexes would affect their transfection B-property efficiency I-property and uptake mechanism , the saxs has been used as a major tool to characterize the nanostructures of self - assembled lipid - dna complexes . [SEP]
[CLS] these characterizations have helped to establish correlations between the structure of lipid - dna complexes and their biological function . [SEP]
[CLS] moreover , the structural information also has provided deeper insights into rational optimization of formulation and transfection B-property efficiency I-property . [SEP]
[CLS] depending on the lipid B-material composition , the ratio of dna to lipids B-material and the solution conditions , lipids B-material and dna can form different structures in solution . [SEP]
[CLS] radler et al . used saxs to characterize the structure of cationic B-material liposomes B-nanoparticle ( cl ) in complex with dna . [SEP]
[CLS] the liposomes B-nanoparticle consisted of binary mixtures of neutral lipids B-material dopc ( dioleoyl phosphatidylcholine ) and cationic B-material lipids B-material dotap ( dioleoyl trimethylammonium propane ) with a 1 : 1 ratio . [SEP]
[CLS] they observed that addition of either linear λ - phage or plasmid dna to the cl resulted in a transition from liposomes B-nanoparticle to a highly ordered multilamellar ( l α c ) structure with dna sandwiched between cationic B-material bilayers ( figure 17 ( a ) ) . [SEP]
[CLS] in a different study , koltover et al . substituted dopc with dope ( dioleoyl phosphatidylethanolamine ) to form a mixture of dotap and dopc . [SEP]
[CLS] at certain compositions , addition of dna molecules into the system led to a completely different structure . [SEP]
[CLS] dna was found to be coated by cationic B-material lipid B-material monolayers I-material and form a columnar inverted hexagonal lattice ( h ii c ) ( figure 17 ( b ) ) . [SEP]
[CLS] in addition to the composition and rigidity of the membrane , such different formations of structures were found to depend on the shape of lipid B-material molecules . [SEP]
[CLS] because dotap and dopc are cylindrical in shape , a mixture of these two gave zero spontaneous curvature and resulted in a multilamellar structure . [SEP]
[CLS] however , since dope has a cone - like shape , leading to negative curvature , the mixture of dotap and dope favored the formation of a columnar inverted hexagonal shape . [SEP]
[CLS] such findings also explained the correlation between the nanostructure of cl - dna complexes and transfection B-property efficiency I-property . [SEP]
[CLS] liposomes B-nanoparticle containing dope have been found to have good transfection B-property efficiency I-property , since they formed a h ii c structure that fused well with the cell B-material membrane , while liposomes B-nanoparticle containing dopc and adopting l α c structures had poor transfection B-property efficiency I-property because they fused poorly with the cell B-material membrane . [SEP]
[CLS] besides monovalent cationic B-material lipids B-material , multivalent cationic B-material lipids B-material have also been reported to form lamellar and micellar conformations in complex with dna . [SEP]
[CLS] in contrast , selfassembled anionic lipids B-material ( als ) and dna complexes have been found to organize into different structures than the cl - dna complex ( figure 17 ( d - f ) ) . [SEP]
[CLS] at low membrane charge densities , al - dna formed a lamellar structure of alternating dna and membrane layers , while at high membrane charge densities , dna was expelled from the complex and a lamellar stack of membranes was formed . [SEP]
[CLS] more interestingly , at high concentration of zn 2 + , the system formed an inverted hexagonal phase in which dna strands were coated with divalent cations B-material and wrapped with anionic membrane monolayers to form hexagonal arrays . [SEP]
[CLS] in a more recent study , ewert et al . discovered another new shape of cl - dna complexes . [SEP]
[CLS] they synthesized a highly charged multivalent cationic B-material lipid B-material ( with 16 positive charges ) with a dendritic headgroup , mvlbg2 , and investigated the structure of the lipid - dna complex by saxs . [SEP]
[CLS] at a narrow range of composition around 25 mol % mvlbg2 and 75 mol % dopc , the complex exhibited the hexagonal phase ( h i c ) , in which hexagonally arranged tubular lipid B-material micelles B-material were surrounded by dna rods with honeycomb symmetry ( figure 17 ( c ) ) . [SEP]
[CLS] in another recent study , the discovery that short complementary b - form dna ( with 6 to 20 base pairs in length ) could exhibit nematic and columnar liquid crystal phases has led to a study of the packing of short dna with nonpairing overhangs in cl - dna complexes . [SEP]
[CLS] employing saxs , bouxsein et al . found that the formation of nematic liquid crystal phases in the cl - dna system was influenced by many parameters , such as the length of dna and the length of nonsticky ends . [SEP]
[CLS] such structure may find its use in delivery of short anisotropic biomolecules with nanoparticle B-nanoparticle membranes for gene silencing in the future . [SEP]
[CLS] as our understandings of the structures of lipid B-material - dna complexes increases with the help of saxs , lipid - based delivery approach will be more optimized and better designed for therapeutic applications . [SEP]
[CLS] in this review , we have summarized the progress made in using synchrotron - based spectroscopic techniques for the characterization of nucleic B-material acids I-material and related nucleic acidsbased or - templated nanomaterials B-material . [SEP]
[CLS] in comparison to the study of other molecules such as organic polymers B-material and proteins B-material , the use synchrotron - based spectroscopic techniques to study nucleic B-material acids I-material is relatively new and cases of studies are not as many . [SEP]
[CLS] despite of this situation , the power of synchrotron - based spectroscopic methods has already been demonstrated to reveal many interesting properties such as the electronic structures of nucleobases and dsdna , the secondary structure of dna molecules , the orientation of surface - bound nucleic B-material acids I-material , the metal - binding sites in nucleic acids , conformations of nucleic B-material acids I-material in solution including intermediates B-property in rna folding and non - canonical structures of dna and rna molecules . [SEP]
[CLS] in addition , time - resolved synchrotron x - ray footprinting in studying the folding of nucleic B-material acid I-material structures is also described . [SEP]
[CLS] finally , application of synchrotron radiation in studying dna - based functional materials , including dna nanostructures , such as dna 3d origami structures and dna - functionalized nanoparticle B-nanoparticle 3d assemblies , as well as dnalipid interactions have also been covered . [SEP]
[CLS] these results have provided deeper insights into the structure and function of nucleic B-material acids I-material , allowing their applications as building blocks for biochemical , biomedical and bionanotechnological applications . [SEP]
[CLS] with so many synchrotron - based spectroscopic techniques available , the choice of a particular method will depend on molecules to be studied and information to be obtained . [SEP]
[CLS] to take full advantages of these techniques for nucleic B-material acid I-material research , further exploring the capability of these methods is required , including optimization of the conditions under which the data are collected , accumulation of reference data for fingerprint comparison and development of theoretical framework to interpret the results . [SEP]
[CLS] with the continuous development of synchrotron facilities aiming at providing light sources with higher flux , higher brightness and more continuous tunability , these synchrotron - based techniques can find even wider range of uses as non - destructive , high - resolution , real - time and in situ analytical methods in biochemical , biomedical and nanomaterial B-material characterizations of even more nucleic B-material acids I-material . [SEP]
[CLS] synchrotron - based techniques for characterizing nucleic B-material acids I-material and nucleic acid - based nanomaterials B-material ( adapted with permission from ref . copyright ( 2011 ) nature publishing group ) . [SEP]
[CLS] the relationship between energy transitions and x - ray absorption edges ( adapted with permission from ref . copyright ( 2000 ) american physical society ) . [SEP]
[CLS] srcd spectra for g - quadruplexes of different lengths , showing signal is linearly correlated with length ( adapted with permission from ref . copyright ( 2010 ) wiley periodicals , inc ) . [SEP]
[CLS] illustration of hg 2 + - induced icd signal intensity change of dna - swcnts . [SEP]
[CLS] strong icd signal was observed for dna wrapped around swcnts . [SEP]
[CLS] when the hg 2 + ions B-material bind to the dna bases , part of the dna disassociates from the swcnts , leading to a significant decrease in the icd signal ( adapted with permission from ref . copyright ( 2008 ) american chemical society ) . [SEP]
[CLS] nitrogen B-material ( a ) and carbon B-material ( b ) k - edge nexafs spectra from pure dna and mixed dna / mcu monolayers on gold B-material at normal ( 90° ) and glancing ( 20° ) incident x - ray angles with increasing backfill time . [SEP]
[CLS] in nitrogen B-material k - edge nexafs spectra ( a ) , the increase in polarization dependence indicates that dna bases are oriented more parallel to the surface than bases in the pure dna monolayer and that ssdna oligomers reorient on average toward a more upright orientation on the surface upon mcu addition . [SEP]
[CLS] in the carbon B-material k - edge nexafs spectra ( b ) , the decrease in the intensity of the π * c = c and σ * c - nh peaks with longer mcu backfill time is consistent with dna displacement from the surface ( adapted with permission from ref . copyright ( 2006 ) american chemical society ) . [SEP]
[CLS] and the sample with 3 mm of hg and 3 mm duplex dna ( blue ) ( adapted with permission from ref . copyright ( 2009 ) elsevier ) . [SEP]
[CLS] ( a ) the tetrahymena ribozyme . [SEP]
[CLS] the p1 - p13 represent different paired secondary structure elements in the ribozyme , and the long range pairings p13 and p14 are indicated with arrows . [SEP]
[CLS] the boxed portion is the ribozyme core B-material , which is most highly conserved . [SEP]
[CLS] the core B-material is largely protected from solvent in the presence of mg 2 + . [SEP]
[CLS] ( b ) a model in which compaction of the ribozyme is much faster than the overall folding to the native state ( adapted with permission from ref . copyright ( 2000 ) nature publishing group ) . [SEP]
[CLS] in the i eq structure , the t - loop is extended whereas in the native structure it forms a compact loop ( adapted with permission from ref . copyright ( 2005 ) elsevier ) . adapted with permission from ref . copyright ( 2009 ) elsevier ) [SEP]
[CLS] residues in p5c were protected most rapidly , followed by the nucleotides in the interior of the p4 - p6 domain . [SEP]
[CLS] in comparison , nucleotides involved in interactions with p2 - p2 . 1 and the p3 - p9 domain were protected more slowly , and the ordering of the catalytic core B-material occurred even slower , on the timescale of several minutes ( adapted with permission from ref . copy right ( 1998 ) american association for the advancement of science ) . [SEP]
[CLS] copyright ( 2008 ) oxford university press ) [SEP]
[CLS] peak indicate an increase of the interparticle distance with increasing l ( adapted with permission from ref . copyright ( 2012 ) american chemical society ) . [SEP]
[CLS] ( a ) gold B-material nanoparticle - dna conjugates can be programmed to assemble into singlecomponent assembly system ( fcc ) using one dna sequence , or binary - component assembly system ( bcc ) using two different linkers ( adapted with permission from ref . [SEP]
[CLS] copyright ( 2008 ) nature publishing group . ) [SEP]
[CLS] ( b ) a typical example of an in situ saxs measurement for probing the internal structure of gold B-material nanoparticle - dna conjugates as temperature changes . [SEP]
[CLS] ( c ) structural factors for gold B-material nanoparticle - dna conjugates with bcc structure ( b and c adapted with permission from ref . copyright ( 2008 ) nature publishing group ) . [SEP]
[CLS] copyright ( 2010 ) wiley - vch verlag gmbh & co . kgaa , weinheim ) . . copyright ( 1998 ) american association for the advancement of science ) . [SEP]
[CLS] 3 . ( a ) different g - quadruplex structures with strand directions indicated by red ( up ) or blue ( down ) . [SEP]
[CLS] the number in parentheses is the number of individual strands in the complex . [SEP]
[CLS] ( b ) srcd spectra for g - quadruplexes of different lengths , showing signal is linearly correlated with length ( adapted with permission from ref . copyright ( 2010 ) wiley periodicals , inc ) . [SEP]
[CLS] ( a ) schematic representation of the interaction of the dna sensor and the target metal B-material . [SEP]
[CLS] ( b ) proposed binding mode of mercury B-material . [SEP]
[CLS] ( c ) exafs data measured on the hg control ( black ) and the sample with 3 mm of hg and 3 mm duplex dna ( blue ) ( adapted with permission from ref . copyright ( 2009 ) elsevier ) . [SEP]
[CLS] 8 . ( a ) secondary structure of the rnase p from bacillus subtilis , and its crystal structure containing the four - way junction ( green ) , the core B-material ( red ) , the j11 / 12 junction ( purple ) , and the tl - receptor B-material ( black ) . [SEP]
[CLS] broken lines represent tertiary interactions , while continuous lines indicate stacking interactions in the native structure . [SEP]
[CLS] the t - loop in the core B-material is composed of five nucleotides , and is of particular importance to the folding intermediate B-property . [SEP]
[CLS] ( b ) comparison between the structure of intermediate B-property ( i eq ) and the native structure ( n ) . [SEP]
[CLS] in the i eq structure , the t - loop is extended whereas in the native structure it forms a compact loop ( adapted with permission from ref . copyright ( 2005 ) elsevier ) . [SEP]
[CLS] ( a ) low - resolution structures of vci - ii riboswitch under different solution conditions , including average unfolded conformation ( blue ) , conformation in the presence of 10 mm magnesium B-material and absence of glycine B-material ( green ) , and glycine - bound structure ( red ) . [SEP]
[CLS] ( b - d ) reconstructed density of ( b ) the yeast trna phe , ( c ) the 24 bp dna duplex , and ( d ) the p4 - p6 domain of the tetrahymena group i intron . [SEP]
[CLS] atomic resolution structure ( black sticks ) and reconstructed density ( colored transparent surfaces ) are superimposed ( adapted with permission from ref . copyright ( 2007 ) elsevier and copyright ( 2007 ) international union of crystallography ) . [SEP]
[CLS] 10 . [SEP]
[CLS] structural characterization of srb2m with saxs . [SEP]
[CLS] ( a ) experimental x - ray scattering patterns from srb2m alone ( curve 1 ) , srb2m - sulfohodamine b complex ( curve 2 ) , and srb2m with pbv ( curve 3 ) . [SEP]
[CLS] ( b ) ab initio models of srb2m with ( 1 ) and without ( 2 ) pbv . [SEP]
[CLS] the monomer B-material model of srb2m is shown in ( b1 ) in magenta , and the dimer model of srb2m is shown in ( b2 ) , with one monomer B-material in magenta and the other one in green . [SEP]
[CLS] the right and bottom slides show the model rotated by 90° around the y and x axes , respectively ( scale bar = 2nm . adapted with permission from ref . copyright ( 2009 ) elsevier ) . [SEP]
[CLS] 11 . [SEP]
[CLS] hydroxyl radical footprinting of rna . [SEP]
[CLS] ( a ) hydroxyl radicals ( • oh ) generated by synchrotron radiation attack backbones of unfolded ( top left ) and folded ( top right ) rna molecules in solution . [SEP]
[CLS] due to formation of tertiary structures , folded rna exhibits decreased solvent accessibility compared to unfold rna . [SEP]
[CLS] sequences buried inside the folded structures ( shown in red ) have less chance to be attacked by • oh , giving rise to less cleavage in these buried regions . [SEP]
[CLS] ( b ) cleavage products can be separated by gel B-technique electrophoresis I-technique . [SEP]
[CLS] unfolded rna is cleaved uniformly , and shows a ladder of bands with even intensity on the gel ( bottom left ) . [SEP]
[CLS] in contrast , the bands of folded rna have several regions with less intensity , corresponding to sequences with decreased solvent accessibility and inhibited cleavage . [SEP]
[CLS] 12 . [SEP]
[CLS] synchrotron - generated hydroxyl radical footprinting of the tetrahymena l - 21 ribozyme . [SEP]
[CLS] ( a ) secondary structure of the ribozyme . [SEP]
[CLS] lettered bases were protected from hydroxyl radical cleavage in 10 mm mg 2 + at 42°c . [SEP]
[CLS] colored regions are regions with similar folding rates , with their rate constants labeled in numbers . [SEP]
[CLS] ( b ) front and back views of a space - filling model of the p4 - p6 domain of the ribozyme . [SEP]
[CLS] ( c ) a model for the early steps of the mg 2 + dependent folding of the ribozyme . [SEP]
[CLS] residues in p5c were protected most rapidly , followed by the nucleotides in the interior of the p4 - p6 domain . [SEP]
[CLS] in comparison , nucleotides involved in interactions with p2 - p2 . 1 and the p3 - p9 domain were protected more slowly , and the ordering of the catalytic core B-material occurred even slower , on the timescale of several minutes ( adapted with permission from ref . copy right ( 1998 ) american association for the advancement of science ) . [SEP]
[CLS] 13 . saxs data and 3d model for the dna cage . [SEP]
[CLS] ( a ) experimental data ( points ) and model fit ( solid lines ) . [SEP]
[CLS] ( b ) octahedral model obtained from the saxs data . [SEP]
[CLS] ( c ) overlay of saxs model and cryo - tem reconstruction in three different views ( adapted with permission from ref . copyright ( 2008 ) oxford university press ) . [SEP]
[CLS] 14 . ( a ) schematic of a dimer of gold B-nanoparticle nanoparticles I-nanoparticle connected by multiple ssdna linkers ( pink strand ) . [SEP]
[CLS] l denotes the number of polythymine bases ( t ) in the ssdna linker , excluding the recognition ends . [SEP]
[CLS] ( b ) computed distribution of the surface - to - surface distance r for a dimer linked by either one ( black line ) or four chains ( red line ) for linker length l = 10 bases . [SEP]
[CLS] ( c ) representative 2d saxs pattern of dimers for l = 0 and l = 75 . [SEP]
[CLS] ( d ) structural factors for the dimer systems with different linker lengths l . [SEP]
[CLS] monotonical shifts of the first s ( q ) peak indicate an increase of the interparticle distance with increasing l ( adapted with permission from ref . copyright ( 2012 ) american chemical society ) . [SEP]
[CLS] 15 . [SEP]
[CLS] 16 . soft crystallization of poly ( dt ) 15 - capped nanoparticles B-nanoparticle in the contact - line region . [SEP]
[CLS] ( a ) series of 1d saxs patterns recorded over drying time . [SEP]
[CLS] ( b ) schematic showing the maintenance of the fcc lattice during corona deformation ( adapted with permission from ref . copyright ( 2010 ) wiley - vch verlag gmbh & co . kgaa , weinheim ) . [SEP]
[CLS] 17 . [SEP]
[CLS] schematics of cationic B-material lipid - dna ( cl - dna ) complexes ( a - c ) and anionic lipid - dna ( al - dna ) complexes ( d - f ) . [SEP]
[CLS] ( a ) lamellar l α c phase of cl - dna complexes ; ( b ) columnar inverted hexagonal h ii c phase of cl - dna complexes ; ( c ) h i c phase of cl - dna complexes , in which tubular lipid B-material micelles B-material arrange in a hexagonal lattice while dna rods arrange on a honeycomb lattice in the interstices of the lipid B-material micelle B-material arrangement ; ( d ) condensed dna - ion - al lamellar structure with alternating layers of dna and anionic membranes glued together by divalent cations B-material ; ( e ) condensed ion - al membrane lamellar structure ; ( f ) 2d inverted hexagonal structure of al - dna complexes , in which hexagonal arrays of divalent cations B-material coated dna strands wrapped in the anionic membrane monolayer tubes ( adapted with permission from ref . copyright ( 1998 ) american association for the advancement of science ) . [SEP]
[CLS] to understand the effect of three - dimensional oligonucleotide structure on protein B-material corona I-material formation , we studied the identity and quantity of human serum proteins B-material that bind to spherical nucleic B-material acid I-material ( sna ) nanoparticle B-nanoparticle conjugates . [SEP]
[CLS] snas exhibit cellular uptake properties that are remarkably different from those of linear nucleic B-material acids I-material , which have been related to their interaction with certain classes of proteins B-material . [SEP]
[CLS] through a proteomic analysis , this work shows that the protein binding properties of snas are sequence - specific and supports the conclusion that the oligonucleotide tertiary structure can significantly alter the chemical composition of the sna protein B-material corona I-material . [SEP]
[CLS] this knowledge will impact our understanding of how nucleic acid - based nanostructures , and snas in particular , function in complex biological milieu . [SEP]
[CLS] spherical nucleic B-material acids I-material ( snas ) are a unique class of nanoparticles B-nanoparticle ( nps B-nanoparticle ) , often consisting of a np B-nanoparticle core B-material that is densely functionalized with highly oriented oligonucleotides . [SEP]
[CLS] the architecture of these structures leads to novel properties that are different from those of their linear counterparts and enables their use in a wide variety of therapeutic and diagnostic applications . [SEP]
[CLS] unlike the linear form of dna , snas exhibit high cellular uptake without the use of additional transfection reagents through enhanced binding of class a scavenger receptors on the cell B-material surface . [SEP]
[CLS] additionally , the dense layer of oligonucleotides on snas makes them resistant to degradation and provides increased stability compared to the linear form . [SEP]
[CLS] these differences between snas and linear nucleic B-material acids I-material highlight the need to understand protein - sna interactions in complex biological media , ranging from in vitro to in vivo systems . [SEP]
[CLS] immediately upon intravenous administration , particles encounter a multitude of serum proteins B-material that often form a corona around the nanoparticle B-nanoparticle and may significantly alter its in vivo behavior . [SEP]
[CLS] in particular , the binding B-event of I-event opsonin I-event proteins I-event leads to recognition and sequestration by macrophages , which causes decreased blood residence times and lower accumulation in target tissue . [SEP]
[CLS] herein , for the first time , we explore the interaction between blood serum and snas , focusing on the number and type of proteins B-material that bind to snas as a function of dna sequence . [SEP]
[CLS] specifically , we look at the role of g - rich sequences , which are known to facilitate interactions with scavenger receptors , an important component in the mechanism of sna cellular internalization . [SEP]
[CLS] based on the dense and highly oriented structure of oligonucleotides , we hypothesized that the formation of tertiary dna structures would be enhanced on the sna scaffold , and alter its serum protein B-material interactions . [SEP]
[CLS] in particular , we investigated the effect of the sna architecture on the formation of g - quadruplexes . [SEP]
[CLS] we designed gold B-nanoparticle nanoparticle I-nanoparticle ( aunp B-nanoparticle ) core snas with a 3 ′ thiol - modified guanine - rich ( g - rich ) sequence ( scheme 1 ) to form gquadruplexes consisting of g - quartets that are hydrogen B-material - bonded through non - watson crick base pairing and are stabilized by metal B-material cations I-material ( figure 1a ) . [SEP]
[CLS] we first wanted to compare the formation of g - quadruplexes on the snawith the linear dna form using circular dichroism ( cd ) spectroscopy B-technique . [SEP]
[CLS] both g - rich snas and g - rich dna exhibit a peak at 265 nm and a trough at 240 nm , which is indicative of parallel g - quadruplex formation . [SEP]
[CLS] this suggests that g - quadruplexes on snas primarily form between neighboring dna strands on the same aunp B-nanoparticle , rather than between dna strands on multiple aunps B-nanoparticle . [SEP]
[CLS] the sna structure supports this , because the dna on the sna is highly oriented . [SEP]
[CLS] further , g - rich snas exhibit a higher degree of ellipticity , suggesting that the g - quadruplex characteristic is enhanced by the sna scaffold , which brings pre - oriented dna strands in close proximity ( figure 1 ) . [SEP]
[CLS] the thermal stabilities of g - quadruplexes formed on the sna and with linear g - rich dnawere analyzed by variable - temperature cd spectroscopy B-technique . [SEP]
[CLS] notably , at physiological temperature ( 37°c ) , the characteristic parallel g - quadruplex cd spectrum disappears and is replaced by signatures diagnostic of single - stranded dna . [SEP]
[CLS] in contrast , the cd spectra of grich snas maintain the signatures associated with g - quadruplexes at elevated temperatures ( figure 1c and d ) . [SEP]
[CLS] to further probe this , the melting temperatures of g - quadruplexes on snas and linear dna were determined by measuring the ellipticity of each sample at 260 nm from 20 - 90°c and determining the fraction of quadruplex remaining at each temperature . [SEP]
[CLS] the results indicate a significant shift in the melting temperature from 34 . 5°c for linear g - rich dna to 51 . 5°c for g - rich snas ( figure 1b ) , confirming that the stabilities of g - quadruplexes are enhanced considerably through the sna structure . [SEP]
[CLS] based on the presence of stable g - quadruplexes on g - rich snas , it was hypothesized that g - rich snas would exhibit different interactions with proteins B-material upon exposure to serum . [SEP]
[CLS] indeed it is known that class a scavenger receptors recognize and bind g - quadruplexes better than single - stranded dna . [SEP]
[CLS] as a control , snas with a poly - t sequence ( t 40 ) were used to probe the effect of the tertiary g - quadruplex structures on protein B-material corona I-material formation . [SEP]
[CLS] g - rich and poly - t snas were incubated B-technique in 10 % human serum ( hs ) for 24 h at 37°c . [SEP]
[CLS] the excess , unbound proteins B-material were removed by washing with pbs . [SEP]
[CLS] protein - coated nps B-nanoparticle were then characterized using dynamic B-technique light I-technique scattering I-technique ( dls ) to investigate the increase in sna size associated with protein B-material corona I-material formation ( figure 2a ) . [SEP]
[CLS] it was found that both g - rich and poly - t snas become larger in the presence of hs , corresponding to the presence of adsorbed proteins B-material . [SEP]
[CLS] interestingly , the increase in size of g - rich snas was larger than the increase in size of poly - t snas ; 185±19 nm versus 34±5 nm for g - rich and poly - t snas , respectively . [SEP]
[CLS] this is likely due to two factors : first , g - rich snas bind more serum proteins B-material ( see below ) ; second , the proteins B-material that adsorb onto g - rich snas may facilitate clustering through protein - protein interactions . [SEP]
[CLS] to further explore this , we quantified the amount of proteins B-material adsorbed onto g - rich and poly - t snas . [SEP]
[CLS] this was accomplished by isolating the proteins B-material that were bound to the snas following incubation B-technique in 10 % hs . [SEP]
[CLS] first , excess , unbound proteins B-material were removed by centrifugation . [SEP]
[CLS] snas ( 1 pmole ) were then treated with 1 % sodium B-material dodecyl sulfate ( sds ) and heated for 5 min to denature and release the bound proteins B-material . [SEP]
[CLS] the amount of protein B-material in these samples was then quantified using the bicinchoninic acid ( bca ) assay . [SEP]
[CLS] it was found that 4 . 4±10 −12 ±3 . 5 × 10 −13 µg protein B-material / np B-nanoparticle adsorbs B-property to g - rich snas whereas 1 . 6±10 −12 ±3 . 8 × 10 −13 µg protein B-material / np B-nanoparticle adsorbs B-property to poly - t snas . [SEP]
[CLS] this corresponds to 40±3 proteins B-material / sna and 15±3 proteins B-material / sna that adsorb onto g - rich and poly - t snas , respectively , assuming an average protein B-material mass of 66 . 5 kda obtained from serum albumin ( figure 2b ) . [SEP]
[CLS] denaturing polyacrylamide B-technique gel I-technique electrophoresis I-technique ( page ) analysis was subsequently performed to characterize the protein B-material corona I-material composition of g - rich and poly - t snas . [SEP]
[CLS] proteins B-material adsorbed onto g - rich and poly - t snas were isolated from snas ( 3 pmole ) following incubation B-technique in 10 % hs at 37°c for 24 h . [SEP]
[CLS] the proteins B-material were then separated by page and imaged using a fluorescent B-property coomassie protein B-material stain ( figure 2c ) . [SEP]
[CLS] comparison of the band intensities from the resulting image confirms that indeed , more total protein B-material adsorbs B-property onto the surface of g - rich snas than onto poly - t snas . [SEP]
[CLS] in addition , comparison of the number of bands of proteins B-material isolated from g - rich snas to those isolated from poly - t snas indicates that more types of proteins B-material bind g - rich snas . [SEP]
[CLS] to identify the specific proteins B-material that adsorb onto g - rich and poly - t snas , we performed mass spectrometry of trypsin - digested protein B-material samples isolated from g - rich and poly - t snas following incubation B-technique in 10 % hs ( tables s3 and s4 ) . [SEP]
[CLS] in total , 82 proteins B-material were isolated from g - rich snas , whereas 54 were isolated from poly - t snas . [SEP]
[CLS] of these proteins B-material , 49 were common between both g - rich and poly - t snas ( figure 3a ) . [SEP]
[CLS] these results confirm that more types of proteins B-material bind g - rich snas than poly - t snas , which is consistent with page analysis . [SEP]
[CLS] the proteins B-material that were identified by mass spectrometry were grouped according to their function : blood B-event coagulation I-event , immune system , lipid B-material transport , molecular transport , and others . [SEP]
[CLS] this revealed that many of the proteins B-material that bind exclusively to g - rich snas are components of the immune response , and that nearly twice as many immune system proteins B-material adsorb B-property onto g - rich snas than poly - t snas ( figure 3b ) . [SEP]
[CLS] these results confirm that the protein B-material corona I-material on g - rich and poly - t snas differs not only in quantity but also composition . [SEP]
[CLS] to quantify the relative amounts of specific proteins B-material bound to g - rich and poly - t snas , western blotting analysis was performed for five proteins B-material : apolipoprotein b100 , factor h , transferrin , complement c3b , and serum albumin ( figure 3c ) . [SEP]
[CLS] these proteins B-material were chosen to represent a range of protein B-material classes , as well as some of the most highly abundant serum proteins B-material . [SEP]
[CLS] the proteins B-material adsorbed onto g - rich and poly - t snas ( 3 pmole ) were analyzed , and the relative amount of each protein B-material was quantified using densitometry . [SEP]
[CLS] three of the five proteins B-material analyzed adsorb onto g - rich snas more than poly - t snas ( figure 3d ) . [SEP]
[CLS] in particular , about three ( apolipoprotein b100 ) , six ( complement factor h ) , and four ( complement c3b ) times as much protein B-material adsorbs B-property onto g - rich snas than onto poly - t snas . [SEP]
[CLS] this result suggests that these proteins B-material may exhibit a higher affinity for g - rich snas than poly - t snas . [SEP]
[CLS] to test this , we used a modified elisa assay to characterize the binding affinity of g - rich and poly - t sequences in their sna and linear forms to complement factor h . [SEP]
[CLS] the results indicate that g - rich snas bind significantly more to complement factor h than linear g - rich dna , which had no appreciable binding ( figure 3e ) . [SEP]
[CLS] this may be explained by the enhancement of g - quadruplex formation on the sna scaffold . [SEP]
[CLS] in addition , the increased affinity of complement factor hfor g - rich snas compared to their poly - t sna counterparts correlates with the observed differences in protein B-material corona I-material composition between g - rich and poly - t snas . [SEP]
[CLS] the presence of opsonin proteins B-material in the protein B-material corona I-material is known to facilitate the interaction of nanoparticles B-nanoparticle with macrophages . [SEP]
[CLS] one such protein B-material , complement c3 , is recognized by complement receptors on the macrophage cell B-material surface , which leads to phagocytosis B-event and subsequent removal from the bloodstream . [SEP]
[CLS] based on the observed increase in complement c3b adsorption onto g - rich snas , we hypothesized that the uptake of g - rich snas by macrophages would be higher than that of poly - t snas . [SEP]
[CLS] to test this , we quantified the uptake of g - rich and poly - t snas in a phagocytic macrophage cell B-material line . [SEP]
[CLS] raw 264 . 7 macrophage cells B-material were treated with one of the following conditions : 1 nm g - rich sna , 1 nm protein - coated g - rich snas , 1 nm poly - t snas , or 1 nm protein - coated poly - t snas for 15 min , 30 min , or 1 h . [SEP]
[CLS] the cells B-material were counted and digested with strong acid for determination of gold B-material content by inductively coupled plasma mass spectrometry to quantify uptake and association ( figure 4 ) . [SEP]
[CLS] initially , the association of g - rich snas with and without protein B-material coating B-material is about twice as much as the association of poly - t snas to macrophages . [SEP]
[CLS] this is expected , since snas are known to be internalized through class a scavenger receptor - I-event mediated I-event endocytosis I-event , and poly - g is a natural ligand for class a scavenger receptors . [SEP]
[CLS] for all three incubation B-technique times , uptake and association of poly - t snas to macrophages is not significantly altered by the presence of a protein B-material coating B-material . [SEP]
[CLS] in contrast , macrophage uptake of protein - coated g - rich snas increases after 30 min by more than two - fold compared to uncoated g - rich snas . [SEP]
[CLS] we suspect that the difference in uptake is due to the higher amount of opsonin protein B-material complement c3b present on g - rich snas , which flags it for phagocytosis B-event ( figure 3c ) . [SEP]
[CLS] this effect is lessened after a 1 h incubation B-technique with macrophages , suggesting that g - rich snas with a hs protein B-material corona I-material may saturate cell B-material surface I-material receptors I-material after a 30 min incubation B-technique . [SEP]
[CLS] this is consistent with previous work that reported typical langmuir binding isotherms of sna uptake , indicating that the mechanism is limited by receptor B-material - ligand interactions . [SEP]
[CLS] the increase in cellular uptake and association of g - rich snas suggests that they are recognized by macrophages to a greater extent than poly - t snas , which may cause them to be rapidly cleared from the blood stream in vivo . [SEP]
[CLS] in conclusion , we have investigated the role of sequence on the protein B-event binding I-event properties of snas . [SEP]
[CLS] in particular , we have shown that the sna architecture enhances the formation of gquadruplexes compared to a linear counterpart , which changes the types of proteins B-material it can interact with in serum . [SEP]
[CLS] this protein B-material corona I-material alters snauptake in a phagocytic macrophage cell B-material line , suggesting that g - rich and poly - t snas may exhibit altered biodistribution and pharmacokinetic profiles in vivo . [SEP]
[CLS] specifically , this work contributes to the design rules for synthesizing therapeutically active snas and our understanding of how they will interact with proteins B-material , and function in complex biological media . [SEP]
[CLS] most notably , whereas g - rich sequences may lead to greater cellular internalization , they also lead to greater macrophage uptake , which could cause increased clearance from the blood stream , a consideration that must be taken into account in all subsequent in vivo studies involving snas . [SEP]
[CLS] characterization of protein B-material adsorption onto g - rich and poly - t snas following incubation B-technique in 10 % human serum for 24 h at 37°c . [SEP]
[CLS] a ) size of g - rich and poly - t snas before and after protein B-material adsorption as determined by dls . [SEP]
[CLS] b ) quantity of protein B-material adsorbed onto g - rich and poly - t snas determined using the bca assay . [SEP]
[CLS] c ) page analysis of proteins B-material adsorbed onto g - rich and poly - t snas . [SEP]
[CLS] note that the heavy band in hs is serum albumin . [SEP]
[CLS] association of raw 264 . 7 mouse macrophages after 15 min , 30 min , and 1 h incubations B-technique with g - rich and poly - t snas with and without a hs protein B-material corona I-material . [SEP]
[CLS] a ) g - quartet structure stabilized by m + ( na + , k + , etc . ) b ) cd spectroscopy B-technique was used to measure the melting temperature ( t m ) of g - quadruplexes formed on the sna and with linear g - rich dna . [SEP]
[CLS] the full cd spectra for c ) g - rich dna and d ) g - rich snas are shown at increasing temperatures , demonstrating the enhanced thermal stability of g - quadruplexes formed on the sna scaffold . [SEP]
[CLS] 3 . analysis of the types of proteins B-material that adsorb onto g - rich and poly - t snas from 10 % human serum after incubation B-technique for 24 h at 37°c [SEP]
[CLS] a ) venn diagram indicating the number of distinct proteins B-material identified by mass spectrometry that adsorb onto g - rich and poly - t snas , categorized by class ( b ) . [SEP]
[CLS] c and d ) western blotting analysis was used to quantify the relative abundance of proteins B-material bound to g - rich and poly - t snas . [SEP]
[CLS] e ) a modified elisa assay was used to study the affinity of linear and sna forms of g - rich and poly - t dna to complement factor h . [SEP]
[CLS] rapid progress in identifying disease biomarkers B-property has increased the importance of creating highperformance detection technologies . [SEP]
[CLS] over the last decade , the design of many detection platforms has focused on either the nano or micro length scale . [SEP]
[CLS] here , we review recent strategies that combine nano - and microscale materials and devices to produce large improvements in detection sensitivity , speed and accuracy , allowing previously undetectable biomarkers B-property to be identified in clinical samples . [SEP]
[CLS] microsensors that incorporate nanoscale features can now rapidly detect diseaserelated nucleic B-material acids I-material expressed in patient samples . [SEP]
[CLS] new microdevices that separate large clinical samples into nanocompartments allow precise quantitation of analytes , and microfluidic systems that utilize nanoscale binding events can detect rare cancer cells B-material in the bloodstream more accurately than before . [SEP]
[CLS] these advances will lead to faster and more reliable clinical diagnostic devices . [SEP]
[CLS] increasingly , advances in medicine rely on understanding the multi - molecular causes , effects and signatures of diseases . [SEP]
[CLS] personalized therapies targeted to highly specific disease [SEP]
[CLS] the detection of biological molecules involves phenomena occurring across different length scales ( fig . 1 ) . [SEP]
[CLS] for example , the regime of biomolecular recognition lies between 1 nm and 10 nm . [SEP]
[CLS] the binding regions of nucleic B-material acids I-material having sequence - specific roles in biology are in the range of 20 base pairs and above , corresponding to 6 nm and greater . [SEP]
[CLS] protein - protein B-material interactions also occur on the nanometre length scale . [SEP]
[CLS] between 10 μm and 100 μm corresponds to the regime of molecular diffusion in solution . [SEP]
[CLS] the size and shape of the biomolecular analytes of interest , combined with physiological temperatures , dictate that in the minutes - to - hours timescale appropriate for rapid biomolecular detection , typical large biological analyte molecules can diffuse 10 - 100 μm . [SEP]
[CLS] the regime of clinical sample size is millimetres to centimetres ( or microlitres to millilitres ) . [SEP]
[CLS] for many applications where the analyte of interest is rare , it is important to sample large volumes . [SEP]
[CLS] the detection of bacterial pathogens to diagnose bloodstream infections and the analysis of circulating tumour cells B-material ( ctcs ) are two examples of such applications where only a few cells B-material may be present in a millilitre of blood , necessitating that several millilitres must be sampled . [SEP]
[CLS] over the last two decades , the increasing availability of bottom - up and top - down strategies for generating nanomaterials B-material , combined with expanded methodologies that allowed conjugation of biological receptors , have prompted a significant focus on miniaturized detectors . [SEP]
[CLS] furthermore , microfluidic devices have become increasingly sophisticated . [SEP]
[CLS] these efforts have produced many examples of high - performance sensing systems , and several microfluidic platforms have been commercialized for research applications . [SEP]
[CLS] early achievements in this area also highlighted the limitations of these approaches when used in isolation . [SEP]
[CLS] while the ability of nanoscale detectors to detect biological molecules with excellent sensitivity became well documented , it was recognized that sensors with very small surface areas could not efficiently and rapidly capture and detect large , slow - moving analyte molecules within clinical samples . [SEP]
[CLS] microfluidic devices that automate the steps required for biomolecular detection using methods such as the polymerase chain reaction or immunoassays served to complement , rather than supplant , diagnostic technologies that offer complete solutions in themselves . [SEP]
[CLS] recently , combining microscale systems with nanomaterial - based strategies has permitted new types of biomolecular analysis to be developed and high levels of sensitivity to be validated with clinical specimens . [SEP]
[CLS] furthermore , using microscale devices to divide large samples into nanoscale aliquots has provided new ways to quantitate biomolecules . [SEP]
[CLS] moreover , microscale devices can leverage nanoscale binding events to sense rare analytes and cells B-material at parts per million levels . [SEP]
[CLS] in this review , we explore these concepts and their impact on advances in protein B-material biomarker B-property analysis , nucleic B-material acid I-material detection and rare - cell B-material analysis . [SEP]
[CLS] to be effective at analysing clinical samples , biomolecular detection systems must achieve high levels of sensitivity , equivalent to low limits of detection . [SEP]
[CLS] stringent specificity is also required to ensure accurate discrimination among the abundance of biomolecules found in a sample . [SEP]
[CLS] these levels of performance must be maintained in heterogeneous samples such as the complex biological matrices of blood , urine and saliva , a requirement that adds a significant challenge in light of the need to overcome nonspecific binding . [SEP]
[CLS] furthermore , to enable routine use of diagnostic test results , rapid turnaround on the timescale of a physician office visit ( ~ 20 minutes ) is highly desirable . [SEP]
[CLS] the enzyme - linked immunosorbent assay ( elisa ) is the laboratory workhorse technology for protein B-material biomarker B-property detection . [SEP]
[CLS] this technique , which uses an antibody B-material sandwich assay format , can achieve clinically relevant levels of specificity and accurately report on levels of a target protein B-material analyte . [SEP]
[CLS] however , because of the technique ' s moderate ( picomolar ) sensitivity , it fails to detect even lower , but potentially clinically relevant levels of biomarkers B-property often found in many samples of interest ( for example , femtomolar to attomolar concentrations ) . [SEP]
[CLS] indeed , many potential markers of cancer , cardiovascular disease and neurodegenerative disease are present at these low levels in affected patients . [SEP]
[CLS] a grand challenge for the protein B-material detection field has thus been to improve sensitivity dramatically relative to the picomolar sensitivity of elisa . [SEP]
[CLS] assays carried out on solid surfaces or in a laminar flow format concentrate the signal required for readout into a specific area ; however , the target molecules in a sample must come into direct contact for a binding event to take place . [SEP]
[CLS] this type of two - dimensional approach becomes limited by the slow diffusion of biomolecules . [SEP]
[CLS] the polymerase chain reaction ( pcr ) , along with other enzymatic amplification methods that copy target molecules until they can be detected by fluorescence B-property or other macroscale techniques , is the present - day gold B-material standard for nucleic B-material acid I-material analysis . [SEP]
[CLS] the sensitivity and specificity of pcr allow detection at single - digit copy numbers and specificity at the singlenucleotide level . [SEP]
[CLS] the level of automation for pcr - based assays achieved in several commercial platforms has enabled moderate - complexity labs to conduct sophisticated molecular diagnostic testing . [SEP]
[CLS] however , the multiple - hours turnaround times and the need for lab skills to obtain accurate answers with pcr assays have not allowed them to be used in point - of - care settings . [SEP]
[CLS] for molecular diagnostics to extend their reach into the clinic , complete , sample - to - answer test automation will be required . [SEP]
[CLS] this penetration to the clinic will demand short turnaround times compatible with physician office workflow . [SEP]
[CLS] achieving these capabilities is particularly important for indications such as infectious disease where the spread and severity of an infection can be limited with a rapid diagnosis . [SEP]
[CLS] therefore , any new nucleic B-material acid I-material biosensing technologies must be simple , fast , highly sensitive and specific . [SEP]
[CLS] another key challenge to bioanalysis relates to implementing protein B-material and nucleic B-material acid I-material analysis when rare cells B-material must be isolated from clinical samples . [SEP]
[CLS] for example , the analysis of ctcs may allow for non - invasive sampling of markers that define a tumour ' s phenotype and metastatic potential . [SEP]
[CLS] however , these cells B-material are present at concentrations as low as 5 cells B-material per millilitre , and are therefore outnumbered by billions of normal cells B-material in one millilitre of blood . [SEP]
[CLS] commercially available platforms for ctc immunocapture have , despite the known limitations that cause many types of ctc to evade detection , brought ctc enumeration into mainstream cancer research . [SEP]
[CLS] advanced platforms with improved sensitivities and expanded versatility ( for example , capture based on new markers , or even marker - free capture ) are required to bring ctc analysis into routine clinical cancer management . [SEP]
[CLS] achieving a clinically relevant combination of sensitivity , specificity and speed in protein B-material detection has recently seen rapid progress through the use of materials of different length scales . [SEP]
[CLS] nanomaterials B-material ( for example , nanoparticles B-nanoparticle ) have high surface - to - volume ratios and their surfaces are therefore well suited for the binding of reporter groups for biomolecular detection assays ; however , microscale materials with lower surface - to - volume ratios that possess a larger core B-material of active material B-material are better choices for manipulations such as magnetic B-property capture . [SEP]
[CLS] hence , combining these materials to detect bioanalytes brings together features that promote efficient binding of target analytes while also facilitating the collection of captured molecules . [SEP]
[CLS] a means of bringing together engineered micro - and nanoparticles B-nanoparticle for high - performance biomarker B-property detection is illustrated by a class of assays that use dna molecules as biobarcodes ( fig . 2a ) . [SEP]
[CLS] because their sequences are connected with specific analytes when the assay is designed , the dna molecules serve as barcodes that report on what analyte has been detected . [SEP]
[CLS] the first component of the capture is a microparticle with a magnetic B-property core B-material , and the second is a spherical nucleic B-material acid I-material ( sna ) [SEP]
[CLS] snas are gold B-nanoparticle nanoparticles I-nanoparticle functionalized with dna molecules that can also be decorated with different functionalities , in this case a monoclonal B-material antibody I-material . [SEP]
[CLS] even at low concentrations , the complex can - once the target protein B-material is sandwiched between the magnetic B-property microparticle and dna - coated gold B-nanoparticle nanoparticle I-nanoparticle - rapidly be isolated using a magnetic B-property field . [SEP]
[CLS] subsequently , the gold B-nanoparticle nanoparticle I-nanoparticle conjugate that is part of the sandwich complex is rapidly dissolved , thereby releasing the dna barcode strands . [SEP]
[CLS] mapping a specific dna sequence onto each protein B-material target of interest enables multiplexing , wherein the sequence is then detected using established microarray methods . [SEP]
[CLS] the efficient capture of targets , enabled by the ability of the snas to interrogate the sample in three dimensions and combined with protein - to - dna amplification , allows for femtomolar , and in some cases even attomolar , limits of detection . [SEP]
[CLS] this type of assay has been used for the detection of the hiv - 1 p24 gag protein B-material , and was shown to have excellent analytical ( fig . 2b ) and clinical ( fig . 2c ) sensitivity . [SEP]
[CLS] another example highlighting the sensitivity of this approach involved a clinical study that sought to correlate levels of the prostate - specific antigen ( psa ) with prostate cancer recurrence in men post - prostatectomy . [SEP]
[CLS] because the assay has a limit of detection 300 times lower than the most sensitive elisa assay available commercially , it was possible to obtain accurate psa measurements on patients who could then be informed that their low psa levels were stable and that it was likely that their cancer would not recur . [SEP]
[CLS] this was true for more than half of the patient pool ; with the other half , the enhanced sensitivity allowed detection of increasing levels of psa , which are linked to recurrence . [SEP]
[CLS] the study also suggested that the assay could be used to monitor patient response to adjuvant or salvage therapies . [SEP]
[CLS] the bio - barcode strategy has also been used to track markers of alzheimer disease in cerebral spinal fluid , a sample matrix in which the protein B-material marker concentration is too low ( < 1 pm ) to be detected using conventional elisa and immunoassay technologies . [SEP]
[CLS] the assay was used to analyse the presence of amyloid - β - derived diffusible ligands ( addls ) in cerebral spinal fluid from patients who had been definitively diagnosed with the disease post - mortem . [SEP]
[CLS] addls are potential soluble B-property pathogenic alzheimer disease markers . [SEP]
[CLS] a 30person study showed that all subjects , both with and without alzheimer disease , had measurable levels of addl , and that all but two of the patients with alzheimer disease had statistically elevated levels of addl compared with samples from healthy age - matched controls . [SEP]
[CLS] the ability to track levels of addl in cerebral spinal fluid , and potentially also in blood , offers a path to using precise diagnostic tools to track the responses of patients who have alzheimer disease to new therapies . [SEP]
[CLS] using nanoscale reporter groups combined with microscale capture agents has produced significant advances in the sensitivity that can be achieved for the detection of clinically relevant protein B-material biomarkers B-property . [SEP]
[CLS] this capability will be useful in a clinical context to monitor disease recurrence and to utilize biomarkers B-property that are present at very low levels . [SEP]
[CLS] an interesting and important question remains open and of interest to this field : what is the lowest concentration at which clinically relevant biomarkers B-property can be present in patient samples ? [SEP]
[CLS] since most biomarker B-property discovery efforts are performed with very small samples , it remains possible that rare , yet highly specific biomarkers B-property , could be missed in light of today ' s sensitivity limits . [SEP]
[CLS] as discovery methods turn up rarer molecules , diagnostic technologies will be required to continue to analyse ever larger samples and further improve detection limits . [SEP]
[CLS] protein B-material biomarkers B-property are important tools for the non - invasive screening and diagnosis of disease , but in many cases it is also essential to analyse nucleic B-material acid I-material sequences . [SEP]
[CLS] analysing genetic mutations and alterations that are implicated in cancer subtyping , bacterial antibiotic resistance and adverse reactions to many drugs are all examples of diagnostic applications where nucleic B-material acid I-material sequences must be delineated . [SEP]
[CLS] today , the analysis of panels of nucleic B-material acids I-material is achieved predominantly in centralized laboratories . [SEP]
[CLS] most commercial assays rely on the enzymatic amplification of a target sequence , followed by readout of the amplified sequence using an optical method . [SEP]
[CLS] a major unmet challenge for the field is the rapid detection of nucleic B-material acids I-material ( that is , in under 30 minutes ) with sample - to - answer automation - an achievement that would enable analysis outside of sophisticated labs . [SEP]
[CLS] realization of this goal would provide a powerful solution for the diagnosis of infectious diseases and for other indications where treatment decisions are time sensitive . [SEP]
[CLS] as an alternative to nucleic B-material acid I-material detection approaches based on enzymatic amplification , strategies based on amplification - free direct detection methods have received significant attention as they may represent a better solution for testing outside of the laboratory environment . [SEP]
[CLS] eliminating amplification may provide a way to speed the delivery of test results , and also simplify assay workflow to enable straightforward automation . [SEP]
[CLS] for example , several integrated circuit - based strategies have sought to use electronic transduction schemes for sensitive direct nucleic B-material acid I-material analysis . [SEP]
[CLS] by functionalizing silicon B-material nanowires B-nanoparticle with nucleic B-material acid I-material probes , field - effect transistors can be made that have conductivities that depend on the presence of a complementary target sequence . [SEP]
[CLS] target hybridization can be monitored quantitatively using this approach as a function of time , and small changes in sequence can also be detected [SEP]
[CLS] these devices combine high levels of performance with simple electronic devices that can be mass manufactured using the same infrastructure leveraged by the consumer electronics industry . [SEP]
[CLS] direct electrochemical readout of specific nucleic B-material acid I-material sequences represents another solution to the rapid molecular analysis challenge . [SEP]
[CLS] electrochemical readout differs from electronic readout because it uses the flow of current at the surface of a sensor to determine the presence or absence of an analyte , rather than detecting a change in the properties of an electronic material B-material . [SEP]
[CLS] in general , electrochemical detection schemes involve the recruitment of redox - active reporter groups to the surface of a sensor in response to the binding of a specific analyte . [SEP]
[CLS] several different approaches to electrochemical nucleic B-material acid I-material detection have been pursued , including methods that use sandwich - type assays to deliver reporter groups , enzymatic labels to produce amplified currents , molecular beacon - like e - dna assays , bio - barcode assays and non - covalent reporter systems [SEP]
[CLS] in designing electrode - based detectors for electrochemical sensing , it is particularly important to consider the implications of the nano - to - micro length scale . [SEP]
[CLS] in contrast to the particle - based approach mentioned above , electrode sensors are tethered to a measurement system during target binding and therefore must employ an alternative strategy to thoroughly interrogate a three - dimensional sample . [SEP]
[CLS] it is advantageous to miniaturize electrodes to minimize signal - to - noise , but detectors that are too small will not experience enough collisions with slow - moving analytes to attain high sensitivity . [SEP]
[CLS] indeed , rationally engineering the structures of electrochemical sensing elements both at the nanometre and micrometre scales has recently been shown to enable improvements in both the sensitivity and speed of nucleic B-material acid I-material detection , permitting detection of clinically relevant levels ( fm ) of nucleic B-material acids I-material in 30 minutes . [SEP]
[CLS] patterned microelectrodes on the surface of a glass ( fig . 3a ) or silicon B-material chip are generated by metal B-material electrodeposition ( fig . 3b ) , and then a finely nanostructured surface is created using a second electrodeposition step ( fig . 3c ) . [SEP]
[CLS] by creating a 100 - μm sensor footprint , the latest approaches promote collisions with slow - moving messenger rna ( mrna ) molecules in solution , thereby removing the diffusional limitations of other chip - based sensors that suffer from low collisional frequencies [SEP]
[CLS] the nanoscale roughness implemented in the coating B-material of these large sensors promotes the display of probes in conformations that promote efficient binding of target nucleic B-material acids I-material . [SEP]
[CLS] fundamental investigations have shown that nucleic B-material acid I-material probe molecules immobilized on nanostructured microelectrode ( nme ) surfaces exhibit an angle of deflection ( fig . 3d ) that limits steric interactions between probes , thereby enhancing the rate and efficiency of hybridization . [SEP]
[CLS] the nmes have been coupled with an electrocatalytic reporter strategy for the readout of target sequences ; electrocatalysis is triggered when a negatively charged sequence binds to the sensor surface that in turn attracts a positively charged redox reporter . [SEP]
[CLS] the electrochemical reduction of this reporter is made catalytic by the inclusion of a second electron acceptor that regenerates the cationic B-material reporter . [SEP]
[CLS] the electrocatalytic approach permits direct analysis of mrnas that serve as markers of pathogenic bacteria and associated antibiotic resistance down to concentrations as low as 1 cell B-material per microlitre ( refs 33 , 38 ; fig . 3e ) within 30 minutes , a level of sensitivity not achieved previously with such fast turnaround . [SEP]
[CLS] this strategy does not require sample clean - up , another feature that speeds the delivery of results [SEP]
[CLS] methods employing enzymatic amplification or active concentrating of samples lag behind this sample - to - answer turnaround time , with the commercial real - time pcr system possessing the highest level of automation typically delivering results in 2 - 3 hours . [SEP]
[CLS] electrochemical nucleic B-material acid I-material analysis carried out using nmes has also been applied to the quantitation of a range of analytes including small molecules and proteins B-material , and biomarkers B-property including cancer - related gene fusions and micrornas [SEP]
[CLS] in view of its low limit of detection , the strategy also shows promise in the analysis of gene expression in rare ctcs isolated from the bloodstreams of patients with cancer [SEP]
[CLS] dna - based nanostructures represent another means to enhance the sensitivity of electrochemical nucleic B-material acid I-material detection . [SEP]
[CLS] tetrahedral structures made of dna oligonucleotides display probes with well - defined distances to promote accessibility and overall detection sensitivity ( fig . 3f ) . [SEP]
[CLS] attomolar concentrations of micrornas were detected using this approach , indicating that the display of probe sequences on both organic and inorganic nanostructures produce high levels of sensitivity . [SEP]
[CLS] nanomolar levels of small - molecule and protein B-material analytes can also be detected using sensors based on dna nanostructures [SEP]
[CLS] while advances in multi - length - scale engineering have improved the sensitivity and speed of nucleic B-material acid I-material detection , key challenges that remain include the demonstration of the robustness of direct nucleic B-material acid I-material detection strategies in a clinical setting and to evaluate whether the diverse range of biological backgrounds encountered in clinical samples will interfere with approaches where trace levels of specific sequences are recruited to electrochemical sensors . [SEP]
[CLS] while eliminating front - end sample processing of biological samples simplifies the instrumentation required significantly , it renders the requirement of excellent specificity and sensitivity even more stringent . [SEP]
[CLS] while the methods discussed thus far are fast , sensitive and highly specific , there are limits to their capacity to quantitate biological analytes accurately over a large dynamic range of concentration . [SEP]
[CLS] several technologies have been developed recently that implement digital methods to address the quantitation challenge . [SEP]
[CLS] in the digital approach , macroscopic patient samples , typically possessing millilitre to microlitre volumes , are subdivided into many much smaller subvolumes , typically in the nanolitre to picolitre range . [SEP]
[CLS] dividing samples in this manner ensures that , on average , only a small integer number of target molecules exist within each compartment [SEP]
[CLS] as a result , the occupancy of compartments may be described using poisson statistics , ideally in such a way that each compartment contains either zero or one molecule of the target analyte ( fig . 4a ) . [SEP]
[CLS] nucleic B-material acid I-material amplification of the contents of these subvolumes can be used to provide either a binary outcome ( hence the digital nomenclature ) , and the concentration of the target analyte can be calculated from this measured distribution . [SEP]
[CLS] in principle , digital approaches provide a wide dynamic range in the determination of target concentrations compared with traditional bulk ensemble measurements . [SEP]
[CLS] the size of the compartments can be varied across the micro - to millilitre length scales to provide an expanded dynamic range . [SEP]
[CLS] microfluidic implementation of this approach has enabled quantitative biomolecular analysis for a variety of applications . [SEP]
[CLS] genetic alterations known as copy - number variations can be monitored to understand how individual patients respond to cancer drugs , viral nucleic B-material acids I-material can be quantitated for infectious disease monitoring , and single - cell gene expression profiling can be carried out to facilitate the identification of biological heterogeneity [SEP]
[CLS] it has also been utilized to quantify unequal expression of the two copies of each gene found in the diploid human genome and profile low levels of residual disease biomarkers B-property that may remain after cancer treatment . [SEP]
[CLS] in prenatal diagnostics , it can provide non - invasive detection of genetic defects , an approach complementary to advanced sequencing strategies . [SEP]
[CLS] digital amplification may also provide an effective solution for the quantification of nucleic B-material acid I-material targets in limited - resource settings - such as a remote clinic , in the field , home or any other location where the sophisticated equipment required for pcr is unavailable . [SEP]
[CLS] making reliable , quantitative measurements using cost - effective devices will require merging simple microfluidic approaches for sample preparation with amplification strategies that are straightforward to implement . [SEP]
[CLS] equipment - free compartmentalization of molecules is a microfluidic strategy particularly appropriate for limited - resource settings , as it eliminates the need for pumps [SEP]
[CLS] furthermore , isothermal amplification chemistry also simplifies the workflow needed to detect nucleic targets by removing the requirement for thermocycling and is a good candidate for lab - free testing . [SEP]
[CLS] technologies combining these approaches have been validated in initial studies , wherein viral nucleic B-material acids I-material of hiv and the hepatitis c virus were partitioned in microcompartments , quantified and equivalence was shown between pcr and isothermal amplification chemistries ( fig . 4b ) . [SEP]
[CLS] these studies were performed using a ' slipchip ' , a glass substrate patterned with microcompartments of varying sizes that can be filled with sample and amplification reagents via relative rotation of a similarly patterned substrate . [SEP]
[CLS] robustness , that is , consistent performance despite variation in external conditions , is a crucial requirement in low - resource settings and one of the key advantages of digital singlemolecule counting approaches [SEP]
[CLS] because of its reliance on binary outputs , the digital format gives isothermal amplification chemistries an unexpected high degree of robustness with respect to perturbations in temperature , reaction time and imaging quality . [SEP]
[CLS] digital isothermal assays have been read using a mobile B-property phone camera that captures an image of a chip and transmits the pattern of the positive and negative compartments wirelessly to a cloud server for analysis [SEP]
[CLS] a recently reported approach that uses microwells , rather than microfluidics , also applies the same digital logic to detect proteins B-material . [SEP]
[CLS] here , micrometre - sized magnetic B-property beads with a capture antibody B-material bound to their surface are added to a patient sample ( fig . 5a , b ) . [SEP]
[CLS] because the number of beads added exceeds the number of molecules in the sample by nearly an order of magnitude , the molecules bind to the beads as described by a poisson distribution [SEP]
[CLS] most beads do not capture any molecules while about 10 % will capture only a single molecule [SEP]
[CLS] as in elisa , a sandwich is formed in which the protein B-material bridges the capture and detection antibodies B-material , and thus any bead that has captured the target of interest carries an enzyme . [SEP]
[CLS] the beads are then distributed into 40 - femtolitre microwells , with each microwell sized to fit a single bead . [SEP]
[CLS] a fluorogenic substrate is added to the bead - loaded microwell array and fluorescent B-property product accumulates in wells containing an enzyme . [SEP]
[CLS] quantitation of the number of the original target protein B-material molecules is thus obtained by counting the number of fluorescent B-property wells . [SEP]
[CLS] femtomolar concentrations of an analyte within a sample can be detected , compared with low picomolar ( pm ) detection limits for the best commercial protein B-material assays using the same reagents . [SEP]
[CLS] this single - molecule - array strategy can also be used to detect synthetic nucleic B-material acids I-material present at femtomolar concentrations without the need for target amplification , and was also adapted to detect bacterial dna at attomolar levels [SEP]
[CLS] the approach has also been used to detect tau protein B-material in samples from patients with alzheimer disease and brain injury [SEP]
[CLS] the resulting data , when combined with measurements of abeta42 levels , can predict cognitive outcome after brain hypoxia due to myocardial infarction . [SEP]
[CLS] the strategy has also been employed to detect toxins , to detect early hiv infection , to predict recurrence of prostate cancer , and to measure inflammatory markers in patients with crohn disease . [SEP]
[CLS] the digital elisa method has also been used to measure the low ( pg ml −1 ) levels of psa in patients with prostate cancer who have undergone radical prostatectomies - these levels cannot be measured with a conventional elisa ( fig . 5c ) . [SEP]
[CLS] the quantitative nature of digital approaches brings new precision to the measurement of disease - related biomarkers B-property , and enriches the precision of information content that can be obtained with clinical samples . [SEP]
[CLS] it will enable biomolecular markers to be detected and monitored , thereby allowing response to therapy to be followed , and disease progression to be tracked accurately . [SEP]
[CLS] however , clinical interpretation of the results will be more complicated and greater clinical validation may be required for the implementation of new quantitative tests that are currently performed on a qualitative basis . [SEP]
[CLS] while molecular biomarker B-property detection is integral to effective clinical diagnostic technologies , another important set of challenges relates to the capture and analysis of specific types of intact cell B-material . [SEP]
[CLS] the concentration of ctcs in the bloodstream of patients who have cancer as well as phenotypic status offers important information that may provide valuable diagnostic and prognostic insights . [SEP]
[CLS] for this application , attaining high levels of sensitivity and specificity is a challenge . [SEP]
[CLS] ctcs can be present at very low levels in blood ( 1 - 10 cells B-material per millilitre ) , and are overwhelmingly outnumbered by normal blood cells B-material a billion - fold . [SEP]
[CLS] early technologies developed for ctc isolation and enumeration relied on tagging cells B-material with magnetic B-nanoparticle nanoparticles I-nanoparticle displaying an antibody B-material specific for the epithelial cell B-event adhesion I-event molecule ( epcam ) , which is a marker present on the surfaces of many cancer cells B-material . [SEP]
[CLS] cellsearch , an instrument approved by the food and drug administration for monitoring treatment efficacy using this approach , is considered by many to be the present - day gold B-material standard for ctc counting . [SEP]
[CLS] while the analytical performance of cellsearch as assessed using cell B-material lines is reasonable ( with a reported analytical sensitivity of 1 ctc in a 7 . 5 - ml sample ) , it typically identifies ctcs in the bloodstream for less than 50 % of patients with cancer with metastatic disease [SEP]
[CLS] as a result , it is primarily used only as a prognostic tool . [SEP]
[CLS] a first step in advancing next - generation ctc systems was to improve the efficiency of capture in patient samples . [SEP]
[CLS] sophisticated microfluidic devices that feature epcam - coated surfaces have proven to be effective for non - destructive ctc capture . [SEP]
[CLS] by displaying a nanoscale capture agent , for example an antibody B-material to epcam , on a microfluidic device , capture efficiencies increased significantly and led to ctc detection sensitivities in patient samples that were improved by a factor of 2 - 3 times relative to cellsearch . [SEP]
[CLS] the utility of these microfluidic devices has been shown for monitoring prostate cancer and the identification of potential drug targets based on expression profiling of ctcs in pancreatic cancer . [SEP]
[CLS] microfluidics - enabled strategies that rely on differences in deformability , size [SEP]
[CLS] or density of ctcs relative to other cells B-material in the bloodstream have also been developed for the sensitive isolation of rare ctcs . [SEP]
[CLS] affinity - based methods for isolating and separating cells B-material remain powerful tools in light of the heterogeneity of ctcs and the possibility that only certain subpopulations may be clinically relevant . [SEP]
[CLS] the use of antibody B-material cocktails to capture both the epithelial and mesenchymal phenotypes of ctcs showed the first demonstration of the epithelial - to - mesenchymal transition in patients with breast cancer . [SEP]
[CLS] work on advanced ctc capture systems has increasingly focused on expanding the types of surface marker that are used for cell B-material selection . [SEP]
[CLS] recently , several ctc capture systems have been reported that can use markers other than epcam for capture and / or analysis , or that rely on negative selection so that a priori knowledge of the surface expression characteristics of ctcs is not required [SEP]
[CLS] for example a microfluidic inertial focusing platform - the ictc chip ( fig . 6a , b ) - combines several types of separation [SEP]
[CLS] it depletes smaller red blood cells B-material and platelets in the first microfluidic stage using a mechanical filter . [SEP]
[CLS] the system then allows ctcs and leukocytes to be sorted using either positive or negative selection via the attachment of magnetic B-property microparticles to the remaining cells B-material , and a magnetic B-property field directs the cells B-material to different isolation chambers . [SEP]
[CLS] for positive selection of ctcs , a marker such as epcam can be used to bind magnetic B-property beads to ctcs for isolation , while for negative selection , leukocyctes and granulocytes can be labelled with a cocktail of antibodies B-material to separate them from the ctcs . [SEP]
[CLS] this approach was shown to be effective with patient samples having low ctc loads . [SEP]
[CLS] for samples where < 30 ctcs were detected in a 7 . 5 - ml sample using the ichip , cellsearch often did not report any ctcs ( fig . 6c ) . [SEP]
[CLS] an additional recent study documented the use of a micro - hall detector for the simultaneous analysis of several different markers . [SEP]
[CLS] this approach does not capture ctcs selectively , but instead identifies cells B-material with a particular marker profile in an unpurified sample , thereby avoiding the loss of cells B-material , which is often incurred in purification . [SEP]
[CLS] magnetic B-nanoparticle nanoparticles I-nanoparticle with varying magnetization B-property properties are functionalized with different antibodies B-material to surface antigens , and the presence of specific particles is analysed with a micro - hall detector . [SEP]
[CLS] this approach increased ctc detection frequency in patient samples by 25 % over what could be achieved with cellsearch . [SEP]
[CLS] another recent study documented a highly versatile ensembledecision aliquot ranking approach in which any antibody B-material of choice can be employed to analyse the marker profile of a ctc . [SEP]
[CLS] multiple antibodies B-material are labelled with different flurophores , incubated B-technique with ctc - containing blood , and the sample is pumped through a microfluidic channel , where fluorescent B-property cells B-material are counted . [SEP]
[CLS] this system also outperformed cellsearch and detected ctcs in 82 out of 90 patients with stage - iv metastatic breast cancer ( 91 % ) , while cellsearch only detected cells B-material in 44 % of the patients . [SEP]
[CLS] significant progress has been made in the isolation and analysis of ctcs and promising advances that combine microscale engineering with nanoscale binding events are on the horizon . [SEP]
[CLS] advanced methods for intracellular rna expression may play an important role in ctc characterization . [SEP]
[CLS] engineered B-nanoparticle nanoparticles I-nanoparticle coated with oligonucleotide probes , for example , can enter cells B-material , bind to an mrna sequence of interest and concomitantly release a fluorophore - labelled sequence . [SEP]
[CLS] this has the potential to search for ctcs based on unique genetic signatures . [SEP]
[CLS] progress towards sensitive , specific , rapid , automated diagnostics has been brisk and impressive ; nevertheless , significant opportunities to address more fully the goals of this field still exist , and multi - length - scale engineering is likely to remain important . [SEP]
[CLS] the degree of automation varies from platform to platform , and typically is less complete in more complex assays . [SEP]
[CLS] full automation of biomolecular detection platforms will broaden the uptake of new detection capabilities and promote efficiency in resource - constrained laboratories . [SEP]
[CLS] better sample processing interfaces that introduce samples into analysis platforms with a high level of automation will facilitate the generation of accurate results even in the absence of highly trained operators . [SEP]
[CLS] the platforms discussed above often focus on the automation of key elements of the sample processing chain ( for example , automated sample processing or readout ) , but ultimately retain manual steps to connect them . [SEP]
[CLS] an automated workflow with no human intervention between the introduction of a sample and data presentation is the goal for most assays so that they can be used outside of laboratory facilities . [SEP]
[CLS] furthermore , although much progress has been made in multiplexing , with the technologies reviewed here finding successful application in the detection and even quantitation of multiple biomarkers B-property , further advances are needed . [SEP]
[CLS] it is projected that research into genetic fingerprints of disease will produce a demand for tests employing hundreds of biomarkers B-property . [SEP]
[CLS] in addition , tools for pre - clinical research that seek to enable the discovery and winnowing down of new gene expression fingerprints will benefit from rapid , automated and low - cost analysis of thousands of markers from libraries of prospective targets . [SEP]
[CLS] technologies that scale to this order of biomarker B-property multiplexing will be in demand , and given the importance of miniaturized diagnostic devices , combinations of micro - and nanofabrication will be an important tool . [SEP]
[CLS] knowledge of the molecular basis of disease is advancing rapidly , with molecular targets emerging in many disease states that represent promising targets for earlier detection and targeted treatment . [SEP]
[CLS] as summarized in this review , technologies that can detect and quantitate protein B-material and nucleic B-material acid I-material biomarkers B-property with the desired level of accuracy , cost and automation will be critical for the integration of these markers into routine clinical practice . [SEP]
[CLS] the advances highlighted herein all perform at high levels because they leverage the advantages of working on several length scales to achieve high levels of sensitivity and specificity . [SEP]
[CLS] uniting features of both nano - and microscale materials brings together compelling capabilities for analyte capture and analysis , and combining microdevices with materials that allow for tagging of molecules and cells B-material at the nanoscale has yielded devices with superior performance . [SEP]
[CLS] these advances provide new means of searching for the markers of cancer , and neurodegenerative and infectious diseases . [SEP]
[CLS] bioanalytical detection approaches monitor binding events between analytes of interest and specific receptors that occur on the nanoscale . [SEP]
[CLS] nanomaterials B-material are ideal for biomolecular display because their dimensions approach the molecular scale , which promotes binding behaviour similar to that observed in solution - based processes . [SEP]
[CLS] for reference , a molecule of cholesterol is shown on the left ; this molecule is nanometre - sized . [SEP]
[CLS] a dna molecule ( red / blue ) and a protein B-material molecule ( green / blue ) , both structures that have sizes on the order of tens of nanometres are also depicted . [SEP]
[CLS] a bacteriophage ( purple ) , bacterial cell B-material ( green ) and human cell B-material ( pink / blue ) span the hundreds of nanometres to micrometre length scale . [SEP]
[CLS] the microscale length regime is also important for biomolecular detection because most large molecules can only diffuse micrometres while detection is in progress . [SEP]
[CLS] devices can straightforwardly be engineered on this length scale , which facilitates the analysis of cells B-material and processing of macroscale clinical samples ( depicted as a test tube ) . [SEP]
[CLS] nucleic B-material acid I-material amplification of the targets in a much smaller volume than during bulk amplification gives a signal that rises above the background level and above the signal arising from non - specific amplification . [SEP]
[CLS] the quantitation of positive and negative wells allows a highly precise ratio of two analytes to be determined . [SEP]
[CLS] this approach provides a wider dynamic range than traditional bulk ensemble measurements when determining the concentrations of target molecules . [SEP]
[CLS] conclusionsb , quantitation of viral rna within a slipchip microfluidic device containing wells of different sizes via reverse transcription B-event and pcr . [SEP]
[CLS] a slipchip is a piece of plastic or glass patterned with an array of microcompartments that can be filled with sample , and then using a similarly patterned top plate , amplification reagents can be introduced into each well by slipping the position of the two substrates . [SEP]
[CLS] after the amplification is complete , fluorescent B-property products ( green ) can be detected visually , and the number of positive wells reports on the concentration of a target analyte . [SEP]
[CLS] at very low concentrations of rna , the large wells have a higher probability of capturing and amplifying a molecule of rna ( larger green spots ) . [SEP]
[CLS] at high concentrations of rna , small wells capture and amplify rna ( small green spots ) . [SEP]
[CLS] quantification is performed over the range of concentrations from where several large wells turn green up to where several small wells remain dark . [SEP]
[CLS] panel b is reproduced with permission from ref . 64 , © 2011 american chemical society . [SEP]
[CLS] a , a target antigen incubated B-technique with magnetic B-property beads ( blue circle ) binds to capture antibodies B-material ( orange y shape ) on the bead surface . [SEP]
[CLS] biotinylated detection antibodies B-material and an enzymatic reporter ( β - galactosidase in this case ) form a sandwich complex . [SEP]
[CLS] b , once deposited in an array of microwells , a fluorescent B-property substrate is added to the sample , and wells are quantitated to analyse levels of protein B-material biomarkers B-property . [SEP]
[CLS] c , analysis of clinical samples for psa levels . [SEP]
[CLS] concentrations of psa were measured in serum samples from patients who had undergone a radical prostatectomy ( blue circles ) , healthy control samples ( red squares ) and bio - rad psa control samples ( black triangles ) determined using a digital elisa . [SEP]
[CLS] all samples yielded levels above the detection limit of the digital method ( red line ) . [SEP]
[CLS] in contrast , the detection limit of a commercially available elisa test ( green line ) is too high to provide comparable data . [SEP]
[CLS] panel c is reproduced from ref . 68 , 2010 nature publishing group . [SEP]
[CLS] this allows white blood cells B-material ( wbcs ) and ctcs to be separated from abundant red blood cells B-material ( rbcs ) . [SEP]
[CLS] either the wbcs or ctcs can be labelled with magnetic B-property particles , and the application of a magnetic B-property force ( b ) then forces the different cells B-material to exit the chip via different outlets where they can be isolated as an enriched population . [SEP]
[CLS] c , ctc levels isolated from blood samples collected from patients with metastatic breast , prostate , pancreatic , colorectal , or lung cancer . [SEP]
[CLS] the samples were analysed with the ichip or the gold B-material - standard cellsearch method . [SEP]
[CLS] significantly higher levels of ctcs were observed when the ichip was used , and many samples that appear negative for ctcs when cellsearch was used for analysis were found to have significant levels of cancer cells B-material with the ichip . [SEP]
[CLS] this indicates that ctc counting is much more accurate with this new approach and enables ctc monitoring even in patients with low counts . [SEP]
[CLS] panels b , c adapted from ref . 95 , © 2013 american association for the advancement of science . [SEP]
[CLS] 1 . length scales of interest for biomolecular detection [SEP]
[CLS] the bio - barcode assay combines micro - and nanoparticles B-nanoparticle for biomolecular detectiona , a protein B-material target ( green diamond ) is bound by a magnetic B-property microparticle ( grey ) functionalized with a specific antibody B-material ( green y - shape ) . [SEP]
[CLS] the microparticles are probed with gold B-nanoparticle nanoparticles I-nanoparticle ( yellow ) that are functionalized with antibodies B-material ( green ) and barcode sequences ( blue ) . [SEP]
[CLS] after isolation , the barcodes are released and detected on an array . [SEP]
[CLS] the pattern of barcode binding on the array displaying a variety of barcode - binding sequences reports on the presence or absence of particular analytes . [SEP]
[CLS] the same approach can be used to detect specific dna sequences ( bottom ) . [SEP]
[CLS] figure 2 . b , data showing hiv p24 gag protein B-material is detected at picogram levels using the bio - barcode assay described in a . nc , noncomplementary control . [SEP]
[CLS] error bars represent standard deviations . [SEP]
[CLS] c , clinical data showing sensitivity and specificity of the bio - barcode assay as compared to the gold B-material standard ( pcr ) . [SEP]
[CLS] the x - axis values correspond to levels of hiv rna in blood samples drawn from 146 patients measured by pcr . [SEP]
[CLS] the y axis displays p24 protein B-material concentrations in these same specimens measured by the new barcode assay . [SEP]
[CLS] each red dot represents a data point collected from a patient sample , and grey bars indicate the median and interquartile range . [SEP]
[CLS] this data set shows excellent correspondence between the gold B-material - standard pcr method used to detect hiv rna and the new barcode assay that measures p24 protein B-material , and there is strong statistical [SEP]
[CLS] 3 . advances in rapid nucleic B-material acid I-material detection based on micro - to - nanoscale engineering of electrode propertiesa , a lithographically patterned chip that serves as a substrate for the patterning of nanostructured microelectrodes ( nmes ) . [SEP]
[CLS] figure 3 . b , nmes combine the advantage of a large surface area with a finely nanostructured surface . [SEP]
[CLS] gold B-material is electroplated on the surface of the chip through apertures etched in a passivation layer . [SEP]
[CLS] probe molecules ( blue lines ) are attached to the electrodes , and after a target - containing sample ( red lines ) is introduced , reporter groups are incubated B-technique with the sensor to give an electrochemical readout . [SEP]
[CLS] c , electron micrographs showing nmes have 100 - μm footprints ( top ) , but feature nanoscale roughness on the order of 10 nm ( bottom ) . [SEP]
[CLS] d , nanoscale roughness promotes the creation of a deflection angle that decreases steric interference between probe molecules ( blue ) immobilized with a thiolated tether ( black ) on a highly nanostructured surface ( orange ) . [SEP]
[CLS] the grey ovals represent the zone within which the probe strand has additional space to interact with a target sequence because [SEP]
[CLS] dividing samples into nano - to femtolitre volumes allows highly accurate quantitation of clinically relevant targets a , in digital single - molecule amplification , target molecules ( blue and red ) are compartmentalized into smaller volumes . [SEP]
[CLS] nucleic B-material acid I-material amplification of the targets in a much smaller volume than during bulk amplification gives a signal that rises above the background level and above the signal arising from non - specific amplification . [SEP]
[CLS] the quantitation of positive and negative wells allows a highly precise ratio of two analytes to be determined . [SEP]
[CLS] this approach provides a wider dynamic range than traditional bulk ensemble measurements when determining the concentrations of target molecules . [SEP]
[CLS] figure 4 . b , quantitation of viral rna within a slipchip microfluidic device containing wells of different sizes via reverse transcription B-event and pcr . [SEP]
[CLS] a slipchip is a piece of plastic or glass patterned with an array of microcompartments that can be filled with sample , and then using a similarly patterned top plate , amplification reagents can be introduced into each well by slipping the position of the two substrates . [SEP]
[CLS] after the amplification is complete , fluorescent B-property products ( green ) can be detected visually , and the number of positive wells reports on the concentration of a target analyte . [SEP]
[CLS] at very low concentrations of rna , the large wells have a higher probability of capturing and amplifying a molecule of rna ( larger green spots ) . [SEP]
[CLS] microwells separate analytes into femtolitre compartments for quantitative protein B-material detection [SEP]
[CLS] 6 . advanced microdevices for circulating tumour cell B-material isolation a , ctcs ( green ) are recognized by antibody - functionalized magnetic B-property particles ( mnps ; grey ) . [SEP]
[CLS] figure 6 . b , a microengineered ctc isolation chip allows negative and positive selection for ctcs by manipulating the flow of microparticle - tagged cells B-material . [SEP]
[CLS] whole blood is injected into the device , and passed through an array of microposts that separate the cells B-material based on size . [SEP]
[CLS] this allows white blood cells B-material ( wbcs ) and ctcs to be separated from abundant red blood cells B-material ( rbcs ) . [SEP]
[CLS] either the wbcs or ctcs can be labelled with magnetic B-property particles , and the application of a magnetic B-property force ( b ) then forces the different cells B-material to exit the chip via [SEP]
[CLS] we report a novel spherical nucleic B-material acid I-material ( sna ) gold nanoparticle B-nanoparticle conjugate , termed the sticky - flare , which enables facile quantification of rna expression in live cells B-material and spatiotemporal analysis of rna transport and localization . [SEP]
[CLS] the sticky - flare is capable of entering live cells B-material without the need for transfection agents and recognizing target rna transcripts B-event in a sequence - specific manner . [SEP]
[CLS] on recognition , the sticky - flare transfers a fluorophore - conjugated reporter to the transcript B-event , resulting in a turning on of fluorescence B-property in a quantifiable manner and the fluorescent B-property labeling of targeted transcripts B-event . [SEP]
[CLS] the latter allows the rna to be tracked via fluorescence B-technique microscopy I-technique as it is transported throughout the cell B-material . [SEP]
[CLS] we use this novel nanoconjugate to analyze the expression level and spatial distribution of β - actin mrna in hela cells B-material and to observe the real - time transport of β - actin mrna in mouse embryonic fibroblasts . [SEP]
[CLS] furthermore , we investigate the application of stickyflares for tracking transcripts B-event that undergo more extensive compartmentalization by fluorophore - labeling u1 small nuclear rna and observing its distribution in the nucleus of live cells B-material . [SEP]
[CLS] recently , the localization of mrna has been identified as an essential process for a number of cellular functions , including restricting the production of certain proteins B-material to specific compartments within cells B-material . [SEP]
[CLS] for instance , synaptic potentiation , the basis of learning and memory , relies on the local translation of specific mrnas in pre - and postsynaptic compartments . [SEP]
[CLS] likewise , the misregulation of rna distribution is associated with many disorders , including mental retardation , autism , and cancer metastasis B-event . [SEP]
[CLS] however , despite the significant role of mrna transport and localization in cellular function , the available methods to visualize these phenomena are severely limited . [SEP]
[CLS] for example , fish , the most commonly used technique to analyze spatial distribution of rna , requires fixation and permeabilization of cells B-material before analysis . [SEP]
[CLS] as a result , analysis of dynamic rna distribution is restricted to a single snapshot in time . [SEP]
[CLS] with such a limitation , understanding the translocation of rna with respect to time , cell B-material cycle , or external stimulus is difficult if not impossible . [SEP]
[CLS] furthermore , fixed cell B-material analysis is a lengthy and highly specialized procedure due to the number of steps necessary to prepare a sample . [SEP]
[CLS] fixation , permeabilization , blocking , and staining processes each require optimization and vary based on cell B-material type and treatment conditions , rendering fish prohibitively complicated in many cases . [SEP]
[CLS] likewise , live cell analysis platforms such as molecular beacons require toxic B-property transfection techniques , such as microinjection or lipid B-material transfection , and are rapidly sequestered to the nucleus on cellular entry . [SEP]
[CLS] recently more sophisticated live cell analyses have been developed that use genetic engineering to introduce exogenous hybrid genes via viral or plasmid transfection . [SEP]
[CLS] these hybrid transcripts B-event include added aptamer motifs that bind to subsequently introduced or coexpressed fluorescent B-property reporters such as malachite green , gfp , or gfp derivatives . [SEP]
[CLS] such techniques offer dynamic visualization of rna ; however , the reliance of genetic engineering and exogenous hybrid rna sequences makes such techniques highly involved and does not allow for the quantification of endogenous gene expression . [SEP]
[CLS] thus , to accurately study the dynamics of endogenous rna , a new type of analysis platform is required . [SEP]
[CLS] ideally such a platform should be capable of both quantifying transcript B-event expression and tracking intracellular transcripts B-event in real time , without the need for transfection agents or specialized techniques . [SEP]
[CLS] herein we present such a platform and use the newly developed construct to perform rna quantification and real - time analysis of dynamic rna translocation . [SEP]
[CLS] previously our group introduced the concept of the nanoflare , a spherical nucleic B-material acid I-material ( sna ) conjugate capable of quantifying rna concentration in live cells B-material with single cell resolution . [SEP]
[CLS] the nanoflare consists of a 13 - nm gold B-nanoparticle nanoparticle I-nanoparticle core B-material functionalized with a densely packed , highly oriented shell B-material of oligonucleotides designed to be antisense to a target rna transcript B-event . [SEP]
[CLS] a fluorophore - conjugated reporter strand , termed the flare sequence , is subsequently hybridized to the antisense oligonucleotides via complementary base pairing . [SEP]
[CLS] hybridization of the flare sequence holds the fluorophore in close proximity to the gold B-material core B-material of the sna , effectively quenching fluorescence B-property . [SEP]
[CLS] however , upon cellular entry the antisense capture sequences of the nanoflare bind to targeted transcripts B-event , forming longer , more stable duplexes . [SEP]
[CLS] these binding events displace the flares from the gold B-material surface , resulting in a discrete " turning on " of fluorescence B-property , the extent of which is quantifiably related to the expression level of the target rna . [SEP]
[CLS] importantly , the nanoflare enters live cells B-material via receptormediated endocytosis B-event without the need for harmful transfection techniques and with negligible cytotoxicity B-property and immunogenicity B-property . [SEP]
[CLS] as a result , the nanoflare has grown into a [SEP]
[CLS] proper function of rna is critical to the health and maintenance of a cell B-material , and its misregulation plays a critical role in the development of many disorders . [SEP]
[CLS] to date , the ability to study rna has been severely limited ; many analytical techniques focus only on quantifying expression levels of transcripts B-event and are not capable of reporting on intracellular localization , which has emerged as a critical component of rna function . [SEP]
[CLS] similarly , techniques capable of probing rna localization only offer snapshots in fixed cells B-material . [SEP]
[CLS] herein we introduce a novel nanoconjugate , termed the stickyflare , capable of reporting on both of these critical components in live cells B-material , thereby gaining a more complete picture or rna function than any analytical technique to date . [SEP]
[CLS] powerful and prolific tool in biology and medical diagnostics , with [UNK] , 600 unique forms commercially available today ( sold under the smartflare trade name ) . [SEP]
[CLS] conventional nanoflares do not allow one to determine the spatial distribution of rna within the cell B-material . [SEP]
[CLS] indeed , nanoflaretranscript binding results in a displacement of the fluorophorelabeled sequence from the nanoparticle B-nanoparticle construct , which spatially separates the fluorescent B-property reporter and transcript B-event ( fig . 1 , upper ) . [SEP]
[CLS] however , with a simple design change where the flare sequence is made longer and complementary to the target rna transcript B-event , a new construct can be realized that can track and quantify rna within a cell B-material ( fig . 1 , lower ) . [SEP]
[CLS] herein , we report the synthesis and development of such a construct , termed the sticky - flare , and investigate its use as a platform for rna quantification and realtime tracking of transcripts B-event as they are transported within live hela and mouse embryonic fibroblast ( mef ) cells B-material . [SEP]
[CLS] these constructs and their activity in cells B-material represent the only way of measuring both the quantity and location of mrna in live cells B-material . [SEP]
[CLS] nucleic B-material acid I-material targets in a sequence - specific manner . [SEP]
[CLS] solutions of β - actin targeting sticky - flares ( 1 nm ) were evaluated before and after the addition of fully - complementary targets , ranging from 5 nm to 2 μm in pbs . [SEP]
[CLS] on addition of a complementary target , a resulting increase in fluorescence B-property was observed in a manner proportional to the concentration of target added , showing the sticky - flares can be used to quantify the concentration of complementary sequences in vitro . [SEP]
[CLS] importantly , the addition of noncomplementary target ( 2 μm ) had no measurable effect ( fig . 2a ) . [SEP]
[CLS] next , β - actin sticky - flares were evaluated in a cell B-material culture model by flow B-technique cytometry I-technique . [SEP]
[CLS] hela human cervical cancer cells B-material were treated with either control ( nontargeting ) sirna ( 50 nm ) delivered via lipofectamine rnaimax transfection agent , anti - β - actin sirna with rnaimax , or rnaimax alone for 24 h , after which the media was replaced with media containing sticky - flares targeting either β - actin mrna or u1 short nuclear rna ( negative control ) . [SEP]
[CLS] after an additional 18 - h incubation B-technique , the fluorescence B-property of each cell B-material was quantified . [SEP]
[CLS] with β - actin sticky - flares , we detected significant knockdown of rna expression levels compared with cells B-material treated with control sirna or transfection agent alone , whereas the control sticky - flares showed no significant difference with any treatment ( fig . 2b and fig . s1 ) . [SEP]
[CLS] intracellular rna tracking by sticky - flares [SEP]
[CLS] our ability to track the spatial distribution of rna with sticky - flares was evaluated using confocal microscopy B-technique . [SEP]
[CLS] two genes with disparate intracellular function and localization patterns were chosen to analyze spatial distribution within cells B-material : β - actin mrna and u1 small nuclear rna ( snrna ) . [SEP]
[CLS] in previous reports , β - actin mrna was found to localize at the growth cones of lamellae in embryonic fibroblasts . [SEP]
[CLS] in contrast , u1 snrna is imported into the nucleus , where it acts as a key component of the spliceosome . [SEP]
[CLS] mefs were cultured in glass - bottomed cell B-material culture chambers with sticky - flares for 12 h , after which the cells B-material were treated with a nuclear stain ( hoechst ) for 10 min and imaged in live form . [SEP]
[CLS] fluorescence B-property from cells B-material treated with β - actin sticky - flares exhibited punctate fluorescence B-property throughout the cell B-material body and a demonstrable preference for the growth cone region of lamellae extensions ( fig . 3 ) . [SEP]
[CLS] additional highly fluorescent B-property regions can be seen within the lamellae extensions , marking β - actin rna being actively transported to and from the growth cone . [SEP]
[CLS] this active transport is further analyzed below . [SEP]
[CLS] importantly , sticky - flares are not limited to use in live cells B-material and can be used to verify rna localization in fixed cells B-material as a convenient control . [SEP]
[CLS] therefore , fixed and permeabilized mefs were treated with sticky - flares , confirming the growth cone specific localization observed in live cells B-material ( fig . s2 ) . [SEP]
[CLS] contrastingly , mefs treated with sticky - flares targeting u1 snrna distinctly showed internuclear fluorescence B-property . [SEP]
[CLS] importantly , sna constructs are sequestered to the cytosol of live cells B-material and cannot enter the nucleus on their own . [SEP]
[CLS] thus , the internuclear fluorescence B-property of u1 sticky - flares indicates specific release from the nanoparticle B-nanoparticle surface and subsequent transport of the fluorescentlabeled rna target into the nucleus . [SEP]
[CLS] beyond studying the final localization of mrna strands , the facile , noninvasive nature of the sticky - flare allows for real - time observation of dynamic rna translocation in live cells B-material . [SEP]
[CLS] to demonstrate this , mefs treated with β - actin sticky - flares were imaged every 10 s with a confocal microscope for a total of 10 min . [SEP]
[CLS] when the plane of imaging was focused on lamellae , transport of β - actin mrna was observed primarily ( but not exclusively ) in the distal direction toward the growth cone ( fig . 4 and movie s1 ) . [SEP]
[CLS] furthermore , when focused directly at the body of the cell B-material , rna dynamics become even more evident , with hundreds of fluorophore - labeled β - actin sequences being transported throughout the cell B-material ( movie s2 ) . [SEP]
[CLS] similar analyses were performed in hela cells B-material . [SEP]
[CLS] in this case , β - actin sticky - flares exhibited a starkly different intracellular distribution , showing a high degree of colocalization with mitochondria ( fig . 5 ) . [SEP]
[CLS] this colocalization of β - actin mrna and mitochondria in hela cells B-material has not been demonstrated before , and the reason behind such highly spatially restricted expression is not known . [SEP]
[CLS] however , this distribution closely parallels the mitochondrial localization of k - ras and gapdh in hela cells B-material , which has been reported in prior work . [SEP]
[CLS] to observe dynamic rna movement and to further probe the β - actin / mitochondrial colocalization , fluorescence B-property was monitored in hela cells B-material starved by culturing in eagle ' s balanced salt B-material solution . [SEP]
[CLS] on starvation , the mitochondrial network collapses and mitochondria and rna both migrated toward the perinuclear region , forming a more punctate expression pattern and maintaining colocalization over the time observed ( movie s3 ) . [SEP]
[CLS] we synthesized and introduced a novel nanoconjugate termed the sticky - flare , which is a result of redesigning the conventional nanoflare or smartflare to extend the capabilities of the unique sna platform . [SEP]
[CLS] the sticky - flare uses the same targeting strategy of recognizing and quantifying rna targets in live cells B-material but has the additional functionality of being able to bind to target transcripts B-event , enabling further analysis of rna transport and localization . [SEP]
[CLS] as such , this is the first demonstration , to our knowledge , of a single platform that enables a complete analysis of rna function in live cells B-material and overcomes many limitations of previous analytical techniques . [SEP]
[CLS] due to the similar nature of the nanoflare and sticky - flare constructs [SEP]
[CLS] , future studies will explore the development of multiplexed sticky - flare constructs that would be capable of targeting multiple transcripts B-event at once , to allow for an internal control in rna quantification and for simultaneous tracking of multiple transcripts B-event . [SEP]
[CLS] we anticipate the sticky - flare will be a valuable tool for investigating proper rna function and its misregulation in disease , and will make such studies accessible to a broader community , given the ease of its application in cell B-material culture . [SEP]
[CLS] further , the sticky - flare may improve analyses in other model systems where asymmetric rna expression is an essential component , such as embryonic development , tissue and organ regeneration , and neurobiology . [SEP]
[CLS] sticky - flare synthesis . [SEP]
[CLS] oligonucleotides were synthesized using standard solid - phase phosphoramidite chemistry ( expedite 8909 nucleotide synthesis system ; abi ) . [SEP]
[CLS] all reagents were purchased from glen research . [SEP]
[CLS] oligonucleotides were purified by reverse - phase hplc . [SEP]
[CLS] the oligonucleotide sequences used in this study are as follows : nontargeting alkyl - thiol B-material , cgt cta cct tcg cgc aaa aaa a - alkane thiol B-material ; nontargeting flare , cy5 - gcg cga agg tag acg gag tcg gtc ga ; β - actin alkyl - thiol B-material , ccg gca tgt gca a aaa aaa a - alkane thiol B-material ; β - actin flare , cy5 - ttg cac atg ccg gag ccg ttg tcg acg a ; β - actin target , tcg tcg aca acg gct ccg gca tgt gca a ; u1 alkyl - thiol B-material , tat cca ttg cac tcc gg aaa aaa a - sh ; u1 flare , cy5 - ccg gag tgc aat gga taa gcc tcg cc ; u1 target , ggc gag gct tat cca ttg cac tcc gg . [SEP]
[CLS] to prepare the dna - functionalized sna conjugates , alkylthiol - terminated oligonucleotides ( 3 μm ) were combined with citrate - capped 13 - nm gold B-material particles ( 13 nm ) and 0 . 01 % tween - 20 and incubated B-technique for 1 h at room temperature . [SEP]
[CLS] next , phosphate buffer ( ph = 7 . 4 ) and sodium B-material chloride I-material were added to a final concentration of 5 and 150 mm , respectively , and incubated B-technique overnight . [SEP]
[CLS] then , sodium B-material chloride I-material was added in 0 . 05 - m increments over 3 h to achieve a final nacl concentration of 300 mm , and the particles were shaken at room temperature for 4 h . [SEP]
[CLS] finally , the conjugates were purified by centrifugation and redispersed in pbs . [SEP]
[CLS] flares were hybridized on the purified dna - gold nanoparticless by adding a stoichiometric equivalent of 10 flares per nanoparticle B-nanoparticle . [SEP]
[CLS] the solution was then heated to 70 °c and slowly cooled to room temperature overnight to facilitate hybridization . [SEP]
[CLS] the resulting sticky - flares were then sterilized using a 0 . 2 - μm acetate syringe filter ( ge healthcare ) to prevent cell B-material contamination and stored at 4 °c . [SEP]
[CLS] ex vivo quantification of sequence - specific target recognition . [SEP]
[CLS] sticky - flares were incubated B-technique in pbs ( gibco ) at a concentration of 1 nm . subsequently , fully complementary oligonucleotide targets ( dna ) were added to the solution at the concentrations listed in the text above . [SEP]
[CLS] this mixture was allowed to incubate B-technique at room temperature for 5 min , after which the resulting fluorescence B-property was quantified via a biotek synergy h4 fluorescence B-property plate reader . [SEP]
[CLS] cell B-material culture and sticky - flare treatment . [SEP]
[CLS] hela cells B-material were cultured in dmem ( gibco ) supplemented with 10 % ( vol / vol ) fbs and 1 % penicillin / streptomycin . [SEP]
[CLS] gene knockdown was effected by treating with 50 nm anti - β - actin ( santa cruz biotechnology ) for 24 h with lipofectamine rnaimax according to the recommended protocol . [SEP]
[CLS] control sirna experiments ( santa cruz biotechnology ) were conducted in a similar manner . [SEP]
[CLS] cells B-material were then washed once with pbs and further incubated B-technique with 400 pm sticky - flares in optimem ( gibco ) for an additional 24 h . [SEP]
[CLS] fluorescence B-property of trypsinized cells B-material was quantified by using a guava easycyte ht flow cytometer ( millipore ) . [SEP]
[CLS] confocal microscopy B-technique was performed with zeiss 510 ( zeiss ) and sp5 ( leica ) confocal microscopes . [SEP]
[CLS] mitochondria were stained using celllight mitochondria - gfp ( life technologies ) . [SEP]
[CLS] fixing and permeabilization of mefs was performed by treating cells B-material with 4 % ( vol / vol ) paraformaldehyde for 10 min , followed by 0 . 1 % triton - x for 5 min . [SEP]
[CLS] cells B-material were then rinsed three times with pbs and treated with either 1 nm sticky - flares or 100 nm antisense dna strands for 1 h . [SEP]
[CLS] following this incubation B-technique , cells B-material were rinsed a further three times with pbs and imaged in the same fashion as live cells B-material . [SEP]
[CLS] acknowledgments . [SEP]
[CLS] confocal analysis was performed at the quantitative bioelemental imaging center ( northwestern university ) . [SEP]
[CLS] this project was supported by national institute of arthritis and musculoskeletal and skin diseases grants r01ar060810 and r21ar062898 and center for cancer nanotechnology excellence initiative of the national institutes of health under award u54 ca151880 . [SEP]
[CLS] p . s . r . is grateful to the national science foundation for a graduate research fellowship under grant dge - 1324585 , and w . e . b . is grateful for the ryan fellowship from northwestern university . [SEP]
[CLS] | in situ hybridization | rna detection | rna localization | nanotechnology t he study of rna is a critical component of biological research and in the diagnosis and treatment of disease . [SEP]
[CLS] evaluation of target recognition by sticky - flares . [SEP]
[CLS] sticky - flares were first evaluated in vitro for their utility in quantifying complementary [SEP]
[CLS] characterization of sequence - specific target recognition and quantification using the sticky - flare . [SEP]
[CLS] ( a ) in vitro assay quantifying the release of fluorescent B-property flares resulting from sequence - specific recognition of a complementary target over a range of concentrations in pbs buffer and the lack of recognition when presented with a high concentration ( 2 μm ) of a noncomplementary target ( sticky - flare concentration = 1 nm ; sequence information given in methods ) . [SEP]
[CLS] ( b ) ( upper ) representative confocal images of hela cells B-material treated with β - actin sticky - flares ; ( upper left ) cells B-material treated with rnaimax ( life technologies ) but no sirna and ( upper right ) cells B-material treated with 50 nm sirna designed to knockdown β - actin delivered via rnaimax . [SEP]
[CLS] ( lower ) graph of β - actin knockdown in hela cells B-material measured using sticky - flares combined with flow B-technique cytometry I-technique . [SEP]
[CLS] cells B-material treated with control sirna ( incorrect sequence for knocking down β - actin or any known gene ) yields cells B-material with the highest average fluorescence B-property . [SEP]
[CLS] when cells B-material are treated with β - actin sirna ( 50 nm ) , they exhibit an [UNK] % reduction in average fluorescence B-property ( p < 0 . 001 ) . [SEP]
[CLS] the bars on the far left are control experiments probing fluorescence B-property from cells B-material treated with stickyflares that are not specific for β - actin mrna or any known gene ; they represent the background fluorescence B-property from a sticky - flare experiment . [SEP]
[CLS] 1 . schematic of rna recognition by the nanoflare ( upper ) and sticky - flare ( lower ) . [SEP]
[CLS] on recognition , the nanoflare binds to rna targets and releases a fluorophore - labeled oligonucleotide reporter ( flare ) to float freely in the cytoplasm . [SEP]
[CLS] in contrast , flares from the sticky - flare are longer and complementary to rna targets , which allows them to bind transcripts B-event and act as fluorescent B-property labels for intracellular tracking . [SEP]
[CLS] rna localization in mefs . [SEP]
[CLS] β - actin targeting sticky - flares localize to the growth cone of growing lamellae , where β - actin rna is found . [SEP]
[CLS] contrastingly , sticky - flares targeting the u1 nuclear rna localize to the nucleus . [SEP]
[CLS] red , cy5 sticky - flare ; blue , nuclear stain ( hoechst ) [SEP]
[CLS] observation of dynamic β - actin mrna transport in mef cells B-material . [SEP]
[CLS] endogenously expressed β - actin mrna is transported distally toward the growth cone ( red , cy5 - labeled sticky - flare ) . [SEP]
[CLS] dashed boxes indicate the labeled rna being tracked . [SEP]
[CLS] each panel indicates a 50 - s advancement . [SEP]
[CLS] β - actin mrna colocalizes with mitochondria in hela cells B-material . [SEP]
[CLS] red , cy5 - labeled sticky - flare ; green , mitochondrial marker ; blue , nuclear stain ( hoechst ) [SEP]
[CLS] ribozymes are highly structured rna sequences that can be tailored to recognize and cleave specific stretches of mrna . [SEP]
[CLS] their current therapeutic efficacy remains low due to their large size and structural instability compared to shorter therapeutically relevant rna such as small interfering rna ( sirna ) and microrna ( mirna ) . [SEP]
[CLS] herein , a synthetic strategy that makes use of the spherical nucleic B-material acid I-material ( sna ) architecture to stabilize ribozymes and transfect them into live cells B-material is reported . [SEP]
[CLS] the properties of this novel ribozyme sna are characterized in the context of the targeted knockdown of o 6 - methylguanine - dna methyltransferase ( mgmt ) , a dna repair protein B-material involved in chemotherapeutic resistance of solid tumors B-material , foremost glioblastoma multiforme ( gbm ) . [SEP]
[CLS] data showing the direct cleavage of full - length mgmt mrna , knockdown of mgmt protein B-material , and increased sensitization of gbm cells B-material to therapy - mediated apoptosis B-event , independent of transfection agents , provide compelling evidence for the promising properties of this new chemical architecture . [SEP]
[CLS] ribozymes are rna sequences containing a highly conserved catalytic loop capable of cleaving mrna . [SEP]
[CLS] along with a high degree of sequence specificity for their substrates , ribozymes offer an attractive alternative to other therapeutic nucleic B-material acid I-material approaches as they are able to directly cleave specific stretches of mrna , rather than recruiting enzymes to mrna for cleavage . [SEP]
[CLS] ribozymes have therefore attracted significant attention as potential therapeutic gene silencing agents . [SEP]
[CLS] however , one of the greatest hurdles preventing ribozymes from becoming a viable gene silencing modality is their susceptibility to chemical degradation and strict requirement for proper folding to recognize and cleave their intended substrate . [SEP]
[CLS] due to their relatively fragile structures , ribozymes are often delivered into cells B-material only after extensive chemical modification and encapsulation in liposomes B-nanoparticle , block copolymers , assembly into larger rna scaffolds , or through plasmid - directed expression . [SEP]
[CLS] the aforementioned approaches can be synthetically challenging , and , depending on the ribozyme sequence , can cause substantially lower catalytic cleavage efficiencies . [SEP]
[CLS] due to such hurdles , researchers have explored alternative approaches for targeting rna substrates , including larger rna cleaving protein - nanoparticle B-nanoparticle conjugates . [SEP]
[CLS] in an effort to develop a straight forward method of creating functional ribozyme structures , without having to undertake extensive chemical modification of the rna , and ensure catalytic activity , we investigated the concept of incorporating a ribozyme into a spherical nucleic B-material acid I-material ( sna ) architecture . [SEP]
[CLS] snas are typically composed of spherical B-nanoparticle nanoparticles I-nanoparticle densely functionalized with oligonucleotides ( either dna or rna ) , and have been shown to increase nucleic B-material acid I-material stability in cellular environments by limiting the extent of degradation caused by endogenous nucleases . [SEP]
[CLS] using an enzymatic ligation reaction , a ribozyme can be covalently attached to a sna surface without chemical changes introduced into the rna ' s sequence . [SEP]
[CLS] the resulting composite architecture combines the unique rna structural stabilization properties offered by snas with the ribozyme ' s ability to target and catalytically cleave a specific mrna sequence ( figure 1 ) . [SEP]
[CLS] such an approach provides the potential for the ribozyme ' s downstream biological function to be retained , as it is the ligase ' s sequence recognition that serves as the basis of assembly at the nanoparticle B-nanoparticle ' s surface rather than chemical modification of the ribozyme itself . [SEP]
[CLS] herein we describe ribozyme - sna conjugates that exhibit high cellular uptake , comparable to that observed by previous classes of snas , and which allow the ribozyme to remain catalytically active and stable as shown through a combination of in vitro rna cleavage and rt - pcr assays , respectively . [SEP]
[CLS] the ribozyme sequence used for this study was designed to target the mrna for o methylguanine - dna methyltransferase ( mgmt ) . [SEP]
[CLS] mgmt was chosen as a target for the ribozyme - snas due to its critical role in regulating therapy susceptibility of glioblastoma multiforme ( gbm ) . [SEP]
[CLS] in gbm , a highly aggressive and uniformly deadly brain cancer , higher expression of mgmt protein B-material has been linked to resistance to temozolomide ( tmz ) therapy . [SEP]
[CLS] tmz , an alkylating chemotherapeutic , induces apoptosis B-event in cancer cells B-material by methylating the o 6 - position of guanine in dna . [SEP]
[CLS] these o 6 - methylguanine lesions can bind thymine rather than cytosine , leading to futile mismatch repair cycles and eventual apoptosis B-event . [SEP]
[CLS] when mgmt is present , it is able to repair o 6 - methylguanine lesions induced by tmz , causing resistance and reducing overall clinical efficacy . [SEP]
[CLS] in order to sensitize gbm cells B-material to tmz - induced apoptosis B-event , we sought to lower the expression of mgmt protein B-material in glioma cells B-material through ribozyme - sna directed cleavage of mgmt mrna . [SEP]
[CLS] the ribozyme sequence used in the construction of the ribozyme - snas was adapted from a truncated mgmt - targeting ribozyme designed by citti and coworkers . [SEP]
[CLS] the sequence contains a hammerhead type structure which upon binding a divalent ion B-material in its cleavage reaction site , cleaves an rna bound to its recognition region . [SEP]
[CLS] all ribozymes used in this study were transcribed enzymatically , ligated to a dna - sna nanoconjugate , and delivered to cells B-material without auxiliary transfection agents . [SEP]
[CLS] in order to initially evaluate the ribozyme - sna ' s ability to recognize and cleave the rna transcript B-event encoding mgmt , the ribozyme - snas were incubated B-technique with a short stretch ( 13 bases ) of rna derived from the full - length mgmt mrna . [SEP]
[CLS] the 13mer rna substrate was modified at its 3 ′ end with a fluorescein and at its 5 ′ end with cy5 for visualization of the cleaved products by gel B-technique electrophoresis I-technique . [SEP]
[CLS] 20 nm ribozyme - snas ( carrying 20±1 ribozymes per particle ) were incubated B-technique with the 13mer rna substrate overnight at 37 °c in the presence or absence of 40 mm mgcl 2 , an essential cofactor for ribozyme - catalyzed cleavage . [SEP]
[CLS] after 12 h , the ribozyme - snas were centrifuged and removed from the reaction , and the supernatant was run on a denaturing polyacrylamide gel to evaluate the extent of rna cleavage . [SEP]
[CLS] as compared to the background degradation observed for the 13mer rna substrate ( figure 2 , first lane ) , it is evident that both the free ribozyme and the ribozyme - snas are capable of cleaving the intended substrate to a similar extent in a mg 2 + dependent fashion . [SEP]
[CLS] therefore the ribozyme - sna is as effective as the free ribozyme in cleaving a 13mer rna substrate in vitro ( figure 2 ) , suggesting that the tertiary structure of the ribozyme is sufficiently retained post enzymatic ligation . [SEP]
[CLS] these data functionally confirm circular dichroism studies , which identified elements of a characteristic a - fold rna sequence within the ribozyme - sna ( figure 1b ) . [SEP]
[CLS] it was then investigated whether or not the ribozyme - sna could successfully transfect cells B-material much like native snas but in manner that retains its enzymatic activity . [SEP]
[CLS] using a cy5 fluorophore - labeled ribozyme - sna , it was shown that the ribozyme - tethered sna could indeed enter cells B-material whereas a free cy5 - ribozyme showed insignificant uptake by cells B-material ( figure 3 ) . [SEP]
[CLS] as the cellular environment is rich in nucleolytic enzymes , a major concern is the prolonged stability of the ribozyme . [SEP]
[CLS] since the ribozymes used in this study are unmodified , we investigated whether the sna could shield or protect the ribozymes from degradation , as previously observed for sirna - and dna - snas . [SEP]
[CLS] to study this , an assay consisting of reverse transcription B-event followed by pcr ( rt - pcr ) was developed to determine the relative stability of the ribozyme pre - and post - exposure to serum nucleases . [SEP]
[CLS] the ribozyme - snas were incubated B-technique with 40 % fetal bovine serum ( fbs ) for 1 h at 37 °c in an effort to expose the ribozymes to nuclease - mediated cleavage . [SEP]
[CLS] the particles were then washed and subjected to the rt - pcr assay . [SEP]
[CLS] pcr amplification of the full - length ribozyme was present on the sna surface despite exposure to relatively harsh enzymatic cocktails containing common nucleases ( figure s1 ) . [SEP]
[CLS] once it was confirmed that the ribozyme ' s function was retained after ligation to the sna , and that the sna architecture conferred enhanced uptake and stability , the ribozyme - snas were assessed for their ability to knockdown mgmt protein B-material expression in glioma cells B-material . [SEP]
[CLS] the extent of mgmt knockdown was assessed via western B-technique blot I-technique analysis I-technique . [SEP]
[CLS] ribozyme - snas were incubated B-technique with t98g glioma cells B-material overnight , and protein B-material lysates were harvested for immunoblotting after 48 to 96 h . [SEP]
[CLS] representative gels are shown in figure 4 along with the averaged results quantified by densitometry ( n = 3 ) . [SEP]
[CLS] treatment with the mgmt - targeting ribozyme - snas resulted in over 75 % knockdown of mgmt protein B-material without transfection agents . [SEP]
[CLS] after testing the degree of mgmt knockdown , the ribozyme snas were evaluated for their ability to amplify apoptotic responses to tmz treatment . [SEP]
[CLS] to this end , we determined effector caspase - 3 / 7 activation and activity by a devdase assay ( using the caspase - 3 / 7 peptidyl substrate ac - devd - amc ; biovision , milpitas , ca ) , and western B-technique blot I-technique analysis I-technique using antibodies B-material specific for active caspase - 3 and caspase - 7 . [SEP]
[CLS] lysates from ribozyme - sna - treated glioma cells B-material showed a considerable increase in devdase activity and effector caspase activation when compared to both the transfected free ribozyme and the control ribozyme - sna ( figure 5a - c ) . [SEP]
[CLS] in summary , we have prepared a novel ribozyme - sna that exhibits the ability to transfect cell B-material membranes and retain its enzymatic activity , once inside cells B-material . [SEP]
[CLS] the approach and chemical architecture bypass the need for chemical modifications to the rna or external transfection agents , both of which limit conventional ribozyme utility . [SEP]
[CLS] importantly , the sna architecture maintains an external environment conducive to retaining ribozyme functionality and enhances the stability of traditionally unstable nucleic B-material acids I-material . [SEP]
[CLS] furthermore , the observation that ribozyme - snas can effectively knockdown mgmt in gbm cells B-material and subsequently sensitize these cells B-material to tmz - mediated apoptosis B-event bode well for developing such structures as experimental therapies for gbm . [SEP]
[CLS] such biological results , including the ability to target and knockdown a central dna repair protein B-material and chemoresistance mechanism , provides functional confirmation of the structural advantages afforded by the sna architecture . [SEP]
[CLS] taken together , this study suggests that enzymatic assembly of rna on snas can be extended to larger , highly structured rnas as a way to retain their native function , an essential factor in the design of nucleic acid - based therapeutics . [SEP]
[CLS] the blue trace indicates the presence of an a form rna sequence 20 at the particle surface as seen by the slightly blue shifted peak at 275 nm , along with the minima at 210 nm . [SEP]
[CLS] c ) aga - rose gel shift assay showing the larger size of the gold B-nanoparticle nanoparticles I-nanoparticle post ligation and covalent attachment of the ribozyme to the sna surface . [SEP]
[CLS] changes in particle size were also confirmed using dynamic B-technique light I-technique scattering I-technique ( table s2 ) . [SEP]
[CLS] assembly of ribozyme - snas using enzymatic ligation a ) schematic depicting the ligation approach for constructing ribozyme - snas . [SEP]
[CLS] figure 1 . b ) circular dichroism of ribozyme - snas . [SEP]
[CLS] orange trace showing b - form dna at the sna surface as indicated by the maxima at 277 nm and the minima at 246 nm ( broadening attributed to interactions with the gold B-nanoparticle nanoparticle I-nanoparticle surface ) . [SEP]
[CLS] the blue trace indicates the presence of an a form rna sequence 20 at the particle surface as seen by the slightly blue shifted peak at 275 nm , along with the minima at 210 nm . c ) aga - rose gel shift assay showing the larger size of the gold B-nanoparticle nanoparticles I-nanoparticle post ligation and covalent attachment of the ribozyme to the sna surface . [SEP]
[CLS] changes in particle size were also confirmed using dynamic B-technique light I-technique scattering I-technique ( tables2 ) . [SEP]
[CLS] in vitro cleavage assay for ribozyme - snas an 8 % denaturing polyacrylamide gel indicating the cleavage of the ribozyme ' s rna substrate under various conditions of salt B-material and time . [SEP]
[CLS] the 13mer rna substrate is cleaved in the presence of the ribozyme - sna when mgcl 2 is present . [SEP]
[CLS] the third and fourth lanes differ in total incubation B-technique time , 12 h versus 24 h , respectively . [SEP]
[CLS] 3 . confocal images of ribozyme - sna uptake into t98g glioma cells B-material comparison of cellular entry as a function of cy5 emission for free cy5 - ribozymes versus cy5 - ribozyme - snas , indicating a greater degree of uptake for sna - tethered ribozymes . [SEP]
[CLS] figure 3 . a ) cy5 channel for free ribozyme b ) brightfield images with cy5 channel overlay showing t98g cells B-material post incubation B-technique with free cy5 - labeled ribozyme . [SEP]
[CLS] c ) cy5 channel showing uptake of cy5 labeled ribozyme - snas into t98g cells B-material . [SEP]
[CLS] d ) brightfield image with cy5 channel overlay of t98g cells B-material post treatment with cy5 labeled ribozyme - snas . [SEP]
[CLS] no transfection agents were used for either free cy5 - ribozymes or cy5 - ribozyme - snas . [SEP]
[CLS] scale bar is 20 μm [SEP]
[CLS] silencing of mgmt by ribozyme - snas a ) t98g cells B-material were treated with control or mgmt - targeting free ribozyme , free ribozyme combined with lipofectamine ( lp ) , or ribozyme - snas and the effects on mgmt protein B-material levels were investigated by western blots . [SEP]
[CLS] figure 4 . b ) quantification of mgmt expression by densitometric analysis ( n = 3 ) . [SEP]
[CLS] caspase activation and activity triggered by ribozyme - snas a ) representative western B-technique blot I-technique analysis I-technique showing the ability of ribozyme - snas to activate effector caspases - 3 / 7 as seen by cleavage ( cl . ) of either caspase . [SEP]
[CLS] figure 5 . b ) averaged caspase activation for control snas versus mgmt - targeting ribozyme - snas with tmz treatment in t98g cells B-material via western blot . [SEP]
[CLS] ribozyme - snas considerably increased caspase activation relative to the control snas . [SEP]
[CLS] c ) quantification of caspase activation by ribozyme - snas in the presence of tmz compared to control snas using a fluorescent B-property peptide B-material reporter assay . [SEP]
[CLS] tmz is dissolved in dmso for all experiments . [SEP]
[CLS] detection of protein B-material expression by mri requires a high payload of gd ( iii ) per protein B-event binding I-event event . [SEP]
[CLS] presented here is a targeted audna nanoparticle B-nanoparticle capable of delivering several hundred gd ( iii ) chelates to the halotag reporter protein B-material . [SEP]
[CLS] incubating B-technique this particle with halotag - expressing cells B-material produced a 9 . 4 contrast - to - noise ratio compared to non - expressing cells B-material . [SEP]
[CLS] the field of molecular imaging is motivated by the need for techniques that enable in vivo visualization of biochemical processes , biomarkers B-property , and gene expression . [SEP]
[CLS] magnetic B-property resonance imaging ( mri ) is an appealing modality for molecular imaging because it provides excellent spatial resolution ( < 100 μm ) , detailed anatomical information , and does not require exposing the subject to potentially harmful ionizing B-property radiation . [SEP]
[CLS] where native mr contrast B-technique is insufficient , contrast B-technique agents I-technique ( cas ) , such as those based on paramagnetic B-property gadolinium B-material , are used to shorten water B-material proton relaxation times , increasing image contrast . [SEP]
[CLS] however , the low sensitivity of gd ( iii ) cas has limited their utility in molecular imaging due to the high concentrations required to produce contrast ( 10 - 100 μm ) . [SEP]
[CLS] crucially , many biomolecules are present at concentrations ( 0 . 1 - 1 μm ) that are below the detection limit of gd ( iii ) cas . [SEP]
[CLS] to date , molecular imaging using gd ( iii ) has been limited to a small number of biomarkers B-property present at high concentrations in vivo . [SEP]
[CLS] the low sensitivity of gd ( iii ) cas has made it challenging to develop mr reporter genes . [SEP]
[CLS] many of these genes are from endogenous proteins B-material , produce negative contrast ( bright - todark ) , and generate only modest contrast overall . [SEP]
[CLS] furthermore , none of the genes in these systems have been integrated into existing reporter gene platforms . [SEP]
[CLS] as such , their utility is limited because they require a unique genetic element dedicated solely to mr detection . [SEP]
[CLS] an ideal reporter platform for mr monitoring of gene expression presents extracellularly , integrates into an existing reporter gene platform , provides irreversible binding of molecular probes , and contains the necessary signal amplification to overcome the low sensitivity of gd ( iii ) probes . [SEP]
[CLS] the halotag reporter gene system addresses these challenges . [SEP]
[CLS] halotag is an engineered haloalkane delahogenase that can be expressed on the outer surface of the plasma membrane . [SEP]
[CLS] the enzyme active site has been modified to catalyze covalent bond formation with terminal haloalkanes , promoting superior probe retention . [SEP]
[CLS] because haloalkanes are virtually absent from eukaryotic systems , halotag and its targeting group create an orthogonal binding pair . [SEP]
[CLS] furthermore , halotag can readily form functional fusions with a variety of proteins B-material . [SEP]
[CLS] the specificity and versatility of the halotag system make it attractive as an mr reporter gene . [SEP]
[CLS] in addition , it operates as a variable - output reporter gene , whereby the researcher can select the nature of the output by choosing the appropriate halotag - targeted agent . [SEP]
[CLS] for this reason , a variety of imaging agents , including fluorophores , pet agents , mr agents , and quantum B-nanoparticle dots I-nanoparticle have been successfully targeted to halotag . [SEP]
[CLS] however , coupling halotag expression to the production of t 1 contrast demands significant signal amplification . [SEP]
[CLS] spherical nucleic B-material acids I-material ( snas ) have the potential to address this design requirement . [SEP]
[CLS] extensive work on over 100 cell B-material types showed that snas exhibit high biocompatibility B-property and low toxicity B-property in vitro and in vivo . [SEP]
[CLS] furthermore , previous work with snas developed a multiplexing strategy to deliver a high payload of gd ( iii ) chelates . [SEP]
[CLS] in this case , the snas were not targeted and their cellular uptake was a result of snas binding B-event to I-event scavenger I-event receptors I-event on the cell B-material surface . [SEP]
[CLS] although snas can be targeted using antibodies B-material or aptamers , there is no precedent for sna targeting using small molecule ligands . [SEP]
[CLS] we demonstrate that halotag - dependent mr contrast enhancement can be achieved by using a ht - targeted audna - gd ( iii ) nanoparticle B-nanoparticle . [SEP]
[CLS] halotag - targeted audna - gd ( iii ) nanoparticles B-nanoparticle were synthesized according to scheme 1 . [SEP]
[CLS] a 24 - mer polydeoxythymidine ( dt ) oligonucleotide bearing a protected 3 ′ thiol B-material and a 5 ′ terminal haloalkane ( ha ) moiety for halotag binding was synthesized ( scheme s1 and s2 ) . [SEP]
[CLS] the oligonucleotide included modified dt bases bearing terminal alkyne functionality at five positions internal to each strand . [SEP]
[CLS] using a gd ( iii ) chelate bearing an azide functionality , a cu ( i ) - catalyzed 1 , 3 dipolar cycloaddition was conducted to produce the complete halotag - targeted gd ( iii ) dna ( scheme s3 ) . [SEP]
[CLS] the purified oligonucleotide was deprotected to expose the 3 ′ thiol B-material and conjugated to gold B-nanoparticle nanoparticles I-nanoparticle using a salt aging procedure . [SEP]
[CLS] the density of oligonucleotide loading on the particle surface was determined by calculation of the gd / au ratio using inductively coupled plasma mass spectrometry ( icp - ms ) . [SEP]
[CLS] results indicate that the average loading of dna was 100 ± 10 strands per particle , yielding a gd ( iii ) - chelate payload of 500 ± 60 per particle . the t 1 relaxivity ( r 1 ) was measured to be 16 ± 3 mm −1 s −1 per gd ( iii ) at 37 °c and 1 . 41 t , and the t 2 relaxivity ( r 2 ) was measured to be 28 ± 3 mm −1 s −1 per gd ( iii ) ( fig . s3 and s4 ) . [SEP]
[CLS] we hypothesized that this degree of signal amplification would enable visualization of surface receptors that would be below the detection limit of individual gd ( iii ) chelates . [SEP]
[CLS] the u - 2 os ht - ecs ( ht + ) cell B-material line constitutively expresses extracellular halotag . [SEP]
[CLS] flow B-technique cytometry I-technique was used to quantify the number of halotag proteins B-material expressed on the outer surface of the plasma membrane by using cell - impermeable halotag - targeted alexafluor488 dye . [SEP]
[CLS] unlike antibody - based cell B-material surface stains , each halotag protein B-material binds irreversibly to only one molecule of alexafluor488 . [SEP]
[CLS] therefore , the number of halotag proteins B-material present on the surface of these cells B-material could be quantified by fluorescence B-property . [SEP]
[CLS] the ht + cell B-material line was observed to express 1 , 800 , 000 ± 500 , 000 copies of halotag on its surface ( fig . s5 ) . [SEP]
[CLS] using the common volume approximation of 2 pl / cell B-material , this yields a concentration of 1 . 6 ± 0 . 4 μm halotag that is accessible to the cell B-material surface . [SEP]
[CLS] though this concentration of halotag corresponds to the top decile of protein B-material expression in the mammalian cell B-material , a gd ( iii ) agent bound to halotag in one - to - one stoichiometry would still fail to achieve a detectable concentration ( fig . s9 ) . [SEP]
[CLS] to directly observe audna - gd ( iii ) - ha binding to halotag , transmission B-technique electron I-technique microscopy I-technique ( tem ) was used to identify membrane binding . [SEP]
[CLS] a change in the subcellular localization of audna - gd ( iii ) - ha was observed when comparing ht + cells B-material to otherwise identical cells B-material that do not express halotag ( u - 2 os ( ht - ) ) ( fig . 1a , 1b , and s10 ) . [SEP]
[CLS] both cell B-material lines showed intracellular clusters of nanoparticles B-nanoparticle , which are likely endosomes or lysosomes ( fig 1b ) . [SEP]
[CLS] this observation is consistent with the previously proposed mechanism of scavenger receptor B-material uptake . [SEP]
[CLS] however , only ht + cells B-material showed large numbers of particles adjacent to the plasma membrane ( fig . 1a ) . [SEP]
[CLS] flow B-technique cytometry I-technique was used to measure binding of audna - gd ( iii ) - ha to halotag on the plasma membrane . [SEP]
[CLS] ht + cells B-material were first incubated B-technique with audna - gd ( iii ) - ha , followed by labeling with halotag - targeted alexafluor488 . [SEP]
[CLS] when audna - gd ( iii ) - ha binds to halotag on the cell B-material surface , fewer sites remain for alexafluor488 binding . [SEP]
[CLS] therefore , audna - gd ( iii ) - ha binding to halotag was monitored by the loss of alexafluor488 fluorescence B-property in both a time and dose dependent manner ( fig . 1c and 1d ) . [SEP]
[CLS] after a 24 - hour incubation B-technique period , halotag binding was observed to plateau at an incubation B-technique concentration of 40 nm nanoparticles B-nanoparticle , with greater than 60 % binding as low as 10 nm ( fig . 1e ) . [SEP]
[CLS] in addition , halotag was saturated after 8 hours of incubation B-technique at 40 nm ( fig . 1f and s11 ) . [SEP]
[CLS] the binding kinetics of audna - gd ( iii ) - ha are slower than halotagtargeted small molecules . [SEP]
[CLS] this is likely the result of a complex protein B-material corona I-material that forms around nanoparticles B-nanoparticle when exposed to serum proteins B-material and reduces access to targeting groups . [SEP]
[CLS] importantly , these data suggest that saturated cells B-material will have an average of 1 , 730 , 000 nanoparticles B-nanoparticle associated with the cell B-material as a result of halotag binding . [SEP]
[CLS] if each particle contributes 500 gd ( iii ) chelates , we predict that halotag saturation will result in 1 . 5 fmol gd ( iii ) / cell B-material . [SEP]
[CLS] this concentration is an order of magnitude above the most conservative estimates for the detection limit . [SEP]
[CLS] to determine the accuracy of these uptake approximations , ht− and ht + cells B-material were incubated B-technique with audna - gd ( iii ) - ha and gd ( iii ) uptake was measured using icp - ms . [SEP]
[CLS] these data can be used to determine the signal contributions that depend on the expression of halotag and untargeted uptake of audna - gd ( iii ) - ha . [SEP]
[CLS] it is likely that untargeted uptake of audna - gd ( iii ) - ha is due to audna nanoparticles binding B-event scavenger I-event receptors I-event , as previously reported . [SEP]
[CLS] ht− cells B-material display saturation for both incubation B-technique concentration and time , which is the expected trend as available scavenger receptors are depleted ( fig . 2a and 2b ) . [SEP]
[CLS] the gd ( iii ) uptake values for ht + cells B-material continue to increase beyond the measured values for ht− cells B-material at several time points and concentrations ( fig . 2a and 2b ) . [SEP]
[CLS] after 8 hours of incubation B-technique with 40 nm audna - gd ( iii ) - ha , ht + cells B-material accumulated threefold more gd ( iii ) than ht− cells B-material ( fig . 2b ) . [SEP]
[CLS] halotag expression resulted in an additional accumulation of 1 . 16±0 . 08 fmol gd ( iii ) / cell B-material over ht− cells B-material . [SEP]
[CLS] this value is very close to the predicted value of 1 . 5 fmol gd ( iii ) / cell B-material calculated from the expression level of halotag ( fig . 1 ) . [SEP]
[CLS] a likely explanation for the reduced uptake is slow degradation of the nanoparticles B-nanoparticle over the course of the incubation B-technique . [SEP]
[CLS] while audna nanoparticles B-nanoparticle are resistant to the activity of dnase , the reaction still proceeds at a measurable rate . [SEP]
[CLS] cell pellet mr images were taken to verify that the additional uptake conferred by halotag expression would effectively translate to contrast in an mr image acquired at 7 t . as expected , both cells B-material lines showed an increase in signal intensity over unlabeled cells B-material after incubation B-technique with audna - gd ( iii ) - ha ( fig . 2c ) . [SEP]
[CLS] significantly , halotag - expressing cells B-material are clearly distinguishable from cells B-material that do not express halotag . [SEP]
[CLS] this contrast enhancement was apparent in both the greyscale t 1 weighted image ( figure s7 ) and the corresponding intensity map ( fig . 2c ) . [SEP]
[CLS] the observed t 1 shortening was significant both for the image slice shown and for the average of 4 slices ( fig . 2c and s5 ) . [SEP]
[CLS] from this image the contrast - to - noise ratio ( cnr ) between ht + and ht− cells B-material was calculated to be 9 . 4 . [SEP]
[CLS] based on the clinical standard for mr imaging , cnr values above 5 are considered to be visually " obvious . [SEP]
[CLS] " we have shown that a gd ( iii ) - conjugated audna nanoparticle B-nanoparticle can be targeted the halotag protein B-material and produce detectable mr contrast . [SEP]
[CLS] the signal amplification afforded by nanoparticle B-nanoparticle targeting enabled delivery of gd ( iii ) at millimolar cellular concentrations for a 2 pl cell B-material . [SEP]
[CLS] as a result , halotag ' s reporter gene functionality has been expanded to include an mr output . [SEP]
[CLS] beyond halotag , this technology has shown that in principle , cell B-material surface I-material receptors I-material can be detected using a t 1 contrast B-technique agent I-technique . [SEP]
[CLS] the straightforward chemistry and broad applicability of sna ' s suggests that these nanoparticles B-nanoparticle can be easily adapted to other surface receptors that bind to a small molecule ligands . [SEP]
[CLS] schematic of audna - gd ( iii ) - ha binding to halotag on the cell B-material surface . [SEP]
[CLS] each particle delivers a high payload of gd ( iii ) to a single protein B-material . [SEP]
[CLS] the nanoparticle B-nanoparticle consists of a 15 nm gold B-material core B-material that is bound to several copies of single stranded dna . [SEP]
[CLS] each strand contains five covalently attached gd ( iii ) complexes . [SEP]
[CLS] the 3 ′ end is functionalized with a thiol B-material for gold B-material binding and the 5 ′ end is modified to include a haloalkane ( ha ) moiety for halotag targeting . [SEP]
[CLS] azide functionalized gd ( iii ) chelates are used to label the dna with gd ( iii ) . [SEP]
[CLS] audna - gd ( iii ) - ha nanoparticles B-nanoparticle bind the halotag protein B-material on the cell B-material surface . [SEP]
[CLS] fig . 1 . s 1a and 1b display the transmission B-technique electron I-technique microscopy I-technique images of ht + cells B-material and ht− cells B-material , respectively , after incubation B-technique with 20 nm audna - gd ( iii ) - ha . [SEP]
[CLS] the expression of surface halotag changes the subcellular localization of audna− gd ( iii ) - ha nanoparticles B-nanoparticle . [SEP]
[CLS] audna− gd ( iii ) - ha nanoparticle B-nanoparticle binding to halotag can be monitored by flow B-technique cytometry I-technique . [SEP]
[CLS] halotag expressing cells B-material labeled with halotag - targeted alexafluor488 after incubating B-technique with audna - gd ( iii ) - ha at c ) various concentrations for 24 hours or for d ) various times at 40 nm . the percent of halotag proteins B-material bound to nanoparticles B-nanoparticle can be extracted from this data . [SEP]
[CLS] s 1e and 1f show the binding curves corresponding to the concentration gradient and time course respectively . [SEP]
[CLS] cellular uptake of gd ( iii ) is measured for both ht− ( black bars ) and ht + ( white bars ) cell B-material lines using icp - ms . [SEP]
[CLS] fig . 2 . a ) the concentration dependence of uptake was measured using a 24 hour incubation B-technique for each concentration . [SEP]
[CLS] b ) cells B-material were incubated B-technique with 40 nm nanoparticles B-nanoparticle for the indicated time . [SEP]
[CLS] the expression of halotag results in measurably higher uptake of gd ( iii ) . [SEP]
[CLS] error bars show standard error of the mean . [SEP]
[CLS] c ) halotag - dependent contrast enhancement is clearly visible after incubation B-technique with audna - gd ( iii ) - ha at a concentration of 52 nm nanoparticles B-nanoparticle for 8 hours . [SEP]
[CLS] gradient bar denotes signal intensity . [SEP]
[CLS] non - viral methods have been explored as the replacement of viral systems for their low toxicity B-property and immunogenicity B-property . [SEP]
[CLS] however , they have yet to reach levels competitive to their viral counterparts . [SEP]
[CLS] in this paper , we combined physical and chemical methods to improve the performance of polyplex delivery of dna and sirna . [SEP]
[CLS] specifically , gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) were used to carry polyplex ( a chemical approach ) while electroporation ( a physical approach ) was applied for fast and direct cytosolic delivery . [SEP]
[CLS] in this hybrid approach , cationic B-material polymer B-material molecules condense and / or protect genetic probes as usual while aunps B-nanoparticle help fix polycations to reduce their cytotoxicity B-property and promote the transfection B-property efficiency I-property of electroporation . [SEP]
[CLS] aunps B-nanoparticle of various sizes were first coated with polyethylenimine ( pei ) , which were further conjugated with dna plasmids or sirna molecules to form aunps B-nanoparticle - polyplex . [SEP]
[CLS] the hybrid nanoparticles B-nanoparticle were then mixed with cells B-material and introduced into cell B-material cytosol by electroporation . [SEP]
[CLS] the delivery efficiency was evaluated with both model anchor cells B-material ( i . e . , nih 3t3 ) and suspension cells B-material ( i . e . , k562 ) , together with their impact on cell B-property viability I-property . [SEP]
[CLS] we found that aunp B-nanoparticle - polyplex showed 1 . 5 ~ 2 folds improvement on the transfection B-property efficiency I-property with no significant increase of toxicity B-property when compared to free plasmid delivery by electroporation alone . [SEP]
[CLS] such a combination of physical and chemical delivery concept may stimulate further exploration in the delivery of various therapeutic materials for both in vitro and in vivo applications . [SEP]
[CLS] gene induction and / or inhibition provide an invaluable approach to understand gene function , control cellular signals , and develop new therapeutic technologies [SEP]
[CLS] having safe and effective delivery tools is the key to achieving its full potential . [SEP]
[CLS] viral transduction is stable and efficienct , but still has high risk of oncogenesis and inflammation [SEP]
[CLS] this stimulates the rapid growth of nonviral delivery systems . [SEP]
[CLS] in chemical - mediated nonviral delivery systems , potent therapeutic molecules are condensed and / or protected by cationic B-material chemicals via forming polymer B-material - dna complexes ( polyplex ) or lipid - dna complexes ( lipoplex ) to overcome multiple delivery barriers B-property . [SEP]
[CLS] they serve as the favorable alternative to their virus - mediated counterparts and have been successfully tested for both in vitro and in vivo delivery of plasmids , oligonucleotides , ribozyme , and small interfering rnas [SEP]
[CLS] however , many of these systems still suffer insufficient delivery efficiency and cell B-property viability I-property , which often ties with their poor nanoparticle B-nanoparticle quality , slow and inefficient cellular uptake and endosome escape , and serious cytotoxicity B-property from free cationic B-material molecules after the unpacking of lipoplex or polyplex . [SEP]
[CLS] as captured cationic B-material molecules are found much less toxic B-property than their free counterparts , nanoparticles B-nanoparticle have been introduced to help fix cationic B-material polymer B-material [SEP]
[CLS] this was also found helpful to produce nanoparticles B-nanoparticle with much narrow size distribution . [SEP]
[CLS] gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) are favored in these applications for their good biocompatibility B-property and multiple functionalities ( i . e . , targeting , therapeutic , and imaging ) . [SEP]
[CLS] however , issues like ineffective cellular internalization remain . [SEP]
[CLS] herein we introduce the use of electroporation to bypass the slow and inefficient endocytosis B-event process by directly delivering therapeutic probes into cell B-material cytosol . [SEP]
[CLS] electroporation is a physical delivery approach in which cells B-material are imposed with short electrical pulses to create temporary pathways on the cell B-material membrane to facilitate the cellular uptake [SEP]
[CLS] it has been widely used to either evaluate the therapeutic performance of exogenous probes or study their trafficking inside cells B-material [SEP]
[CLS] a simple combination of lipoplex nanoparticles B-nanoparticle and electroporation has been explored early in the delivery of oligonucleotides in the format of lipoplex . [SEP]
[CLS] however , negative impacts on both the delivery efficiency and the cell B-property viability I-property were found [SEP]
[CLS] it was believed that the destroyed complex structure during electroporation released a large number of free cationic B-material molecules , which significantly lower the overall cell B-property viability I-property . [SEP]
[CLS] to avoid similar situation , we first immobilized cationic B-material polymer B-material on aunps B-nanoparticle and then allowed conjugation with negatively charged therapeutic probes to form aunps B-nanoparticle - polyplex complex . [SEP]
[CLS] in addition to the help on retaining cationic B-material polymer B-material on the surface , the presence of aunps B-nanoparticle also enhances the electroporation performance with focused electric pulses and localized poration , which was proved beneficial for not only the recovery of treated cells B-material to gain high cell viability I-property , but also the uptake of probes from multiple sites to facilitate the cytosolic delivery . [SEP]
[CLS] specifically , cationic B-material polymer B-material , polyethylenimine ( pei ) , was immobilized on aunps B-nanoparticle by electrostatic interactions ( figure 1 ) . [SEP]
[CLS] dna plasmids or sirna probes were then conjugated with pei molecules to form aunps B-nanoparticle - polyplex . [SEP]
[CLS] the complex nanoparticles B-nanoparticle were then mixed with cells B-material for electroporation . [SEP]
[CLS] the delivery enhancement was evaluated by the cell B-property viability I-property and the transfection B-property efficiency I-property . [SEP]
[CLS] branched pei ( mw = 25kda ) , gold B-nanoparticle nanoparticles I-nanoparticle of 5 - 40 nm were obtained from sigma - aldrich . [SEP]
[CLS] the concentration of 1x aunps B-nanoparticle refers to the stock solution , which has 0 . 01 wt % of au ( 0 . 1 mg / ml ) while the actual particle number varies with the size of aunps B-nanoparticle . [SEP]
[CLS] other concentrations of aunps B-nanoparticle were prepared by either concentrating or diluting from the stock solution . [SEP]
[CLS] dna plasmids with gwiz [UNK] gfp and gwiz [UNK] luc reporter genes were purchased from aldevron , inc . ( fargo , nd ) . [SEP]
[CLS] small interfering rna ( sirna ) used for silencing gfp ( expressed by pmaxgfp purchased from lonza ) and luciferase genes were synthesized by thermo scientific ( pittsburgh , pa ) and the sequences were as follows : sirna for gfp silence , sense strand , 5 ′ - cgcaugaccaacaagaugauu - 3 ′ ; antisense strand , 5 ′ - ucaucuuguuggucaugcggc - 3 ′ ; luciferase gl3 duplex ( luc - sirna ) , sense strand , 5 ′ - cuuacgcugaguacuucga - 3 ′ ; antisense strand , 5 ′ - ucgaaguacucagcguaag - 3 ′ . [SEP]
[CLS] all other chemicals were purchased from sigma - aldrich and the cell B-material culture reagents were purchased from life technologies ( carlsbad , ca ) unless specified . [SEP]
[CLS] to prepare aunps B-nanoparticle / pei polyplex , 500μl 0 . 01wt % of aunps B-nanoparticle was added into 500μl 0 . 5mg / ml pei ( ph7 . 0 ) . [SEP]
[CLS] the original citric acid terminated surface of aunps B-nanoparticle facilitates the deposition of pei molecules through electrostatic interactions . [SEP]
[CLS] the incubation B-technique was performed at room temperature for 20 min and the extra pei was removed by centrifuging at 15000×g for 10 min . [SEP]
[CLS] the pei coated aunps B-nanoparticle were resuspended in desirable amount of pbs ( ph = 7 . 0 ) and 5μl of nucleic B-material acid I-material solution ( with a concentration of 5mg / ml ) was added to aunps B-nanoparticle / pei of varying concentrations . [SEP]
[CLS] the resulting mixture was mixed by pipetting and further incubated B-technique at room temperature for 20 min . [SEP]
[CLS] nih / 3t3 cells B-material ( atcc , crl - 1658 ) were grown and maintained in high glucose dmem supplemented with 10 % newborn calf serum ( ncs ) , 1 % penicillin and streptomycin , 1 % lglutamine and 1 % sodium B-material pyruvate . [SEP]
[CLS] k562 cells B-material ( atcc , ccl - 243 ) were routinely cultured in rpmi 1640 supplemented with 10 % ncs , 100 u / ml penicillin , 100 μg / ml streptomycin , and 100 μg / ml l - glutamine B-material . [SEP]
[CLS] all cultures were maintained at 37° c with 5 % co2 and 100 % relative humidity . [SEP]
[CLS] nih / 3t3 or k562 cells B-material were first centrifuged and resuspended in fresh opti - mem i ( a serum free medium ) at a density of 0 . 5×10 6 cells B-material / ml . [SEP]
[CLS] samples were then mixed with polyplexes of various concentrations and sizes . [SEP]
[CLS] cell B-material electroporation was done with a commercial instrument ( ecm 830 , harvard apparatus ) in cuvettes with a 2 - mm gap , each containing a 100 μl sample solution . [SEP]
[CLS] the electroporation conditions are established from previous work as follows : 125v , 10 ms with a single unipolar pulse [SEP]
[CLS] after electroporation , samples were transferred to 6 - well cell B-material culture plates , incubated B-technique in fresh medium for another 24 hr and then harvested for analysis . [SEP]
[CLS] the transfection B-property efficiency I-property of gwiz [UNK] gfp plasmids was evaluated both qualitatively by visualizing the number of cells B-material with green fluorescence B-property within a representative area selected from the entire culture surface under an inverted fluorescence B-property microscope ( olympus , japan ) and quantitatively by counting cells B-material using a four - color flow B-technique cytometry I-technique system ( facs calibur , bd biosciences , ca ) 24 hr post transfection . [SEP]
[CLS] briefly , an amount of 1 . 5×10 6 cells B-material / ml was collected and the percentage of gfp - positive cells B-material was calculated quantitatively via flow cytometer . [SEP]
[CLS] the unstained samples were run first to adjust the voltage setting and compensation of the flow cytometer . [SEP]
[CLS] then the tested samples were processed by cellquest . [SEP]
[CLS] at least 10 , 000 events were collected for each sample . [SEP]
[CLS] the gfp down regulation efficiency was determined by agilent 2100 bioanalyzer ( agilent technologies , santa clara , ca ) . [SEP]
[CLS] the fluorescence B-property intensity of gfp was measured using cell B-material assay module with live cells B-material stained with carboxy - naphthofluorescein ( cbnf ) . [SEP]
[CLS] the results were analyzed with agilent 2100 expert software and 500 - 1 , 500 events were counted for each sample . [SEP]
[CLS] the luc - sirna down regulation efficiency was quantified by one - glo [UNK] luciferase assay system ( promega , madison , wi ) . [SEP]
[CLS] 100ul one - glo [UNK] reagent was added to the cell B-material growth in 100ul of medium in 96 - well plate . [SEP]
[CLS] luminescence B-property was measured with a plate reader ( fluostar optima , bmg labtech , germany ) after 10 min incubation B-technique at room temperature for complete cell B-material lysis . [SEP]
[CLS] the cell B-property viability I-property was evaluated by an mts cell B-material proliferation assay ( promega , madison , wi ) . [SEP]
[CLS] briefly , the cells B-material in 100 μl / well of medium were transferred to a 96 - well plate and incubated B-technique . [SEP]
[CLS] 20 μl of celltiter 96 aqueous one solution ( promega , madison , wi ) was added to each well and cells B-material were incubated B-technique at 37°c for another 4 hr . [SEP]
[CLS] absorbance was measured at 492 nm on an automated plate reader ( elx 800 , biotek , vt ) . [SEP]
[CLS] data points were represented as the mean ± standard deviation ( sd ) of triplicates , unless otherwise indicated . [SEP]
[CLS] the distribution of aunps B-nanoparticle - polyplex in 3t3 cells B-material were examined using an inverted fluorescent B-property microscopy B-technique . [SEP]
[CLS] as aunps B-nanoparticle are well known to quench the fluorescent B-property signal from proximal fluroprobes , a sandwich design of fluorophore - labeled aunps B-nanoparticle ( fnp , from nanopartz , inc , having alexa fluor 546 ) with fluorophores separated from the gold B-material surface by polymer B-material spacers B-material , were used to circumvent this problem . [SEP]
[CLS] plasmids were stained with yoyo - 1 iodide B-material ( life technology ) with a ratio of 100bp / dye . [SEP]
[CLS] the mixture of cells B-material with nucleic B-material acids I-material , aunps B-nanoparticle or aunps B-nanoparticle - polyplex were washed twice with 1x pbs ( ph7 . 0 ) , followed by fixation with 4 % paraformaldehyde for 30 min . [SEP]
[CLS] nuclei were stained with 20 μm of dapi for 10 min at room temperature . [SEP]
[CLS] cells B-material from each sample were then mounted on cover glass slides . [SEP]
[CLS] images of phase contrast , green ( nucleic B-material acids I-material ) , red ( aunps B-nanoparticle ) and blue ( nuclei ) fluorescence B-property channels were taken on an olympus 1×51 inverted microscope ( olympus , japan ) with a 100x objectives . [SEP]
[CLS] in our nomenclature , symbols like " a / b " or " a−b " means materials a and b are conjugated together through electrostatic interactions after incubation B-technique ; " a + b " means a and b are simply mixed without incubation B-technique before further treatment . [SEP]
[CLS] current polyplex delivery vehicles B-material have not yet shown competitive delivery advantages over natural virus - based counterparts . [SEP]
[CLS] this is at least partially attributed to their heterogeneous assembly conditions and poor synthetic quality of nanoparticles B-nanoparticle ( i . e . , relatively large variations in size , structure , and component quantity ) . [SEP]
[CLS] as in aunps B-nanoparticle - polyplex synthesis , cationic B-material polymer B-material molecules ( e . g . , pei ) were first immobilized on the surface of aunps B-nanoparticle , their amount in individual aunps B-nanoparticle - polyplex should be more uniform than those synthesized through dynamic complexation of free charged agents . [SEP]
[CLS] this further determines the total dosage of genetic probes which are condensed on aunps B-nanoparticle - polyplex later on . [SEP]
[CLS] therefore , the introduction of aunps B-nanoparticle in polyplex not only helps fix free or dissociated polycations on a solid surface , but also provides better management on molecule assembly and multipleagent packaging . [SEP]
[CLS] as the consequence , nanoparticles B-nanoparticle of better quality are produced . [SEP]
[CLS] as shown in afm images in figure 2 , more homogeneous morphology was found for aunpspolyplex than polyplex synthesized via vortex mixing ( figure 2e ) [SEP]
[CLS] their size was also much smaller and more uniform , which was further confirmed with quantitative measurements using dynamic B-technique light I-technique scattering I-technique ( figure 2f ) . [SEP]
[CLS] except for aunps B-nanoparticle - polyplex synthesized from 5 nm aunps B-nanoparticle , the average size o aunps B-nanoparticle - polyplex with various original sizes fell between 100 - 200 nm , an appropriate size range of nanoparticles B-nanoparticle for efficient cellular uptake . [SEP]
[CLS] as the size of dna plasmids used in this study is much bigger than that of aunps B-nanoparticle , the same dna molecules are suspected to interact with multiple aunps B-nanoparticle - pei nanoparticles B-nanoparticle simultaneously ( as shown schematically in supplemental figure s1 ) . [SEP]
[CLS] therefore , clusters ( or aggregates from conjugation networking ) of aunps B-nanoparticle - pei - dna , instead of many individual aunps B-nanoparticle - polyplex nanoparticles B-nanoparticle with assembly structure schematically shown in figure 1 , are more likely formed . [SEP]
[CLS] with smaller size and higher mobility B-property , aunps B-nanoparticle of 5 nm allow easier occurrence of such conjugation networking than other aunps B-nanoparticle with larger original size . [SEP]
[CLS] as the results , such stable clusters might become the dominated population when small aunps B-nanoparticle are used in aunps B-nanoparticle - polyplex synthesis . [SEP]
[CLS] to verify that pei molecules retain on aunps B-nanoparticle during electroporation , fluorescence B-property probes were tagged to track the locations and fate of aunps B-nanoparticle - polyplex during the cellular uptake . [SEP]
[CLS] as aunps B-nanoparticle quench fluorescent B-property signal from proximal fluroprobes , fluorophore - labeled aunps B-nanoparticle ( fnp ) with polymer B-material spacer B-material separating fluorophores from the gold B-material surface were used . [SEP]
[CLS] these nanoparticles B-nanoparticle are also carboxylated to match similar interaction capacity as those with citric acid terminated surface . [SEP]
[CLS] after conjugating with pei molecules , fluoroprobes are pushed back to the gold B-material surface and therefore , the fluorescent B-property signal of fnp is quenched again unless most immobilized pei molecules are gone . [SEP]
[CLS] when dna plasmids are condensed on fnp , the pei layer underneath serves as the new thick spacer B-material so that the yoyo - 1 labeled dna probes become visible and are used to track the uptake of aunps B-nanoparticle - polyplex . [SEP]
[CLS] compared to the untreated sample ( figure 3a ) , samples of simply mixing cells B-material and yoyo - 1 labeled dna plasmids ( figure 3b ) or fnp ( figure 3c ) have weak fluorescence B-property spots visible . [SEP]
[CLS] this is attributed to their tiny particle size and limited fluorescence B-property signal when staying as individual nanoparticles B-nanoparticle . [SEP]
[CLS] after capped with a layer of pei , even such weak fluorescence B-property signals disappeared ( figure 3d ) , which confirmed that the original fluorophores were pushed back close to the gold B-material surface . [SEP]
[CLS] as the pei layer serves as the new spacer B-material layer , the condensed dna plasmids labeled with yoyo - 1 exhibited similar fluorescent B-property signal as free dna plasmids ( showing weak green fluorescence B-property spots in figure 3e ) , indicating that the location of aunps B-nanoparticle - polyplex was mainly outside cells B-material . [SEP]
[CLS] after electroporation , stronger fluorescent B-property signals were generally seen in electroporated cells B-material with naked plasmids and fnp as nanoparticles B-nanoparticle accumulated in cells B-material ( figures 3f - 3g ) . [SEP]
[CLS] no fluorescence B-property signal was observed for the sample using pei - coated fnp ( figure 3h ) , though similar accumulation of aunps B-nanoparticle - pei nanoparticles B-nanoparticle were clearly observed in the phase contrast image . [SEP]
[CLS] this suggests that the electric pulses did not break the interactions between aunps B-nanoparticle and pei molecules . [SEP]
[CLS] for yoyo - 1 labeled aunps B-nanoparticle - pei - dna polyplex , strong green fluorescence B-property signal was shown inside cells B-material ( figure 3i ) . [SEP]
[CLS] as samples were fixed immediately after electroporation , this clearly indicates the similar quick and direct cytosolic delivery of plasmids by electroporation . [SEP]
[CLS] we further explored the delivery of dna plasmids from aunps B-nanoparticle - polyplex by electroporation . [SEP]
[CLS] the electroporation was done with nih 3t3 cells B-material with a btx system using pwizgfp plasmids and the following pulse scheme was applied : 125 v ( 625 v / cm ) , single 10 ms pulse . [SEP]
[CLS] successful transfection was observed 24 hr after electroporation in all four cases : electroporation with dna alone ( no aunps B-nanoparticle ) , with aunps B-nanoparticle + dna , with polyplex ( no aunps B-nanoparticle ) , and with aunps B-nanoparticle - polyplex , as shown in figure 4 . [SEP]
[CLS] however , a simple combination of electroporation and polyplex showed significantly negative impact on both the delivery efficiency and cell B-property viability I-property ( figure 4b ) , which is consistent with an early observation for lipoplex delivery using a similar approach . [SEP]
[CLS] the poor - quality and loose structure of polyplex might have been destroyed to release a large number of free cationic B-material pei molecules . [SEP]
[CLS] these free positively charged macromolecules , together with additional harsh electric pulses , further lowered the overall cell B-property viability I-property and transfection when compared to the electroporation of naked plasmids ( figure 4a ) . [SEP]
[CLS] electroporation delivery of plasmids together with aunps B-nanoparticle and aunps B-nanoparticle - polyplex showed better gfp expression ( figures 4c - 4d ) , which confirmed again our early observation the enhancement of aunps B-nanoparticle to electroporation performance [SEP]
[CLS] more quantitative comparison was done by counting the percentage of gfppositive cells B-material using flow B-technique cytometry I-technique ( figure 5 ) . [SEP]
[CLS] efficiency B-property of I-property pgfp transfection I-property from aunps B-nanoparticle - polyplex ( using 5 nm aunps B-nanoparticle at a concentration of 1x or 0 . 1 mg / ml ) was about one and half folds of that using naked plasmids and a simple mixture of dna and aunps B-nanoparticle ( dna alone : 34 . 8 % ±2 . 0 % ; 5nm aunps B-nanoparticle and dna : 44 . 4 % ; 5 nm aunps B-nanoparticle - polyplex : 53 . 4 % ) . [SEP]
[CLS] when aunps B-nanoparticle of larger size ( 10 - 40 nm for their original size ) were used , the transfection B-property efficiency I-property was further enhanced to about two folds of that using naked dna alone . [SEP]
[CLS] as for comparison , the gfp transfection using electroporation with polyplex was only about one third of that standard electroporation . [SEP]
[CLS] some loss on the cell B-property viability I-property ( i . e . , ~ 10 % ) was observed in aunps B-nanoparticle - polyplex electroporation samples than that using naked dna . [SEP]
[CLS] but it is worth - while the sacrifice for using electroporation to bypass the endocytosis B-event delivery route with direct cytosol delivery and 1 . 5 - 2 folds increase on the transfection B-property efficiency I-property . [SEP]
[CLS] when comparing to a cell B-property viability I-property of ~ 40 % ( i . e . , less than half of the standard electroporation of naked dna , ~ 90 % ) that using polyplex in electroporation , our approach of introducing aunps B-nanoparticle to fix free or dissociated pei is effective . [SEP]
[CLS] this observation was also endorsed with further complex cytotoxicity B-property analysis without electroporation ( see supplemental figure s2 ) . [SEP]
[CLS] in this delivery improvement , the aunps B-nanoparticle core B-material helps enhance the electroporation performance from two different aspects 49 : ( 1 ) reducing the resistance of the electroporation buffer solution so that the local pulse strength on cells B-material is enhanced ; ( 2 ) serving as virtual microelectrodes to locally porate cells B-material with limited area from many different sites . [SEP]
[CLS] because of their high conductivity ( ~ 4 . 5×10 6 s / m ) , aunps B-nanoparticle dispersed in buffer and cytoplasm ( the conductivity is ~ 0 . 3 - 1 . 5 s / m ) help greatly reduce the potential drop there so that the majority of the electric voltage is imposed on the cell B-material membrane indeed . [SEP]
[CLS] the cell B-material membrane disruption could therefore be done more effectively without altering the cell B-material physiological conditions ( i . e . , salt B-material concentration ) or losing cell B-property viability I-property . [SEP]
[CLS] with the electric field converges in the vicinity of aunps B-nanoparticle , they work like many virtual nanoelectrodes to cause localized poration . [SEP]
[CLS] different from the traditional bulk electroporation with two large breakdown locations , multiple small poration sites are formed after adding aunps B-nanoparticle that benefits not only the recovery of the cell B-material membrane , but also the cytosolic delivery of plasmids from multiple sites . [SEP]
[CLS] the contribution of aunps B-nanoparticle - polyplex to the transfection improvement of dna plasmids is also multifactorial : ( 1 ) they help fix pei on the surface of aunps B-nanoparticle to significantly reduce the toxicity B-property caused by the presence of free and / or dissociated cationic B-material polymer B-material molecules in polyplex ( see supplemental figure s2 ) ; ( 2 ) they also effectively produce polyplex nanoparticles B-nanoparticle with smaller average size than the naked dna plasmids and narrower size distribution when compared to that from vortex mixing synthesis ( figure 2f ) ; ( 3 ) the pei molecules in aunps B-nanoparticle - polyplex help protect dna plasmids and condense them near the vicinity of cell B-material membrane to promote the cytosolic delivery and also later nuclear transport . [SEP]
[CLS] these facts offer aunp B-nanoparticle - polyplexes advantages over the use of the mixture of aunp B-nanoparticle and naked dna in electroporation ( figure 5 ) as well as many traditional transfection approaches ( see supplemental figure s3 ) on the transfection B-property efficiency I-property and cell B-property viability I-property . [SEP]
[CLS] the slight loss on cell B-property viability I-property ( in figure 5b ) probably results from the presence of some free pei molecules to the electroporated cells B-material . [SEP]
[CLS] small interfering rna ( sirna ) is now widely used to down regulate specific gene expression in cells B-material with their complementary nucleotide sequence . [SEP]
[CLS] to demonstrate the effectiveness of aunps B-nanoparticle - polyplex electroporation to sirna delivery , we chose sirna with specific sequences to silence the expression of gfp and luciferase when co - transfecting with pgfp and pluc by electroporation . [SEP]
[CLS] as shown in figure 6a , clearly less gfp expression was seen when co - delivering pmaxgfp and the corresponding sirna . [SEP]
[CLS] aunps B-nanoparticle - polyplex ( sirna ) helped turn off more gfp expression than that using free sirna ( figure 6b ) . [SEP]
[CLS] similar down regulation performance was also found when co - transfecting pluc and the corresponding sirna gl3 , as shown in figure 6c . [SEP]
[CLS] compared to the interference result of free sirna gl3 , additional ~ 15 % further drop of luciferase signal was found when sirna molecules were conjugated in aunps B-nanoparticle - polyplex . [SEP]
[CLS] because sirna have much smaller size than plasmid dna , neither the delivery of free sirna nor aunps B-nanoparticle - polyplex to cell B-material cytosol through electroporation is very challenging . [SEP]
[CLS] however , their quick denature feature makes the delivery focus of sirna delivery often on their protection from enzyme degradation . [SEP]
[CLS] therefore , sirna delivery with aunps B-nanoparticle - polyplex electroporation could be more beneficial when used for in vivo delivery . [SEP]
[CLS] it is also worth to point out that the enhancement of aunps B-nanoparticle - polyplex to sirna interference performance should be better than what was shown in figure 6 . [SEP]
[CLS] as co - transfection of dna plasmids and sirna approach was adopted here , the interference of sirna to the expression of the targeting reporter gene occurs simultaneously with that particular transgene expression in cells B-material . [SEP]
[CLS] the early presence of copious sirna probes could silence the targeting proteins B-material more efficiently than those that already maintain a sustained concentration level in cells B-material . [SEP]
[CLS] therefore , both free sirna and sirna from aunps B-nanoparticle - polyplex showed efficient down regulation performance here , which allows only limited room to further enhance the interference with aunps B-nanoparticle - polyplex . [SEP]
[CLS] moreover , the presence of aunps B-nanoparticle during polyplex ( sirna ) delivery simultaneously enhanced the actual expression level of gfp or luciferase with the same mechanism demonstrated in figure 5 . [SEP]
[CLS] this means sirna molecules in aunps B-nanoparticle - polyplex must shut off more gpf or luciferase proteins B-material than that using free sirna to reach the similar protein B-material expression level . [SEP]
[CLS] in another word , the enhancement of aunps B-nanoparticle - polyplex to sirna down regulation is actually better than what was shown in figure 6 for their higher starting protein B-material level than that using free sirna probes . [SEP]
[CLS] in summary , we immobilized polyplex on gold B-nanoparticle nanoparticles I-nanoparticle and delivered them into cells B-material through electroporation . [SEP]
[CLS] conjugating with aunps B-nanoparticle helps minimize the cytotoxicity B-property concerns from polyplex after cytoplasmic release while still retains good probe protection . [SEP]
[CLS] it also avoids poor nanoparticle B-nanoparticle quality existing in traditional polyplex synthesis , namely large size and broad size variations , by managing molecule interactions and assembly on the surface of aunps B-nanoparticle . [SEP]
[CLS] combining with electroporation , conjugated polyplex ( aunps B-nanoparticle - polyplex ) showed quick delivery and significant enhancement on the transfection B-property efficiency I-property with no obvious increase of toxicity B-property . [SEP]
[CLS] such a combination of physical and chemical delivery concept may stimulate further exploration in the delivery of various therapeutic materials for both in vitro and in vivo applications . [SEP]
[CLS] the choice of aunps B-nanoparticle in the enhancement of polyplex delivery lies on their high conductivity and excellent biocompatibility B-property . [SEP]
[CLS] their other potential advantages , such as sensing signal enhancement via localized surface plasmon resonance ( lspr ) or surface enhanced raman spectrum ( sers ) , have not yet been investigated with this gene delivery approach . [SEP]
[CLS] but there is promising potential of our approach to integrate both diagnostic and therapeutic functions in one nanosystem ( namely nanotheranostics ) to accomplish both noninvasively tracking the targeting therapeutic probes and measuring their deliver performance simultaneously . [SEP]
[CLS] this surely will help increase our understanding on the regulation mechanism of many therapeutic probes and quick establishment of appropriate strategies to improve their delivery or treatment performance . [SEP]
[CLS] other forms of gold B-material nanostructures , such as nanorod B-nanoparticle , nanoshell B-nanoparticle , or nanowires B-nanoparticle , in principle , could also be used for the similar purposes . [SEP]
[CLS] its success will accelerate and broaden the applications of these the afm images of aunps B-nanoparticle - polyplex morphology with the original size of aunps B-nanoparticle of ( a ) 5nm , ( b ) 10 nm , ( c ) 30 nm , ( d ) 40 nm . [SEP]
[CLS] panel ( e ) is the traditional polyplex synthesized through vortex mixing approach [SEP]
[CLS] panel ( f ) is the quantitative dls particle size measurement . [SEP]
[CLS] 1 . schematic illustration on the procedure of aunps B-nanoparticle - polyplex synthesis and delivery . [SEP]
[CLS] phase contrast and fluorescence B-property microscopic images of distribution and fate of aunpspolyplex when mixing with nih 3t3 cells B-material ( a - e ) and immediately after electroporation ( f - i ) : ( a ) untreated samples ( negative control ) , ( b ) mixture of cells B-material and naked dna plasmids ( green ) ; ( c ) mixture of cells B-material and fnp ( red ) ; ( d ) mixture of cells B-material with fnp / pei nanoparticles B-nanoparticle ; ( e ) mixture of cells B-material with fnp / pei / dna ; ( f ) electroporation with dna alone ( green ) ; ( g ) electroporation with fnp ( red ) ; ( h ) electroporation with fnp / pei ; and ( i ) electroporation with fnp / pei / dna . [SEP]
[CLS] 4 . fluorescence B-property and phase contrast microscopic images of pgfp plasmid transfection to nih 3t3 cells B-material by electroporation naked dna plasmids , a mixture of aunps B-nanoparticle and dna plasmids , polyplex , and aunps B-nanoparticle - polyplex . [SEP]
[CLS] quantitative measurement of electroporation enhanced aunps B-nanoparticle - polyplex delivery performance on 3t3 cells B-material : ( a ) the flow B-technique cytometry I-technique results on transfection B-property efficiency I-property and ( b ) the cell B-property viability I-property via mts assay . [SEP]
[CLS] as comparison , results from electroporation with dna alone , polyplex , and samples of a simple mixing of aunps B-nanoparticle and dna are also shown . [SEP]
[CLS] the error bars correspond to triplicate experiments made with independently produced batches . [SEP]
[CLS] ( * * * p < 0 . 001 ) [SEP]
[CLS] 6 . aunps B-nanoparticle - polyplex electroporation enhanced sirna delivery on k562 cells B-material : ( a ) fluorescence B-property images of and ( b ) intensity measurement on gfp expression level , and ( c ) the luminescence B-property measurement on luciferase expression level for free sirna ( " pmax + sirna " and " pluc + gl3 " ) and sirna from aunps B-nanoparticle - polyplex ( " pmax + aunp B-nanoparticle / pei / sirna " and " pluc + aunp B-nanoparticle / pei / gl3 " ) . [SEP]
[CLS] ( * p < 0 . 05 , * * * p < 0 . 001 ) . [SEP]
[CLS] electroporation figured prominently as an effective nonviral gene delivery approach for its balance on the transfection B-property efficiency I-property and cell B-property viability I-property , no restrictions of probe or cell B-material type , and operation simplicity . [SEP]
[CLS] the commercial electroporation systems have been widely adopted in the past two decades while still carry drawbacks associated with the high applied electric voltage , unsatisfied delivery efficiency , and / or low cell B-property viability I-property . [SEP]
[CLS] by adding highly conductive gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) in electroporation solution , we demonstrated enhanced electroporation performance ( i . e . better dna delivery efficiency and higher cell B-property viability I-property ) on mammalian cells B-material from two different aspects : the free , naked aunps B-nanoparticle reduce the resistance of the electroporation solution so that the local pulse strength on cells B-material was enhanced ; targeting aunps B-nanoparticle ( e . g . , tf - aunps B-nanoparticle ) were brought to the cell membrane to work as virtual microelectrodes to porate cells B-material with limited area from many different sites . [SEP]
[CLS] the enhancement was confirmed with leukemia cells B-material in both a commercial batch electroporation system and a home - made flow - through system using pwizgfp plasmid dna probes . [SEP]
[CLS] such enhancement depends on the size , concentration , and the mixing ratio of free aunps B-nanoparticle / tf - aunps B-nanoparticle . [SEP]
[CLS] an equivalent mixture of free aunps B-nanoparticle and tf - aunps B-nanoparticle exhibited the best enhancement with the transfection B-property efficiency I-property increased 2 - 3 folds at minimum sacrifice of cell B-property viability I-property . [SEP]
[CLS] this new delivery concept , the combination of nanoparticles B-nanoparticle and electroporation technologies , may stimulate various in vitro and in vivo biomedical applications which rely on the efficient delivery of nucleic B-material acids I-material , anticancer B-property drugs , or other therapeutic materials . [SEP]
[CLS] efficient delivery of nucleic B-material acids I-material often plays important roles in the treatment of various diseases . [SEP]
[CLS] its delivery involves the insertion of healthy copies of dna or rna probes in specific cells B-material , which relies on either viral infection or nonviral membrane perturbation . [SEP]
[CLS] viral vectors offer stable and efficient transduction , but have safety concerns associated with oncogenesis , immunogenicity B-property , and toxicity B-property [SEP]
[CLS] nonviral delivery approaches , including chemical and physical approaches , have been explored as replacements to natural viruses , but yet to reach competitive levels to their viral counterpart . [SEP]
[CLS] among nonviral approaches , gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) have been extensively explored in dna or rna delivery for their good biocompatibility B-property and unprecedented combination of therapeutic and imaging capability . [SEP]
[CLS] through the unique gold B-material - thiol B-material chemistry and / or electrostatic interactions , aunps B-nanoparticle help improve the cellular uptake of molecule probes through similar internalization routes ( i . e . , endocytosis B-event ) as other nanoparticles B-nanoparticle . [SEP]
[CLS] the long intracellular delivery barriers B-property generally prevent many nanoparticles B-nanoparticle from reaching cytosol or nucleus . [SEP]
[CLS] with the aid of cell penetrating peptides B-material ( cpps ) , aunps B-nanoparticle - sirna nanoconjugates were successfully demonstrated to reach cytosol directly to improve the delivery efficiency [SEP]
[CLS] however , the low release extent of sirnas from aunps B-nanoparticle still leads to poor transfection B-property efficiency I-property due to the high affinity of aunps B-nanoparticle and therapeutic agents [SEP]
[CLS] compared to nanoparticle - mediated delivery routes , physical approaches have been used in the past two decades to directly deliver drugs or gene probes to desired intracellular locations ( e . g . , cytosol or nucleus ) to attain impressive benefits in various biomedical research and clinic trials . [SEP]
[CLS] among them , electroporation figures prominently for their balance of simplicity , transfection effectiveness , less restrictions on probe or cell B-material type , and operation convenience . [SEP]
[CLS] in electroporation , short , high - voltage electric pulses are applied to surpass the cell B-material membrane capacitance , making the subjected cells B-material transiently permeable . [SEP]
[CLS] the required transmembrane potential ( δv m , in v ) for reversible breakdown of the cell B-material membrane can be estimated by : ( 1 ) where e ext is the electric field strength ( in v / cm ) , r is the radius of cell B-material ( in cm ) , π is the angle between e ext and the membrane surface . [SEP]
[CLS] from equation ( 1 ) , the breakdown sites on the cell B-material membrane appear first at those locations which face the two electrodes ( i . e . , π = 0° and 180° ) . [SEP]
[CLS] current electroporation systems have been reasonably successful while still carrying several major drawbacks which are associated with the high applied electric voltage and / or the lack of uniformity of electric pulses on all treated cells B-material . [SEP]
[CLS] the low electrical conductivity of the electroporation solution ( e . g . , for pbs , it is ~ 1 . 5 s / m ) leads to the consumption of a large percentage of the overall applied voltage across the two planar electrodes and the actual voltage allocated on treated cells B-material is much lower than expected , as illustrated in figure 1a . [SEP]
[CLS] because of the physiological conditions requirements , increasing the ion strength ( e . g . , salt B-material concentration ) of the electroporation buffer is not allowed to avoid such additional voltage consumption . [SEP]
[CLS] to achieve desired probe transfection B-property efficiency I-property , harsh electroporation conditions ( e . g . , high - voltage pulses ) are therefore necessary to ensure enough permeabilization to the majority of treated cells B-material . [SEP]
[CLS] this makes electroporation inevitably accompanied with unwanted effects ( e . g . , strong electrochemical reactions , gas bubble issue , and joule heating ) , which are harmful to the survival of treated cells B-material [SEP]
[CLS] current protocols are often established on the compromise between acceptable transfection B-property efficiency I-property and cell B-property viability I-property . [SEP]
[CLS] the recent introduction of microtechnology in electroporation research devoted to the reduction of these issues through closely patterning electrode pairs [SEP]
[CLS] but these designs often sacrifice some favorite features of electroporation systems , namely simplicity , low - cost , and operation convenience . [SEP]
[CLS] here we present a simple approach to enhance the transfection performance of electroporation that is compatible to most commercial electroporation instruments as well as the emerging micro / nanoelectroporation systems . [SEP]
[CLS] in this new approach , free therapeutic probes ( e . g . , dna plasmids ) are directly introduced into cell B-material cytosol through electroporation while aunps B-nanoparticle are added to locally enhance the electric pulse strength and control the poration area on the cell B-material membrane with minimum operation changes . [SEP]
[CLS] because of their high conductivity ( ~ 4 . 5×10 6 s / m ) , the electric voltage consumed by the electroporation buffer is greatly reduced so that most of the applied electric voltage is truly imposed on cells B-material . [SEP]
[CLS] in addition , as the electric pulses are converged in the vicinity of aunps B-nanoparticle , they work like many virtual microelectrodes when staying around cells B-material with the focused field strength to cause localized poration , as shown in figure 1b . [SEP]
[CLS] different from bulk electroporation with two large breakdown locations at the two poles of cells B-material facing the electrodes , multiple small poration sites are expected to be created on the cell B-material membrane by aunps B-nanoparticle . [SEP]
[CLS] this could benefit not only the recovery of the cell B-material membrane and the survival of cells B-material , but also the uptake opportunity for the subjected probes from multiple sites . [SEP]
[CLS] as aunps B-nanoparticle are also randomly dispersed in the electroporation solution , just like cells B-material themselves , they are expected to be present evenly around cells B-material , which might further reduce the polarization variations associated with suspended state of cells B-material in electroporation . [SEP]
[CLS] to test our hypothesis , we added aunps B-nanoparticle to the electroporation solution , together with mammalian cells B-material and dna plasmids . [SEP]
[CLS] cells B-material were then executed using both a commercial batch - type electroporator ( btx 830 from harvard apparatus ) and a home - made semicontinuous flow electroporator ( sfe ) [SEP]
[CLS] the pulse strength focusing evaluation was done from two aspects : ( i ) execute cells B-material at the presence of aunps B-nanoparticle under standard electroporation conditions , in which the cell B-property viability I-property should get worse as the electric pulse was focused by aunps B-nanoparticle and cells B-material received higher - than - optimal electric pulses ; ( ii ) perform electroporation under less - effective conditions ( i . e . , low - voltage , much benign pulses ) , in which the focusing effect helps gain sufficient electrical strength for better transfection B-property efficiency I-property and / or cell B-property viability I-property . [SEP]
[CLS] a human chronic leukemia cells B-material ( k562 cells B-material ) , a hard - to - transfect cell B-material line , were used in this investigation so that the localized electroporation with controlled polarization area and locations on the cell B-material membrane could also be conveniently evaluated through ligand - receptor affinity binding . [SEP]
[CLS] the electroporation enhancement evaluation was focused on the cell B-property viability I-property and the transfection B-property efficiency I-property of a reporter gene ( wizgfp ) . [SEP]
[CLS] similar enhancement performance was also observed in nih 3t3 cells B-material ( data not shown ) , confirming the broad effectiveness of the enhancement roles aunps B-nanoparticle play in electroporation . [SEP]
[CLS] gold B-nanoparticle nanoparticles I-nanoparticle of 5 nm , 10 nm , and 20 nm were obtained from sigma - aldrich . [SEP]
[CLS] dna plasmids with [UNK] gfp reporter gene were purchased from aldevron , inc . [SEP]
[CLS] the concentration of 1x aunps B-nanoparticle refers to the stock solution , which has 0 . 01 wt % of au ( 0 . 1 mg / ml ) while the actual particle number varies with the size of aunps B-nanoparticle . [SEP]
[CLS] other concentrations of aunps B-nanoparticle were prepared by either concentrating or diluting from the stock solution . [SEP]
[CLS] all other chemicals were purchased from sigma - aldrich and the cell B-material culture reagents were purchased from life technologies ( carlsbad , ca ) unless specified . [SEP]
[CLS] k562 lymphoblast cells B-material were obtained from american type cell B-material culture ( atcc , manassas , va ) . [SEP]
[CLS] k562 cells B-material were cultured in rpmi 1640 media supplemented with 10 % heatinactivated fetal bovine serum ( fbs ) , 100 u / ml penicillin , 100 μg / ml streptomycin , and lglutamine . [SEP]
[CLS] cells B-material were maintained in t - 75 flasks at 37 °c with 5 % co2 and subcultured using 0 . 25 % ( w / v ) trypsin with edta • 4na . [SEP]
[CLS] transferrin ( sigma ) of 10 mg / ml in 50 mm pbs with 5 mm edta was first reacted with traut ' s reagent for 1 hr at room temperature . [SEP]
[CLS] the thiolated protein B-material was then purified by dialysis ( with dialysis cartridge from emd millipore , amicon cartridge , cat no . ufc805024 ) . [SEP]
[CLS] aunps B-nanoparticle of 20 nm was then incubated B-technique with thiolated transferrin at 4°c overnight . [SEP]
[CLS] excess transferrin was removed by repeated centrifugation and washed with pbs . [SEP]
[CLS] the purified tf - aunps B-nanoparticle were used immediately or stored at 4°c . [SEP]
[CLS] k562 cells B-material were first centrifuged and re - suspended in fresh opti - mem i ( a serum free medium ) at a density of 0 . 5×10 7 cells B-material / ml . [SEP]
[CLS] samples were then mixed with aunps B-nanoparticle of various concentrations ( 0 . 01 - 1 . 0 mg / ml ) and sizes ( 5 , 10 , and 20 nm ) and further added 25 μg dna . [SEP]
[CLS] electroporation with a commercial instrument ( ecm 830 , harvard apparatus ) was done in electroporation cuvettes with a 2 - mm gap , each containing a 100 μl sample solution . [SEP]
[CLS] two pulse conditions were used : 125v , 10 ms ( standard electroporation conditions ) and 95 v , 10 ms ( low - voltage pulse , much benign electroporation conditions ) with a single pulse . [SEP]
[CLS] sfe microchannel electroporation was done by pumping cells B-material through a serpentine microchannel ( length × width × depth = 37 . 5 mm × 150 μm × 150 μm ) at a low flow rate ( e . g . , 1 . 8 ml / hr ) and electric pulses ( 76 pulses with each at 16v , 10 ms ) were added through pt electrodes . [SEP]
[CLS] cells B-material were then flushed out with opti - mem i medium and collected for further analysis . [SEP]
[CLS] detailed sfe operation procedure can be found in our early publication . [SEP]
[CLS] after electroporation , samples were transferred to 6 - well cell B-material culture plates , incubated B-technique in fresh medium for another 24 hr , and then harvested for analysis . [SEP]
[CLS] the transfection B-property efficiency I-property of pwizgfp plasmids was evaluated both qualitatively by visualizing the number of cells B-material with green fluorescence B-property within a representative area selected from the entire culture surface under an inverted fluorescence B-property microscope ( olympus , japan ) and quantitatively by counting cells B-material using a four - color flow B-technique cytometry I-technique system ( facs calibur , bd biosciences , ca ) 24 hr post transfection . [SEP]
[CLS] briefly , an amount of 1 . 5×10 6 cells B-material / ml was collected and the percentage of gfp - positive cells B-material was calculated quantitatively via flow cytometer . [SEP]
[CLS] the unstained samples were run first to adjust the voltage setting and compensation of the flow cytometer . [SEP]
[CLS] then the tested samples were processed by cellquest . [SEP]
[CLS] at least 10 , 000 events were collected for each sample . [SEP]
[CLS] the cell B-property viability I-property was evaluated by an mts cell B-material proliferation assay ( promega , madison , wi ) . [SEP]
[CLS] briefly , the cells B-material in 100 μl / well of medium were transferred to a 96 - well plate and incubated B-technique . [SEP]
[CLS] 20 μl of celltiter 96 aqueous one solution ( promega , madison , wi ) was added to each well and the cells B-material were incubated B-technique at 37°c for another 4 hr . [SEP]
[CLS] absorbance was measured at 492 nm on an automated plate reader ( elx 800 , biotek , vt ) . [SEP]
[CLS] data points were represented as the mean ± standard deviation ( sd ) of triplicates , unless otherwise indicated . [SEP]
[CLS] the distribution , cellular binding , and uptake of aunps B-nanoparticle in k562 cells B-material were examined by laser scanning confocal microscopy B-technique . [SEP]
[CLS] the mixture of cells B-material and red fluorescence B-property of aunps B-nanoparticle ( from nanopartz , inc ) was washed twice with 1x pbs , followed by fixation with 4 % paraformaldehyde for 30 min . [SEP]
[CLS] nuclei were stained with 20 μm of dapi for 5 min at room temperature . [SEP]
[CLS] cells B-material from each sample were then mounted on cover glass slides . [SEP]
[CLS] images of phase contrast , red and blue fluorescence B-property channels were taken on a zeiss 510 meta laser scanning confocal microscopy B-technique ( carl zeiss microimaging , inc . , ny ) and then merged images were produced using the lsm imaging software . [SEP]
[CLS] as aunps B-nanoparticle are well known to quench the fluorescent B-property signal from proximal fluroprobes , a sandwich design of aunps B-nanoparticle ( from nanopartz , inc , having fluorophores separated from the gold B-material surface with polymer B-material spacer B-material ) were used to circumvent this problem . [SEP]
[CLS] fluorophore labeled aunps B-nanoparticle with ch 3 group terminated surface ( fnps , 10 nm for gold B-nanoparticle nanoparticle I-nanoparticle core B-material ) were used to represent free , naked aunps B-nanoparticle used in electroporation experiments . [SEP]
[CLS] to visualize and track tf - aunps B-nanoparticle , carboxylated aunps B-nanoparticle ( fnp - cooh ) were used . [SEP]
[CLS] fnp - cooh nanoparticles B-nanoparticle ( 1 mg / ml in pbs ) were first incubated B-technique with 1 - ethyl - 3 - ( 3dimethylaminopropyl ) carbodiimide ( 20mg / ml in pbs ) for 15 min at room temperature . [SEP]
[CLS] transferrin solution ( 0 . 4 mg / ml in pbs ) was then added and incubated B-technique at room temperature for 24 hr to obtain fnps with transferrin targeting probes ( tf - fnps ) . [SEP]
[CLS] tf - fnps particles were then purified by repeated centrifugation and re - suspension in pbs for three times prior to the binding B-event with I-event transferrin I-event receptors I-event ( tfr ) on k562 cell B-material surface . [SEP]
[CLS] a commercial finite - element methods ( fem ) software , comsol ( mathworks , natick , ma ) , was used to calculate the electric field around cell B-material at the presence of a single aunp B-nanoparticle . [SEP]
[CLS] we considered an axial symmetric model with one gold B-nanoparticle nanoparticle I-nanoparticle ( d = 20 nm ) embedded in cell B-material membrane ( 5 nm in thickness ) . [SEP]
[CLS] a k562 cell B-material ( d = 15 μm ) was placed at the center of the left side boundary ( the symmetrical axis ) in the computation domain ( 60 μm × 20 μm ) . [SEP]
[CLS] a polar angle ( π ) with respect to the electric field direction was defined and the gold B-nanoparticle nanoparticle I-nanoparticle was placed at the top of the cell B-material where π = 180° . [SEP]
[CLS] an electric field ( e = 475 v / cm ) was assigned across the top and bottom of the computation domain and the right side boundary was set as insulated wall . [SEP]
[CLS] a three - layer cell B-material model , divided as the external medium , the cell B-material membrane , and the cell B-material cytoplasm , was setup here [SEP]
[CLS] the electric field distribution around the nanoparticle B-nanoparticle and the cell B-material was calculated by solving the laplace equation using comsol : ( 2 ) where σ is electrical conductivity and v is the electrical potential . [SEP]
[CLS] the electric field strength was then determined by . [SEP]
[CLS] in this three - layer cell B-material model , the electrical conductivity of buffer , cytoplasm , membrane , and gold B-material particle was set as 0 . 8 , 0 . 2 , 5×10 −7 , and 4×10 7 s / m , respectively . [SEP]
[CLS] we first did electroporation with k562 cells B-material in both btx and sfe systems , adopting the pulse conditions which were previously optimized with wizgfp plasmids alone : 125 v ( 625 v / cm ) , single 10 ms pulse for the btx system and 16 v ( 1067 v / cm ) , multiple 10 ms pulses for the sfe system . [SEP]
[CLS] successful transfection was observed in all four cases : btx without aunps B-nanoparticle , sfe without aunps B-nanoparticle , btx with aunps B-nanoparticle , and sfe with aunps B-nanoparticle . [SEP]
[CLS] many cells B-material in each case expressed green fluorescence B-property protein B-material ( gfp ) 24 hr after electroporation ( figure 2a ) . [SEP]
[CLS] more quantitative comparison was done by counting the percentage of gfp - positive cells B-material ( figure 2b ) . [SEP]
[CLS] efficiency B-property of I-property pgfp transfection I-property from sfe was generally much better than that from btx ( btx : 27 . 5±1 . 9 % , sfe : 51 . 6±4 . 5 % ) , which is consistent with our earlier observations . [SEP]
[CLS] after adding aunps B-nanoparticle ( 5 nm at a concentration of 5x or 0 . 5 mg / ml ) , the transfection percentage was significantly increased to 50 . 8±6 . 7 % for btx electroporator and to 61 . 1±4 . 8 % for sfe microchannel electroporator , respectively . [SEP]
[CLS] some loss on the cell B-property viability I-property was observed ( from 78 . 9±2 . 9 % to 57 . 4±5 . 1 % for btx electroporator and from 69 . 9±4 . 7 % to 52 . 1±2 . 3 % for sfe microchannel electroporator ) , as shown in figure 2c . [SEP]
[CLS] this is not surprising considering the focusing effect of aunps B-nanoparticle could shift away the electric pulses from the desired strength . [SEP]
[CLS] as mentioned earlier , k562 cells B-material in figure 2 were executed at electroporation conditions optimized without the presence of aunps B-nanoparticle . [SEP]
[CLS] considering the high conductivity of aunps B-nanoparticle , their addition greatly reduced the resistance contributed by the buffer solution so that most electric voltage imposed between the two electrodes was allocated to cells B-material . [SEP]
[CLS] the actual pulse strength on the cell B-material membrane was therefore mitigated to a higher - than - optimal level , resulting in over perturbation to the treated cells B-material . [SEP]
[CLS] such harsh conditions increased the percentage of irreversible breakdown of the cell B-material membrane , making the loss of the cell B-property viability I-property inevitable . [SEP]
[CLS] as this pulse strength focusing effect is generated from the presence of free aunps B-nanoparticle in the electroporation buffer , the cell B-material transfection B-property efficiency I-property can get improved ( when reversible breakdown still dominates ) or become worse ( when irreversible breakdown becomes the dominant - - cells B-material might have probes successfully delivered while not get the subjected transgenes expressed prior to lysis ) , depending on the concentration and size of the added aunps B-nanoparticle . [SEP]
[CLS] from data shown in figure 2c , the field focusing level for 5 nm aunps B-nanoparticle at a concentration of 5x ( 0 . 5 mg / ml ) belonged to the first case ( i . e . , reversible breakdown still dominated ) . [SEP]
[CLS] the transfection of pwizgfp got improved while accompanied with lower cell B-property viability I-property . [SEP]
[CLS] nevertheless , this confirmed the electric field focusing effect of aunps B-nanoparticle to electroporation . [SEP]
[CLS] note : the transfection percentage is defined as the number of transfected cell B-material divided by the number of total living cells B-material 24 hr post transfection in each sample and the cell B-property viability I-property is measured as the ratio of the living cells B-material in each sample to that in the negative control samples . [SEP]
[CLS] these definitations might be different from some others used in literature ( divided by the number of cells B-material initial used or cells survived right after transfection ) and emphasize the fate of all survived cells B-material , though sometimes show low values on the transfection ( or the cell B-property viability I-property ) for their large number of the total living cells B-material . [SEP]
[CLS] our fem simulation confirmed the enhancement effects of aunps B-nanoparticle to electroporation . [SEP]
[CLS] the electrical potential distribution is plotted by colorful contours while the electric field lines through buffer , the gold B-nanoparticle nanoparticle I-nanoparticle , and the cell B-material are shown by blue lines in figure 3 . [SEP]
[CLS] because of the high conductivity , the electric field is clearly focused near the gold B-nanoparticle nanoparticle I-nanoparticle . [SEP]
[CLS] such localized focusing effect could also help attract charged dna molecules from the surrounding area towards the focusing spot and enrich them there . [SEP]
[CLS] as transient pores will form later at the same location , we also compared the total current passing through the pore with and without a gold B-nanoparticle nanoparticle I-nanoparticle around . [SEP]
[CLS] it was found that the current was enhanced 34 % ( from 1 . 77 na to 2 . 38 na ) when a gold B-nanoparticle nanoparticle I-nanoparticle was around ( figure 3c ) . [SEP]
[CLS] this suggests that the charged dna plasmids could transport faster with aunps B-nanoparticle in close proximity and more of them could be delivered into cells B-material before the resealing of the cell B-material membrane . [SEP]
[CLS] as the presence of aunps B-nanoparticle greatly affect the actual pulse strength on treated cells B-material , their size and number presented around each cell B-material are critical to the electric pulse strength focusing level and the resulting electroporation performance ( the transfection B-property efficiency I-property and cell B-property viability I-property ) . [SEP]
[CLS] in the following sections , we evaluated the dependence of the pulse strength focusing effect on the size and concentration of aunps B-nanoparticle . [SEP]
[CLS] aunps B-nanoparticle of various concentrations ( 0 . 1x - 10x of the stock solution ) were used to evaluate the pulse strength focusing effect using the btx electroporator . [SEP]
[CLS] similar to the aforementioned results , when electroporating cells B-material at their standard pulse conditions ( 625v / cm , single 10ms pulse ) , the transgene expression enhancement generally sacrificed some of the cell B-property viability I-property with the increase of the aunps B-nanoparticle concentration ( figure 4a ) . [SEP]
[CLS] after adding 5 nm aunps B-nanoparticle at a concentration of 0 . 1x - 1x ( 1x = 0 . 1mg / ml aunps B-nanoparticle ) , the cell B-property viability I-property retained at the same level ( 71 . 9±5 . 1 % −68 . 9±2 . 9 % ) as in electroporation with naked dna . [SEP]
[CLS] but that value started dropping gradually when more concentrated aunps B-nanoparticle solutions ( 2 . 5x - 10x ) were used . [SEP]
[CLS] such cell B-property viability I-property loss endorsed the field enhancing effect of free aunps B-nanoparticle mentioned earlier . [SEP]
[CLS] because the buffer resistance was reduced when adding aunps B-nanoparticle , the local pulse strength on cells B-material was focused . [SEP]
[CLS] when starting from the standard pulse conditions , some treated cells B-material were over - perturbed to lethal levels . [SEP]
[CLS] when increasing the concentration of aunps B-nanoparticle , this pulse focusing effect got continuously enhanced and more cells B-material were over - polarized or died . [SEP]
[CLS] as the consequence , the cell B-property viability I-property dropped . [SEP]
[CLS] a threshold concentration of aunps B-nanoparticle existed for this pulse - focusing effect : it did not become obvious until the number of aunps B-nanoparticle reaches a certain level ( e . g . , 1x , 7 . 91 × 10 13 particles / ml for 5 nm aunps B-nanoparticle ) . [SEP]
[CLS] further enhancing the pulse strength focusing effect led to loss of cell B-property viability I-property , but beneficial to the improvement of the transfection B-property efficiency I-property . [SEP]
[CLS] for 5 nm aunps B-nanoparticle , the transfection B-property efficiency I-property increased from ~ 25 % ( bulk electroporation with dna only ) to the maximum enhancement of ~ 51 % at the concentration of 5x ( 0 . 5 mg / ml or ~ 3 . 96 × 10 14 5 nm aunps B-nanoparticle / ml ) when increasing aunps B-nanoparticle concentration and started decaying afterwards due to the significant loss on the cell B-property viability I-property ( figure 4b ) . [SEP]
[CLS] as the electric pulses were generally over - focused when concentrated aunps B-nanoparticle were introduced at standard electroporation protocols , such enhancement might be more beneficial when more benign conditions are used ( at these conditions , transfection with naked dna along is less effective as the consequence ) . [SEP]
[CLS] at these conditions , the cell B-property viability I-property is certainly high and the enhancement is mainly contributed to the improvement on the transfection B-property efficiency I-property of molecular probes . [SEP]
[CLS] during our investigation , to ensure the local pulse strength was still effective for the majority of cells B-material , we lowered the overall electric voltage ( while keeping the pulse duration unchanged ) to a minimum field strength that was just enough to transfect a statistically meaningful number of cells B-material with dna probes alone . [SEP]
[CLS] for k562 cells B-material , this minimum condition was 475 v / cm ( 95 v when tested with 2 mm btx cuvettes ) with a 13 . 6±1 . 5 % transfection B-property efficiency I-property of naked dna using the btx electroporator . [SEP]
[CLS] as shown in figure 4c , for all three sizes of aunps B-nanoparticle , similar cell B-property viability I-property ( ±10 % ) was achieved within a broad concentration range of aunps B-nanoparticle ( 0 . 1x - 10x ) . [SEP]
[CLS] different from the enhancement performance at standard conditions , continuous increase on the transfection B-property efficiency I-property was achieved for all cases ( figure 4d ) . [SEP]
[CLS] such improvements were not only significant when compared to that using naked dna ( i . e . , ~ 14 % ) at the same pulse condition , but also much better than the best performance the btx electroporator could achieve with naked dna ( i . e . , ~ 25 % ) . [SEP]
[CLS] this additional gain on the transfection B-property efficiency I-property at benign electroporation conditions further confirmed from another viewpoint the focusing effect of free aunps B-nanoparticle - - low - voltage pulses could be focused to high enough levels to provide the needed transmembrane field strength for better transfection B-property efficiency I-property ( 40 . 0±4 . 1 % for 5 nm , 45 . 2±4 . 0 % for 10 nm , 56 . 1±3 . 3 % for 20 nm ) . [SEP]
[CLS] more important , such delivery enhancement was attained with no or little sacrifice of the cell B-property viability I-property for the application of low - voltage pulses . [SEP]
[CLS] besides the concentration effect , the size of aunps B-nanoparticle also contributes to the reduction of the buffer resistance and the pulse strength focusing level on cells B-material . [SEP]
[CLS] moreover , the size of aunps B-nanoparticle could affect the poration area on the cell B-material membrane if aunps B-nanoparticle are brought close enough . [SEP]
[CLS] as shown in figures 1b and 3b , aunps B-nanoparticle converge the electric field on their two poles and when hang around cells B-material . [SEP]
[CLS] they work as many tiny virtual electrodes with focused pulses pointing towards the cell B-material membrane . [SEP]
[CLS] this could induce the polarization on the cell B-material membrane within limited area ( i . e . , localized poration ) , which has been found to be beneficial for the electroporation performance [SEP]
[CLS] therefore , the electroporation enhancement with various sizes of aunps B-nanoparticle reflects a combination of the pulse strength focusing effect and localized electroporation benefits . [SEP]
[CLS] three different sizes of aunps B-nanoparticle ( 5 nm , 10 nm , and 20 nm ) were tested here and their effects on the transfection B-property efficiency I-property and the cell B-property viability I-property are included in figure 4 . [SEP]
[CLS] similar to aunps B-nanoparticle of 5 nm , aunps B-nanoparticle of 10 nm and 20 nm exhibited similar concentration dependence on the transfection B-property efficiency I-property and the cell B-property viability I-property . [SEP]
[CLS] as the concentration of aunps B-nanoparticle in figure 4 were calculated based on the weight percentage of added aunps B-nanoparticle , their buffer resistance reduction effect or the pulse strength focusing level should be similar when the particle concentration is constant . [SEP]
[CLS] in other words , at the same concentration of aunps B-nanoparticle , the enhancement difference for cases in figure 4 reflected mainly the contribution of various particle sizes to the localized electroporation benefit . [SEP]
[CLS] the enhancement difference on the transfection B-property efficiency I-property among various sizes of aunps B-nanoparticle was marginal at low aunps B-nanoparticle concentrations and became significant only when concentrated aunps B-nanoparticle were used . [SEP]
[CLS] for 625 v / cm pulses , this started from 1x aunps B-nanoparticle , and for 475v / cm pulses , it was from 2 . 5x due to their different pulse strength focusing levels and localized electroporation situations . [SEP]
[CLS] this is reasonable as the pulse strength focusing effect was weak at low aunps B-nanoparticle concentrations . [SEP]
[CLS] similarly , localized electroporation was very limited at low aunps B-nanoparticle concentrations , as only a small portion of total free aunps B-nanoparticle could aggregate around cells B-material . [SEP]
[CLS] in excess of the threshold aunps B-nanoparticle concentration , their contributions on the field focusing effect and localized poration became more pronounced so that the benefit on the transfection B-property efficiency I-property improvement showed up . [SEP]
[CLS] the larger the size of aunps B-nanoparticle , the better transfection B-property efficiency I-property was achieved . [SEP]
[CLS] large aunps B-nanoparticle of a relative lower concentration could also help gain better transfection B-property efficiency I-property than small aunps B-nanoparticle at a higher concentration . [SEP]
[CLS] for example , electroporation with 2 . 5x and 5x aunps B-nanoparticle of 20 nm help achieve similar or even better pgfp transfection than 5x and 7 . 5x aunps B-nanoparticle of 5 nm , respectively ( figures 4b & 4d ) . [SEP]
[CLS] among various particle sizes , the cell B-property viability I-property difference at the same pulse strength focusing level ( i . e . , the same aunps B-nanoparticle concentration ) was marginal in most cases . [SEP]
[CLS] therefore , these aunps B-nanoparticle ( 5 to 20 nm ) are appropriate for the electroporation enhancement without extra addition to the cell B-material toxicity B-property . [SEP]
[CLS] however , these results also suggested that , with free aunps B-nanoparticle ( i . e . , aunps B-nanoparticle that are randomly dispersed in the electroporation buffer ) , the electroporation enhancement was mainly decided by the pulse strength focusing effect or the concentration of aunps B-nanoparticle . [SEP]
[CLS] localized poration only became beneficial at high aunps B-nanoparticle concentration when sufficient aunps B-nanoparticle presented around cells B-material during electroporation . [SEP]
[CLS] but this easily leads to over - perturbation if added aunps B-nanoparticle are all free aunps B-nanoparticle . [SEP]
[CLS] to further enhance the localized poration effect , sufficient number of aunps B-nanoparticle must be brought close to cells B-material through some pre - concentration approaches . [SEP]
[CLS] as many transferrin receptors ( tfr ) are available on the cell B-material membrane of k562 cells B-material , aunps B-nanoparticle were conveniently brought to cells B-material by grafting transferrin ( tf ) molecules on their surface . [SEP]
[CLS] this was done with the help of the high affinity of sulfhydryl groups to the gold B-material surface . [SEP]
[CLS] specifically , sulfhydryl groups were introduced to transferrin molecules by converting a small proportion of their primary B-material amine I-material groups to sulfhydryl groups with traut ' s reagent . [SEP]
[CLS] the modified transferrin with sulfhrdryl groups were then incubated B-technique with free aunps B-nanoparticle to form transferrin aunps B-nanoparticle ( tf - aunps B-nanoparticle ) , as shown in figure 5a . [SEP]
[CLS] to evaluate how this transferrin - targeting mechanism affected the localized electroporation , we incubated B-technique tf - aunps B-nanoparticle of 1x with k562 cells B-material for various incubation B-technique times and compared their performance on transfection enhancement . [SEP]
[CLS] as shown in figure 5b , the best improvement occurred in samples having 4 - hr incubation B-technique time and the transfection B-property efficiency I-property reached 41 . 7±3 . 2 % when compared to that of btx with naked dna ( 26 . 4±1 . 9 % ) and btx with free aunps B-nanoparticle ( 34 . 4±2 . 9 % ) . [SEP]
[CLS] such 50 % or less enhanced performance resulted from the fact that the gradual depletion of mobile B-property aunps B-nanoparticle in the electroporation buffer because of tf - aunps B-nanoparticle grafting on the cell B-material membrane . [SEP]
[CLS] as the consequence , though localized electroporation got improved , the pulse focusing effect from free aunps B-nanoparticle diminished . [SEP]
[CLS] to retain both the pulse strength focusing and localized electroporation advantages , we added free aunps B-nanoparticle to tf - aunps B-nanoparticle at various mixing ratios ( 0 % , 25 % , 50 % , 75 % , and 100 % tf - aunps B-nanoparticle ) . [SEP]
[CLS] based on other pioneering work on tranferrin targeting , it took about 4 hr incubation B-technique to accomplish complete affinity binding of transferrin to the tfrs on cells B-material [SEP]
[CLS] therefore , we first incubated B-technique k562 cells B-material with tf - aunps B-nanoparticle for 4 hr and then added the needed quantity of free aunps B-nanoparticle right before electrporaiton . [SEP]
[CLS] as shown in figure 6a , such a combination showed better enhancement on the transfection B-property efficiency I-property under the standard electroporation conditions with only 1x total aunps B-nanoparticle ( free aunps B-nanoparticle + tf - aunps B-nanoparticle ) while the actual improvement varied with their mixing ratio : a sustained increase was seen on the transfection B-property efficiency I-property when more tf - aunps B-nanoparticle were added until reaching a 50 % / 50 % mixture of free - aunps B-nanoparticle and tf - aunps B-nanoparticle ( the transfection B-property efficiency I-property reached 58 . 2±1 . 8 % ) , followed by some declines . [SEP]
[CLS] this provides ~ 2 . 5 folds increase on the dna tranfection when compared to electroporation with naked dna only . [SEP]
[CLS] considering the low concentration of aunps B-nanoparticle used here ( only 1x ) , the electroporation enhancement with a combination of free aunps B-nanoparticle and tf - aunps B-nanoparticle seems more effective than that using free aunps B-nanoparticle or tf - aunps B-nanoparticle alone . [SEP]
[CLS] the best enhancement came from an appropriate balance on the pulse strength focusing and localized electroporation advantages aunps B-nanoparticle offered . [SEP]
[CLS] it is worth to mention that such a transfection B-property efficiency I-property improvement was achieved without sacrificing much of the cell B-property viability I-property ( figure 6a ) . [SEP]
[CLS] at more benign conditions ( 475v / cm ) , the enhancement was not very obvious , consistent with the free aunps B-nanoparticle enhancement result at low concentrations ( 0 . 1x - 1x ) . [SEP]
[CLS] this insufficient pulse focusing level cannot provide desired transmembrane potential to polarize the majority of cells B-material . [SEP]
[CLS] when more aunps B-nanoparticle were introduced ( e . g . , 5x for the total aunps B-nanoparticle concentration ) , the enhancement on the transfection B-property efficiency I-property became significant for pulses of both 625 v / cm and 475 v / cm and the equivalent mixture of free aunps B-nanoparticle and tf - aunps B-nanoparticle still offered the best transfection B-property efficiency I-property ( figure 6b ) . [SEP]
[CLS] however , because of the overfocusing effect , obvious loss of the cell B-property viability I-property was found for the case with the pulse strength of 625 v / cm , consistent with our early observations . [SEP]
[CLS] we tracked the cellular uptake of aunps B-nanoparticle before and after electroporation for both free , naked aunps B-nanoparticle and tf - aunps B-nanoparticle using confocal microscope and the results were shown in figure 7 . [SEP]
[CLS] as free aunps B-nanoparticle were mixed with cells B-material right before electroporation , the short contact time did not provide enough time to allow endocytosis - based uptake of aunps B-nanoparticle and no obvious fluorescently B-property labeled aunps B-nanoparticle ( fnps ) were observed ( figure 7a ) . [SEP]
[CLS] after electroporation , many fnps were clearly found in cell B-material cytoplasm ( figure 7b ) , indicating that aunps B-nanoparticle transported into cells B-material after electroporation . [SEP]
[CLS] as all samples were fixed shortly after electroporation , we believe the majority of fnps were taken through the transient openings on the cell B-material membrane , not the endocytosis B-event process . [SEP]
[CLS] as mentioned in our fem simulation , aunps B-nanoparticle around transient pores could enhance the cellular uptake of dna plasmids because the increase of electrical current ( see section 3 . 1 ) . [SEP]
[CLS] aunp B-nanoparticle imaging and tracking in vitros 7c and 7d showed the distributions of tf - fnps before and after electroporation ( note : tf - fnps alone , not a mixture with free fnps were used here ) . [SEP]
[CLS] the accumulation of tf - fnps on the cell B-material membrane was clearly found in samples without electroporation treatment ( figure 7c ) , confirming the formation of ligand - receptor B-material bonds after incubation B-technique . [SEP]
[CLS] after electroporation , fnps were also found in the cell B-material cytoplasm ( figure 7d ) , in consistent with what exhibited in free fnps electroporation . [SEP]
[CLS] but unlike in free fnps electroporation sample , many tf - fnps were also found on the cell B-material membrane or regions nearby , suggesting that at least some tf - aunps B-nanoparticle conjugates and the coupling of tf - tfr could survive the electroporation process . [SEP]
[CLS] aunps B-nanoparticle were used to enhance in vitro delivery of dna probes for both batch - type and flowthrough electroporation systems . [SEP]
[CLS] highly conductive free aunps B-nanoparticle were added to the electroporation buffer to reduce the solution resistance so that the pulse strength on cells B-material could be enhanced . [SEP]
[CLS] tf - aunps B-nanoparticle were brought to k562 cells B-material through affinity binding with tfr receptors on the cell B-material membrane , serving as many virtual microelectrodes to locally polarize cells B-material from various sites , each affected only limited area . [SEP]
[CLS] in this way , electroporation was enhanced with better transfection B-property efficiency I-property and the same or higher cell B-property viability I-property . [SEP]
[CLS] with dna plasmids carrying a wizgfp reporter gene , we confirmed the pulse strength focusing effect after adding free aunps B-nanoparticle in the electroporation buffer and investigated its dependence on the particle size , concentration , and electroporation conditions . [SEP]
[CLS] we also evidenced the contributions of localized electroporation with tf - aunps B-nanoparticle . [SEP]
[CLS] an equivalent mixture of free aunps B-nanoparticle and tf - aunps B-nanoparticle was found to provide the best enhancement performance while the optimal concentration of aunps B-nanoparticle was decided by the original pulse conditions . [SEP]
[CLS] this study offers a new approach to improve the delivery efficiency of nucleic B-material acids I-material or anticancer B-property drugs through the combination of nanoparticles B-nanoparticle and electroporation technologies . [SEP]
[CLS] aunps B-nanoparticle were adopted here for their low cost and easy accessibility while other forms of gold B-material nanostructures , such as nanorod B-nanoparticle , nanoshell B-nanoparticle , or nanowires B-nanoparticle , in principle , could be used for similar purposes . [SEP]
[CLS] as these gold B-material nanomaterials B-material have been widely explored in sensing , imaging , diagnosis , and therapeutic applications , our approach demonstrates a new function of these nanomaterials B-material and / or broadens their potentials for multiple - function applications in drug discovery and clinical practice . [SEP]
[CLS] gold B-nanoparticle nanoparticles I-nanoparticle enhancement on electroporation of k562 cells B-material with a commercial batch electroporation ( labeled as " btx " ) system and a home - made semi - continuous microchannel system ( labeled as " microchannel " ) . [SEP]
[CLS] panel dependence of the pulse enhancement on the size and concentration of free aunps B-nanoparticle : panels ( a - b ) are the cell B-property viability I-property ( a ) and the transfection B-property efficiency I-property ( b ) with an overall pulse strength of 625 v / cm and panels ( c - d ) are the results with an overall pulse strength of 475 v / cm ( panel c : the cell B-property viability I-property ; panel d : the transfection B-property efficiency I-property ) . [SEP]
[CLS] the blue and red dash lines refer to the cell B-property viability I-property and the transfection B-property efficiency I-property of electroporation with naked dna at the optimal conditions ( 675 v / cm , single pulse of 10 ms ) , respectively . [SEP]
[CLS] 1x aunps B-nanoparticle refers to 0 . 01 wt % or 0 . 1 mg / ml gold B-material content . [SEP]
[CLS] the combined enhancement of the pulse strength focusing and localized electroporation effects using a mixture of free aunps B-nanoparticle and tf - aunps B-nanoparticle of various mixing ratios with a total aunps B-nanoparticle concentration of 1x ( a ) and 5x ( b ) with the pulse strength of 625 v / cm and 475 v / cm . [SEP]
[CLS] aunps B-nanoparticle of 20nm were used here . [SEP]
[CLS] 1 . schematic illustration on the mechanism of aunps B-nanoparticle enhancement on electroporation : ( a ) the pulse enhancement effect through minimizing the electric voltage consumed by the low conductive electroporation buffer during electroporation . [SEP]
[CLS] by adding highly conductive aunps B-nanoparticle , more percentage of the overall electric voltage across the two electrodes is allocated on cells B-material to have focused pulses when compared to the use of electroporation buffer alone ; ( b ) localized electroporation when aunps B-nanoparticle are brought to the cell B-material membrane through affinity binding B-event with I-event receptors I-event there . [SEP]
[CLS] the electric field is converged on the conductive aunps B-nanoparticle and these aunps B-nanoparticle could serve as virtual electrodes to polarize only limited area on the cell B-material membrane when stay nearby . [SEP]
[CLS] ( a ) exhibits fluorescence B-property and phase contrast microscopic images of pgfp plasmid transfection through btx , btx with aunps B-nanoparticle , microchannel , and microchannel with aunps B-nanoparticle . [SEP]
[CLS] panels ( b ) and ( c ) are quantitative results of the transfection B-property efficiency I-property ( b ) and the cell B-property viability I-property ( c ) . [SEP]
[CLS] the concentration of aunps B-nanoparticle used here is 5x or 0 . 05 wt % ( 0 . 5 mg / ml ) . n = 6 and ( * * * ) represents p < 0 . 005 . [SEP]
[CLS] 3 . simulation of the electric field focusing effect of aunps B-nanoparticle in electroporation . [SEP]
[CLS] ( a ) the model and meshes setup for one gold B-nanoparticle nanoparticle I-nanoparticle embedded in the membrane of a k562 cell B-material . [SEP]
[CLS] ( b ) the calculated electric field lines around the aunp B-nanoparticle . [SEP]
[CLS] ( c ) the electric field around a transient pore on the cell B-material membrane at the presence of one aunp B-nanoparticle around . [SEP]
[CLS] 5 . localized electroporation enhancement with tf - aunps B-nanoparticle : ( a ) schematics of grafting tf - aunps B-nanoparticle as virtual electrodes on the cell B-material membrane , ( b ) the localized enhancement with tf - aunps B-nanoparticle alone at various binding stages . [SEP]
[CLS] the aunps B-nanoparticle used here are 20 nm with 1x concentration ( 0 . 1 mg / ml ) . [SEP]
[CLS] the confocal microscope images of the cellular uptake of aunps B-nanoparticle before and after electroporation : ( a - b ) for free fnps and ( c - d ) for tf - fnps . [SEP]
[CLS] images in panels ( a ) and ( c ) were taken before electroporation and panels ( b ) and ( d ) were after electroporation . [SEP]
[CLS] fnps of 10x or 0 . 1 wt % ( 1 . 0 mg / ml ) were used in all samples and tf - fnps were incubated B-technique with cells B-material for 4 hr . [SEP]
[CLS] a new strategy for synthesizing spherical nucleic B-material acid I-material ( sna ) nanostructures from biodegradable B-property dna block copolymers is reported . [SEP]
[CLS] multiple dna strands are grafted to one end of a polyester chain ( poly - caprolactone ) to generate an amphiphilic B-property dna brush block copolymer ( dbbc ) structure capable of assembling into spherical micelles B-material in aqueous solution . [SEP]
[CLS] these novel dbbcbased micelle - snas exhibit a higher surface density of nucleic B-material acids I-material compared to micelle B-material [SEP]
[CLS] spherical nucleic B-material acids I-material ( snas ) are an emerging class of nanostructure , typically consisting of a nanoparticle B-nanoparticle core B-material densely functionalized with a nucleic B-material acid I-material shell B-material . [SEP]
[CLS] these structures exhibit a wide variety of novel properties that are substantively different from their linear nucleic B-material acid I-material counterparts , making them especially attractive for intracellular applications . [SEP]
[CLS] specifically , they are recognized by class a scavenger receptors and naturally internalized by many cell B-material types via caveolin - mediated endocytosis B-event . [SEP]
[CLS] they have been used as novel probes for measuring intracellular genetic content and as potent gene regulation agents . [SEP]
[CLS] they are especially attractive as gene regulation materials since they do not need ancillary transfection agents such as viruses , peptides B-material , lipids B-material or cationic B-material polymers B-material to cross the cell B-material membrane . [SEP]
[CLS] thus far , snas have been made with a variety of core B-material materials , including many inorganic compositions and several polymer B-material compositions , and there are now a few examples of hollow sna structures that are held together by cross - linked dna or silica B-material shells I-material . [SEP]
[CLS] although the nanoparticle B-nanoparticle core B-material typically plays little role in determining the general chemical and biological properties of the corresponding snas , it can be a major concern when such structures are being considered as therapeutic candidates . [SEP]
[CLS] indeed , a significant synthetic challenge for the chemistry community is to design totally biocompatible B-property and biodegradable B-property snas that exhibit the hallmark properties of conventional snas with inorganic B-material cores I-material . [SEP]
[CLS] one approach has been to use liposomal B-nanoparticle architectures as cores B-material , but an alternative approach that may provide even greater tailorability is the use of polymer B-material micelle B-material architectures . [SEP]
[CLS] although a variety of nucleic acid polymer micelle B-material architectures have been reported , none have been developed and optimized with regard to general sna features and intracellular biological activity . indeed , a few studies involving polymer B-material micelle B-material structures for intracellular gene regulation have been undertaken , but they either required a cationic B-material transfection agent ( e . g . polyethyleneimine ) for transfection or were constructed using non - degradable polymers B-material . [SEP]
[CLS] to date , there are no biodegradable B-property dna block copolymer ( dbc ) based micelle B-material structures that have been shown to exhibit the natural cellular uptake properties of snas , and therefore the use of such structures with respect to intracellular and therapeutic applications has been limited . [SEP]
[CLS] importantly , sna - like properties are directly related to the orientation and high density of nucleic B-material acids I-material on the nanoparticle B-nanoparticle surface . [SEP]
[CLS] to overcome the problem of low cell B-material internalization associated with dna block copolymer micelle B-material structures , we identified the need to synthesize micelle B-material snas with a dense layer of nucleic B-material acids I-material on the polymer B-material core B-material surface . [SEP]
[CLS] herein , we describe a strategy for preparing dbc micelle - snas with a biodegradable B-property core B-material and a dense layer of highly oriented oligonucleotides projecting from the surface of the aforementioned core B-material . [SEP]
[CLS] traditionally , approaches to synthesizing dbc micelle B-material architectures have involved the linear coupling of nucleic B-material acids I-material to hydrophobic B-property polymer B-material blocks ( scheme 1a ) . [SEP]
[CLS] we have found this approach does not reliably yield structures with a nucleic B-material acid I-material density high enough and suitable for optimal sna cellular transfection capabilities ( vide infra ) . therefore , our synthetic approach relies on the generation of a dna - brush block copolymer ( dbbc ) based micelle B-material structure ( scheme 1b ) , which was pioneered by gianneschi et al . in the context of non - degradable constructs for materials assembly purposes . [SEP]
[CLS] in our synthetic approach , the key components of the dbbc micelle B-material structures are prepared by grafting multiple dna strands onto the terminal segment of a diblock copolymer consisting of polycaprolactone ( pcl ; pcl is a component in fda - approved therapeutics ) and azide - modified pcl via copperfree click chemistry to form a dbbc macromolecule . [SEP]
[CLS] then , when transferred from an organic solvent mixture consisting of 1 : 1 dmso / dmf into an aqueous solution , the assynthesized amphiphilic B-property dna - brush block copolymers assemble into ~ 40 nm diameter spherical micelles B-material ( scheme 1b ) . [SEP]
[CLS] for comparative purposes , we also prepared a similarly sized micelle B-material structure from a linear dbc component ( scheme 1a ) . [SEP]
[CLS] in a typical micelle - sna synthesis ( figure s1 ) , α - chloro - ε - caprolactone ( α - cl - εcl ) prepared by the baeyer - villager oxidation of α - chlorocyclohexanone was employed as a monomer B-material for polymerization . [SEP]
[CLS] the diblock copolymer poly ( α - cl - εcl - b - εcl ) was synthesized from the monomer B-material , α - cl - εcl , and commercially available polycaprolactone ( m w = ~ 14 kda ) as the macro - initiator ( see supporting information ) . [SEP]
[CLS] the pendant chlorides B-material of the as - synthesized poly ( α - cl - εcl - b - εcl ) were converted into azides by treatment with sodium B-material azide in dmf overnight at room temperature . [SEP]
[CLS] 1 h nmr spectroscopy B-technique was used to characterize the product , and the spectrum suggested the as - synthesized diblock copolymer contains on average 15 n 3 groups , as determined by integration ( peak a and g in figure s2 ) . [SEP]
[CLS] in addition , ft - ir spectroscopy B-technique of the azide - containing pcl diblock copolymer showed the characteristic azide band at 2106 cm −1 ( n = n = n stretch , figure s3 ) . [SEP]
[CLS] gel permeation chromatography B-technique ( gpc ) analysis is also consistent with block copolymer formation as evidenced by an increase in molecular weight from ~ 14 to ~ 16 kda ( figure s4 ) . [SEP]
[CLS] when substituted with azide groups , the poly ( α - n 3 - εcl - b - εcl ) exhibits a slightly shorter retention time than that of poly ( α - cl - εcl - b - εcl ) in the gpc analysis . [SEP]
[CLS] finally , poly ( α - n 3 - εcl - b - εcl ) and an excess of cyclooctyne - terminated dna strands were added to an organic solvent mixture ( 1 : 1 dmso : dmf ) to initiate the copper - free click reaction of dna strands onto the azide - modified block , resulting in the formation of the dna - g - pclb - pcl macromolecule . [SEP]
[CLS] for comparative purposes , a linear dbc was also synthesized ( figure s1 ) . [SEP]
[CLS] instead of using 14k pcl as the macro - initiator , an azide - terminated poly ( ethylene oxide B-material ) with 4 ethylene glycol units was employed to initiate the caprolactone polymerization to synthesize a peo - b - pcl block copolymer with a comparable molecular weight ( ~ 18 kda based on 1 h nmr integration , figure s2 ) . [SEP]
[CLS] again , click chemistry was used to conjugate dna to the as - synthesized peo - b - pcl block copolymer and generate the linear dna - b - peo - b - pcl block copolymer ( figure s2 ) . [SEP]
[CLS] after the dna conjugation reaction , both the dna brush block copolymer micelle B-material - snas ( dbbc - snas ) and the linear dna block copolymer micelle B-material - snas ( ldbc - snas ) were prepared and purified according to literature protocols used for analogous linear non - biodegradeable B-property structures . [SEP]
[CLS] in this process , excess dna was removed by an ultrafiltration device with a mwco of 50 kda membrane . [SEP]
[CLS] the amount of dna conjugated to the polymer B-material was determined by measuring the absorbance of the micelle - sna solutions at 260 nm . [SEP]
[CLS] based on the total amount of polymer B-material used for conjugation , it was determined that an average of 10 dna strands were successfully conjugated onto each polymer B-material chain . [SEP]
[CLS] moreover , polyacrylamide B-technique gel I-technique electrophoresis I-technique , under denaturing conditions , was used to analyze the resulting dna - g - pclb - pcl macromolecules . [SEP]
[CLS] as shown in figure s5 , the bands representing dbbc conjugates with multiple dna strands can be clearly identified on the gel when excess dna was used for the conjugation . [SEP]
[CLS] with purified samples , multiple techniques were employed to characterize these two polycaprolactone - based snas ( figure 1 ) . [SEP]
[CLS] gel B-technique electrophoresis I-technique ( 1 % agarose gel , figure 1a ) showed a single major band for each sample on the gel image , indicating the micelle - snas have a narrow size distribution . [SEP]
[CLS] compared to unmodified dna of the same sequence , the electrophoretic B-property mobilities I-property of the micelle - snas are greatly decreased , consistent with the significant difference in size and overall charge . [SEP]
[CLS] notably , the electrophoretic B-property mobility I-property of sna made from the brush architecture is slightly lower than that of one made from the linear structure , indicating that the former sna is slightly larger . [SEP]
[CLS] the size distributions of the micelle - snas were also probed by dynamic B-technique light I-technique scattering I-technique ( dls ) , which indicated an average hydrodynamic diameter of ~ 44 nm for the snas derived from the brush architecture and ~ 40 nm for ones derived from the linear architecture ( figure 1b ) , consistent with the gel analysis . [SEP]
[CLS] meanwhile , zeta B-property potential I-property analysis gave values of −48 . 5 ± 3 . 7 mv and −27 . 9 ± 3 . 2 mv , respectively ( figure s6 ) , consistent with the micelle B-material derived from the brush architecture having a higher density of dna . [SEP]
[CLS] furthermore , the micelle B-material - snas were cast on mica and imaged by atomic B-technique force I-technique microscopy I-technique in dry form ( figures s7 ) ; in both cases , spherical structures were readily apparent . [SEP]
[CLS] to visualize the morphology of the micelle - snas in an environment closer to the one in which they are prepared , cryogenic transmission B-technique electron I-technique microscopy I-technique ( cryo - tem ) was used ( figure 1d ) . [SEP]
[CLS] in this method , a very thin layer of micelle - sna - containing solution was quickly frozen and directly imaged . [SEP]
[CLS] the cryo - tem imaging avoids dehydration - induced artifacts , allowing the fully intact nanoparticles B-nanoparticle to be visualized . [SEP]
[CLS] indeed , round 30 - 50 nm diameter particles of both dbbc - snas and ldbc - snas were observed , which are consistent with the dls size data . [SEP]
[CLS] since the density of dna on the particle surface is a key factor that leads to its sna - like properties , it is important to determine nucleic B-material acid I-material surface coverage . [SEP]
[CLS] in contrast with conventional aunp B-nanoparticle - snas , where particle concentration can be easily determined by measuring the plasmon resonance peak associated with aunps B-nanoparticle , it is difficult to directly determine the micelle B-material particle concentration using spectrophotometric methods . [SEP]
[CLS] alternatively , we used nanoparticle B-nanoparticle tracking analysis ( nta ) to determine the particle concentrations for micelle - snas ( see details in supplementary information , figure s8 and table s2 ) . [SEP]
[CLS] importantly , the dna loading for the brush block copolymer based micelle B-material - snas ( 302 strands / particle ; 22 . 2 pmol / cm 2 ) was significantly higher than the linear block copolymer based micelle B-material - sna ( 190 strands / particle ; 15 . 6 pmol / cm 2 ) , which is comparable to aunp B-nanoparticle - based snas of similar sizes ( 300 strands / particle for 30 nm aunp B-nanoparticle cores B-material ; corresponding to a dna density of 17 . 6 pmol / cm 2 ) . [SEP]
[CLS] another important feature of sna structures is their cooperative hybridization properties , arising from the association of multiple dna strands between each particle . [SEP]
[CLS] this results in a sharp melting transition during the thermal denaturation of hybridized complementary particles . [SEP]
[CLS] generally , higher dna loading results in higher melting temperatures and sharper transitions under comparable conditions . [SEP]
[CLS] the thermal denaturation of micelle B-material - snas was carried out by hybridizing both brush and linear block copolymer based micelle B-material - snas with aunp B-nanoparticle - snas containing complementary sticky ends , respectively , and then monitoring the absorbance change at 260 nm with gradual heating ( figure 1c and figure s9 ) . [SEP]
[CLS] around the melting temperature , extinction dramatically increased due to cooperative dna dehybridization . [SEP]
[CLS] as shown in figure 1c , the melting curves for both types of micelle - snas when hybridized with aunp B-nanoparticle - snas were sharp and elevated , with the tm of the dbbc - based micelle B-material - sna ( 39 . 5 °c ) being almost 3 degrees higher than the linear dbc - based micelle B-material - sna ( 36 . 9 °c ) . [SEP]
[CLS] the higher melting temperature for the dbbc - based micelle B-material - sna is consistent with a structure with a higher surface density of nucleic B-material acids I-material . [SEP]
[CLS] with well - characterized micelle B-material - snas in hand , we evaluated their biological function with regard to in vitro cell B-material uptake studies ( figure 2 ) . [SEP]
[CLS] both micelle - snas exhibit the ability to enter cells B-material without the assistance of cationic B-material transfection reagents , but with different efficiencies . [SEP]
[CLS] for example , confocal microscopy B-technique was employed to reveal that fluoresceinlabelled micelle - snas ( 1 μm total dna ) enter hela cells B-material after 16 h of incubation B-technique . [SEP]
[CLS] notably , the green fluorescence B-property from micelle B-material - snas consisting of brush block copolymers was significantly more intense than that of the linear block copolymer based micelle B-material - snas ( figure 2d , e ) , indicating more efficient uptake of the former as opposed to the latter . [SEP]
[CLS] this result was also confirmed by quantifying the fluorescence B-property intensity of the cell B-material population using flow B-technique cytometry I-technique . [SEP]
[CLS] although incubated B-technique with equal amounts of total dna , the mean fluorescence B-property intensity of cells B-material treated with brush block copolymer based micelle B-material - snas was almost twice that of brush block copolymer based micelle B-material - snas ( figure 2f ) , presumably due to their higher surface density of nucleic B-material acids I-material [SEP]
[CLS] compared to the positive control which utilized lipofectamine® 2000 as the transfection agent , dbbc - snas show slightly lower but comparable transfection B-property efficiency I-property without any co - carrier . [SEP]
[CLS] we further studied the intracellular location of dbbc - snas as a function of incubation B-technique time under conditions where hela cells B-material were continuously incubated B-technique with fluorescein - labelled micelle B-material - snas ( 1 μm total dna ) . [SEP]
[CLS] sna - aunp B-nanoparticle conjugates traffic through the endocytic pathway into late endosomes and reside there without accumulating in lysosomes . [SEP]
[CLS] it is hypothesized that the dbbc - snas follow the same route upon cellular entry due to their similar architecture . in these experiments , the dbbc - snas carry a green fluorophore ( fluorescein ) , and the cells B-material are stained with a complementary alexa dye - labeled marker of interest ( red ) . [SEP]
[CLS] as shown in figure 3 , strong colocalization of snas with rab9 ( a protein B-material which preferentially localizes in late endosomes ) was observed after 6 h of incubation B-technique , and colocalization persisted 24 h post - incubation B-technique . [SEP]
[CLS] importantly , we did not observe appreciable colocalization between the fluorescent B-property signals of dbbc - snas and markers for lysosomes ( lamp - 1 ) over a 24 h period of time . [SEP]
[CLS] from these data , we conclude that snas primarily remain inside late endosomes and do not migrate beyond this point to the lysosome . [SEP]
[CLS] these observations confirm that within a typical cell B-material doubling time ( 23 - 24 h for hela ) , the micelle - snas likely employ the same intracellular trafficking pathway as aunp B-nanoparticle - based snas . [SEP]
[CLS] we further tested the intracellular gene regulation capacity of the dbbc - based micelle - snas in specialty c166 mouse endothelial cells B-material over - expressing enhanced green fluorescent B-property protein B-material ( egfp ) . [SEP]
[CLS] the dna strands on the micelle B-material - snas were designed as an antisense sequence against the egfp mrna . [SEP]
[CLS] after transfection and further incubation B-technique for 48 h , egfp knockdown is readily apparent by fluorescence B-technique microscopy I-technique ( figure 4a , b ) . [SEP]
[CLS] importantly , the quantification of egfp protein B-material expression via western blot shows a concomitant ~ 52 % knockdown ( figure 4c ) , consistent with the conclusion that the micelle - sna can effectively bind the cytosolic mrna target and alter the expression of its associated protein B-material . [SEP]
[CLS] compared to ldbc - snas at the same condition , dbbc - snas are more effective at regulating gene expression via the antisense mechanism ( figure 4d ) . [SEP]
[CLS] when the dbbc - based micelle - snas are modified with a scrambled dna sequence , egfp expression does not reduce at all concentrations tested ( 0 . 5 - 2 μm of total dna ) , indicating the gene knockdown effect is sequence - specific ( figure s10 ) . [SEP]
[CLS] to further validate that our new constructs may have significant potential for biomedical applications , we tested the biodegradation B-property and cellular toxicity B-property of dbbc - snas . [SEP]
[CLS] during the transfection and intracellular trafficking process , snas will encounter quite different chemical environments , such as varied ph values and the presence of different enzymes in serum , endosomes , and lysosomes . [SEP]
[CLS] this may cause the pcl core B-material of the micelle B-material particles to degrade at a different rate depending on their location . [SEP]
[CLS] to evaluate their biodegradability B-property , we incubated B-technique the dbbc - snas in buffers of varied ph values to mimic different intracellular environments . [SEP]
[CLS] as shown by agarose B-technique gel I-technique electrophoresis I-technique , component single - stranded dna appeared after 24 h incubation B-technique , indicating the degradation of micelle - snas ( figure 5a ) . [SEP]
[CLS] meanwhile , gel bands representing the dbbc - snas became smeared and diffused along increased incubation B-technique time ( figure 5b ) . [SEP]
[CLS] notably , micelle B-material - snas degraded faster in lower ph buffer conditions , pointing to the possibilities of their faster degradation when trafficking to intracellular vesicles with low ph , such as the endosomes . [SEP]
[CLS] the cellular toxicity B-property of dbbc - based micelle - snas was analyzed using the standard mtt B-technique assay I-technique ( figure 6 ) . [SEP]
[CLS] even with an incubation B-technique time of up to 4 days , we found that cell B-property viability I-property remained essentially unchanged . [SEP]
[CLS] this confirms that the as - synthesized micelle - snas with biocompatible B-property and biodegradable B-property polycaprolactone cores B-material do not have measurable cellular toxicity B-property under these conditions . [SEP]
[CLS] in conclusion , we have developed a novel strategy to construct a dna - brush block copolymer based micelle B-material - sna with increased dna surface density , which endows the new construct with properties similar to the well - established aunp B-nanoparticle - snas . [SEP]
[CLS] compared to the micelle B-material - snas constructed from linear block copolymers , the dbbc - based micelle B-material - snas exhibit a higher surface density of nucleic B-material acids I-material , a more negatively charged surface , a higher melting temperature , a cooperative melting profile , and more effective transfection agentfree cellular uptake . [SEP]
[CLS] importantly , the micelle B-material - snas derived from the dna - brush block copolymer show effective target gene knockdown in vitro . [SEP]
[CLS] it is worthwhile noting that since the polymer B-material core B-material can gradually degrade under physiological conditions due to acid - catalyzed or esterase - catalyzed cleavage of ester bonds , these constructs open new avenues towards the controllable , continuous release of nucleic B-material acids I-material as regulatory agents for intracellular biological processes . [SEP]
[CLS] generally , in addition to pcl , a wide variety of polyesters , such as pla and plga can be employed to initiate polymerization and then form dbbc - based snas consisting of different types of polyester cores B-material . [SEP]
[CLS] as such , the rate of particle degradation and nucleic B-material acid I-material release may be tuned by judicious monomer B-material selection . [SEP]
[CLS] we anticipate that this synthetic approach can be extended to a wide variety of polyesters to generate a new class of nanostructured B-material materials I-material with highly tailorable properties and efficacies for various nucleicacid based therapeutic approaches . [SEP]
[CLS] all dna synthesis was carried out on a bioautomation mm48 dna synthesizer according to the standard manufacturer trityl - on protocol . [SEP]
[CLS] all reagents for oligonucleotide synthesis were purchased from glen research ( sterling , va ) and used following manufacturer ' s instructions . [SEP]
[CLS] ac - dc and dmf - dg phosphoramidites were used to enable room - temperature deprotection of the nucleobases . [SEP]
[CLS] α - chlorocyclohexanone , sodium B-material azide , n , ndimethylformamide ( dmf ) , tin ( ii ) octanoate , caprolactone ( cl ) , and all other solvents were purchased from sigma - aldrich ( st . louis , mo ) and used without further purification . [SEP]
[CLS] mchloroperoxybenzoic acid ( mcpba ) was purchased from fluka ( buchs , switzerland ) . [SEP]
[CLS] the monomer B-material α - chloro - ε - caprolactone was prepared by the baeyer - villager oxidation of αchlorocyclohexanone using excess mcpba . [SEP]
[CLS] after purification , 0 . 5 g monomer B-material was mixed with 3 . 0 g commercially available polycaprolactone ( m w = ~ 14 kda , as macro - initiator ) in dry toluene ( 10 ml ) . [SEP]
[CLS] the mixture was dried by repeated ( 3× ) azeotropic distillation with toluene . [SEP]
[CLS] then , the solution was heated to 130 °c in a preheated oil bath and stirred under n 2 . [SEP]
[CLS] the reaction mixture was stirred for 5 min at 130 °c and then one drop of tin B-material ( ii ) octanoate was added via syringe . [SEP]
[CLS] the temperature was maintained at 130 °c for another 3 hours . [SEP]
[CLS] the resulting viscous mixture was rapidly cooled , upon which it solidified . [SEP]
[CLS] the crude polymer B-material was purified by repeated dissolution in dichloromethane and heptane precipitation ( 3× ) , resulting in a white solid after drying . [SEP]
[CLS] then the pendant chlorides B-material of the as - synthesized poly ( α - cl - εcl - b - εcl ) were converted into azides by treatment with sodium B-material azide in dmf overnight at room temperature . [SEP]
[CLS] after removal of dmf in vacuo , 15 ml of toluene was added , and the remaining nan B-technique 3 was removed by centrifugation ( 4000 rpm at 25 °c for 20 min ) . [SEP]
[CLS] the diblock copolymer was recovered by precipitation in heptane . [SEP]
[CLS] after washing with heptane multiple times , the solid precipitate was collected by vacuum filtration and dried under reduced pressure ( see more details in supporting information ) . [SEP]
[CLS] azide - terminated oligo ( ethylene oxide B-material ) with 4 peg units ( c 8 h 16 o 4 n 3 , 109 mg , 0 . 5 mmol ) was weighed into a dry flask and ε - caprolactone ( 8 . 5 g with m / i ratio of ~ 150 : 1 ) was subsequently added . [SEP]
[CLS] the synthesis and purification of single azide - terminated peo - pcl diblock copolymer follow the same procedures for poly ( α - cl - εcl - b - εcl ) as mentioned above with similar isolated yield of the desired product . [SEP]
[CLS] for each azide - containing polymer B-material , the same procedure was followed . [SEP]
[CLS] first , the polymer B-material was dissolved in a 1 : 1 dmso / dmf solution , and then a large excess of dbco - dna dissolved in dmso was added to the polymer B-material solution and incubated B-technique at 40 °c overnight . [SEP]
[CLS] after conjugation , the resulting linear and brush dbc samples were used immediately for micelle B-material formation . [SEP]
[CLS] briefly , dbcs dissolved in an organic solvent ( dmso / dmf , 1 : 1 ) were loaded in a dialysis bag with a 50 kda m w cutoff membrane and the solution was dialyzed against deionized B-material water I-material for 12 hours . [SEP]
[CLS] after removal of large aggregates by filtration through a 0 . 2 μm syringe filter , the micelle - sna conjugates were purified using an amicon ultra - 15 ultrafiltration device ( millipore , mwco 50 kda ) to remove excess free dna . [SEP]
[CLS] 1 h nmr spectra were recorded on a bruker avance 400 mhz nmr spectrometer and referenced internally to residual proton signals in the denatured solvents . [SEP]
[CLS] ftir spectra were collected on a perkin - elmer spectrum 100 ftir spectrometer using disposable kbr plates . [SEP]
[CLS] gel permeation chromatography B-technique ( gpc ) was carried out in thf at 32 °c at 1 ml / min with a viscotek tdamax liquid chromatograph ( malvern instruments , uk ) equipped with a programmable autosampler and refractive B-property index I-property detector . [SEP]
[CLS] afm images were obtained with a bruker dimension icon atomic force microscope in a tapping mode under ambient conditions . [SEP]
[CLS] dls hydrodynamic size and zeta B-property potential I-property measurements were collected on a malvern zetasizer nano - zs ( malvern instruments , uk ) with a laser wavelength of 633 nm . [SEP]
[CLS] cryogenic tem images were collected on a jem 1230 microscope ( jeol ) at an accelerating voltage of 80 kv . nta measurements were performed with a nanosight lm10 ( nanosight , amesbury , uk ) equipped with a sample chamber with a 638 nm laser and a viton fluoroelastomer o - ring . [SEP]
[CLS] confocal fluorescence B-property images of the cells B-material treated with micelle - snas were collected with a zeiss lsm 510 inverted confocal scanning microscope . [SEP]
[CLS] cells B-material were cultured at 37 °c and 5 % co 2 in dmem supplemented with 10 % fbs and 1 % streptomycin / penicillin . [SEP]
[CLS] for cellular uptake study , hela cells B-material were seeded in a 6 - well plate 24 h prior to treatment and incubated B-technique with micelle B-material - snas derived from linear or brush block copolymer structures , respectively . [SEP]
[CLS] right before flow B-technique cytometry I-technique analysis , cells B-material were trypsinized , washed , suspended in 0 . 5 ml 1× phosphate - buffered saline ( pbs ) and fixed by addition of 0 . 5 ml of 3 . 7 % formaldehyde in pbs . [SEP]
[CLS] the fluorescence B-property intensity from fluorescein ( excitation wavelength at 488 nm , emission wavelength at 520 nm ) of 10 , 000 cells B-material was collected using a bd lsr ii flow cytometer . [SEP]
[CLS] for western blotting , c166 cells B-material overexpressing egfp were transfected with micelle - snas ( with 0 . 5 , 1 , and 2 μm of total dna in optimem ) overnight and further incubate B-technique for another 2 days after medium change . [SEP]
[CLS] protein B-material lysate with equal amount of total protein B-material were fractionated by 4 - 20 % precast gradient gel ( bio - rad ) . [SEP]
[CLS] the intact gel was then transferred to a nitrocellulose membrane ( thermo scientific ) and blocked in odyssey blocking buffer ( li - cor biosciences ) . [SEP]
[CLS] proteins B-material were detected with primary antibodies B-material against actin ( 1 : 500 ) ( santa cruz biotechnology ) , egfp ( 1 : 1000 ) ( clontech laboratories inc ) followed by irdye 680 secondary antibodies B-material ( 1 : 10 , 000 ) ( li - cor biosciences ) diluted in pbst containing 5 % non - fat milk . [SEP]
[CLS] the desired bands were visualized using the odyssey® clx infrared imaging system ( li - cor biosciences ) . [SEP]
[CLS] cells B-material were seeded in a 35 mm fluorodish and incubated B-technique with fluorescein - labelled dbbc - snas ( with total dna 1 μmol ) in complete dmem for different time points . [SEP]
[CLS] cells B-material were rinsed with pbs , fixed in 3 . 7 % pfa in pbs for 15 min , and imaged under a zeiss lsm 510 inverted confocal scanning microscope . [SEP]
[CLS] the excitation wavelength of fluorescein - labelled dbbc - snas was 488 nm , and the corresponding emission filter was 500 - 550 nm . [SEP]
[CLS] to track the colocalization of snas with intracellular proteins B-material , after incubation B-technique with fluoresceinlabelled dbbc - snas ( with total [ dna ] = 1 μmol ) for different durations of time , cells B-material were rinsed with pbs , fixed in 3 . 7 % pfa in pbs , and permeated with 0 . 1 % triton x - 100 for 3min . [SEP]
[CLS] after blocking with 5 % bsa in pbs for 1 h , cells B-material were stained with a primary antibody B-material against the protein B-material marker of interest at 5 μg / ml ( 1 % bsa in pbs ) overnight at 4 °c . [SEP]
[CLS] after rinses with 0 . 05 % tween - 20 in pbs , cells B-material were stained with an alexa fluor 633labeled secondary antibody B-material ( invitrogen alexa fluor 633 goat anti - rabbit or mouse igg ( h + l ) at 1 μg / ml ( 1 % bsa in pbs ) for 1 hour at rt . [SEP]
[CLS] the excitation wavelength of the secondary antibody B-material was 633 nm , and the corresponding emission filter was 660 - 710 nm . [SEP]
[CLS] the primary antibodies B-material include rabbit against rab9 ( abcam ab179815 ) , mouse against lamp1 ( santa cruz sc - 20011 ) . [SEP]
[CLS] to visualize the amount of nucleic B-material acids I-material released from dbbc - snas under different ph conditions an agarose gel experiment was carried out . [SEP]
[CLS] briefly , fluorescein - labeled dbbc - snas containing 10 μm total dna were diluted 1 : 1 with the following buffer solutions ( 100 mm each ) : hepes buffer , ph 5 . 5 ; mes buffer , ph 6 . 0 , pbs buffer , ph 7 . 4 . [SEP]
[CLS] as a control , a sample was prepared with the aunp B-nanoparticle functionalized with the same oligonucleotides ( anti - egfp antisense dna ) diluted 1 : 1 with the buffers described above , respectively . [SEP]
[CLS] the solutions were allowed to stand at rt for 18 , 24 , 48 , and 72 hours , then were analyzed by agarose B-technique gel I-technique electrophoresis I-technique ( 1 % agarose ) . [SEP]
[CLS] the agarose gels were imaged on a fujifilm fla - 5100 gel imager with a 473 nm laser to visualize the fluorescein - labeled dna . [SEP]
[CLS] confocal microscopy B-technique of fluorescein - labelled dbbc - sna and immunofluorescence staining of organelle markers ( red , labelled by alexa fluor 663 ) . [SEP]
[CLS] biomarkers B-property are rab9 ( for late endosomes ) and lamp - 1 ( for lysosomes ) . [SEP]
[CLS] note that most dbbc - snas colocalize with late endosomes during the incubation B-technique in hela cells B-material . [SEP]
[CLS] there is no significant colocalization of dbbc - snas with lysosomes , which is consistent with the behaviour of aunp B-nanoparticle - snas . [SEP]
[CLS] cellular toxicity B-property of dbbc - based micelle - snas analyzed by a standard mtt B-technique assay I-technique . [SEP]
[CLS] cells B-material treated with dbbc - snas ( red ) , ldbc - snas ( blue ) , and single stranded dna ( black ) at the same total dna concentration ( 2 μm ) . [SEP]
[CLS] viable cells B-material with active metabolism convert mtt into a colored formazan product with an absorbance maximum near 570 nm , and cell B-property viability I-property was quantified by normalization of the absorbance at 570 nm to non - treated cells B-material . [SEP]
[CLS] error bar are standard deviation of absorbance at 570 nm from 3 independent wells of cells B-material in a 96 - well plate . [SEP]
[CLS] 1 . schematic showing the synthesis of dna grafted block copolymer - based micelle B-material snas . [SEP]
[CLS] ( a ) the synthesis of the linear dna - b - peo - b - pcl block copolymer and the corresponding formation of micelle - snas ( ldbc - snas ) . [SEP]
[CLS] ( b ) the synthesis of the brush dna - g - pcl - b - pcl block copolymer and the formation of micelle B-material snas ( dbbc - snas ) with a higher surface density of nucleic B-material acids I-material . [SEP]
[CLS] characterization of as - synthesized polycaprolactone - based micelle B-material - snas . [SEP]
[CLS] ( a ) 1 % agarose B-technique gel I-technique electrophoresis I-technique ; nucleic B-material acids I-material were stained with ethidium bromide B-material ; ( b ) a typical dls measurement of micelle B-material - snas derived from ( top ) linear and ( bottom ) brush block copolymer structures ; ( c ) melting transition behaviour for micelle B-material - snas hybridized to complementary 15 nm aunp B-nanoparticle - snas ; ( black trace ) micelle B-material - snas made from linear structures and ( red trace ) micelles B-material made from the brush architecture ; ( d ) cryogenic tem images of micelle B-material - snas derived from brush block copolymer . [SEP]
[CLS] both dbbc - sna and ldbc - sna nanoparticles B-nanoparticle remain intact under cryogenic conditions and are relatively uniform in size with diameters ~ 30 - 50 nm . [SEP]
[CLS] cellular uptake of micelle B-material - snas . [SEP]
[CLS] ( a - e ) fluorescence B-property micrograph of hela cells B-material incubated B-technique with different forms of nucleic B-material acids I-material at a total dna concentration of 1 μm for 16 h . [SEP]
[CLS] dna strands were labelled at the 5 ' - end with fluorescein and the dye molecules are located at the outside terminus of the micelle B-material - sna structure . [SEP]
[CLS] figure 2 . a ) negative control , cells B-material without dna incubation B-technique ; b ) single stranded fluorescein - labelled dna ( fluo - dna ) ; c ) positive control , single stranded fluo - dna transfected with lipofectamine® 2000 ; d ) micelle B-material - snas derived from linear block copolymer structures ; e ) micelle - snas derived from brush block copolymer structures . [SEP]
[CLS] in the fluorescence B-property images , micelle - snas assembled from the brush block copolymer show significantly higher uptake compared to those derived from the linear analog or component single stranded dna ; ( f ) fluorescence - activated cell B-material sorting ( facs ) analysis of the cells B-material when incubated B-technique with different forms of nucleic B-material acids I-material . [SEP]
[CLS] facs data also confirm the brush block copolymer based micelle B-material - sna has higher cell B-material uptake efficiency than that of the linear block copolymer based micelle B-material - sna . [SEP]
[CLS] single stranded dna and dna transfected by conventional lipofectamine® 2000 were used as controls . [SEP]
[CLS] 4 . gene regulation by dbbc - based micelle - snas . [SEP]
[CLS] ( a ) fluorescence B-property micrograph of c166 mouse endothelial cells B-material that highly express the egfp protein B-material before dbbc - based micelle - sna treatment . [SEP]
[CLS] ( b ) fluorescence B-property micrograph of c166 mouse endothelial cells B-material after dbbc - sna treatment . [SEP]
[CLS] the green fluorescence B-property was significantly suppressed due to the egfp gene knockdown . [SEP]
[CLS] the micelle B-material - snas were equipped with anti - egfp sequence and the cells B-material were cultured for another 2 days after sna transfection . [SEP]
[CLS] ( c ) western blotting of egfp expression in c166 cells B-material after treatment with anti - egfp dbbc - based micelle - snas and single stranded dna samples under various total dna concentrations . [SEP]
[CLS] ( d ) western blotting of egfp expression in c166 cells B-material after treatment with anti - egfp dbbc - snas and control samples under total dna concentrations of 2 μm . the positive control sample was antisense dna transfected by conventional lipofectamine® 2000 . [SEP]
[CLS] actin was used as an internal reference . [SEP]
[CLS] 5 . the ph - dependent degradation of dbbc - based micelle - snas . [SEP]
[CLS] samples were incubated B-technique in 50 mm mes buffer ( ph 5 . 5 ) , 50 mm hepes buffer ( ph 6 . 0 ) , or 1x pbs ( ph 7 . 4 ) . [SEP]
[CLS] agarose B-technique gel I-technique electrophoresis I-technique was used to monitor the degradation process . [SEP]
[CLS] figure 5 . a ) degradation of dbbcbased micelle B-material - snas after 24 hour incubation B-technique under different ph buffer conditions . [SEP]
[CLS] note that a significant amount of single stranded dna can be observed in the gel image ; b ) the mobility B-property and shape of the dbbc - based sna bands over incubation B-technique time under different buffer conditions . [SEP]
[CLS] in the first 24 hours , the sna bands look relatively sharp . [SEP]
[CLS] however , as the incubation B-technique time increases , the bands become smeared and diffuse , indicating the gradual degradation of the entire micellar structures . [SEP]
[CLS] note that the micelles B-material held at lower ph ( 5 . 5 ) suffer considerably more degradation after 72 hours . [SEP]
[CLS] c . a . m . acknowledges support from the center for cancer nanotechnology excellence ( ccne ) initiative of national institutes of health ( nih ) awards u54 ca119341 and u54 ca151880 , dixon translational research grants initiative , and the defense advanced research planning agency award n66001 - 11 - 1 - 4189 . [SEP]
[CLS] l . h . was a howard hughes medical institute international student research fellow [SEP]
[CLS] c . h . j . c . acknowledges a postdoctoral research fellowship from the croucher foundation . [SEP]
[CLS] the electron B-technique microscopy I-technique work was performed at the biological imaging facility ( bif ) and the electron probe instrumentation center ( epic ) at northwestern university . [SEP]
[CLS] we also acknowledge dr . natalia chernyak for providing the azide - terminated oligo ( ethylene oxide B-material ) initiator . [SEP]
[CLS] dna is proving to be a powerful scaffold to construct molecularly precise designer dna devices . [SEP]
[CLS] recent trends reveal their ever - increasing deployment within living systems as delivery devices that not only probe but also program and reprogram a cell B-material , or even whole organisms . [SEP]
[CLS] given that dna is highly immunogenic B-property , we outline the molecular , cellular and organismal response pathways that designer nucleic B-material acid I-material nanodevices B-nanoparticle are likely to elicit in living systems . [SEP]
[CLS] we address safety issues applicable when such designer dna nanodevices B-nanoparticle interact with the immune system . [SEP]
[CLS] in light of this , we discuss possible molecular programming strategies that could be integrated with such designer nucleic B-material acid I-material scaffolds to either evade or stimulate the host response with a view to optimizing and widening their applications in higher organisms . [SEP]
[CLS] deoxyribonucleic B-material acid I-material ( dna ) is central to encoding genetic information in eukaryotic and prokaryotic cells B-material , as well as in bacteriophages and viruses . [SEP]
[CLS] dna also possesses rich diversity in structure and enzymatic function 1 . [SEP]
[CLS] it is thus an ideal molecular material B-material in which to integrate versatile chemical functionalities 1 , 2 . [SEP]
[CLS] dna is abundant in circulating blood in animals as a result of its release from dying cells B-material ; cells B-material therefore constantly come in contact with dna from extraneous sources ( referred to as foreign dna ) that they distinguish as ' non - self ' . [SEP]
[CLS] this is distinct from endogenous ' self - dna ' or the cell B-material ' s own genomic dna present within its nucleus . [SEP]
[CLS] mechanisms that differentiate ' self ' from ' nonself ' dna are usually based on simple chemical modifications , for example , cytosine methylation of dna 3 . [SEP]
[CLS] exogenous dna can even function as mobile B-property genetic elements and is responsible for the acquisition of genetic traits 4 . [SEP]
[CLS] cells B-material are also capable of recombining non - self dna with self - dna . [SEP]
[CLS] a proportion of non - self dna is harmful , such as that derived from lytic bacteriophages and viruses , and consequently , mechanisms to rapidly detect and eliminate it have also evolved 5 , 6 . [SEP]
[CLS] the interactions of foreign or synthetic dna with biological systems are therefore multilayered , complex and lead to different outcomes . [SEP]
[CLS] dna has been widely exploited in living systems for bio medical applications as duplex dna ( fig . 1a ) in the form of a synthetic carrier of genetic instructions , such as circularized plasmid dna ( fig . 1b ) , and its nanostructured forms ( fig . 1c - g ) . [SEP]
[CLS] nanoparticulate complexes of plasmid dna with non - immunogenic B-property polymers B-material such as chitosan B-material ( fig . 1c ) have been used in vivo for gene delivery 7 . [SEP]
[CLS] such nanoparticles B-nanoparticle are distinct from structural motifs formed by specific sequences of dna such as the g - quadruplex 8 or i - motif 9 ( fig . 1d ) . [SEP]
[CLS] spherical nucleic B-material acids I-material ( sna ; fig . 1e ) represent another distinct class of nanostructured dna where single - stranded ( ss ) or double - stranded ( ds ) dna are displayed on the surface of inorganic B-nanoparticle nanoparticles I-nanoparticle 10 . [SEP]
[CLS] importantly , due to its molecular programmability , dna can be assembled into rationally designed , structurally precise architectures at the nanoscale that are popularly referred to as designer dna architectures 1 . [SEP]
[CLS] designer dna nanodevices B-nanoparticle are fabricated using either dna tiling or dna origami . [SEP]
[CLS] dna tiling exploits hierarchical assembly of structured dna motifs called tiles using sticky - ended cohesion 1 ( fig . 1f ) . [SEP]
[CLS] dna origami , on the other hand , utilizes multiple , short ' staple ' strands that hybridize with domains on a large viral - genome - derived ssdna scaffold , folding it into precise superarchitectures 11 ( fig . 1g ) . [SEP]
[CLS] importantly , designer dna architectures present great potential to probe and program living systems 2 . [SEP]
[CLS] however , before their successful and wide implementation in higher organisms , it is important to consider the various cellular and systemic responses that such dna architectures might elicit . [SEP]
[CLS] in this perspective , we discuss examples of dna nanodevices B-nanoparticle that have been deployed in multicellular organisms and their modes of introduction . [SEP]
[CLS] even though dna is a natural biopolymer B-material , when present at the wrong place at the wrong time it can elicit a strong inflammatory reaction . [SEP]
[CLS] we summarize how cells B-material respond to exogenous or endogenous dna to better understand and possibly anticipate host responses to designer dna nanodevices B-nanoparticle . [SEP]
[CLS] finally , we outline potential design considerations and immediate challenges that impact the delivery of dna nanodevices B-nanoparticle and the corresponding host response with a view to improving tolerance , thereby promoting their wider use . [SEP]
[CLS] cells B-material protect self - dna against foreign dna using surveillance systems that detect non - self dna and trigger mechanisms for their swift elimination . [SEP]
[CLS] for example , in bacteria and archaea , sequence - independent and sequence - specific restriction mechanisms 5 , 6 , and the memory - based adaptable crispr / cas ( clustered regularly interspaced short palindromic repeats / crispr associated complex ) 12 systems [SEP]
[CLS] biological responses to dna in higher organismsdirect endonuclease activity against foreign dna . [SEP]
[CLS] thus dna - based early immune systems evolved from bacteria , diversified in eukaryotes , and are at the heart of mammalian responses to natural dna . [SEP]
[CLS] europe pmc funders author manuscripts dna viruses and bacteriophages are detected by direct binding of their dna to one or more receptors in the host cell B-material they infect . [SEP]
[CLS] therefore when exposed to natural or synthetic nucleic B-material acids I-material , cells B-material respond as they do to viral infection 13 . [SEP]
[CLS] eukaryotic cells B-material respond to dna in two ways ; ( i ) by upregulating interferon ( ifn ) genes , and ( ii ) stimulating caspase - 1 protease activity through inflammasome signalling pathways 14 . [SEP]
[CLS] detection of dna converges on upregulation and secretion of type i ifns ( ifn - α and ifn - β ) 14 , 15 . [SEP]
[CLS] secreted ifns induce the expression of a large number of ifn - stimulated genes , many of which have broad antiviral B-property and inflammatory roles 15 . [SEP]
[CLS] dna - dependent activation of caspase - 1 inflammasomes results in the release of cytokines , such as interleukin - 1β and interleukin - 18 , which have antimicrobial B-property as well as pro - inflammatory properties 16 , 17 . [SEP]
[CLS] defined inflammatory responses to dna facilitate the long - term adaptive immune response to infection . [SEP]
[CLS] this feature is exploited in dna - based vaccines to boost immunity 18 . [SEP]
[CLS] in other settings , however , chronic expression of ifns and activation of caspase - 1 cause inflammatory tissue damage in the host 19 . [SEP]
[CLS] thus , like natural dna , synthetic dna nanodevices B-nanoparticle too are likely to elicit an overall inflammatory and antiviral B-property - like response from the host . [SEP]
[CLS] host cells B-material detect exogenous dna that is extracellular , vacuolar or cytoplasmic using > 20 different receptor B-material proteins B-material 14 , 19 . [SEP]
[CLS] the majority elicit ifn secretion , and one ( aim2 ) directly activates caspase - 1 ( ref . ; fig . 2 ) . [SEP]
[CLS] only major dna receptors are discussed here to provide an overall view of the complex system . [SEP]
[CLS] for example , dsdna in the cytosol is bound by receptors such as ifi16 , ddx41 , cgas , mre11 and dna - pk ; all induce ifns via a protein B-material called sting 14 , 21 ( fig . 2 ) . [SEP]
[CLS] on binding B-event cytosolic I-event dna I-event , cgas synthesizes a newly identified second messenger - 2 ' , 3 ' - cgamp ( cyclic [ g ( 2 ' , 5 ' ) pa ( 3 ' , 5 ' ) p ] ) - that allosterically activates sting to enhance ifn responses 22 - 24 ( fig . 2 ) . [SEP]
[CLS] interestingly , a : trich dsdna in the cytosol can be transcribed by rna polymerase iii into 5 ' ppp - containing rna that induces ifn expression by binding the rig - i dsrna receptor B-material 25 . [SEP]
[CLS] the protein B-material lrrfip1 binds cytosolic dna and induces ifns via a novel β - catenin pathway 26 . [SEP]
[CLS] post endocytosis B-event into vacuoles , dsdna containing umethylated cpg motifs is detected by the tlr9 receptor B-material that induces the expression of ifns independently of sting 14 , 21 ( fig . 2 ) [SEP]
[CLS] little is known about nucleotide modifications that affect receptor B-event binding I-event and downstream signalling . [SEP]
[CLS] one could speculate that self - dna is protected from cytosolic dna sensors via compartmentalization into the nucleus , whereas rna sensors detect specific ' foreign ' chemical modifications of rna distinguishing it from host mrna . [SEP]
[CLS] differential methylation and secondary structures of dsdna elicit differential responses via the tlr9 receptor B-material . [SEP]
[CLS] the 2 ' - deoxyribose sugar is an important determining factor in the context of backbone chemistry ( phosphodiester versus phosphorothioate ) in two ways . [SEP]
[CLS] first , ribose sugars are not tolerated on tlr9 ligands . [SEP]
[CLS] second , abasic as well as non - cpg - motif - containing 2 ' deoxyribose phosphodiester chains activate tlr9 , whereas similar phosphorothioate chains inhibit tlr9 27 , 28 . [SEP]
[CLS] in the case of the cytosolic aim2 receptor B-material , sequence - independent detection of ~ 80 bp dsdna results in its oligomerization and activation of caspase - 1 ( ref . [SEP]
[CLS] therefore successful dna scaffolds must not only fly under the radar to avoid activating antiviral - like inflammatory responses , but must also be amenable to natural and safe disposal from biological systems post - deployment . [SEP]
[CLS] thus dna nanodevices B-nanoparticle need to incorporate design principles to navigate the multilayered detection and defense machinery of higher organisms . [SEP]
[CLS] a selection of designer dna nanodevices B-nanoparticle have been used either as drug - delivery vehicles B-material or diagnostic probes in living systems 2 . [SEP]
[CLS] although several nanodevices B-nanoparticle have been applied to cells B-material in culture , their application in multicellular organisms has only just emerged . [SEP]
[CLS] this is primarily due to major molecular barriers B-property faced in vivo such as ( i ) efficient delivery and targeting to the site of interest , ( ii ) stabilityof externally introduced dna nanodevices B-nanoparticle , and ( iii ) their potential toxicity B-property in the host organism . [SEP]
[CLS] of the limited number of nanodevices B-nanoparticle deployed in vivo , the dna icosahedron and tetrahedron present good case studies to discuss these molecular barriers B-property 45 , 46 . [SEP]
[CLS] currently , in vivo delivery strategies predominantly rely on injections to target dna nanodevices B-nanoparticle to specific cell B-material types . [SEP]
[CLS] the first study of a designer dna architecture in a multicellular living organism used a ph - sensitive dna nanodevice B-nanoparticle , the i - switch , which was microinjected into caenorhabditis elegans ; post - injection , the i - switch was targeted to specific scavenger cells B-material that displayed cell - surface anionic ligand - binding receptors ( fig . 3a ; left panel ) . [SEP]
[CLS] once internalized , it was shown that the nanodevice B-nanoparticle could probe endosomal maturation 47 . [SEP]
[CLS] this same strategy was exploited to introduce a cargo - loaded dna icosahedron to scavenger cells B-material , where both cargo functionality and device integrity were quantitatively preserved post - delivery 45 , 48 ( fig . 3a ; right panel ) . [SEP]
[CLS] in mammalian systems , dna architectures have been delivered intravenously . [SEP]
[CLS] a general design principle for targeting the nanostructure to the site of interest exploits the display of specific ligands on the nanodevice B-nanoparticle that enables its binding to a cell - specific endogenous receptor B-material , leading to its cellular uptake . [SEP]
[CLS] by exploiting the overexpression of the folate B-material receptor I-material on cancerous cells B-material , it was possible to target tetrahedral dna nanoparticles B-nanoparticle bearing folate moieties and short interfering rna ( sirna ) to xenograft tumours in nude mice 46 ( fig . 3b ; left panel ) . [SEP]
[CLS] post - internalization , the sirna reduced the expression level of a target gene 46 . [SEP]
[CLS] however , despite their enrichment in the tumour , some dna nanoparticles could also be uptaken by non - cancerous cells B-material bearing the folate B-material receptor I-material , given its ubiquitous expression across tissues 49 . [SEP]
[CLS] nevertheless , this strategy seems promising to target selected malignancies . [SEP]
[CLS] another example is the in vivo delivery of a tetrahedral dna device displaying antigens and adjuvants to produce antibodies B-material 50 . [SEP]
[CLS] a model antigen ( streptavidin ) and adjuvant ( cpg deoxyoligonucleotides ) were assembled on a dna tetrahedron and injected into a mouse . [SEP]
[CLS] identification of the dna - streptavidin - cpg complex by circulating macrophages and dendritic cells B-material in the bloodstream led to the production of anti bodies against streptavidin 50 ( fig . 3b ; right panel ) . [SEP]
[CLS] among diverse delivery modes , oral administration is popular in various model organisms , yet surprisingly has not been adopted thus far for dna nanostructures , probably due to the high susceptibility of dna to acid - catalysed depurination and nucleases , and its low efficiency in traversing tissue barriers B-property . [SEP]
[CLS] often , therapeutic proteins B-material or peptides B-material are packaged within liposomes B-nanoparticle , nano particles , dendrimers B-nanoparticle or micelles B-material to protect against proteolysis and low gastrointestinal ph 51 , 52 . [SEP]
[CLS] intranasal delivery presents exciting possibilities . [SEP]
[CLS] the high surface area and permeability of the nasal endo thelial membrane promote rapid absorption and decreased metabolism of the applied nanoparticles B-nanoparticle 53 . [SEP]
[CLS] further , intranasal administration is user friendly and relatively non - invasive . [SEP]
[CLS] it also provides access to the mammalian central nervous system , through mechanisms that are not yet well understood 54 . [SEP]
[CLS] for example , plasmid dna coated with a polycationic lysine B-material derivative has been delivered intranasally in rats where it led to green fluorescent B-property protein B-material expression in the brain 55 . [SEP]
[CLS] other routes to access a specific tissue involve direct injections at the target site , most notably injections of chitosan - dna nanoparticles B-nanoparticle into the mouse eye 56 , 57 and lung 58 as well as the rat brain 59 and liver 60 . [SEP]
[CLS] direct tissue injections present good potential as they provide high local concentrations and therefore minimize toxicity B-property , but are invasive and require special expertise . [SEP]
[CLS] importantly , regardless of the delivery route , dna nanostructures are likely to elicit an immune response that would need to be either exploited or mitigated depending on the functionality of the architecture - for example , vaccine or therapeutic cargo . [SEP]
[CLS] without robust targeting technology , there is a risk of nonspecific nanodevice B-nanoparticle delivery to undesired sites . [SEP]
[CLS] cell - specific targeting of dna nanodevices B-nanoparticle has been achieved using specificligand - receptor B-material pairs , where the dna architecture enters cells B-material via receptor - mediated endocytosis B-event 61 . [SEP]
[CLS] newer strategies that leverage cancer cell B-material surface - expressed biomarkers B-property 62 , endogenously expressed tissue - specific receptors 45 , 47 , synthetic recombinant antibodies B-material 63 or aptamers 64 need to be devised to achieve efficient targeting . [SEP]
[CLS] for example , the erbb receptor B-material could be a promising route for anti - cancer dna nanodevices B-nanoparticle in the future 62 . [SEP]
[CLS] the anionic ligand - binding B-event receptors that have high affinity for dna present another viable option . [SEP]
[CLS] synthetic protein - dna partners also present exciting possibilities . [SEP]
[CLS] for example , a recombinant antibody B-material fused to furin , a trans - golgi network trafficking protein B-material , has been used to achieve organelle targeting in cells B-material 63 . [SEP]
[CLS] this recombinant antibody B-material recognized a specific 8 - mer dsdna sequence on the nanostructure and was able to target it to the trans - golgi network 63 . [SEP]
[CLS] although mechanisms of cytosolic delivery of dna via receptormediated endocytosis B-event , as seen with lipid - complexed dna 65 , remain unclear , these could be leveraged in future to deliver dna nanodevices B-nanoparticle cytoplasmically . [SEP]
[CLS] nanodevices B-nanoparticle enter cells B-material through different endocytic mechanisms , and how these different entry mechanisms could affect efficacy and stability of dna devices remains to be addressed . [SEP]
[CLS] given that there are several mechanisms that dispose of superfluous dna to maintain homeostasis , the efficacy and stability of the dna nanodevice B-nanoparticle need to match the application for which it is designed . [SEP]
[CLS] systemic circulatory stability depends on complex factors such as digestion by extracellular dnases , size and possibly shape of dna nanodevices B-nanoparticle , tissue uptake and removal from circulation by the kidneys and liver . [SEP]
[CLS] while it is known that size or shape of nanostructured gold B-material , carbon B-material , silica , dendrimers B-nanoparticle , quantum B-nanoparticle dots I-nanoparticle , liposomes B-nanoparticle and polymeric particles affect their in vivo clearance times 52 and uptake 66 , analogous studies on designer dna nano structures remain to be performed . [SEP]
[CLS] cellular stability of dna nanodevices B-nanoparticle could be altered by tuning delivery pathways that circumvent lysosomal delivery 61 . [SEP]
[CLS] further , partially dissociated nanostructures could trigger stronger immune responses , greater off - target effects and elevated toxicities B-property . [SEP]
[CLS] thus nanostructure stability and nanodevice B-nanoparticle uptake pathways will cumulatively impact their bioavailability B-property . [SEP]
[CLS] one of the earliest studies that addressed dna nanodevice B-nanoparticle stability in a multicellular organism exploited the scavenger cells B-material of c . elegans to investigate dna nanostructure clearance by lysosomal degradation 48 . [SEP]
[CLS] here , a dna duplex displaying two single - stranded domains showed an in vivo half - life of 8 hours . [SEP]
[CLS] reducing the number of single - stranded domains on the nanostructure increased its half - life to 11 hours . [SEP]
[CLS] on the other hand , an architecture such as a dna icosahedron , devoid of free termini , was recalcitrant to lysosomal degradation over 24 hours of investigation 48 . [SEP]
[CLS] it has also been shown that fluorescently B-property labelled dna tetrahedra remained stable up to 48 hours post - transfection in cultured human embryonic kidney ( hek ) cells B-material 67 . [SEP]
[CLS] one of the reasons for increased in vivo stability of architectures such as the icosahedron and tetrahedron is that the mg 2 + requirement 67 , 68 for their structural integrity is relatively low and corresponds to physiological concentrations ( 1 - 2 mm mg 2 + ) 69 . [SEP]
[CLS] in this case , the mg 2 + is needed to primarily stabilize their constituent n - way junctions . [SEP]
[CLS] however , this is not the case for most dna origami - based structures , where phage ssdna is folded into dna helices that are packed lengthwise 11 . [SEP]
[CLS] such packing requires high concentrations the current knowledge of host responses to dsdna can be leveraged to design dna nanodevices B-nanoparticle that either evade or activate the immune response by tuning their in vivo stability . [SEP]
[CLS] fully ligated architectures , with minimal single strands and free termini , might show enhanced stability and resistance to cellular nucleases ( fig . 4 ( i ) ) . [SEP]
[CLS] this was borne out in vitro on ligated polyhedra 76 and later confirmed in vivo 48 . [SEP]
[CLS] second , within a given architecture , the spatial position and accessibility of a sequence governs its vulnerability to nuclease digestion . [SEP]
[CLS] it has been shown that sequences placed near three - way junctions in a tetrahedron were more resistant to nucleases 77 . [SEP]
[CLS] this implies that the stability of a nanostructure can be tuned by varying the degree of accessibility of its vulnerable sites to either enhance its in vivo half - life or to promote its degradation to facilitate a payload release ( fig . 4 ( ii ) ; blue region ) . [SEP]
[CLS] third , chemical modifications B-event of I-event the I-event dna I-event scaffold could also make it recalcitrant to degradation . [SEP]
[CLS] for instance , the stability of a triangular dna prism in fbs could be enhanced using hexaethylene glycol - and hexanediol - modified phosphoramidites at the 5 ' and 3 ' termini of component oligonucleotides 78 . [SEP]
[CLS] in addition , a covalently linked base pair between 5 - formyluracil and 5 - aminocytosine via schiff base formation has been developed ( fig . 4 ( iii ) ; top chemical structure ) . [SEP]
[CLS] dna duplexes containing this base pair are stable under denaturing conditions and dissociate only at high temperatures 79 . [SEP]
[CLS] another novel strategy that crosslinks duplex dna to enhance structural stability utilizesan aniline - derived dna heterobase and ( hydroxy ) benzaldehyde . [SEP]
[CLS] this is similar to an a - t base pair that can be reversibly crosslinked ( fig . 4 ( iii ) ; bottom chemical structure ) and was used to enhance the half - life of a dna duplex in vitro 80 . [SEP]
[CLS] another strategy to evade enzymatic cleavage of dna nano - structures is to use unnatural nucleic B-material acid I-material analogues that have modified chirality such as l - dna 81 ( fig . 4 ( iv ) ; left chemical structure ) , modified backbones such as peptide B-material nucleic B-material acid I-material 82 ( fig . 4 ( iv ) ; right chemical structure ; right chemical structure ) , or modified sugar residues such as locked nucleic B-material acids I-material ( lna ) , morpholinos and bridged nucleic B-material acids I-material ( pna ) 83 . [SEP]
[CLS] these unnatural analogues are not substrates for endo genous nucleases and could prolong nanodevice B-nanoparticle lifetimes , yet affording essentially identical functionality . [SEP]
[CLS] for instance , it has been shown that the incorporation of 2 ' fluoro rna in the viral phi29 dna - packaging rna sequence yields an rna device that was stable in rnase a as well as in fbs over 36 hours and , further , could effectively gear the phi29 motor to package the viral dna and produce infectious particles 84 . [SEP]
[CLS] chimaeric nucleic B-material acid I-material duplexes , such as pna - dna 85 , rna - dna 86 or lna - dna 87 , can be further used to fabricate architectures with enhanced stabilities . [SEP]
[CLS] such approaches however , are yet to be embraced widely in the field given the prohibitively high cost of their syntheses . [SEP]
[CLS] an important property of nanostructured dna scaffolds is their propensity to induce antibody B-material responses . [SEP]
[CLS] an example is the use of a tetrahedral dna nanodevice B-nanoparticle as an adjuvant in vertebrates that produced antibodies B-material against an antigen of choice 50 . [SEP]
[CLS] another example is a 30 - helix , hollow dna origami tube , which when coated with cpg motifs potently activated immune cells B-material in a tlr9 - dependent manner 88 . [SEP]
[CLS] this has also been observed with cpg - motifs presented on sna that elicited better antibody B-material responses and tumour regression in mice compared with their ssdna counterparts 33 . [SEP]
[CLS] given that such immunostimulatory or immunoregulatory dna nanodevices B-nanoparticle outperform unstructured or polymer B-material - coated stimulatory dna , they could become important in prophylaxis , therapeutics and possibly even transform vaccine biology 89 . [SEP]
[CLS] conversely , dna nanodevices B-nanoparticle could also be designed to potentially suppress immune responses , which would be useful in contexts such as uncontrolled inflammation . [SEP]
[CLS] regulatory t cells B-material are suppressor cells B-material that dampen immune responses and can be elicited using optimized cpg - free , polymer - complexed dna nanostructures 32 , 90 . [SEP]
[CLS] in addition , single - stranded phosphorothioate oligonucleotides with four ttaggg motifs can directly bind aim2 and ifi16 and prevent inflammasome signalling , and may be a starting point to find specific inhibitors of these pathways 91 . [SEP]
[CLS] while the above applications leverage immune responses in complex mammalian systems , simpler invertebrate models offer the power of genetics for more fundamental studies on the interaction of nanostructured dna scaffolds with the innate immune system . [SEP]
[CLS] for example , c . elegans or drosophila melanogaster , which lack acquired immunity and some of the dna receptors found in mammals but maintain humoral and innate immune responses , are excellent test beds to apply dna nanodevices B-nanoparticle as probes and tools 92 , 93 . [SEP]
[CLS] however , the genetically tractable zebrafish ( danio rerio ) , which harbours both an innate and adaptive immune response as well as the ifn - response system , may eventually prove to be a better model system 94 , 95 . [SEP]
[CLS] importantly , it is also known that not all dna sequences are equally immunogenic B-property . [SEP]
[CLS] therefore , screens to identify sequences of low ( or enhanced ) immunogenicity B-property should better inform nanostructure design to fine - tune immune responses ( fig . 4 ( v ) , ( vi ) ) . [SEP]
[CLS] further , immune responses are likely to be species - specific , as has been observed in the case of differential immunomodulatory effects of dna on human versus mouse cells B-material 28 . [SEP]
[CLS] we envision that the next generation of biologically smart dna nano devices would need to incorporate an additional layer of design . [SEP]
[CLS] these would leverage size and shape information to achieve precise targeting and clearance properties , exploiting sequences with customdesigned host immune responses . [SEP]
[CLS] such nanodevices B-nanoparticle could act as smart cargo carriers that , as a function of molecular logic , can either be destabilized or remodelled to release their cargo . [SEP]
[CLS] although there are rare examples of conditional cargo release from dna nano - devices albeit in vitro 96 - 98 , there is paucity of information on the effects of dna sequence , nanostructure size and shape on their immunogenicity B-property and in vivo clearance of the resultant dna nanostructures in any organism . [SEP]
[CLS] these data would improve our understanding of basic dna sequence requirements to develop design criteria that can rationally tune biological nanodevice B-nanoparticle functionality such as stability or immunogenicity B-property . [SEP]
[CLS] even the latest sequence design programmes 99 , 100 consider only architectural shape and flexibility of dna nanodevices B-nanoparticle . [SEP]
[CLS] such next - generation designer dna nanodevices B-nanoparticle with customized host responses would find tantalizing applications in drugdelivery . [SEP]
[CLS] given that responses of individual subjects to dna differ greatly , one can envisage dna nanodevices B-nanoparticle engineered for personalized immunotherapy . [SEP]
[CLS] the capacity to cell - specifically deliver dna architectures and / or achieve molecular - logic - guided cargo release could enable cellular programming and reprogramming . [SEP]
[CLS] more excitingly , in the longer term , the delivery of dna or rna cargo encoding transcription B-event programs , which bypass the immune system , could be used to remodel synapses to alter learning and memory , or for tissue regeneration , or building organoids from single cells B-material . [SEP]
[CLS] to fully harness the potential of the dna scaffold in vivo , the next layer of design in dna nanodevices B-nanoparticle needs strategies to leverage endogenous surveillance mechanisms to maximally tap nano device functionality . [SEP]
[CLS] in addition , cytosolic dna receptors might detect oxidation - damaged dna more readily 30 . the [SEP]
[CLS] cellular responses to dna also depend on the cell B-material type involved ; plasmacytoid dendritic cells B-material ( pdcs ) , myeloid dcs ( mdcs ) and macrophages and t - or b - lymphocytes express different sensors for dna and thus respond differently 31 . [SEP]
[CLS] for example , in the case of tlr9 , pdcs secrete large amounts of ifn - α when exposed to cpg oligonucleotides , which spontaneously form nano - particles as a result of g - tetrad formation ( called class a cpg ) 28 , 30 . [SEP]
[CLS] tlr9 activation of b cells B-material by cpg oligonucleotides devoid of complex secondary structures ( class b cpg ) leads to cell B-material proliferation 28 . [SEP]
[CLS] further , mdcs that detect non - cpg dsdna via tlr9 trigger suppressor t - cell B-material responses 32 . [SEP]
[CLS] cpg dna when presented in synthetic motifs such as snas ( fig . 1e ) are far more potent stimulators of tlr9 responses than the corresponding ssdna . [SEP]
[CLS] this underscores the interplay of dna structure and cell B-material type in generating an activating or suppressive biological response 33 . homeostatic [SEP]
[CLS] mechanisms prevent unnecessary activation of dna responses by prompt disposal of cellular dna at the end of its life cycle 34 , 35 ( fig . 2 ) . [SEP]
[CLS] the importance of dnases is revealed in hereditary autoinflammatory syndromes where high levels of circulating dna are seen , such as systemic lupus erythematosus 36 , aicardi - goutieres syndrome and rheumatoid arthritis 38 . [SEP]
[CLS] mutations in dnase i ( encoded by the gene dnase1 ) and dnase iii ( encoded by trex1 ) result in increased circulating dna and ifn expression 37 , 39 , 40 . [SEP]
[CLS] dnase i , a secreted enzyme , degrades dna from ingested food as well as that in blood . [SEP]
[CLS] reduced dnase i activity can result in lupus , a disease that shows features of increased ifn production 41 , 42 . [SEP]
[CLS] similarly , loss of trex1 results in the accumulation of ~ 60 b long ssdna , which drives ifn - dependent pathology 37 , 39 . [SEP]
[CLS] likewise , dnase ii ( encoded by dnase2a ) deficiency in mice results in ifn - driven autoimmunity , which is reversible if mice are also deficient in ifn receptors or sting 43 , 44 . [SEP]
[CLS] 10 - 80 mm ) of divalent cations B-material , such as mg 2 + , to lower the debye screening length and stabilize the overall architecture in vitro 70 , 71 . [SEP]
[CLS] when introduced into biological systems , where physiological mg 2 + concentrations are at least tenfold lower 69 , these architectures are destabilized . [SEP]
[CLS] as shown by hahn et al . , dna origami - based octahedra , nanotubes B-nanoparticle and nanorods B-nanoparticle show low stability in fetal bovine serum ( fbs ) as well as in cell B-material lines such as mouse 3t3 fibroblasts , hek - 293 and human h441 adenocarcinoma 72 . [SEP]
[CLS] further , the low yields of complex origami - based nanostructures results in molecularly heterogenous populations in bulk 73 , 74 and pose additional impediments to more widespread applications in vivo . [SEP]
[CLS] importantly , increased stability has been demonstrated by incorporating a surface lipid B-material coating B-material on dna origami - based octahedra in a mouse model 75 , highlighting a strategy to increase the in vivo stability of dna origami - based nano devices ( fig . 4 ( i ) ) . [SEP]
[CLS] 1 . dna and its various nanostructured forms . [SEP]
[CLS] figure 1 . a , duplex dna is widely exploited in therapeutics and biomedical applications in the form of diverse nanostructures . [SEP]
[CLS] b , circularized elements such as plasmids are routinely used for gene expression . [SEP]
[CLS] c , plasmids are often complexed with non - immunogenic B-property polymers B-material such as chitosan B-material or polyethylene glycol ( green ) to enhance gene delivery . [SEP]
[CLS] d , structural motifs such as g - quadruplexes are formed by specific sequences of dna in response to chemical triggers such as ions B-material and ph . e , spherical nucleic B-material acids I-material are fabricated by immobilizing single - stranded or duplex dna ( green strands ) on the surface of inorganic B-nanoparticle nanoparticles I-nanoparticle ( brown core B-material ) . [SEP]
[CLS] f , designer dna nanodevices B-nanoparticle are formed by assembling rationally designed dna motifs with sticky ends ( top ) . [SEP]
[CLS] the dna buckyball B-nanoparticle structure ( bottom ) is ~ 80 nm in diameter . [SEP]
[CLS] g , dna origami - based nanodevices B-nanoparticle use several staple strands ( blue and orange ) to fold a large viral genome - derived dna strand ( grey ) into defined super - architectures that can be used to deliver molecular payloads ( yellow and pink ) . figure reproduced with permission from : d , ref . 8 , nature publishing group ; e , ref . 10 , american chemical society ; f , ref . 101 , nature publishing group ; g , ref . 96 , aaas . [SEP]
[CLS] reproduced with permission from : d , ref . 8 , nature publishing group ; e , ref . 10 , american chemical society ; f , ref . 101 , nature publishing group ; g , ref . 96 , aaas . [SEP]
[CLS] natural mechanisms to detect and dispose of foreign dna by host cells B-material . [SEP]
[CLS] several cytosolic receptors , such as ifi16 , mre11 , dnapk , ddx41 and cgas , detect dna in the cytosol , directly bind duplex dna ( dsdna ) and trigger transcription B-event of type i interferons ( ifns ) , which in turn upregulate various interferon - stimulated genes ( isgs ) . [SEP]
[CLS] these cytosolic receptors induce ifns via the protein B-material sting . [SEP]
[CLS] figure 2 . cgas binds dna and synthesizes a novel second messenger - 2 ' , 3 ' - cgamp ( cgamp ) - that activates sting . [SEP]
[CLS] rna polymerase iii ( pol iii ) transcribes dna into rna that binds the rna receptor B-material rig - i , and induces ifns [SEP]
[CLS] 3 . targeting and delivery of dna nanostructures in vivo [SEP]
[CLS] figure 3 . a , microinjection - mediated introduction of a ph - sensitive dna nanodevice B-nanoparticle ( left ) and a cargo - loaded dna icosahedron ( yellow spheres ; right ) in c . elegans uses the anionic ligand - binding B-event receptors I-event ( red , bottom ) to achieve cell - specific targeting . [SEP]
[CLS] the red star and red and blue circles represent fluorophores . [SEP]
[CLS] b , the dna tetrahedron , bearing folate moieties ( grey triangles ) and sirna ( purple duplex ) on its surface ( left ) , was targeted to murine tumours overexpressing the folate B-material receptor I-material ( red ) . [SEP]
[CLS] the dna tetrahedron was also used as a scaffold to display streptavidin ( red ovals ) as an antigen and single - stranded cpg oligonucleotides ( purple strands ) as an adjuvant ( right ) , which was introduced via venous injections into the mouse bloodstream . [SEP]
[CLS] internalization of the dna tetrahedron into b - cells B-material ( blue ) and macrophages ( green ) leads to downstream activation of t cells B-material ( red ) , which in turn activate synthetic antibody B-material production against streptavidin by b cells B-material . [SEP]
[CLS] reproduced with permission from : a ( left ) , ref . 47 , nature publishing group ; a ( right ) , ref . 45 , nature publishing group ; b ( left ) , ref . 46 , nature publishing group ; b ( right ) , ref . 50 , american chemical society . [SEP]
[CLS] strategies to tune stability of designer dna nanodevices B-nanoparticle to tailor host immune responses . [SEP]
[CLS] dna architectures devoid of nicks or single - stranded domains show increased stability in vivo , which can be enhanced by encapsulation in non - immunogenic B-property polymers B-material ( i ) . [SEP]
[CLS] positioning nuclease - sensitive sequences ( blue region ; ii ) near n - way junctions decreases susceptibility to nucleases ( blunt blue arrows ) . [SEP]
[CLS] incorporation of modified , chemically crosslinkable nucleobases ( green region ; iii ) could enhance duplex stability in vivo . [SEP]
[CLS] top and bottom chemical structures show examples of crosslinkable nucleobases 79 , 80 . [SEP]
[CLS] non - natural nucleic B-material acid I-material analogues ( red ; iv ) , such as l - dna ( left chemical structure ) and peptide B-material nucleic B-material acid I-material ( right chemical structure ) , are not substrates for endogenous nucleases . [SEP]
[CLS] immunogenicity B-property of a designer dna nanodevice B-nanoparticle could be lowered by structurally shielding sequences of high immunogenicity B-property ( magenta regions ; v ) , and exposing sequences of low immunogenicity B-property ( orange regions ; vi ) to the external milieu . [SEP]
[CLS] part ( i ) reproduced with permission from ref . 102 , aaas . [SEP]
[CLS] nanomanufacturing , the commercially - scalable and economically - sustainable mass production of nanoscale materials and devices , represents the tangible outcome of the nanotechnology revolution . [SEP]
[CLS] in contrast to those used in nanofabrication for research purposes , nanomanufacturing processes must satisfy the additional constraints of cost , throughput , and time to market . [SEP]
[CLS] taking silicon B-material integrated circuit manufacturing as a baseline , we consider the factors involved in matching processes with products , examining the characteristics and potential of top - down and bottom - up processes , and their combination . [SEP]
[CLS] we also discuss how a careful assessment of the way in which function can be made to follow form can enable high - volume manufacturing of nanoscale structures with the desired useful , and exciting , properties . [SEP]
[CLS] and displays . [SEP]
[CLS] , , however , to realize the potential benefits of all of these diverse applications we must develop efficient , cost - effective and robust nanomanufacturing methods . [SEP]
[CLS] nanomanufacturing is a term whose usage varies with the approach , and rationale for the choice of approach , for fabricating 1 , 2 or 3 - dimensional nanostructures . [SEP]
[CLS] it can mean making small features on larger objects , ( e . g . integrated circuit [ ic ] fabrication ) , making nanoscale objects with special properties ( e . g . quantum B-nanoparticle dot I-nanoparticle synthesis ) , assembling nanoscale objects into more complex structures ( e . g . dna origami - directed assembly ) , incorporating nanoscale objects into larger objects to enable special functionality ( e . g . graphene into electronic devices or into liquor distillation apparatuses ) , , and using nanotechnology to manufacture nanoscale structures ( e . g . dip - pen nanolithography ) . [SEP]
[CLS] , given these varied interpretations , we must define our use of the terms " nanomanufacturing " and " nanofabrication " before proceeding further . [SEP]
[CLS] they are often used interchangeably , but , in the interest of adding a level of precision to the conversation , we will draw a distinction between them . [SEP]
[CLS] first , we note that here we employ a broad definition of nanofabrication that includes both conventional , top - down methods , such as those used in the production of semiconductors , as well as bottom - up methods such as chemical synthesis and selfassembly . [SEP]
[CLS] for the purposes of this discussion , we distinguish between nanofabrication and nanomanufacturing using the criterion of economic viability , suggested by the connotations of industrial scale and profitability associated with the word " manufacturing " . [SEP]
[CLS] nanomanufacturing , as we define it here , therefore has the salient characteristic of being a source of money , while nanofabrication , is often a sink . [SEP]
[CLS] in all cases , for a process or technology to be considered manufacturable , the cost of manufacturing and the volumes that can be produced must be consistent with the selling price and total addressable sales market . [SEP]
[CLS] in other words , if it is possible only to produce something in small volumes and at high cost , then it must command a high price ; conversely , if the product fetches a low price , then not only must the cost of production be correspondingly low , but the volumes required by the market must be large enough in order to make the enterprise economically self - sustaining . [SEP]
[CLS] , mathematically this relationship follows from the fact that the yearly revenue generated by a given tool or process is the selling price of the product ( vertical axis in fig . 1 ) multiplied by the amount of product generated per year . [SEP]
[CLS] the rate of product generation is most conveniently represented in terms of the throughput ( horizontal axis in fig . 1 ) . [SEP]
[CLS] a majority of the products generated by or processes used in nanomanufacturing concern what are essentially thin - film or quasi two - dimensional structures . [SEP]
[CLS] we therefore choose to represent the amount of product generated in units of area , specifically meters squared . [SEP]
[CLS] for example , the thickness of integrated circuits , hard drives , photovoltaics , sensors , and coatings B-material is extremely small compared to their area and so the amount of area correlates directly to the amount of product . [SEP]
[CLS] while this is not always the case , such as for catalysts B-property , nanoparticles B-nanoparticle and nanotubes B-nanoparticle , which can be made volumetrically , these are often used to cover or coat B-material a given area and so the area metric for production capacity in this case can be taken to refer to the " as used " or " as applied " area . [SEP]
[CLS] it is clear from fig . 1 that either a high throughput or a high selling price is required to achieve a given yearly revenue . [SEP]
[CLS] for example , a yearly revenue of $ 1 million can be obtained with a throughput of 10 −12 m 2 • s −1 only if the selling price is on the order of $ 10 billion per meter squared but the same revenue follows from a few cents per meter squared if the throughput is on the order of 1 m 2 • s −1 . [SEP]
[CLS] of course multiple tools and / or processes can be used to increase revenue but this is economically viable only if money can be made on each tool or process individually . [SEP]
[CLS] to supply some background and indicate the scale of the nanomanufacturing challenge , figure 2 shows the selling price ( $ • m −2 ) versus the annual production ( m 2 ) for a variety of nano - enabled or potentially nano - enabled products . [SEP]
[CLS] the overall global market sizes are also indicated . [SEP]
[CLS] it is interesting to note that the selling price spans five orders of magnitude , the production six , and the market size three . [SEP]
[CLS] although there is no strong correlation between the variables , it interesting to note that there is an overall trend , with smaller - volume products commanding a relatively high price , as would be expected from the simple model shown in figure 1 . [SEP]
[CLS] the diversity of market size , product volume , and price reinforces the idea that it is essential to carefully consider how to optimize the match between process and product . [SEP]
[CLS] in the context of the preceding discussion , the intent of this article is to provide an overview of the current state of and future prospects for nanomanufacturing . [SEP]
[CLS] some of the many nanofabrication techniques under development , , , , may one day be used in nanomanufacturing , but , in order to understand which ones are likely to make the transition to being a revenue source and not a sink , it is necessary to identify how the physical aspects of any given technique affect its ability to generate products that meet the desired functional requirements in a cost - effective manner . [SEP]
[CLS] every product in the market place has a set of " functional requirements " , i . e . , things it must do to be useful to the consumer . [SEP]
[CLS] the sophistication of these functional requirements drives the product specifications in terms of structural complexity , dimensional and compositional accuracy and precision , tolerable defect levels and the degree to which the product can be classified as being active as opposed to passive . [SEP]
[CLS] these product specifications can be used to determine which fabrication approaches potentially have the necessary capabilities . [SEP]
[CLS] the final choice of nanomanufacturing technology must be driven by the cost of ownership , which depends critically on the characteristics of the manufacturing process , including yield and throughput . [SEP]
[CLS] here we focus on nanomanufacturing primarily as it pertains to the creation of structures with a relatively high degree of functionality and structural complexity and hierarchy . [SEP]
[CLS] we do not address the production of nanomaterials B-material , nor the challenges associated with introducing them into the marketplace . [SEP]
[CLS] the majority of those challenges , apart from those connected with metrology , , are common to materials in general . [SEP]
[CLS] the rest of this article is organized as follows : first we examine the two broad classes of nanofabrication processes : top - down , i . e . , deterministic processes , and bottom - up , i . e . , stochastic processes ; then we consider the combination of top - down with bottom - up ; next we explore what might be possible by adding driven dissipative processes ; and finally we discuss the importance of design for nanomanufacturing . [SEP]
[CLS] at each step we illustrate the areas of applicability of the various approaches through examples . [SEP]
[CLS] before we begin our discussion of these two approaches , we must clarify our use of the words " deterministic " and " stochastic " . [SEP]
[CLS] deterministic manufacturing processes are designed to produce outcomes that exhibit very narrow distributions of the product performance mean and variation . [SEP]
[CLS] however , this does not mean that there cannot be significant randomness at the atomic or nanometer scale . [SEP]
[CLS] stochastic processes , while statistical in nature , can , when averaged over the large numbers of nanostructures typically involved , also exhibit narrow distributions of the product performance mean and variation . [SEP]
[CLS] another way of looking at this ( figure 3 ) is to consider the length scales over which variations occur . [SEP]
[CLS] short - range precision and accuracy may be excellent for some stochastic processes , such as protein B-material synthesis and folding , but tend to degrade rapidly at length scales larger than an individual unit . [SEP]
[CLS] in contrast , deterministic methods may be disordered at the atomic or nanometer scale , but can have exceptional long - range accuracy and precision . [SEP]
[CLS] as noted above , the product ' s functional requirements are used to determine if a given approach might be suitable . [SEP]
[CLS] top - down processes are fundamentally deterministic , in that order is imposed on the system by the action of external forces . [SEP]
[CLS] as gordon moore observed in his seminal 1965 paper , this means that yield is not dictated by equilibrium thermodynamic considerations - in contrast to , for example , chemical reactions . [SEP]
[CLS] yield in top - down processes can therefore can be engineered essentially to any desired degree consistent with physical laws : current ic manufacturing processes produce functioning devices with error rates below roughly 1 in 10 12 and have generally far fewer than 0 . 1 defects per centimeter squared . [SEP]
[CLS] it follows that , as long as there is no fundamental physical limit and the relevant economic drivers apply , the capabilities of top - down fabrication processes will tend to evolve according to typical learning curves . , , [SEP]
[CLS] integrated circuit manufacturing represents the apogee of top - down control over matter , yielding devices with unprecedented and ever - increasing levels of functionality in ever smaller spaces . [SEP]
[CLS] the six decades following the invention of the ic have seen the evolution of photolithography to the point where a modern photolithography tool , operating with an immersion lens at a wavelength of 193 nm , is capable of printing , at a resolution of 38 nm , 10 12 features per second . [SEP]
[CLS] it does this while maintaining control over the feature size to within 10 % , and the ability to overlay thirty or more layers with respect to one another to within an uncertainty of less than 5 . 5 nm . [SEP]
[CLS] , , this degree of control has of course also been enabled by concomitant progress in etch and deposition technologies and photoresist chemistry . [SEP]
[CLS] , the ability to print features which are so much smaller than the wavelength of the exposing radiation requires careful engineering of the mask pattern which in the end has no simple relationship to the features being printed on the wafer . [SEP]
[CLS] , this type of mask engineering must be combined with simultaneous optimization and precise control of the illumination incident on the mask to create the required intensity distribution at the wafer . [SEP]
[CLS] , in fact , the complexity of the coupled illumination - mask diffraction problem is so great that each future generation of chips relies on the computing power made available by the current generation of devices to solve it and , for all but the highest - volume devices , the mask cost is the dominant factor in the cost of ownership . [SEP]
[CLS] , in addition , multiple masks may be required to print the features for a single level when double - or multiple - patterning approaches are used to achieve the desired feature density . [SEP]
[CLS] , , , , as noted above , it is the economic advantage gained by increasing integration that drives the technological progress in ic production , and it is this economic advantage that will determine whether or not the current incarnation of photolithography gives way to extreme ultraviolet lithography ( euv ) , which operates at a wavelength of 13 . 5 nm . [SEP]
[CLS] the use of this much shorter wavelength brings many additional complexities , and therefore higher costs , as far as the lithography tool is concerned , but can , in principle , reduce the mask complexity thereby reducing the overall cost of ownership . [SEP]
[CLS] , the final result will depend on whether a suitable combination of resist sensitivity and illumination power can be reached to deliver economically viable throughputs . [SEP]
[CLS] alternatively , the drive for greater circuit densities and functionalities may be satisfied by the use of 3d approaches . [SEP]
[CLS] , , , , , as impressive as the current state of the art is , it is important to point out that photolithography is only as good as it needs to be . [SEP]
[CLS] in particular , for integrated circuits to function , it is only the relative placement of individual circuit levels across the chip that must be controlled . [SEP]
[CLS] variations in the overall size and shape of the chip up to a few percent are acceptable as long as the level - to - level overlay is maintained . [SEP]
[CLS] thus , for ics , absolute accuracy is not critical . [SEP]
[CLS] on the other hand , with the increasing emphasis on combining nanophotonic and nanomechanical devices with conventional ics , absolute accuracy will be extremely important since both nanophotonic and nanomechanical devices typically are required to operate at a fixed external wavelength or frequency . [SEP]
[CLS] for example , an on - chip nanophotonic device structure such as an add - drop filter comprising ring resonators , waveguides and / or gratings operating in the 1550 nm telecommunications band with a 30 ghz channel separation ( ≈ 0 . 3 nm wavelength difference ) nominally requires dimensions and / or periodicities accurate to within a fraction of the wavelength difference , i . e . a few picometers . [SEP]
[CLS] these requirements are beyond the current capabilities of photolithography and necessitate an increase in the overall device complexity to include systems which can tune them to match a given external wavelength or frequency . [SEP]
[CLS] even a perfectly fabricated device will require tuning to compensate for thermal effects which shift the operating wavelength . [SEP]
[CLS] embossing processes have been extended to the nanoscale , opening up a range of new applications . [SEP]
[CLS] nanoimprint lithography , , , , can , in principle , not only produce features at the size needed in integrated circuit production , but , because the imprint template is replicated precisely , can do so without those complexities inherent in sub - wavelength optical lithography mentioned above : the features on the template look exactly like the features on the substrate . [SEP]
[CLS] however , this means that the patterned area on the imprint mask is the same size as the patterned area on the wafer ( it is therefore described as a 1× mask ) and fabricating a 1× mask to the required degree of precision is not trivial . [SEP]
[CLS] pattern placement capabilities close to those of photolithography tools have been achieved by introducing schemes that use controlled deformation of the imprint template . [SEP]
[CLS] , , , such schemes , coupled with improvements in template fabrication processes , are currently suitable for ic manufacturing at feature sizes of ≈ 20 nm ( i . e . the 2× nm nodes ) . [SEP]
[CLS] , , , another potential benefit comes from the ability of nanoimprint to print multiple pattern levels simultaneously , which can result in a significant reduction in the number of process steps needed to complete a device . [SEP]
[CLS] , , , , aside from meeting overlay requirements , the principal obstacle facing the technology is for it to achieve the same throughput and low level of defects as photolithography . [SEP]
[CLS] nanoimprint ' s ability to fabricate almost arbitrarily small features precisely and accurately means that it is almost uniquely suited to the production of bit - patterned magnetic B-property storage media , , , , which have tolerances on feature size and size variation that are significantly more stringent than those for ics . [SEP]
[CLS] interestingly , although the local pattern placement specification for bit - patterned media ( bpm ) must be better than 1 . 25 nm because the flying read head cannot track glitches in position , the long - range pattern placement requirement is only 10 µm or approximately 0 . 3 % , because the head can track long - range , slowly - varying placement errors . , for nanoimprint to be an appropriate choice of manufacturing technology for bit - patterned media , it is not enough that it can produce small features competitively with photolithography because the price per unit area must be two orders of magnitude lower . [SEP]
[CLS] part of the necessary fabrication cost reduction may be achieved because only a single level needs to be patterned , which reduces the tool complexity and cost as well as the number of fabrication steps , and part by dramatically lowering the amortized mask costs , by using a single master to generate up to 10 000 copies , which can then each generate 10 000 diskdrive platters . [SEP]
[CLS] additional cost reductions come from the much higher defect density that can be tolerated for bpm as opposed to ics ( 1 in 10 4 versus 1 in 10 12 ) because of the availability of read - channel error correction schemes . [SEP]
[CLS] , a less demanding specification for defect density translates into a reduced need for costly defect inspection during manufacturing . [SEP]
[CLS] as the technology evolves and defect levels decrease , it is even being considered for the production of flash memory , which has relatively relaxed overlay requirements , , though defect levels will need to be reduced to ~ 0 . 1 cm −2 . [SEP]
[CLS] an attractive feature of imprint or embossing processes in general is that they can be adapted for use in continuous , roll - to - roll ( r2r ) manufacturing , , , , , , which reduces the cost of fabrication and increases the throughput . [SEP]
[CLS] the cost per meter squared may range between 0 . 1 $ • m −2 and 10 $ • m −2 , depending on whether direct embossing or a thin - film ultraviolet light curing B-property process is used . [SEP]
[CLS] it is difficult to achieve precise overlay and long - range placement accuracy in r2r because of the tendency of the flexible substrate ( called the " web " ) to deform during processing . [SEP]
[CLS] single - level structures , used in applications such as large - area reflective coatings B-material , , , and holographic wrapping paper , do not suffer from these problems . [SEP]
[CLS] extending single - level r2r technology to the nanoscale would enable applications such as organic photovoltaics , , light - management films and wire - grid polarizers for high - contrast displays , , anti - reflective coatings B-material , super - hydrophobic ( philic ) surfaces , as well as those requiring plasmonic activity . [SEP]
[CLS] , , self - aligned imprint lithography uses a multilevel template that produces pre - aligned structures , avoiding level - to - level alignment problems . [SEP]
[CLS] it can be used to generate large - area , low - cost electronics such as display backplanes with minimum feature sizes of 1 µm at web speeds of up to 5 m / min . [SEP]
[CLS] we also note that , while stitching defects are important in displays , other forms of defects are much less significant . [SEP]
[CLS] nanostructured surfaces produced by r2r imprint may also be useful for energy generation and storage if high aspect ratio features can be generated . [SEP]
[CLS] nanoimprint is already used to produce nanoparticles B-nanoparticle for diagnostic and therapeutic applications . [SEP]
[CLS] , , , at first sight , it might appear that high - volume particle - production techniques such as milling , would be by far more economical . [SEP]
[CLS] however , medical and biological applications generally require very precise control over particle size and shape , and the small amount of product needed to achieve the desired effect [ e . g . drug delivery , imaging contrast ] , , , and the high value associated with medical treatments make this an economically viable approach . [SEP]
[CLS] in addition , nanoimprint can be a relatively gentle process , creating patterns without the need for high temperatures or aggressive chemicals , and thus allowing the safe handling of fragile biomolecules . [SEP]
[CLS] perhaps the most exciting aspect of nanoimprint is that it is heir to all the numerous macroscale embodiments of the printing process . [SEP]
[CLS] at the most fundamental level , these all transfer material B-material from a patterned surface to a substrate . [SEP]
[CLS] functional inks are already enabling a revolution in printed lectronics for flexible displays , wearable electronics and the " internet of things " , and there is every reason to suppose that one as profound will occur as inks are developed for nanoscale applications . [SEP]
[CLS] as an example , microcontact printing , , the small - scale analog of flexographic printing , has recently been demonstrated at the nanoscale . [SEP]
[CLS] similarly , nanotransfer printing , analogous to transfer printing on ceramics , enables the fabrication and heterogeneous integration of complex nanostructures made from a wide variety of materials . [SEP]
[CLS] , , , , , , this gives the family of nanoimprint methods the potential to be a true nanomanufacturing platform technology , limited only by the availability of suitable templates , inks , and surface energy control . [SEP]
[CLS] optical lithography and nanoimprint are the dominant top - down nanomanufacturing methods , despite there being a large number of other nanofabrication approaches available . [SEP]
[CLS] at this point , it is worth asking why these other techniques have not made the transition into nanomanufacturing . [SEP]
[CLS] one of the principle obstacles that must be overcome is reaching an economically viable throughput . [SEP]
[CLS] electron - beam lithography , for example , can generate sub - 10 nm features , , , over large areas , with good placement and overlay but , because of its relatively low throughput , it is limited commercially to the production of masks for use in photo - and nanoimprint lithography and device development , , and non - commercially to the production of nanostructures for research and defense purposes . [SEP]
[CLS] the prospects for increasing the throughput of electron - beam systems are severely limited because of the fundamental physics of space - charge effects - the repulsion between neighboring electrons in a single electron column leads to a loss of resolution or blurring of the beam as the beam current increases . , , this limits the maximum beam current , and hence the throughput , that can be attained at a given resolution . [SEP]
[CLS] this inability to scale led to the demise of early programs focused on creating electron - beam systems for ic production . [SEP]
[CLS] , however , the economic pressures associated with leading - edge optical lithography still makes this an active area of research , with newer efforts relying on groups of columns , or targeted towards low - volume applications , under development . [SEP]
[CLS] , , a common response to this type of scaling problem is to propose the use of massively parallel arrays of columns or tips , , to boost the throughput . [SEP]
[CLS] achieving the required feature size control means that the beam size and dose delivered must be well calibrated or dynamically controlled across the array . [SEP]
[CLS] however , the electron source for each column is typically a field emitter for which the beam current is exponentially dependent on the emission area and geometry . [SEP]
[CLS] even if each emitter can be made identical , as soon as they are put into operation the evolution of the nanoscale emission area resulting from phenomena such as surface diffusion , ion B-material bombardment , absorption of contaminants , etc . will cause the emission characteristics for the individual tips to diverge from one another . [SEP]
[CLS] feedback control to remedy this problem is possible in principle , , but has so far proven to be extremely difficult to implement in practice . [SEP]
[CLS] similar considerations apply to many forms of scanning probe lithography , where attempts at parallelization are frustrated by , for example , tip wear . [SEP]
[CLS] , a notable exception to this is dip - pen nanolithography , and its variants , , which , through its ability to directly pattern different types of chemistries , including biological ones , , targets and satisfies a set of constraints different from those relevant to ic device fabrication . [SEP]
[CLS] this characteristic qualifies it as a disruptive technology , , and the same may be true of other fabrication processes that also offer benefits orthogonal to those provided by ic fabrication methods . [SEP]
[CLS] before we leave the discussion of top - down fabrication methods , it is worth considering in which circumstances a particular nanofabrication process might reasonably be associated with nanomanufacturing . [SEP]
[CLS] 4 shows the throughput versus cost for patterning methods used in ic manufacturing . [SEP]
[CLS] as we have described above , optical lithography meets the cost and throughput targets needed for integrated circuit manufacturing , while electron - beam lithography does not . [SEP]
[CLS] however , the masks used in photolithography , and indeed the master templates for all manner of print - based nanomanufacturing , are made using electron - beam lithography . [SEP]
[CLS] similarly , focused ion B-material beam patterning is , by absolute standards , very slow and extraordinarily costly , and would normally be considered only as a nanofabrication approach suitable for research . [SEP]
[CLS] but , when used for high - value operations such as circuit edit and mask repair it is an economically viable part of the ic manufacturing process flow . [SEP]
[CLS] these examples serve to further reinforce the need for a careful analysis to match fabrication techniques with products . [SEP]
[CLS] in contrast to the deterministic nature of top - down processes , bottom - up processes are driven by a combination of thermodynamics and kinetics which then determines the yield of the desired structure . [SEP]
[CLS] the most attractive features of bottom - up nanomanufacturing processes are that there is typically no need for expensive tooling to create nanoscale structures , and scaling to large volumes is potentially straightforward . [SEP]
[CLS] by applying the tools of chemical synthesis , quantum B-nanoparticle dots I-nanoparticle , , plasmonically - active particles , , , carbon B-nanoparticle nanotubes I-nanoparticle , metallic nanowires B-nanoparticle and multifunctional particles for medical applications , , , , have been successfully produced in manufacturing quantities . [SEP]
[CLS] efforts to develop purely bottom - up self - assembly methods to create more complex devices , , , typically rely on engineering the interactions between the various components , placing them in a simple environment and then letting the system evolve to a final state . [SEP]
[CLS] for example , consider the case where there is one relative arrangement of the elemental units that meets the product ' s functional requirements . [SEP]
[CLS] if the energy of this particular arrangement is e and , out of all the possible arrangements , there are n other arrangements all with energy e n close to e which are not the desired structure , and all other arrangements have much higher energy , then the odds of getting the desired structure , assuming the system is in thermal equilibrium , are on the order of where k b t is the thermal energy at the end of the production cycle . [SEP]
[CLS] in general , as the number of units needed to build the target structure increases there are likely to be more arrangements that have energy close to e i . e . , n increases and so the odds of the getting the " right " result decrease . [SEP]
[CLS] hence , unlike top - down manufacturing , the product yield is statistically determined . [SEP]
[CLS] this means that , when high yields ( i . e . high purity ) are needed , costly separation and purification steps are required . [SEP]
[CLS] the expression above represents the best case for a single - step or one - pot process , and is based on the assumption that thermodynamic equilibrium can be reached within a relevant timescale . [SEP]
[CLS] however , as systems become more complex , the phenomenon of kinetic trapping , can prevent them from reaching the desired equilibrium state . [SEP]
[CLS] this effect is most easily understood in terms of the potential energy landscape of the elemental units . [SEP]
[CLS] , if the elementto - element potential energy depends on the relative position and orientation of each unit with respect to each other and there are n elemental units then , up to an overall rotation and translation , the potential energy landscape is a function of all the 3n + 3n = 6n position and rotation coordinates of each elemental unit . [SEP]
[CLS] in general , the 6n dimensional potential energy landscape will have numerous metastable local minima , and only one global minimum which defines the desired assembled structure . [SEP]
[CLS] the challenge in the bottom - up assembly of complex structures is then to engineer the element - to - element interactions , formation of intermediate B-property structures , and process conditions ( e . g . annealing schedule ) so that there is a clear path for the components to follow through the potential energy landscape to the global minimum . [SEP]
[CLS] , alternatively , applications must be targeted that require , for example , only shortrange order and / or allow for a high defect level . [SEP]
[CLS] colloidal self - assembly has been investigated intensively for many years , , , , , because of the potential for colloidal structures to produce photonic bandgap materials , and high - density magnetic B-property recording media . [SEP]
[CLS] early work , , was directed towards trying to use photonic bandgap materials generated in this way for nanophotonic applications . [SEP]
[CLS] however , the difficulty of avoiding kinetic trapping , to achieve the requisite structural perfection , engineering in features such as waveguides , and programmed point defects , and finding cost - effective ways to integrate the structures produced with other photonic devices proved too great to overcome . [SEP]
[CLS] nanophotonic structures are now typically fabricated using top - down methods . , , the same is true of bpm . [SEP]
[CLS] in contrast , self - assembled colloidal structures are ideal candidates for the generation of large - area , low - cost , structural - color materials , , , because the degree of perfection required to meet the functional specification is so much less and the ability to scale production to large areas through roll - to - roll processing is so much greater . [SEP]
[CLS] colloidal self - assembly specifically at the nanoscale has exciting possibilities in terms of generating novel materials by combining nanoparticles B-nanoparticle with different properties into welldefined crystalline structures . [SEP]
[CLS] , here again , it is important to identify applications for which such materials can be integrated into a manufacturing process flow . [SEP]
[CLS] dna is the archetypal self - assembling system , with tremendous flexibility in the types of structures that can be produced , based on single - stranded ( ssdna ) , double - stranded or duplex ( dsdna ) , and more complex supra - molecular assemblies . [SEP]
[CLS] , one - , two - and threedimensional structures can be made , and the ability of other nanoscale objects to be functionalized with dna , combined with the specificity conferred by complementary sequence recognition , means that dna can connect and organize disparate nanostructures to make relatively complex constructs , , , , , , , , , including well - controlled nanoparticle B-nanoparticle crystal lattices , , , , , and even active systems . [SEP]
[CLS] , , , , dna origami is a prime example of the power of dna to control the arrangement of nanoscale objects , providing a molecularly precise " breadboard " to which nanostructures can be attached . [SEP]
[CLS] , , , , , , in addition , dna structures can be responsive to variations in temperature , ionic species / concentration and ph . , , it is also possible to vary the number and strength of dna - mediated interactions between nanoparticles B-nanoparticle , which can lead to interesting stimulus - dependent responses , allowing the creation of new , environmentally - responsive nanostructures . [SEP]
[CLS] , , , , [SEP]
[CLS] the development of dna - based self - assembly is still at a relatively early stage , though progressing rapidly , , and there are few studies on the yield , speed and ultimate levels of complexity that can be achieved in single units and assemblages on substrates . [SEP]
[CLS] , in addition , although the underlying arrangement of dna may be precise , the presence of linker molecules that must be used between the nanostructure and the dna and the fact that dna structures are not perfectly rigid , , , , inevitably lead to a reduction in placement precision . [SEP]
[CLS] the diffusional nature of the assembly process and the typical rate constants for the reactions involved mean that it takes a long time to create complex structures and that the yields for those structures are going to be consistent with chemical synthesis , not top - down fabrication , even though substantial progress is being made in developing optimized annealing schedules and buffer compositions . [SEP]
[CLS] additional improvements may be achieved by controlling the energetics of the structure to guide the assembly process and formation of secondary structure . [SEP]
[CLS] , , , applications , such as the fabrication of vaccines and other biomedically - active structures , , for which 100 % yield and purity , precise placement , and control of multiple levels of structural hierarchy are not prerequisites , therefore need to be identified for this technology to be used in nanomanufacturing . [SEP]
[CLS] finally , it is important to note that , while dna itself is not particularly robust , recent work has shown how structures produced using dna can subsequently be encapsulated in silica - based materials to dramatically improve their environmental stability . [SEP]
[CLS] so far we have discussed the strengths and weaknesses of top - down and bottom - up approaches to nanomanufacturing , but combining the two together can yield the best of both worlds . [SEP]
[CLS] guided or templated self - assembly typically makes use of boundaries created by top - down methods that interact with a system that has an intrinsic structural length scale . [SEP]
[CLS] this latter can arise from the balance between long - range magnetic B-property , electrostatic , or strain energy , , or , as in the case of block copolymers , can come from local interactions built into the molecular structure of the material B-material . [SEP]
[CLS] block copolymers phase separate on the nanoscale , with an intrinsic length scale determined by the molecular weights of the components and a structure determined by their relative volume fractions . [SEP]
[CLS] nanostructures formed in this way can be functional themselves , , , , , can be used to template the formation , or arrangement , , , , , , , of other nanostructures , produce materials responsive to their environment , , or can be used to pattern an underlying material B-material . [SEP]
[CLS] , while using a self - assembled structure as an intermediate B-property step in a patterning process , as opposed to the final functional structure , may seem to introduce unnecessary process complexity , there are other challenges involved in using functional materials directly . [SEP]
[CLS] in the case of diblocks , creating a material B-material that phase separates at the requisite length scale , has the necessary surface energies to assemble in the desired orientation with respect to the substrate , has the correct kinetic behavior to minimize defects , can be coated in thin - film form , all while maintaining the sought - for functionality , gives some idea of the difficulty involved . [SEP]
[CLS] given these factors , it becomes clear that the investment required to develop a suitable material B-material system is only worthwhile for high - volume / high - value applications . [SEP]
[CLS] long - range order can be introduced by using a sparse templating pattern generated by topdown methods , and is a very attractive route to making well - controlled nanoscale features : it greatly relaxes the requirements for the top - down process in terms of feature spacing and throughput and deals with the limitations of the bottom - up assembly process . [SEP]
[CLS] , , in addition , this approach can even be used to generate relatively complex three - dimensional structures , and is therefore being considered for the manufacture of ics , bpm , , , , , , , , and other structures . [SEP]
[CLS] photolithography is limited , not in terms of the smallest feature size that can be produced , , but in its ability to place them close together ( the minimum pitch attainable in a single patterning step is λ / 2na ) . [SEP]
[CLS] by using photolithography to create a guiding pattern and the assembly of a diblock to fill in the details , it is possible to make dense , nanoscale patterns with excellent control . [SEP]
[CLS] , , , similarly , cylindrical or spherical diblocks can be templated by sparse patterns of posts made by electron - beam lithography to form well - ordered arrays of features for bpm . [SEP]
[CLS] in this case the benefit lies in both dramatically reducing the time needed for the electron - beam lithography step and in the ability of the diblock to effectively repair patterning defects and generate much more uniform and dense patterns than could otherwise be produced . [SEP]
[CLS] an interesting feature of this approach is the interplay between the templating pattern and the diblock which , by altering the energetics of the system , can lead to either a reduction or an increase in the defect level . , , there are limits to how sparse a templating pattern can be employed . [SEP]
[CLS] in the bpm case if the guiding features are too far apart , then there is a degeneracy because more than one orientation of the diblock can match the templating pattern , leading to the formation of domain boundaries . [SEP]
[CLS] in lamellar diblocks , as the distance from a directing boundary increases , undulations in the interfaces increase to the point where they lead to an unacceptable level of line - edge roughness for ic fabrication . [SEP]
[CLS] , these limitations represent design constraints , but are unlikely to impede the adoption of this technology in manufacturing . [SEP]
[CLS] the time taken for diblock systems to order can be relatively long and increases with increasing molecular weight , , but recent work using hightemperature , , , , solvent annealing , , or a combination , , indicates that this is unlikely to be a serious issue . [SEP]
[CLS] reducing chain entanglement with brush block copolymers is also an effective strategy . [SEP]
[CLS] , however , these types of directed self - assembly processes are restricted in terms of the amount of information that can be added to the system and are capable only of producing single harmonics of the templating structure . [SEP]
[CLS] additional patterning steps will therefore always be needed to create the kind of structural complexity necessary for logic devices . [SEP]
[CLS] finally , although the number of equilibrium defects is expected to be negligible , , eliminating defects related to the templating structures is still challenging , and is becoming more so as feature sizes decrease , requiring smaller molecular weight diblocks with smaller domain sizes operating closer to the order - disorder transition . [SEP]
[CLS] this latter effect is leading to the search for materials with higher flory - huggins interaction parameters ( χ ) , , , or for smallmolecule additives that can be used to drive phase separation . [SEP]
[CLS] , , , , , [SEP]
[CLS] one difficulty in controlling the assembly of nanoscale objects is finding interactions that are strong enough to manipulate them , and that scale well to small dimensions . [SEP]
[CLS] capillary interactions can satisfy these requirements , and have been used to create a variety of interesting structures . [SEP]
[CLS] , in particular , the capillary interactions that occur at a fluid interface on a patterned substrate can be used to assemble nanoparticles B-nanoparticle precisely and with high yield onto lithographically patterned features . [SEP]
[CLS] , , the convective flows that are set up at a meniscus can make the process quite efficient , by concentrating nanoparticles B-nanoparticle at the fluid - substrate contact line , , , , , , , , - the so - called coffee - stain effect . [SEP]
[CLS] beyond the need to control the contact angle between substrate and fluid within a fairly forgiving range , this process is agnostic with regard to the nature of the substrate and the nanoparticles B-nanoparticle and can so be used to assemble a wide variety of materials without the need for any kind of harsh processing involving solvents , acids / bases or energetic plasmas . [SEP]
[CLS] additionally , once assembled onto a templating substrate , the nanoparticle B-nanoparticle structures can readily be transferred to a different material B-material . [SEP]
[CLS] , unfortunately , the maximum linear contact line speeds achieved so far are only ~ 1 µm • s −1 to mm • s −1 , , , , which , even if used in a roll - to - roll process with a meter wide web , translate into an areal throughput of 10 −6 m 2 • s −1 to 10 −3 m 2 • s −1 [SEP]
[CLS] the limiting factors are the overall concentration of nanoparticles B-nanoparticle , which cannot be increased indefinitely without causing deposition in un - patterned areas , and the evaporation rate of the carrier fluid , which also cannot be increased dramatically . [SEP]
[CLS] interestingly , the combination of a roll - to - roll patterned substrate with inkjet printing has been used to create color filters with a precision far better than can be achieved with inkjet alone . [SEP]
[CLS] although currently only being used for micron - sized features , this is potentially a highly extensible approach . [SEP]
[CLS] unlike the self - assembling systems described above , which simply " fall down " a free - energy landscape to a stable equilibrium , damped driven systems require energy input in order to form and maintain a self - organized structure . [SEP]
[CLS] the belousov - zhabotinsky reaction , is a wellknown example , but the archetype is a living being . [SEP]
[CLS] all living things require energy inputthe driver - or they die and decay . [SEP]
[CLS] the input energy is dissipated as work and heat - the damping . [SEP]
[CLS] this constant flow of energy through the system maintains it in its self - organized form , for example in all aspects of intracellular transport . [SEP]
[CLS] , , although we have learned to harness living systems to manufacture everything from alcohol B-material to spider silk , in contrast to systems at equilibrium , very little is known about the general principles governing dampeddriven , self - organizing systems , , preventing us from creating our own . [SEP]
[CLS] in this context one particular biological process is worth discussing : protein B-material folding . [SEP]
[CLS] key parts of the folding process require energy input which is dissipated as heat and so this is a damped driven process . [SEP]
[CLS] , , folding occurs much more rapidly than would be expected if it was purely a stochastic approach to equilibrium as when colloidal particles are annealed into crystalline structures . [SEP]
[CLS] protein B-material folding is not a random walk but rather a quasi - deterministic trajectory across the potential energy landscape directed both by the internal structure of the molecule as well as by the action of external , atp - driven , " chaperone " molecules , known as chaperonins . [SEP]
[CLS] , the concept of using both internal structure combined with external control may be the most effective way forward for creating more complex , functional nanostructures cost - effectively . [SEP]
[CLS] so far , we have discussed how various nanomanufacturing approaches may or may not be suited to the economically sustainable production of functional structures and devices . [SEP]
[CLS] it is also important to remember that there are often a number of different structures that will yield similar functionality . [SEP]
[CLS] choosing the right form can make the difference between something remaining a laboratory curiosity or becoming a product . [SEP]
[CLS] as a case in point , consider optical metamaterials : the first demonstrations of negative index behavior in the microwave were achieved using macroscale features , such as split - ring resonators , fabricated using simple , scalable , printed - circuit board methods . [SEP]
[CLS] subsequent attempts to make materials active at visible wavelengths used the same geometries , replicated at scales 10 −4 to 10 −5 smaller using electron - beam lithography . [SEP]
[CLS] , although these successfully demonstrated the desired functionality , fabrication , integration and scaling remain challenging , and the cost of such structures is prohibitive ( $ 10 6 • m −2 to $ 10 9 • m −2 , depending on whether variable shaped - beam or gaussian beam patterning is used ) . [SEP]
[CLS] a more complete understanding of nanoscale light - matter interactions has led to a new generation of optical metamaterials that comprise alternating layers of metals B-material and dielectrics . [SEP]
[CLS] , , these are eminently manufacturable over large areas and at low cost using conventional thin - film deposition processes . [SEP]
[CLS] as a point of comparison , the types of films made this way , such as those used for touch screens are ~ $ 10 • m −2 . [SEP]
[CLS] a similar development process has occurred in the quest for gecko - type dry adhesives . [SEP]
[CLS] early attempts focused on faithful replication of the biological structure via complex lithography to achieve the desired function . [SEP]
[CLS] more recent work has used the same design principles as the natural system , but in the form of a composite textile that can be made simply , using standard methods . [SEP]
[CLS] this type of device structure / fabrication process evolution is familiar in the semiconductor industry . [SEP]
[CLS] circuit size reduction often takes place first via a " dumb shrink " or through the use of representative test structures , rapidly followed by an optimized device structure and process redesign that incorporates any new physics and fabrication constraints whose importance has been elucidated during the initial learning phase . , [SEP]
[CLS] the term nanomanufacturing covers a host of different materials , devices , products and processes and is simply too broad to cover in detail in a short article . [SEP]
[CLS] in this brief survey we have therefore endeavored to highlight the importance of matching the process to the functional requirements of the product , and especially of including economic viability as the distinguishing criterion that separates nanomanufacturing from nanofabrication . [SEP]
[CLS] we have also illustrated how a deep understanding of the link between form and function can lead to manufacturability . [SEP]
[CLS] as we consider the range of products that involve some form of nanomanufacturing , some framework is necessary to enable comparisons between them . [SEP]
[CLS] in figure 5 we introduce such a framework . [SEP]
[CLS] the horizontal axis is again the selling price in $ • m −2 . [SEP]
[CLS] the vertical axis represents the complexity of the item being produced . [SEP]
[CLS] the complexity is captured by a compound term representing the information content of the product and the precision and perfection with which it must be produced in order to satisfy the functional requirements . [SEP]
[CLS] we choose the kolmogorov complexity parameter , k , as the measure of the information content of an object - it can be thought of as the number of bits required to specify the desired structure . [SEP]
[CLS] the precision is determined by dividing the maximum coherence length of the object , ξ max , by the product of the minimum feature size , d min , and the fractional tolerance , f , of that minimum feature size . [SEP]
[CLS] the perfection , p , is the maximum allowable fraction of defective components , number density of defects ( " defectivity " in ic manufacturing ) , or concentration of impurities B-property . [SEP]
[CLS] the combination of ( ξ max / ( f • d min • p ) ) represents the difficulty of producing the item to the required standard . [SEP]
[CLS] putting this all together , we define the manufacturing complexity , m c , as log 10 [ k • ξ max / ( f • d min • p ) ] . [SEP]
[CLS] as an example , only a few bits are needed to specify a tio 2 nanoparticle B-nanoparticle . [SEP]
[CLS] we need only a kilobit or so to define the composition , size , and acceptable size distribution . [SEP]
[CLS] no long - range structural coherence is necessary , and the allowable fraction of defective particles is most likely a few percent for most applications . [SEP]
[CLS] this leads to an m c of 5 . [SEP]
[CLS] in contrast , the cad data for an integrated circuit is many gigabits ( 10 9 ) , spatial coherence may be required over 1 cm for feature sizes of ≈ 10 nm , which must be produced with a size variation of no more than 10 % , and a defect level of 10 −12 is required . [SEP]
[CLS] m c for an ic is therefore ≈ 28 . [SEP]
[CLS] in general , as m c increases , so does the functionality of the product and the price it commands . [SEP]
[CLS] thus , near the origin , we find bulk nanomaterials B-material , at intermediate B-property levels of complexity and price , functional structures , and at the highest point , integrated circuits . [SEP]
[CLS] while there is a strong correlation between price and manufacturing complexity , it is instructive to consider some of the outliers . [SEP]
[CLS] a blu - ray disk , for example , is cheap to manufacture , but commands a relatively high price because of the information , e . g . a movie , that it contains . [SEP]
[CLS] we label the upper left quadrant of the graph " bits " to indicate that in this region selling price is dominated by information content . [SEP]
[CLS] in contrast , the price of gold B-nanoparticle nanoparticles I-nanoparticle functionalized with a biomolecule is dominated by the cost of that particular molecule . [SEP]
[CLS] we therefore label the lower right quadrant " atoms B-material " to indicate that the price is controlled by the cost of some scarce material B-material . [SEP]
[CLS] finally , we note that both the selling price and manufacturing complexity may change . [SEP]
[CLS] for example , once production is scaled , manufacturing costs may drop , or competitors may enter a market and drive prices down . [SEP]
[CLS] alternatively , as discussed above , a more complete understanding of how structure is related to function may allow both for a reduction in information content and the use of lessexpensive production technology . [SEP]
[CLS] in terms of their complexity or functionality versus their cost per unit area , it appears as though there are two distinct areas where current activity is located . [SEP]
[CLS] as might be expected , there is a concentration of devices and structures concerned with information processing , storage and transmission at the high - value end , while bulk materials and structures with limited structural complexity and functionality occupy the low - value end . [SEP]
[CLS] interestingly , there is , to date , very little in the way of nanosystems with an intermediate B-property degree of complexity and functionality , with high - value nanostructures engineered for theranostic applications representing the present state of the art . [SEP]
[CLS] at this point it is worth considering why this might be the case . [SEP]
[CLS] the ability to produce complex , hierarchical systems depends on the availability of physical components that have a high degree of modularity , the organization of those components into functional building blocks , and a design process that allows for multiple , high levels of abstraction . [SEP]
[CLS] the basic component of digital integrated circuits , the transistor , is physically modular , and can be configured into modular functional units ( arithmetic logic units ) , enabling the design to proceed at increasingly high levels of abstraction . [SEP]
[CLS] interestingly , the techniques developed for managing complexity in integrated circuits are now being applied to biological systems . [SEP]
[CLS] the lack of similarly modular physical and logical basic units , reflected by a lack of standard process modules and minimal design automation , respectively , explains why microelectromechanical systems ( mems ) and nanophotonic systems have yet to evolve to the same level of sophistication as integrated circuits . [SEP]
[CLS] in contrast , the rapid progress in dna - based nanotechnology flows from the fact that it offers a system optimized over 3 . 5 billion years that already exhibits the desired structural and functional modularity . [SEP]
[CLS] ultimately , the ability to manufacture , i . e . , create a sustainable business , depends on satisfying both technical constraints - including throughput , process yield , throughput and flexibility , energy consumption , and environmental sustainability - and a host of constraints relating to cost - including capital equipment expenditure , profit margin , equipment depreciation timescale , market volume , and market cycle time . [SEP]
[CLS] finding the optimum tradeoff in such a multi - dimensional parameter space is challenging . [SEP]
[CLS] frequently , the relevant variables can only be poorly estimated and , as illustrated above , the rapid pace of improvement in basic understanding can suddenly transform something from a laboratory curiosity into a potential product . [SEP]
[CLS] so , what does this all mean ? as yogi berra observed , " it ' s tough to make predictions , especially about the future . " , but what follows is our best guess . [SEP]
[CLS] the technology to fabricate integrated circuits will continue to evolve in capability and cost , but will remain uneconomic for low value - per - unit - area , high - volume products . [SEP]
[CLS] the family of lithographic technologies , such as nanoimprint , whose development has been driven in large part by the semiconductor industry , will be scaled to suit a variety of cost structures and so will find a wide range of applications , especially for those structures requiring only a single patterned layer . [SEP]
[CLS] bottomup self - assembly will have a role in the production of simple functional materials that are used in high volumes and must be inexpensive , while directed assembly allows for the imposition of longer - range order and hierarchy that will be important for some applications . [SEP]
[CLS] perhaps the most exciting prospect is that of creating dynamical nanoscale systems that are capable of exhibiting much richer structures and functionality . [SEP]
[CLS] whether this is achieved by learning how to control and engineer biological systems directly , or by building systems based on the same principles , remains to be seen , but will undoubtedly be disruptive and quite probably revolutionary . [SEP]
[CLS] refer to web version on pubmed central for supplementary material B-material . [SEP]
[CLS] vocabulary nanofabricationany and all techniques for creating nanoscale structures nanomanufacturing the scaled - up , reliable , and commercially - viable production of nanoscale materials , structures , devices , and systems top - down far - from - equilibrium processes in which structure is created deterministically by the introduction of information and the imposition of external forces , fields , or other actions bottom - up processes in which structure is determined predominantly by information intrinsic to the system itself , such as the specific sequence of chemical bonds or amino B-material acids I-material , such processes are inherently statistical or stochastic in nature and result in thermodynamically - stable structures and devices self - assembly the observed behavior of a system for which an ordered state is thermodynamically favorable and kinetically accessible damped - driven systems , processes or structures that require some type of input , such as energy , to form and maintain their structure ( the driven part ) , and which produce heat or entropy as part of their existence ( the damped part ) , the prime example being living organisms . [SEP]
[CLS] log - log plot of the product selling price ( $ • m −2 ) versus single tool throughput ( m 2 • s −1 ) with contours showing the relationship between throughput and selling price for different yearly revenue levels . [SEP]
[CLS] optical lithography as used for integrated circuit manufacturing is an example of a low - throughput process used to make a very high value product , leading to large revenues . [SEP]
[CLS] flexography , used for newsprint production , is a very high throughput process manufacturing a low value product , leading to more modest per - tool revenues . [SEP]
[CLS] illustration of the role of stochastic processes in controlling structural precision in top - down , bottom - up and damped - driven assembly . [SEP]
[CLS] placement of individual atoms B-material , such as dopants , within semiconductor devices is effectively only as good as the device dimension , with current manufacturing methods . [SEP]
[CLS] i . e . , an atom B-material is only constrained to lie somewhere within the " box " created . [SEP]
[CLS] placement of edges is typically a fraction of feature size , and is uniform within the length scale of a circuit . [SEP]
[CLS] systems , such as diblock copolymers , which selfassemble with no guiding pattern , show excellent short - range order that decays exponentially with distance . [SEP]
[CLS] the placement of individual molecules within a domain is again controlled by the size of the box . i . e . , the domain . [SEP]
[CLS] control over individual atom B-material placement is greatest in biomolecules , where it is specified by atomic relationships in , for example , amino B-material acids I-material , and then by the hierarchy of secondary and tertiary structure . [SEP]
[CLS] placement precision between biomolecules not bound together decays rapidly as a function of separation . [SEP]
[CLS] the plot shows a measure of manufacturing complexity , m c , as a function of cost per unit area ( dollars per square meter ) . [SEP]
[CLS] we define manufacturing complexity ( m c ) as log 10 [ k • ξ max / ( d min • f • p ) ] , where k is the kolmogorov complexity , ( ξ max / d min ) is a measure of the maximum distance over which spatial coherence must be maintained , compared to the minimum feature size , the fractional tolerance , f , is the maximum allowable variation in feature size , and the perfection , p , is the maximum fraction of defective components or concentration of impurities B-property that can be permitted . [SEP]
[CLS] the cost may be dominated by the information content ( bits ) , as in the case of a blu - ray disk , or by the material B-material ( atoms B-material ) as for a protein - functionalized nanoparticle B-nanoparticle [ see text for details ] . [SEP]
[CLS] 2 . log - log plot of the approximate product selling price ( $ • m −2 ) versus global annual production ( m 2 ) for a variety of nano - enabled , or potentially nano - enabled products . [SEP]
[CLS] approximate market sizes ( 2014 ) are show next to each point ( the si contains the information we used to estimate each data point ) . [SEP]
[CLS] 4 . throughput ( m 2 • s −1 ) versus cost ( $ • m −2 ) for top - down patterning techniques used in integrated circuit manufacturing . [SEP]
[CLS] despite nine orders of magnitude variation in cost and throughput , each technique falls into the nanomanufacturing , rather than nanofabrication , category in this context . [SEP]
[CLS] while not capable of the same performance in terms of placement accuracy , roll - to - roll nanoimprint lithography is included as a point of comparison with another high - throughput nanoscale patterning technique . [SEP]
[CLS] note the strong negative correlation between throughput and cost . [SEP]
[CLS] hydrogen B-material peroxide is a product of cellular respiration and is one of the most important reactive oxygen B-material species endogenously generated by cells B-material . [SEP]
[CLS] h 2 o 2 plays fundamental roles in multiple physiological and pathological B-event processes I-event , with the controlled generation of h 2 o 2 essential to maintaining homeostasis and hence cell B-material survival . [SEP]
[CLS] [SEP]
[CLS] an overproduction of h 2 o 2 results in oxidative stress that causes a range of diseases , including neurodegeneration , diabetes , and cancer . [SEP]
[CLS] [SEP]
[CLS] the importance of h 2 o 2 in biological processes necessitates the development of real - time sensing platforms for h 2 o 2 . [SEP]
[CLS] recently , fluorescent B-property probes have been designed for h 2 o 2 sensing , including small molecule fluorescent B-property sensors , synthetic cell B-material - penetrating peptides B-material , genetically encoded fluorescent B-property proteins B-material , and nanozymes . [SEP]
[CLS] however , the transient nature of ros and the similarity of h 2 o 2 with other ros in terms of size and oxidative properties make peroxide sensing challenging . [SEP]
[CLS] hybrid nanomaterials B-material integrating synthetic nanomaterials B-material with biomolecules including proteins B-material and nucleic B-material acids I-material are important tools for chemical and biosensing . [SEP]
[CLS] these systems incorporate key elements of biomolecular function with the unique physical structural attributes of nanomaterials B-material . [SEP]
[CLS] a substantial body of research exists on nucleic B-material acid and peptide - functionalized nanomaterials B-material , with protein - based systems rapidly emerging . [SEP]
[CLS] these systems generally use native proteins B-material for sensing applications , providing recognition elements ( e . g . antibodies B-material ) as well as enzymatic function . [SEP]
[CLS] genetic engineering of proteins B-material provides a tool for creating np B-nanoparticle - protein B-material sensors with improved sensing selectivity and stability , however the complicated genetic fusion process and limited functional B-material group I-material options for proteins B-material provide limitations to this approach . [SEP]
[CLS] chemical modification of proteins B-material is an appealing alternative for endowing proteins B-material with useful non - natural functionality and tunable properties . [SEP]
[CLS] we reasoned that integrating the strategies of chemically modified proteins B-material with nanoparticle B-nanoparticle surface engineering would yield hybrid nanomaterials B-material with synergetic functionality . [SEP]
[CLS] we report here the creation of h 2 o 2responsive np B-nanoparticle - protein B-material conjugates and their ability to selectively monitor endogenous h 2 o 2 production in live cells B-material . [SEP]
[CLS] in this study , we modify green fluorescent B-property protein B-material ( gfp ) with boronate B-material functionality at the primary B-material amine I-material of lysine B-material residues to provide a family of phenylboronate - functionalized gfps ( pb - gfps ) . [SEP]
[CLS] these proteins B-material react with galactosefunctionalized gold B-nanoparticle nanoparticles I-nanoparticle ( aunp B-nanoparticle - gal ) via boronate B-material ester formation ( figure 1 ) . [SEP]
[CLS] by tuning the extent of boronate B-material modification of gfp through convenient chemical modifications , we demonstrate that the conjugation of pb - gfp with aunp B-nanoparticle - gal quenches the fluorescence B-property of gfp depending on the number of boronate B-material moieties conjugated to gfp . [SEP]
[CLS] the pb - gfp / aunp B-nanoparticle - gal functionalization is peroxide - responsive : h 2 o 2 catalyzes the bioorthogonal oxidation of boronate B-material [SEP]
[CLS] this oxidation disassembles the complex with concomitant restoration of gfp fluorescence B-property ( figure 1 ) , providing selective gfp - based onoff h 2 o 2 detection for real time and in situ cellular oxidative stress monitoring . [SEP]
[CLS] boronate - functionalized gfp was prepared through the reaction of gfp with 4 - nitrophenyl 4 - ( 4 , 4 , 5 , 5 - tetramethyl - 1 , 3 , 2 - dioxaborolan - 2 - yl ) benzyl carbonate B-material ( nbc ) at different molar ratios ( see experimental details in supporting information ) . [SEP]
[CLS] the number of phenylboronic acid ( pb ) conjugated to gfp was determined using matrix - assisted laser desorption / ionization B-property - time of flight ( maldi - tof ) mass spectroscopy B-technique ( figure s1 , supporting information ) . [SEP]
[CLS] in this study , gfp conjugated with 5 , 9 , and 20 pb per protein B-material molecule , designated as pb5 - gfp , pb9 - gfp , and pb20 - gfp , respectively , were complexed with gold B-nanoparticle nanoparticles I-nanoparticle ( 2 nm core B-material ) functionalized with galactose ligands ( aunp B-nanoparticle - gal ) that were prepared through the place - exchange reaction of 1 - pentanethiol protected gold B-nanoparticle nanoparticles I-nanoparticle ( aunp B-nanoparticle - c5 ) and galactose ligands ( supporting information ) . [SEP]
[CLS] the aunps B-nanoparticle were characterized using transmission B-technique electron I-technique microscopy I-technique ( tem ) and dynamic B-technique light I-technique scattering I-technique ( dls ) analysis ( figure s2 , supporting information ) . [SEP]
[CLS] in addition , gfp without pb conjugation ( gfp ) was used as a negative control to evaluate the effect of pb conjugation on enhancing protein - np B-nanoparticle interaction . [SEP]
[CLS] the attachment between aunp B-nanoparticle - gal and pb - gfp was quantified by titrating gfp with aunp B-nanoparticle and measuring the gfp fluorescence B-property intensity change . [SEP]
[CLS] the formation of a stable aunp B-nanoparticle / pb - gfp interface efficiently quenches gfp fluorescence B-property , providing straightforward observation of binding . [SEP]
[CLS] as shown in figure 2 , the fluorescence B-property of gfp solution ( 100 nm protein B-material in pbs ) shows little change with the addition of aunp B-nanoparticle , indicating little binding between gfp and aunp B-nanoparticle , in the presence of high concentration of salt B-material ( 137 mm nacl ) . [SEP]
[CLS] in contrast , pb - gfp fluorescence B-property was quenched with the addition of aunp B-nanoparticle , depending on the number of pb units conjugated to gfp and the amount of aunp B-nanoparticle added to pb - gfp . [SEP]
[CLS] for example , pb20 - gfp fluorescence B-property was quenched up to 90 % at an np B-nanoparticle to protein B-material ratio of 0 . 2 , while 18 % and 70 % gfp emission quenching was observed for pb5 - gfp and pb9 - gfp respectively under the same conditions . [SEP]
[CLS] this enhanced quenching indicates pb20 - gfp protein B-material has a stronger binding affinity with aunp B-nanoparticle than that of pb5 - gfp and pb9 - gfp . [SEP]
[CLS] the affinity and the stern - volmer constant of aunp B-nanoparticle - gal and pb20 - gfp are substantially higher than pb5 - gfp and pb9 - gfp , as determined by the gfp fluorescence B-property titration curve ( table s1 and figure s3 , supporting information ) [SEP]
[CLS] we previously demonstrated that pb modified proteins B-material are ros - responsive , as hydrogen B-material peroxide can cleave the boronate B-material from the conjugated proteins B-material through a bioorthogonal oxidization reaction . [SEP]
[CLS] we confirmed the ros - responsive nature of pb - gfp by treating pb20 - gfp protein B-material with h 2 o 2 , and characterizing pb cleavage using maldi - tof . [SEP]
[CLS] as shown in figure s4 ( supporting information ) , maldi - tof analysis indicated that the treatment of pb20 - gfp ( 30 μm protein B-material ) with 30 mm h 2 o 2 resulted in a loss of fourteen pb groups from pb20 - gfp . [SEP]
[CLS] the disassembly of the aunp B-nanoparticle / pb - gfp by h 2 o 2 was then followed using fluorescence B-technique spectroscopy I-technique . [SEP]
[CLS] as shown in figure 3 , the attachment of aunp B-nanoparticle ( 25 nm ) to pb20 - gfp ( 100 nm protein B-material in pbs ) quenched gfp fluorescence B-property by 90 % . [SEP]
[CLS] treatment of the complex with 1 mm h 2 o 2 for 30 min resulted in essentially complete restoration of gfp fluorescence B-property . [SEP]
[CLS] as shown in figure s5 ( supporting information ) , the fluorescence B-property of aunp B-nanoparticle / pb20 - gfp complex ( 100 nm gfp and 25 nm aunp B-nanoparticle - gal ) increased shortly after adding 1 mm h 2 o 2 , no further gfp fluorescence B-property changed was observed after 25 minutes of incubation B-technique , indicating pb20 - gfp protein B-material was completely released from aunp B-nanoparticle . [SEP]
[CLS] the fast and dynamic process of the h 2 o 2 - regulated aunp B-nanoparticle / pb20 - gfp interface prompted us to study its potential application as an h 2 o 2 assay . [SEP]
[CLS] novel sensing platforms that discriminate different types of ros and are capable of real time monitoring of ros generation are important to study the physiological role of ros and develop new disease diagnostic tools . [SEP]
[CLS] we evaluated the capability of the aunp B-nanoparticle / pb20 - gfp complex for sensitive and selective sensing of h 2 o 2 . [SEP]
[CLS] as shown in figure 4a , treatment of aunp B-nanoparticle / pb20 - gfp complex ( 100 nm pb20 - gfp and 25 nm aunp B-nanoparticle - gal mixed in pbs containing 10 % fetal bovine serum ) with 20 μm h 2 o 2 , restored gfp fluorescence B-property gradually within 2 h of incubation B-technique . [SEP]
[CLS] fast restoration of gfp fluorescence B-property was observed for aunp B-nanoparticle / pb20 - gfp complex treated with a higher concentration of h 2 o 2 . [SEP]
[CLS] for example , 150 μm h 2 o 2 restored gfp fluorescence B-property to more than 90 % after 40 minutes of incubation B-technique . [SEP]
[CLS] the response of the aunp B-nanoparticle / pb20 - gfp complex is very selective toward h 2 o 2 ( figure 4b ) . [SEP]
[CLS] the addition of a variety of biologically relevant ros , including hypochlorite ( clo - ) , h 2 o 2 , tertbutyl hydroperoxide ( tbhp ) , nitroxide radical ( no • ) , and h 2 o 2 in the presence of catalase ( which acts to degrade h 2 o 2 ) , generated no perceptible response from the complex . [SEP]
[CLS] we next explored the utility of aunp B-nanoparticle / pb20 - gfp complex for real time monitoring of cellular oxidative stress . [SEP]
[CLS] we first verified the use of the aunp B-nanoparticle / pb20 - gfp interface for h 2 o 2 sensing in cell B-material culture medium . [SEP]
[CLS] to this end , human t lymphocyte jurkat cells B-material were spiked with different concentrations of h 2 o 2 , followed by an incubation B-technique with aunp B-nanoparticle / pb20 - gfp complex ( 100 nm pb20 - gfp and 25 nm aunp B-nanoparticle - gal ) and pb20 - gfp fluorescence B-property measurement . [SEP]
[CLS] as shown in figure 5a , jurkat cells B-material alone had no effect on disassembling the aunp B-nanoparticle / pb20 - gfp complex during the 2h of incubation B-technique , indicating the high stability of aunp B-nanoparticle / pb20 - gfp interface in biological media . [SEP]
[CLS] the addition of 2 . 5 μm h 2 o 2 resulted in a noticeable increase in fluorescence B-property within 2h , while the gfp fluorescence B-property was 2 . 5 times higher for aunp B-nanoparticle / pb20 - gfp complex spiked with 40 μm h 2 o 2 ( figure 5a ) . [SEP]
[CLS] additionally , the gfp fluorescence B-property is linearly correlated to the concentration of h 2 o 2 spiked to the cell B-material culture medium in the range of 2 . 5 to 40 μm ( figure s6 , supporting information ) . [SEP]
[CLS] we next studied the capability of the aunp B-nanoparticle / pb20 - gfp interface to monitor endogenous h 2 o 2 generated by jurkat cells B-material in the presence of the ros stimulator , phorbol 12myristate - 13 - acetate ( pma ) . [SEP]
[CLS] pma treatment activates napdh oxidase and induces the generation of superoxide , which is converted to ros species including h 2 o 2 . [SEP]
[CLS] as shown in figure 5b , jurkat cells B-material pretreated with 0 . 5 μm pma before aunp B-nanoparticle / pb20 - gfp complex ( 100 nm pb20 - gfp and 25 nm aunp B-nanoparticle - gal ) incubation B-technique resulted in a significant enhancement of gfp fluorescence B-property , compared to the cells B-material without pma stimulation . [SEP]
[CLS] in addition , when 0 . 5 mg / ml catalase was simultaneously added to pma - stimulated jurkat cells B-material , no gfp fluorescence B-property enhancement was observed due to consumption of the generated peroxide . [SEP]
[CLS] by calibrating the gfp fluorescence B-property of pma - stimulated jurkat cells B-material to that of the aunp B-nanoparticle / pb20 - gfp complex in the presence of exogenous h 2 o 2 after 120 minutes incubation B-technique , we estimated that the pma - stimulation generated h 2 o 2 at a rate of 0 . 49 nmol / 10 4 cells B-material / h , consistent with previous reports . [SEP]
[CLS] in summary , we have co - engineered an np B-nanoparticle - protein B-material complex to provide an ros - responsive system for real time h 2 o 2 sensing and monitoring of cellular oxidative stress . [SEP]
[CLS] this complex was created and modulated by the complexation between galactose - decorated gold B-nanoparticle nanoparticles I-nanoparticle and boronate modified protein B-material . [SEP]
[CLS] the assembled aunp B-nanoparticle / pb - gfp interface is highly stable under a physiological environment , with gfp fluorescence B-property generated by h 2 o 2 , providing timely biological dose estimates . [SEP]
[CLS] the responsiveness of this system demonstrates the utility of co - engineering for creating functional and responsive synthetic - biological hybrid nanomaterials B-material , a strategy that can be applied to many biological and physiological challenges . [SEP]
[CLS] fluorescence B-property titration of pb - gfp with aunp B-nanoparticle - gal . [SEP]
[CLS] 100 nm protein B-material was mixed with increased amounts of aunp B-nanoparticle - gal in pbs , and after incubation B-technique for 15 min , the fluorescence B-property intensity of gfp was recorded . [SEP]
[CLS] gfp fluorescence B-property ( λ ex = 475 nm , λ em = 510 nm ) in the presence of aunp B-nanoparticle - gal was normalized to that without aunp B-nanoparticle - gal addition . [SEP]
[CLS] each titration experiment was performed in three replicates . [SEP]
[CLS] the red solid lines represent the best curve fitting using the previously reported method . [SEP]
[CLS] 1 . chemically engineered aunp B-nanoparticle / pb - gfp interface for h 2 o 2 sensing . [SEP]
[CLS] a ) schematic illustrating the interfacing of aunp B-nanoparticle - gal with pb - gfp , and phorbol 12 - myristate - 13 - acetate ( pma ) stimulated in situ h 2 o 2 generation modulated gfp release from the aunp B-nanoparticle / pb - gfp interface . [SEP]
[CLS] b ) reaction mechanism of pb - gfp with h 2 o 2 catalyzed bioorthogonal pb cleavage . [SEP]
[CLS] 3 . fluorescence B-property intensity of pb20 - gfp , and aunp B-nanoparticle - gal / pb20 - gfp complexes before and after addition of h 2 o 2 . [SEP]
[CLS] the aunp B-nanoparticle - gal / pb20 - gfp conjugates were prepared by incubating B-technique pb20 - gfp ( 100 nm ) with a fixed pb20 - gfp to aunp B-nanoparticle - gal ratio ( 4 : 1 ) for 15 min . [SEP]
[CLS] all gfp fluorescence B-property values were presented relative to the pb20 - gfp control . [SEP]
[CLS] error bar represents the standard deviation of three independent studies . [SEP]
[CLS] photograph showing fluorescence B-property of pb20 - gfp , aunp B-nanoparticle - gal / pb20 - gfp complexes , and aunp B-nanoparticle - gal / pb20 - gfp complexes upon addition of h 2 o 2 under ultraviolet light . [SEP]
[CLS] 4 . h 2 o 2 concentration - dependent and selective modulation of the aunp B-nanoparticle - gal / pb20 - gfp interface . [SEP]
[CLS] a ) the fluorescence B-property response of aunp B-nanoparticle - gal / pb20 - gfp ( 100 nm pb20 - gfp ) upon the addition of increasing concentrations of h 2 o 2 in pbs containing 10 % fbs . [SEP]
[CLS] the fluorescence B-property response was presented relative to the pb20 - gfp control . [SEP]
[CLS] b ) time - dependent fluorescence B-property response of aunp B-nanoparticle - gal / pb20 - gfp ( 100 nm pb20 - gfp ) upon addition of h 2 o 2 ( 50 μm ) or other ros ( 100 μm ) as indicated in pbs containing 10 % fbs . [SEP]
[CLS] catalase ( 0 . 5 mg / ml ) was added to verify the role of h 2 o 2 in modulating aunp B-nanoparticle / pb20 - gfp [SEP]
[CLS] 5 . detection of h 2 o 2 using the aunp B-nanoparticle - gal / pb20 - gfp in cellular environment . [SEP]
[CLS] ( a ) timedependent fluorescence B-property response of aunp B-nanoparticle - gal / pb20 - gfp complex ( 100 nm of pb20 - gfp ) added to jurkat cells B-material spiked with different concentrations of h 2 o 2 . [SEP]
[CLS] the fluorescence B-property response was presented relative to the aunp B-nanoparticle - gal / pb20 - gfp controls . [SEP]
[CLS] ( b ) the fluorescence B-property response of aunp B-nanoparticle - gal / pb20 - gfp ( 100 nm of pb20 - gfp ) incubated B-technique with jurkat cells B-material stimulated with pma ( 0 . 5 μm ) , or incubated B-technique with catalase ( 0 . 5 mg / ml ) and pma ( 0 . 5 μm ) [SEP]
[CLS] nucleic B-material acids I-material ( nas ) hold significant potential for the treatment of several diseases . [SEP]
[CLS] topical delivery of nas for the treatment of skin diseases is especially advantageous since it bypasses the challenges associated with systemic administration which suffers from enzymatic degradation , systemic toxicity B-property and lack of targeting to skin . [SEP]
[CLS] however , the skin ' s protective barrier B-property function limits the delivery of nas into skin after topical application . [SEP]
[CLS] here , we highlight strategies for enhancing delivery of nas into skin , and provide evidence that translation of topical na therapies could have a transformative impact on the treatment of skin diseases . [SEP]
[CLS] nucleic B-material acids I-material ( nas ) hold great potential for the treatment of various diseases , and there has been a significant amount of both academic as well as commercial interest in a variety of na - based therapeutics including genes , antisense oligodeoxynucleotides ( odns ) , sirna , aptamers , and cpg oligonucleotides . [SEP]
[CLS] however , translation of these platforms to the clinic has been significantly limited by challenges associated with delivering nas to the diseased site . [SEP]
[CLS] enzymatic degradation in the blood , rapid clearance from systemic circulation , to whom correspondence should be addressed : prof . samir mitragotri ( samir @ engineering . ucsb . edu ) . [SEP]
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[CLS] the outermost layer of skin , the sc , is primarily responsible for its barrier B-property function . [SEP]
[CLS] the sc is a thin layer only 10 - 20 µm thick that is made up of corneocytes . [SEP]
[CLS] corneocytes are anucleate cells B-material heavily enriched with intracellular keratin filaments . [SEP]
[CLS] corneocytes are held together in a " brick and mortar " structure by a lipid B-material matrix composed of ceramides , free fatty acids , and cholesterol . [SEP]
[CLS] materials traversing the skin barrier B-property must , therefore , diffuse through the tortuous lipid B-material channels , and / or traverse transcellularly through corneocytes , or enter the skin through hair follicles or sweat ducts ( fig . 1 ) . [SEP]
[CLS] transport within the lipid B-material bilayers I-material , however , is the most common mode of passage through the skin . [SEP]
[CLS] this results in the exclusion of most foreign materials , and more specifically , renders passage of large , hydrophilic B-property molecules ( > 500 da and log p o / w < 1 . 5 ) such as nas ( typically > > 10 , 000 da , log p o / w < 0 ) to virtually negligible levels without some form of enhancement startegy . [SEP]
[CLS] the layer underlying the sc , the epidermis , can also serve as another transport barrier B-property . [SEP]
[CLS] the epidermis is the first viable tissue layer of the skin where the pathology of several dermatological disorders resides . [SEP]
[CLS] the epidermis is 50 - 100 µm thick and is composed primarily of keratinocytes . [SEP]
[CLS] as keratinocytes migrate upward from deeper portions of the epidermis to the sc they gradually begin to keratinize and secrete lipids B-material that eventually form the sc bilayers . [SEP]
[CLS] this process continues as keratinocytes terminally differentiate into corneocytes and serves to rejuvenate the sc from underneath while the outermost layer of the sc sloughs away . [SEP]
[CLS] within the epidermis , keratinocytes are held together by cell - cell tight junctions . [SEP]
[CLS] in the epidermis , claudin - 1 , claudin - 4 , occludin , and zonula occludens - 1 are responsible for inhibiting paracellular transport . [SEP]
[CLS] this makes transport of nas , other large macromolecules , and drug carriers such as nanoparticles B-nanoparticle and na - lipid complexes difficult both vertically , deeper into the skin , as well as laterally from the site of administration to peripheral areas of the skin . [SEP]
[CLS] for effective treatment , both the sc as well as epidermal transport barriers B-property must be overcome to deliver nas to all areas of the disease . [SEP]
[CLS] over the years , a large number of strategies have been devised for perturbing the sc to enhance the delivery of drugs into and through the skin . [SEP]
[CLS] these strategies can be generally categorized into three main groups : physical , active , and passive methods . [SEP]
[CLS] the advantages and disadvantages of each class of perturbation methods are summarized in table 2 . [SEP]
[CLS] their use for the delivery of nas is described below . [SEP]
[CLS] microneedles - intradermal injections are the simplest and most direct method for delivering nas into the skin . [SEP]
[CLS] here , the barrier B-property properties of the sc are overcome completely by injecting nas directly into the viable tissue layers of the skin . [SEP]
[CLS] intradermal injections are typically used for evaluating efficacy of nas or other cutaneous therapeutics , or as the positive control for evaluating dermal delivery technologies , however , the downsides of intradermal injection for treating skin disease are overwhelming . [SEP]
[CLS] needle - phobia is a serious concern for a large number of children as well as adults leading to significant patient noncompliance . [SEP]
[CLS] moreover , intradermal injections are limited only to the site of application , and injection into multiple sites during a single administration is challenging . [SEP]
[CLS] to avoid many of these drawbacks , microneedle arrays have been developed . [SEP]
[CLS] microneedle arrays comprise needles that are only 100 - 700 µm in length ( fig . 2 ) . [SEP]
[CLS] when placed on the skin , their sharp tips allow easy insertion into the stratum corneum , while the short length ensures adequate penetration into the skin without disrupting nerves in deeper skin tissue . [SEP]
[CLS] microneedles have been used extensively for the delivery of nas . [SEP]
[CLS] mikszta et al . used microneedle arrays to deliver plasmid dna encoding a hepatitis b surface antigen for immunizationin , and they showed extensive immunological response in mice . [SEP]
[CLS] antibody B-material titers following application of microneedle arrays were significantly higher and less variable than when delivered using either intradermal or intramuscular injection . [SEP]
[CLS] chabri et al . used microneedles to deliver cationic B-material lipid - dna complexes ( ~ 100 nm diameter ) into the skin . [SEP]
[CLS] ding et al . demonstrated successful immunization of mice with co - administration of diphtheria toxoid and cpg oligonucleotide delivered by microneedle array , and gonzalez - gonzalez et al . demonstrated effective delivery of anti - luciferase sirna and gene silencing in luciferase expressing transgenic mice [SEP]
[CLS] microporation - microporation is another technique that employs physical disruption of the sc for delivery of large therapeutics or therapeutic carriers . [SEP]
[CLS] an array of resistive elements can be placed on the skin . [SEP]
[CLS] an electric current pulsed through the array results in localized ablation of corneocytes in contact with the array . [SEP]
[CLS] alternatively , erbium : yttrium - aluminum - garnet ( er : yag ) laser arrays can be used for localized ablation of the sc and epidermis . [SEP]
[CLS] similar to microneedle arrays , microporation has gained considerable interest over the last decade . [SEP]
[CLS] for example , lee et al . used laser microporation to deliver antisense oligonucleotide as well as plasmid dna into the skin . [SEP]
[CLS] delivery of antisense oligonucleotide was enhanced 3 - 30 fold compared to intact skin in vitro . [SEP]
[CLS] in addition , expression of gfp in nude mice was enhanced 160 fold after application of gfp plasmid dna . [SEP]
[CLS] the amount of enhancement correlated with both the laser fluency as well as the size of oligonucleotide . [SEP]
[CLS] the same group also showed enhanced delivery of sirna . [SEP]
[CLS] sirna delivery into skin was enhanced 3 . 5 fold and localized mainly in the dermis . [SEP]
[CLS] hessenberger et al . used laser microporation to deliver cpg oligonucleotides into the skin and successfully protected against immune response to grass pollen in mice . [SEP]
[CLS] they used the precise laser epidermal system ( pantec biosolutions ) which creates well - defined arrays of micropores in the skin , and allows precise control over the number , density , and depth of the micropores giving the user unprecedented tuneability over the amount and localization of therapeutic delivered . [SEP]
[CLS] electroporation - electroporation can be used to permeabilize the skin and enhance passive diffusion of drug . [SEP]
[CLS] the mechanism of electroporation is quite different from that of electrically - induced microporation . [SEP]
[CLS] electrically - induced microporation utilizes electric fields to induce thermal ablation of sc microstructure creating pores in the skin . [SEP]
[CLS] on the other hand , electroporation is the application of short duration ( < 0 . 5 s ) and high intensity ( < 100 v ) electric pulses to the skin which result in transient permeabilization of the lipid B-material bilayers I-material in the skin and concurrently permeabilize cell B-material membranes of epidermal keratinocytes . [SEP]
[CLS] electroporation is also expected to create aqueous pores through the skin . [SEP]
[CLS] however , electroporation , unlike microporation , acts through non - thermal mechanisms as lipid B-material rearrangement , reduced skin resistance , and enhanced transdermal transport are observed in the absence of a significant temperature rise in the pulse medium . [SEP]
[CLS] using electroporation , regnier et al . showed enhanced permeability of the stratum corneum to a phosphorothioate antisense odn . [SEP]
[CLS] the permeability enhancement lasted up to 1 hr postelectroporation in rat skin in vitro . [SEP]
[CLS] specifically , transport was enhanced > 4 - fold into the sc and > 3 - fold into viable skin tissue when oligonucleotide solution was applied immediately following electroporation . [SEP]
[CLS] further , zhang et al . demonstrated effective gene transfection and expression in the epidermis of human skin . [SEP]
[CLS] iontophoresis - iontophoresis can be used to drive transport of charged drugs like nas . [SEP]
[CLS] applying a continuous low intensity ( < 10 v ) electric field at a constant current has been used extensively to deliver a wide range of charged therapeutics including calcitonin , luteinizing hormone - releasing hormone , and dexamethasone . [SEP]
[CLS] in contrast to electroporation which acts primarily on the skin structure , iontophoresis is not believed to cause major changes to the skin . [SEP]
[CLS] minor structural effects can be observed and may partially contribute to delivery enhancement through sweat ducts and hair follicles . [SEP]
[CLS] nevertheless , iontophoresis is believed to primarily act on the drug itself , driving transport of a charged molecule by means of an applied electric field . [SEP]
[CLS] using iontophoresis , kigasawa et al . demonstrated successful delivery of anti - il - 10 sirna . [SEP]
[CLS] specifically , using an atopic dermatitis model in rats they showed a 73 % reduction in il - 10 mrna levels after treatment with anti - il - 10 sirna and iontophoresis . [SEP]
[CLS] the same group later extended the technique for vaccination as well as treatment of melanoma tumors B-material . [SEP]
[CLS] kigasawa et al . reported enhanced delivery of cpg oligonucleotides into the epidermis and dermis using iontophoresis compared to that by free diffusion . [SEP]
[CLS] further , for the treatment of melanoma tumors B-material in hairless mice , cpg oligonucleotides delivered using iontophoresis resulted in tumor B-material reduction comparable to subcutaneous injection . [SEP]
[CLS] abu hashim et al . also demonstrated the ability to deliver nf - κb decoy odn into skin using iontophoresis for atopic dermatitis treatment . [SEP]
[CLS] while passive diffusion of fitc - nf - κb decoy odn was negligible for all concentrations tested , ~ 100 pmol / cm 2 was delivered after 6 hours of iontophoresis using 0 . 5 ma current density . [SEP]
[CLS] in addition , the authors observed a linear dependence between the flux of odn into the skin with both current density and concentration of drug . [SEP]
[CLS] when the technique was applied for the treatment of atopic dermatitis in mice , the authors noted significant reduction in both tissue swelling and tnfa expression . [SEP]
[CLS] in comparison , treatment with buffer alone , as well as treatment with a scrambled odn sequence , resulted in no significant reduction in either ear swelling or tnfa . [SEP]
[CLS] iontophoresis has also been used to deliver na complexes into the skin . [SEP]
[CLS] brus et al . achieved enhanced delivery of polyethylenimine ( pei ) complexed with odn . [SEP]
[CLS] negatively charged odn was electrostatically complexed with positively charged pei at a charge ratio of 1 : 13 . 3 to create positively charged complexes . [SEP]
[CLS] complexing was shown to protect oligonucleotides from enzymatic degradation and enhance intracellular delivery in epidermal keratinocytes . [SEP]
[CLS] complexes did not show any measureable passive diffusion , however , under constant electrical current the complexes did passage into the skin primarily via the shunt pathway . [SEP]
[CLS] sonophoresis - low - frequency ultrasound has also been demonstrated to transiently permeabilize the sc lipid B-material bilayers I-material , facilitating the delivery of a large number of macromolecules including insulin , bovine serum albumin , and heparin . [SEP]
[CLS] tezel et al . first reported delivery of therapeutic quantities of nas into the skin using sonophoresis . [SEP]
[CLS] delivery of odns into porcine skin in vitro was significantly enhanced by applying 2 . 4 w / cm 2 ultrasound for 10 min ( fig . 3 ) . [SEP]
[CLS] interestingly , enhancement seemed to occur in localized regions of the skin ( fig . 3 ) , however , these regions were not observed to be hair follicles as is commonly reported for iontophoresis - mediated delivery of nas . [SEP]
[CLS] instead , localized penetration zones most likely correspond to localized areas of acoustic cavitation inherent to this technique , and occupied ~ 5 % of skin surface area . [SEP]
[CLS] in addition , the authors showed no observable effect of ultrasound on skin histology . [SEP]
[CLS] na delivery with ultrasound was also shown to treat skin disease in vivo . [SEP]
[CLS] tran et al . demonstrated ~ 30 % reduction in melanoma tumor B-material size when an anti - b - raf sirna liposome formulation was delivered into skin using sonophoresis . [SEP]
[CLS] the liposome B-nanoparticle was postulated to protect sirna from enzymatic degradation in skin , as well as aid intracellular uptake by melanoma cells B-material . [SEP]
[CLS] nanoparticles B-nanoparticle - nanoparticles B-nanoparticle are promising due to their high loading capacity , ability to shield enzymatic degradation , and reduce immunogenicity B-property . [SEP]
[CLS] due to their size , nanoparticles B-nanoparticle are expected to require physical perturbation of the sc and underlying epidermis for effective delivery . [SEP]
[CLS] recently , however , functionalized nanoparticles B-nanoparticle have been shown to passively diffuse through the skin and elicit a therapeutic response . [SEP]
[CLS] for example , siu et al . demonstrated effective delivery of functionalized carbon B-nanoparticle nanotubes I-nanoparticle for the treatment of melanoma in vivo . [SEP]
[CLS] single - walled carbon B-nanoparticle nanotubes I-nanoparticle functionalized with pei were shown to deliver anti - b - raf sirna and result in b - raf silencing and attenuation of tumor B-material growth in a mouse melanoma model . [SEP]
[CLS] ozbas - turan et al . delivered chitosan B-material complexed with βgalactosidase plasmid dna into mouse skin resulting in significant expression of βgalactosidase after 7 days of treatment . [SEP]
[CLS] similarly , cui et al . showed delivery and expression of luciferase plasmid dna when complexed with chitosan B-material . [SEP]
[CLS] liposomes B-nanoparticle - liposomes have also been studied extensively for nucleic B-material acid I-material delivery for the treatment of skin disease . [SEP]
[CLS] for example , desai et al . used cationic B-material lipid B-nanoparticle nanoparticles I-nanoparticle complexed with anti - tnfa sirna and capsaicin to treat psoriasis in vivo . [SEP]
[CLS] treatment resulted in significantly reduced expression of a number of inflammatory cytokines including tnfa , il - 17 , il - 23 , and nf - κb . bracke et al . used ultraflexible liposomes B-nanoparticle loaded with anti - β defensin - 2 to treat psoriasis in vivo . [SEP]
[CLS] plasmid dna can also be delivered using liposomes B-nanoparticle . [SEP]
[CLS] li et al . delivered il - 4 encoding plasmid dna into mouse skin in vivo for the treatment of psoriasis . [SEP]
[CLS] enhanced expression of il - 4 led to suppressed hyperplastic and inflamed vessels in the skin . [SEP]
[CLS] similarly , kim et al . used liposomes B-nanoparticle complexed with antisense odn for il - 13 mrna to treat atopic dermatitis in vivo . [SEP]
[CLS] treatment with liposome B-nanoparticle formulation resulted in a significant reduction in skin thickness and inflammatory cell B-material infiltration . [SEP]
[CLS] spherical nas - highly ordered spherical complexes of nucleic B-material acids I-material ( spherical nucleic B-material acids I-material ) have shown potential for treating skin disease due to their enhanced delivery into skin , internalization into skin cells B-material , and protection of nas from degradation ( fig . 4 ) . [SEP]
[CLS] further , several different sequences of nas can be incorporated into a single construct for multifactorial diseases . [SEP]
[CLS] imaging agents like quantum B-nanoparticle dots I-nanoparticle can be used as the core B-material . [SEP]
[CLS] alternatively , hollow spherical nucleic B-material acids I-material can be prepared for incorporation of additional drug . [SEP]
[CLS] zheng et al . successfully delivered anti - egfr ( epidermal growth B-material factor I-material receptor I-material ) sirna into mouse skin using gold B-material - core B-material nanoparticles B-nanoparticle . [SEP]
[CLS] specifically , gold B-nanoparticle nanoparticles I-nanoparticle coated with a dense layer of highly - ordered and covalently bound sirna resulted in passive transport through intact mouse sc and localized exclusively in the dermis and epidermis . [SEP]
[CLS] after 3 weeks of treatment the authors observed nearly complete knockdown of egfr as well as repression of downstream phosphorylation , and reduction in epidermal thickness . [SEP]
[CLS] spherical nucleic B-material acids I-material also demonstrated the ability to stifle nuclease degradation and efficiently internalize into a large variety of cell B-material types to stimulate gene silencing . [SEP]
[CLS] further , spherical nucleic B-material acids I-material have been used to treat psoriasis as well as aid wound healing in vivo through knockdown of tnfα and ganglioside gm3 synthase , respectively . [SEP]
[CLS] peptides B-material - peptides B-material hold potential as drug delivery vehicles B-material owing to their simplicity , diversity , biocompatibility B-property , and potential for multi - functionality . [SEP]
[CLS] in addition , peptides B-material can be easily screened and selected from phage - display libraries for various functions ( fig . 5 ) . [SEP]
[CLS] phage - display screening offers a powerful tool for high - throughput discovery of novel peptide B-material sequences that can enhance penetration of large cargos into skin . [SEP]
[CLS] moreover , peptide B-material sequences that localize in unique regions of the skin can also be selected to target specific cell B-material types and minimize side effects . [SEP]
[CLS] over the last ten years , several peptides B-material have been identified which possess the ability to enhance transport of nas into the skin and elicit a therapeutic response . [SEP]
[CLS] the first of these peptides B-material discovered using phage - display screening was td - 1 ( acssspskhcg ) . [SEP]
[CLS] lin et al . showed td - 1 could enhance transport of gapdh sirna into viable tissue in the skin , as well as subcutaneous tissue , and silence gapdh expression . [SEP]
[CLS] co - incubation B-technique of gapdh sirna with td - 1 resulted in similar gapdh expression levels as intradermal injection , while expression levels for both methods of application were significantly less than sirna applied on the skin without peptide B-material . [SEP]
[CLS] further , application with td - 1 resulted in significantly reduced levels of target mrna in the skin for up to 3 days and target protein B-material in the skin for up to 7 days . [SEP]
[CLS] hsu and mitragotri identified another peptide B-material using phage - display screening , space peptide B-material ( actgstqhqcg ) , with the ability to not only enhance delivery of sirna across the skin but also enhance intracellular uptake . [SEP]
[CLS] in vivo application of space peptide B-material conjugated to il - 10 sirna or gapdh sirna resulted in ~ 30 % or ~ 45 % knockdown in protein B-material expression , respectively . [SEP]
[CLS] this result is in contrast to the control formulation containing sirna alone which showed negligible gene silencing . [SEP]
[CLS] further , space peptide B-material was shown to be non - toxic B-property at concentrations as high as 10 mg / ml . [SEP]
[CLS] uchida et al . used a dual - peptide B-material system with both tat peptide B-material ( grkkrrqrrrcg ) and at1002 ( fcigrlcg ) to enhance gene silencing in the skin . [SEP]
[CLS] tat peptide B-material and sirna were complexed via electrostatic interactions . [SEP]
[CLS] complexes were then co - incubated B-technique with at1002 and applied on the skin . [SEP]
[CLS] here , tat peptide B-material was used as a cell B-material - penetrating peptide B-material to enhance intracellular delivery , and at1002 was used as a tight - junction modulator to enhance permeation into the epidermis and dermis . [SEP]
[CLS] tat peptide B-material has also been shown to enhance macromolecule transport across the sc . [SEP]
[CLS] the system was later applied to treat atopic dermatitis in mice . [SEP]
[CLS] application of anti - rela sirna complexed with tat peptide B-material and co - incubated B-technique with at1002 led to reductions in ear thickness , clinical skin severity , topical cytokine levels , and serum ige production . [SEP]
[CLS] yi et al . successfully delivered anti - microphthalmia - associated transcription B-event factor ( anti - mitf ) sirna conjugated to td - 1 r8 peptide B-material ( acssspskhcgrrrrrrrr ) for the treatment of patients with melisma . [SEP]
[CLS] treatment with a cream formulation containing td - 1 r8 peptide B-material co - administered with anti - mitf sirna resulted in reductions in tyrosinase , tyrosinase - related protein B-material 1 , and melanocortin 1 receptor B-material leading to measurable inhibition of melanin production and melanocyte apoptosis B-event . [SEP]
[CLS] in fact , after 4 weeks application patients demonstrated a significant lightening of facial hypermelanosis lesions and almost completely restored dark lesions to normal skin color after 12 weeks . [SEP]
[CLS] dendrimers B-nanoparticle - although less studied than peptides B-material for delivering nas into skin , dedrimers B-nanoparticle are similarly advantageous due to their diversity , ease of synthesis , and functional B-material group I-material density . [SEP]
[CLS] bielinska et al . demonstrated the use of dendrimers B-nanoparticle for topical gene delivery . [SEP]
[CLS] dendrimer B-nanoparticle complexes resulted in measurable gene expression of chloramphenicol transacetylase that was ~ 7 - fold higher than with naked plasmid . [SEP]
[CLS] in addition , venuganti et al . used a similar dendrimer B-nanoparticle in combination with iontophoresis to deliver antisense oligonucleotide . [SEP]
[CLS] treatment with antisense oligonucleotide targeting the anti - apoptotic protein B-material , bcl - 2 , resulted in significantly enhanced apoptosis B-event and reduction in tumor B-material size in mice . [SEP]
[CLS] the burden of skin disease is alarming and growing fast . [SEP]
[CLS] 1 out of 3 individuals are estimated to have a skin disease at any given time , resulting in direct healthcare costs over $ 30 billion annually in the us alone . [SEP]
[CLS] symptoms from the ~ 3000 different skin diseases range from itching , redness , and irritation to physical disfigurement , or death . [SEP]
[CLS] furthermore , skin diseases manifest externally leading to severe and even debilitating emotional distress and social prejudice . [SEP]
[CLS] dermatological drugs aimed at treating skin disease reap an estimated $ 24 . 4 billion in annual revenue . [SEP]
[CLS] in addition , many other skin conditions , not considered disease , are of major concern . [SEP]
[CLS] these include cosmetic conditions like wrinkling , cellulite , discoloration , and skin pliancy . [SEP]
[CLS] growing demand for more effective treatments of cosmetic conditions is evidenced by the emergence and growth of the cosmeceutical market which was estimated to be ~ $ 35 billion globally in 2013 . [SEP]
[CLS] topical na therapies have exciting potential to reduce this burden and be transformative in the way we deal with and treat skin disease ( table 3 ) . [SEP]
[CLS] examples are discussed in detail below . [SEP]
[CLS] targeting orphan disease can help speed translation by allowing fast - tracked regulatory processing from orphan drug designation . [SEP]
[CLS] there are several orphan skin diseases with recognized na targets . [SEP]
[CLS] hidradenitis suppurativa ( hurley disease ) is an orphan disease that affects over 100 , 000 people in the us , most commonly women . [SEP]
[CLS] it is a chronic and debilitating disease characterized by painful abscesses , redness , and scarring , leading to significant physical and psychological impairment and reduced quality of life . [SEP]
[CLS] currently , the most common form of treatment is surgical excision of diseased areas of the skin . [SEP]
[CLS] for severe forms of hurley disease there is no fda approved treatment . [SEP]
[CLS] studies into the aetiology and pathogenesis of hurley disease suggest it is a multifactorial inflammatory disease characterized by overexpression of tnfα as well as other inflammatory cytokines like il - 17 and il - 23 . [SEP]
[CLS] treatment of hurley disease with anti - tnfα monoclonal antibodies B-material or other biologics has been shown to be effective . [SEP]
[CLS] tnfα promotes inflammation through recruitment of vascular endothelial cells B-material and immune cells B-material . [SEP]
[CLS] further , it is overly expressed at sites of inflammation while repression generally results in alleviation of inflammation . [SEP]
[CLS] while there is no fda approved drug indication for treating moderate to severe hurley disease , several anti - tnfα biologics are already on the market including etanercept and adalimumab . [SEP]
[CLS] in fact , the anti - tnfα monoclonal B-material antibody I-material , humira ( adalimumab , abbvie inc . ) , was recently granted orphan drug designation by the fda to start clinical trials on moderate to severe hurley disease patients . [SEP]
[CLS] on the other hand , these drugs can only be administered by injection or infusion and systemic side effects limit their long - term use . [SEP]
[CLS] alarmingly , the most common side effect of systemic delivery is the development of other autoimmune diseases as a direct result of treatment due to increased expression of il - 17 and il - 23 as well as other inflammatory cytokines . [SEP]
[CLS] therefore , topical delivery of anti - tnfα sirna or antisense oligonucleotide is an attractive platform to limit off target effects , improve patient compliance , and reduce hospital - related costs . [SEP]
[CLS] even more impactful , a cocktail of topical anti - tnfα , anti - il - 17 , and anti - il - 23 na therapeutics may prove synergistic at alleviating hurley disease symptoms while avoiding adverse reactions . [SEP]
[CLS] epidermolysis bullosa ( eb ) is another rare skin disease with few effective treatments . [SEP]
[CLS] eb affects roughly 30 , 000 people in the us and ~ 500 , 000 people globally . [SEP]
[CLS] eb symptoms typically include severe skin fragility to the point where large segments of skin can be removed from simply scratching or rubbing skin . [SEP]
[CLS] alarmingly , few patients with severe eb survive past the age of 30 due to extensive wounds and infection . [SEP]
[CLS] there is no fda approved drug indication for eb . [SEP]
[CLS] management of eb consists of limiting blister formation from scratching and friction . [SEP]
[CLS] for more effective treatment , studies suggest il - 1β and its receptor B-material may be a valuable target . [SEP]
[CLS] indeed , overexpression of il - 1β has been detected in patients with eb and treatment of eb in mice with anti - il - 1β or il - 1β receptor B-material antagonist resulted in improvement of symptoms . [SEP]
[CLS] further , twi biotechnology inc . was recently granted orphan drug designation for its anti - il - 1β and anti - il - 1β receptor B-material small molecule drug ac - 201 . [SEP]
[CLS] eb can also be targeted at the genetic level . [SEP]
[CLS] one subtype of eb ( epidermolysis bullosa simplex ) , is predominantly caused by dominant - negative mutations in the genes encoding keratin - 5 and keratin - 14 ( k5 and k14 ) . [SEP]
[CLS] sirna therapies that specifically knockdown mutant keratin without affecting wild - type keratins have shown promise . [SEP]
[CLS] a second subtype of eb ( dystrophic epidermolysis bullosa ) is caused by mutations in collagen vii and gene delivery of wild - type collagen vii has shown promise as a therapeutic in phase i / ii clinical trials . [SEP]
[CLS] pachyonychia congenita ( pc ) is a rare genetic autosomal dominant skin disorder that affects 5 , 000 to 10 , 000 people . [SEP]
[CLS] pc may be caused by mutations in either k6a , k6b , k6c , k16 , or k17 . [SEP]
[CLS] the disease typically manifests with calluses and blisters on the feet and palms , as well as discoloration , cysts , and hyperkeratosis of the hair follicles . [SEP]
[CLS] moreover , patients typically live in constant pain . [SEP]
[CLS] due to the large number of different mutations that can result in pc , topical application of personalized na therapies holds the most potential . [SEP]
[CLS] in fact , treatment of patients with topical sirna therapies are showing promise both in mouse models as well as in humans . [SEP]
[CLS] netherton syndrome is an autosomal recessive disorder estimated to affect 1 / 50 , 000 individuals . [SEP]
[CLS] symptoms present at birth or shortly after and include painful peeling and scaling of skin . [SEP]
[CLS] more alarmingly , netherton syndrome manifests significant impairment of skin barrier B-property function leading to life - threatening dehydration and frequent systemic infections . [SEP]
[CLS] netherton syndrome is caused by a mutation of the serine B-material protease inhibitor kazal type 5 ( spink5 ) gene leading to serine B-material protease hyperactivity and excessive protein B-material degradation in skin . [SEP]
[CLS] essentially , protease hyperactivity results in excessive and premature epidermal desquamation . [SEP]
[CLS] netherton syndrome may be treated by down - regulating production of serine B-material proteases in the skin , or alternatively , inducing expression of wild - type serine B-material protease inhibitor . [SEP]
[CLS] due to the significant reduction in skin barrier B-property function , initial treatment with topical nas may not require dermal enhancement . [SEP]
[CLS] instead , technologies to localize drug in the skin such as cell B-material - targeting or skin targeting peptides B-material may be required . [SEP]
[CLS] however , as skin integrity improves more significant enhancement may be required . [SEP]
[CLS] mainstream disease would also benefit from topical na therapies . [SEP]
[CLS] for example , psoriasis is estimated to affect nearly 4 . 5 million adults in the us alone . [SEP]
[CLS] children are also commonly affected by the disease . [SEP]
[CLS] it is a chronic skin disease that presents as severe lesions , redness , and itching and poses a significant burden on patients ' quality of life . [SEP]
[CLS] about 20 - 25 % of patients are estimated to be dissatisfied with their current treatment . [SEP]
[CLS] of even more concern , for patients suffering the most severe form of psoriasis , erythrodermic psoriasis , there is no standard treatment regimen . [SEP]
[CLS] due to the multifactorial nature of psoriasis , na therapies may be advantageous because na cocktails targeting a number of different targets can be formulated and delivered leading to down / up regulation as needed . [SEP]
[CLS] psoriasis is one of the more exhaustively studied forms of inflammatory skin disease and numerous therapeutic targets have been proposed ; a subset is described here . [SEP]
[CLS] similar to hurley disease , tnfα is perhaps the most established drug target for treating severe forms of psoriasis . [SEP]
[CLS] topical delivery of anti - tnfα sirna as well as a sirna cocktail including anti - tnfα and anti - stat3 was shown to be effective at alleviating psoriasis - like symptoms in mice . [SEP]
[CLS] activated t helper cells B-material have also been shown to play a major role in the manifestation of psoriasis , and selective skewing from th1 phenotype to the il - 4 producing th2 phenotype can alleviate psoriasis symptoms . [SEP]
[CLS] further , selective differentiation to the th2 phenotype can be simply induced by subcutaneous injection of il - 4 . [SEP]
[CLS] as an alternative to injection , psoriasis may be alleviated through topical delivery of plasmid dna to induce expression of il - 4 in the skin . [SEP]
[CLS] proof - of - concept has been demonstrated in a k14 - vegf transgenic mouse model of psoriasis . [SEP]
[CLS] atopic dermatitis ( ad ) affects 10 - 20 % of children and 1 - 3 % of adults worldwide . [SEP]
[CLS] ad typically manifests with severe redness and itching , skin lesions , and papules resulting in significant impact on quality of life . [SEP]
[CLS] similar to psoriasis , the inflammatory nature of the disease makes topical na treatment appealing . [SEP]
[CLS] although the exact cause of ad is not known , upregulation of inflammatory pathways such as the nf - κb inflammatory pathway are typically observed , and inhibition of these pathways have shown promise . [SEP]
[CLS] for example , delivery of anti - rela , an important member of the nf - κb inflammatory pathway , resulted in significant improvement in disease symptoms in mice . [SEP]
[CLS] in addition , knockdown of nf - κb with decoy odns also showed therapeutic promise in mice . [SEP]
[CLS] several cytokines are also believed to be associated with the pathogenesis of ad including il - 4 , - 5 , - 10 , and - 13 . [SEP]
[CLS] these cytokines are typically expressed in significantly higher quantities in ad lesions compared to healthy skin , and knockdown of these cytokines and others with topical na therapies has shown promise in mouse models of ad . [SEP]
[CLS] further , suppressing the th2 - type dominated immunological response typical of ad may hold therapeutic promise . [SEP]
[CLS] indeed , delivery of cpg oligonucleotides was shown to significantly reduce ad lesions in mice by shifting from th2 - type dominant immune response to a more balanced th1 / th2 immune response . [SEP]
[CLS] cosmetic conditions would also benefit significantly from topical na therapies developed as either pharmaceuticals or cosmeceuticals . [SEP]
[CLS] however , cosmeceutical development may benefit from faster translation to the clinic due to reduced fda regulations provided claims of the cosmeceutical are appropriately chosen . [SEP]
[CLS] for example , cellulite manifests in 80 - 90 % of postadolescent women . [SEP]
[CLS] it is characterized by an " orange peel " appearance of the skin primarily in the thighs and buttocks . [SEP]
[CLS] while physical burden is not associated with cellulite , significant emotional burden is typically associated with cellulite . [SEP]
[CLS] cellulite pathology is generally not well understood , however , a few na targets that show promise are proposed in literature . [SEP]
[CLS] for example , decreased microcirculation in subcutaneous adipose tissue is believed to play a role in cellulite formation . [SEP]
[CLS] specifically , overexpression of angiotensin - converting enzyme has been linked to higher production of angiotensin ii , a vasoconstrictive peptide B-material , along with lower production of bradykinin , a vasodilatative peptide B-material . [SEP]
[CLS] breakdown of the extracellular matrix ( ecm ) due to enzyme hyperactivity may also play an important role in cellulite . [SEP]
[CLS] normal ecm may potentially be restored by silencing enzymes like collagenase , elastase , and lipase . [SEP]
[CLS] finally , fibrosis and localized inflammation may play an important role in cellulite formation . [SEP]
[CLS] specifically , hypoxic inducible factor - 1 ( hif - 1 ) mutations that minimize its ability to induce fibrosis and inflammation have been shown to be associated with individuals who do not typically have cellulite , or have reduced severity of cellulite , while individuals with wild - type hif - 1 were shown to have a higher probability of more severe presentation of cellulite . [SEP]
[CLS] skin wrinkling is another cosmetic condition that generates significant emotional burden . [SEP]
[CLS] wrinkling affects everyone as they age , however , treatment is most typically sought by postadolescent females . [SEP]
[CLS] unfortunately , few effective treatments exist for the reduction of wrinkles . [SEP]
[CLS] currently , the most common treatment is intradermal injections of botulinum toxin a ( botox ) . [SEP]
[CLS] botox , however , is extremely toxic B-property and results in temporary muscular paralysis at the site of injection . [SEP]
[CLS] further , botox must be administered by a trained physician limiting its use and increasing its cost . [SEP]
[CLS] alternatively , topical na therapies may be beneficial as both a treatment and prevention strategy . [SEP]
[CLS] for example , elastase upregulation has been proposed as an important factor for ultraviolet irradiation induced wrinkle formation . [SEP]
[CLS] inhibition of elastase with n - phenethylphosphonyl - l - leucyl - l - tryptophane resulted in reduced formation of wrinkles in mice exposed to daily doses of ultraviolet irradiation . [SEP]
[CLS] therefore , knockdown of elastase with topically applied nas may be a viable option for the treatment and prevention of skin wrinkling , as well as provide patients with a safer and cheaper alternative to botox injections . [SEP]
[CLS] melasma is a hyperpigmentation disorder that manifests as darkening or browning of the skin , typically on the face . [SEP]
[CLS] melasma disproportionately affects women and disproportionately affects ethnicities with darker skin color , however overall , melasma is generally estimated to account for 4 - 10 % of all dermatological - related doctor visits . [SEP]
[CLS] moreover , melasma is commonly reported to induce significant psychological burden , social impairment , and reduced quality of life . [SEP]
[CLS] the most common treatments are skin lightening agents like tyrosinase inhibitors and spot removing agents like retinoic acid , kojic acid , and azelaic acid . [SEP]
[CLS] however , these are typically either ineffective or result in unsightly white spots when used in the clinic . [SEP]
[CLS] topical na therapies may prove beneficial for treating melasma without adverse side effects associated with current treatments . [SEP]
[CLS] specifically , studies have shown silencing of the microphthalmia - associated transcription B-event factor ( mitf ) gene could be an effective treatment strategy . [SEP]
[CLS] mitf encodes for tyrosinase , tyrosinase - related protein B-material 1 , and melanocortin 1 receptor B-material which are all involved in melanin synthesis . [SEP]
[CLS] topical delivery of anti - mitf sirna has shown promise in human trials . [SEP]
[CLS] despite significant efforts to deliver nas into the skin as well as identifying na targets for treating skin disease , significant hurdles remain . [SEP]
[CLS] three major areas posed serious challenges in the past and still remain the bottleneck to successful translation of topical na therapies : [SEP]
[CLS] ( 1 ) skin delivery , ( 2 ) cellular internalization , and ( 3 ) stability of nas . [SEP]
[CLS] these challenges must be addressed concurrently to develop successful na topical delivery systems for use in humans . [SEP]
[CLS] microneedles and other physical methods are the most effective at enhancing delivery into the skin , and the type and size of therapeutic is not restricted . [SEP]
[CLS] further , large depots can be easily incorporated for sustained release . [SEP]
[CLS] however , significant questions remain in regards to their effectiveness at localized and homogeneous delivery into skin tissue . [SEP]
[CLS] specifically , physical methods inherently result in localized penetration zones . [SEP]
[CLS] they do not address horizontal diffusion of na which is retarded by cell - cell tight junctions in the epidermis . [SEP]
[CLS] active methods such as electroporation and sonophoresis may also be limited by localized penetration zones ( fig . 3 ) . [SEP]
[CLS] the effect of size of penetration area on clinical outcome needs to be determined . [SEP]
[CLS] combination therapies with co - delivery of nas and , for example , tight junction modulators may be required for efficacy in humans . [SEP]
[CLS] also of concern , physical methods do not enhance cellular internalization nor protect nas from degradation in the skin . [SEP]
[CLS] nas need to enter into the cytoplasm or nucleus of cells B-material to elicit a therapeutic response . [SEP]
[CLS] physical methods alone cannot facilitate this enhancement , and therefore , they must inherently be combined with other technologies to enable cell B-material internalization . [SEP]
[CLS] for example , microneedle arrays integrating active methods like electroporation and sonophoresis have been designed to facilitate na delivery enhancement into cells B-material . [SEP]
[CLS] however , incorporating active methods for cell B-material internalization further adds complexity and cost . [SEP]
[CLS] passive methods for cell B-material internalization , for example , tagging nas with cell B-material - penetrating peptides B-material , cell - penetrating dendrimers B-nanoparticle , or cell - penetrating aptamers have the potential to provide affordable means of permeation enhancement . [SEP]
[CLS] in fact , several cell B-material - penetrating peptides B-material are currently in clinical trials which makes them an attractive option [SEP]
[CLS] on the other hand , cell B-material - penetrating peptides B-material combined with physical or active dermal penetration enhancement still neglect the stability issues of nas . [SEP]
[CLS] sirna , odn , and plasmid dna are susceptible to enzymes in the epidermis and dermis which can lead to significant degradation . [SEP]
[CLS] to address this concern , packaging nas in cationic B-material lipid B-material complexes or liposomes B-nanoparticle would both protect the na from enzymatic degradation as well as aid cell B-material internalization . [SEP]
[CLS] in addition , chemical modifications of nas like attaching charge neutral moieties via cleavable or reducible linkers or use of a phosphorothioate backbone has been shown to aid in internalization and protect from degradation , respectively , without compromising function . [SEP]
[CLS] the regulatory hurdles for medical devices and drug - device combinations must also be addressed . [SEP]
[CLS] physical methods are sophisticated and will be highly regulated . [SEP]
[CLS] fabrication , sterilization , and implementation of the device must be demonstrated to result in safe and effective therapy . [SEP]
[CLS] this regulation is in addition to any oversight process for the na itself and further complicates translation of topical na therapies . [SEP]
[CLS] along this line , a lot of effort has been spent over the years to address fabrication concerns . [SEP]
[CLS] for example , methods have been devised to fabricate microneedle devices using steel and other strong materials . [SEP]
[CLS] further , biocompatible B-property and biodegradable B-property microneedles have been proposed . [SEP]
[CLS] many of these methods can be scaled using the same processes used for integrated circuits which affords cost - effective manufacturing as well as the potential for outsourcing manufacturing . [SEP]
[CLS] lacking , however , are studies assessing effects of sterilization and implementation protocols on patient safety . [SEP]
[CLS] for example , all physical methods result in disruption of the skin barrier B-property . [SEP]
[CLS] this disruption will remain until re - epithelialization at the site of ablation can occur . [SEP]
[CLS] while barrier B-property function of the skin is diminished , pathogens may potentially enter the skin and cause infection . [SEP]
[CLS] therefore , the site of application may need to repeatedly be sterilized , or sealed with a sterile bandage , for days or weeks after application of the na therapy . [SEP]
[CLS] depending on the frequency of application as well as the site of application ( e . g . face , hands , feet ) , requiring a permanent or semi - permanent sterile bandage may not be tolerated by the patient . [SEP]
[CLS] prevalence of adverse events from physical ablation of the sc and epidermis must be determined before microneedle arrays and microporation devices see widespread use in the clinic . [SEP]
[CLS] perhaps the most significant drawback of both physical and active methods , however , is limited application area . [SEP]
[CLS] for diseases like hurley disease , eb , moderate to severe psoriasis , skin wrinkling , and many others which can occupy a large percentage of skin surface area , physical and active methods may not be practical . [SEP]
[CLS] in these cases , passive perturbation methods may be most effective . [SEP]
[CLS] passive transport enhancers can be formulated as a cream and applied to all areas of the body , including the face , in a patient friendly manner . [SEP]
[CLS] further , formulations incorporating synergistic combinations of skin penetrating and cell internalizing transport enhancers can be easily envisaged . [SEP]
[CLS] the main limitation of passive methods remains their low transport enhancement relative to physical and active methods . [SEP]
[CLS] further , passive transport enhancers like peptides B-material and dendrimers B-nanoparticle may be limited to use with nas of a certain size like antisense oligonucleotides and sirna as opposed to plasmid dna . [SEP]
[CLS] effort has been spent over the years to identify and understand passive transport enhancers . [SEP]
[CLS] high - throughput screening for chemical enhancers is well - documented and has proven useful for identifying synergistic combinations of chemicals that perturb the skin with minimal irritation . [SEP]
[CLS] along the same lines , peptides B-material can be screened in a high - throughput fashion using phagedisplay ( fig . 5 ) . [SEP]
[CLS] phage libraries applied to the skin are selected based on their ability to transport through the skin . [SEP]
[CLS] after only a few rounds , a library of ~ 10 9 peptide B-material sequences can be screened . [SEP]
[CLS] particularly advantageous , this technique can be used to identify peptides B-material that localize in particular layers of the skin e . g . epidermis or dermis . [SEP]
[CLS] delivery of na therapies to specific locations in the skin could be beneficial to limit off - target effects . [SEP]
[CLS] phage screening is a powerful technique , and an extensive number of phage libraries exist . [SEP]
[CLS] effort should be place on identifying novel peptides B-material with powerful skin - penetrating ability . [SEP]
[CLS] the same methodology could be used to screen for skin - penetrating aptamers , which to the authors ' knowledge has not been attempted to - date , further broadening the space of chemically and structurally distinct passive transporters for delivering nas . [SEP]
[CLS] as new transporters are identified , we can better understand how to overcome the skin barrier B-property to deliver increasingly large cargos . [SEP]
[CLS] for example , the number of skin - penetrating peptides B-material identified thus far have afforded a better understanding of their mechanism of transport enhancement . [SEP]
[CLS] interestingly , td - 1 , tat , and space peptide B-material all seem to bind and interact with keratin in the corneocytes in the skin facilitating transport of cyclosporine a through the transcellular pathway . [SEP]
[CLS] as a result of this work , we posit screening of low molecular weight ligands for affinity to keratin may help identify ligands with improved na delivery . [SEP]
[CLS] it is also conceivable that the mechanism of enhancement is dependent on the cargo delivered , however , similar studies with na cargos are severely lacking . [SEP]
[CLS] concurrently , definitive mechanistic studies to elucidate modes of transport enhancement of nanoparticles B-nanoparticle , liposomes B-nanoparticle , and spherical nucleic B-material acids I-material are lacking and studies that have been reported do not reach adequate consensus . [SEP]
[CLS] alvarez - roman et al . have studied the distribution of polystyrene particles after topical application . [SEP]
[CLS] they observed almost exclusive uptake into the hair follicles . [SEP]
[CLS] follicular uptake was dependent on size of the particles with 20 nm particles accumulating to a larger extent than 200 nm particles . [SEP]
[CLS] similar localization in the hair follicles has been observed for liposomes B-nanoparticle and ultradeformable liposomes B-nanoparticle , as well as titanium B-material dioxide I-material microparticles , micro / nanoemulsion B-nanoparticle droplets , lipoplexes , and solid B-nanoparticle lipid I-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] however , naked nas have also been shown to localize in hair follicles when applied without any nanocarrier which suggests nanocarriers may only hold potential as drug depots for controlled release topical formulations of nas . [SEP]
[CLS] still others have demonstrated dispersed distributions of nanocarriers into viable epidermis and dermis . [SEP]
[CLS] verma et al . demonstrated enhanced drug delivery using liposomes B-nanoparticle and also concluded that size was the most important parameter . [SEP]
[CLS] however , it is important to note that these studies were performed with fluorescent B-property drug and therefore do not distinguish between penetration of intact liposomes B-nanoparticle versus enhancement in drug delivery through simple fluidization of lipid B-material bilayers I-material . [SEP]
[CLS] in fact , kirjavainen et al . concluded that non - fusogenic fluorescently B-property - tagged liposomes B-nanoparticle do not penetrate skin . [SEP]
[CLS] instead , only liposomes B-nanoparticle that were fusogenic with sc lipids B-material were able to enhance drug delivery . [SEP]
[CLS] moreover , fusogenic liposomes B-nanoparticle enhanced penetration of drug applied in free solution subsequent to application of liposomes B-nanoparticle . [SEP]
[CLS] these data suggest liposomes B-nanoparticle may act through incorporation into and fluidization of the lipid B-material bilayers I-material to enhance drug delivery . [SEP]
[CLS] other factors of liposome B-nanoparticle design such as charge , amount of cholesterol , and acyl chain length did not appear to influence drug delivery , in contrast , geusens et al . concluded liposomes B-nanoparticle can penetrate intact skin if flexible enough . [SEP]
[CLS] it is reasonable that liposomes B-nanoparticle that can penetrate via the multilamellar B-material lipid B-material bilayers I-material in the sc . [SEP]
[CLS] others have also reached similar conclusions arguing ultraflexible liposomes B-nanoparticle create hydration - driven transport mechanisms through the skin . [SEP]
[CLS] however , since studies have shown localization of ultraflexible liposomes B-nanoparticle in hair follicles , another possibility is that flexibility does not aid penetration across multilamellar sc but instead enhances transport only across the unilamellar lining of hair follicles . [SEP]
[CLS] if confirmed , this may preclude treatment of regions devoid of hair follicles like palms and foot pads . [SEP]
[CLS] interestingly , spherical nucleic B-material acids I-material , are not be expected to fluidize lipid B-material bilayers I-material , nor are they be expected deform to facilitate passive diffusion through tight intercellular lipid B-material channels in the skin ; therefore , we would expect them to localize in hair follicles like the majority of solid nanoparticles B-nanoparticle > 40 nm studied to date . [SEP]
[CLS] yet , spherical nucleic B-material acids I-material exhibit extensive penetration homogenously into epidermal and dermal tissue how spherical nucleic B-material acids I-material penetrate skin has yet to be reported but should be of immediate interest given the significant potential this platform appears to have for treating skin disease . [SEP]
[CLS] although in vitro testing using franz diffusion cells B-material can be performed easily and quickly to screen large libraries of phage - displayed peptides B-material and aptamers , testing individual formulations of nanocarriers remains tedious . [SEP]
[CLS] thus , studies that continue to identify mechanisms of nanocarrier delivery and important design parameters for delivery enhancement are crucial to advance passive delivery methods . [SEP]
[CLS] on the other hand , passive enhancers can be significantly affected by the state of the sc . [SEP]
[CLS] for example , a liposome B-nanoparticle formulation may be engineered to be non - toxic B-property or non - irritating to intact skin . [SEP]
[CLS] however , when applied on diseased skin , because barrier B-property function is diminished , the same formulation may deliver an irritating or cytotoxic B-property dose . [SEP]
[CLS] in contrast , delivery using physical methods should be minimally affected by the diseased state of skin and active methods can be easily tuned to control the amount of dose delivered . [SEP]
[CLS] formulations of passive enhancers , especially complex formulations , are not as easily tuned . [SEP]
[CLS] this must be considered during the design and implementation of passive enhancers . [SEP]
[CLS] clearly , much is still unknown ; however , translation of na therapies appears to be close . [SEP]
[CLS] near - term challenges include validating safety of microneedle and microporation arrays . [SEP]
[CLS] establishment of regulatory guidelines will help on this front . [SEP]
[CLS] in particular , we posit physical methods for delivering cpg oligonucleotides or other na - based vaccine will be the first realization of topical na therapy in the clinic . [SEP]
[CLS] indeed , the benefits of needle - free immunization are well - established . [SEP]
[CLS] however , despite their superior delivery efficacy , active and physical methods are severely limited by application area , and therefore , are not ideal for local delivery of nas to skin tissue where disease symptoms manifest . [SEP]
[CLS] to this end , long - term challenges include high - throughput screening of passive enhancers , followed by systematic studies to elucidate mechanisms of na delivery . [SEP]
[CLS] a synergistic combination of passive sc , tight junction , and cell B-material membrane perturbers may be required to tackle most forms of skin disease and realize the full transformative potential of topical na therapies in the clinic . [SEP]
[CLS] topical application of nas for the treatment of skin disease is an advantageous route for the translation of na therapies to the clinic . [SEP]
[CLS] topical application offers many advantages over alternative methods of administration including avoidance of many challenges that are current roadblocks to systemic na therapy implementation . [SEP]
[CLS] although the skin does pose a significant barrier B-property to drug delivery , especially to large hydrophilic B-property macromolecules like nas , extensive effort has been spent to overcome this barrier B-property . [SEP]
[CLS] these efforts have been highlighted here , and include the use of microneedles , microporation , electroporation , iontophoresis , sonophoresis , nanoparticles B-nanoparticle , liposomes B-nanoparticle , spherical nucleic B-material acids I-material , peptides B-material , and dendrimers B-nanoparticle . [SEP]
[CLS] translation of these technologies to the clinic is sure to have a transformative impact on the burden of both skin disease as well as cosmetic conditions . [SEP]
[CLS] a large number of genetic and multifactorial inflammatory diseases result in chronic , physical and emotional burden to patients who have few treatment options other than repeated dosing by injection or infusion , or invasive surgery . [SEP]
[CLS] similarly , cosmetic conditions result in significant emotional burden with limited effective treatment options other than invasive surgery . [SEP]
[CLS] future efforts should focus on validating the safety of long - term physical disruption of the skin , better understanding the mechanisms na enhancement into skin via passive methods , and exhaustively screening for novel ligands and synergistic combinations of enhancers to realize the full potential of topical na therapies in the clinic . [SEP]
[CLS] the same strategy could be used to screen for aptamers . [SEP]
[CLS] transport pathways into the skin a : intercellular pathway through lipid B-material bilayers I-material . [SEP]
[CLS] b : transcellular pathway through keratinrich corneocytes . [SEP]
[CLS] c : shunt pathway through hair follicles and sweat ducts . [SEP]
[CLS] microneedle arrays offer controlled length and sharp tips for easy insertion through the sc and into the epidermis ( a ) scanning electron micrograph of a 20×20 silicon B-material microneedle array . [SEP]
[CLS] ( b ) scanning electron micrograph of a microneedle tip . [SEP]
[CLS] figure 2 . reproduced with permission from henry s , et al . [SEP]
[CLS] microfabricated microneedles : a novel approach to transdermal drug delivery . [SEP]
[CLS] journal of pharmaceutical sciences . 1998 ; 87 : 922 - 5 . [SEP]
[CLS] 3 . sonophoresis enhances delivery of na into skin through localized perturbation zones ( a ) top view of skin exposed to sulfarhodamine b and ultrasound . [SEP]
[CLS] ( b ) and ( c ) fitc - na delivery into skin after ultrasound treatment . [SEP]
[CLS] ( b ) cross - section of skin corresponding to a localized perturbation zone . [SEP]
[CLS] ( c ) cross - section of skin corresponding to a non - localized perturbation zone . [SEP]
[CLS] figure 3 . modified with permission from tezel a , et al . [SEP]
[CLS] topical delivery of anti - sense oligonucleotides using low - frequency sonophoresis . [SEP]
[CLS] pharmaceutical research . 2004 ; 21 : 2219 - 25 . [SEP]
[CLS] the anatomy of spherical nucleic B-material acid I-material nanostructures an inorganic B-material core I-material is densely functionalized with oligonucleotides containing three segments : a recognition sequence , a spacer B-material segment , and a chemical - attachment group . [SEP]
[CLS] additionally , other functional groups such as dye molecules , quenchers , modified bases , and drugs can be attached along any segment of the oligonucleotide . [SEP]
[CLS] figure 4 . reproduced with permission from cutler ji , auyeung e , mirkin ca . [SEP]
[CLS] spherical nucleic B-material acids I-material . [SEP]
[CLS] journal of the american chemical society . 2012 ; 134 : 1376 - 91 . [SEP]
[CLS] copyright 2012 american chemical society . [SEP]
[CLS] experimental design to screen for skin - penetrating peptides B-material using phage - display [SEP]
[CLS] peptide - conjugated nanoparticles B-nanoparticle ( nps B-nanoparticle ) have promising potential for applications in biosensing , diagnosis , and therapeutics because of their appropriate size , unique self - assembly , and specific substrate - binding properties . [SEP]
[CLS] however , controlled assembly and selective target binding are difficult to achieve with simple peptides B-material on np B-nanoparticle surfaces because high surface energy makes nps B-nanoparticle prone to self - aggregate and adhere nonspecifically . [SEP]
[CLS] here , we report the self - assembly and gelatin B-material binding properties of collagen mimetic peptide B-material ( cmp ) conjugated gold B-material nps B-nanoparticle ( cmp - nps B-nanoparticle ) . [SEP]
[CLS] we show that the orientation of cmps displayed on the np B-nanoparticle surface can control np B-nanoparticle assembly either by promoting or hindering triple helical folding between cmps of neighboring nps B-nanoparticle . [SEP]
[CLS] we also show that cmp - nps B-nanoparticle can specifically bind to denatured collagen by forming triple - helical hybrids between denatured collagen strands and cmps , demonstrating their potential use for detection and selective removal of gelatin B-material from protein B-material mixtures . [SEP]
[CLS] cmp conjugated nps B-nanoparticle offer a simple and effective method for np B-nanoparticle assembly and for targeting denatured collagens with high specificity . [SEP]
[CLS] therefore , they may lead to new types of functional nanomaterials B-material for detection and study of denatured collagen associated with diseases characterized by high levels of collagen degradation . [SEP]
[CLS] conjugation of biomolecules ( e . g . , dna , proteins B-material and peptides B-material ) to inorganic B-nanoparticle nanoparticles I-nanoparticle ( nps B-nanoparticle ) opened up a broad range of exciting research in photonics , 1 , 2 biocatalysis , 1 , 3 - 5 bioelectronics , biosensors , drug delivery , and gene regulation . [SEP]
[CLS] nps B-nanoparticle used in drug and gene delivery are particularly attractive , since their small size allows for tissue penetration as well as elimination , while still affording a high number of targeting moieties and bioactive molecules on np B-nanoparticle surfaces . [SEP]
[CLS] nps B-nanoparticle in these studies are often conjugated with molecules that have high affinity to specific biomolecules for the purpose of targeting diseased cells B-material or tissues ; however such molecules tend to also exhibit nonspecific affinity to other biomolecules . [SEP]
[CLS] this poses a significant difficulty for clinical applications that involve the use of nps B-nanoparticle in bodily fluid that is comprised of high ionic strengths and numerous surface active biomolecules . [SEP]
[CLS] even peptides B-material that are carefully designed to disperse nps B-nanoparticle do not protect them from aggregation under a wide range of conditions . [SEP]
[CLS] practical applications of np B-nanoparticle targeting rely on not only np B-nanoparticle ' s ability to recognize target molecules , but also its inertness toward nonspecific binding . [SEP]
[CLS] self - assembly is a powerful tool in controlling both the physical and biological properties of nps B-nanoparticle . [SEP]
[CLS] biomolecular recognition , such as dna hybridization , protein B-material - ligand interactions and peptide B-material - peptide interactions have been widely exploited for controlled assembly of nps B-nanoparticle . [SEP]
[CLS] a np B-nanoparticle ' s aggregated state is reported to affect their electro - optical properties and recent studies have shown that the np B-nanoparticle assemblies play an important role in delivery and elimination in vivo . [SEP]
[CLS] for example , the use of dna - modified gold B-material nps B-nanoparticle , which assemble into a superstructure , was reported to improve its accumulation in tumors B-material and at the same time facilitate elimination from the body , because after the accumulation , the assembly can degrade into individual nps B-nanoparticle which are cleared via the kidney . we believe that developing new tools for controlled assembly of nps B-nanoparticle will expand the design of new nano - and mesoscale materials for functional application in biotechnology . [SEP]
[CLS] collagen is the most abundant protein B-material in mammals , and controlling the collagen assembly process in the design of new biomaterials has been a long - standing interest for many researchers . [SEP]
[CLS] in particular , small peptides B-material that mimic the triple helical structure of collagen have been investigated for applications in disease detection and imaging , drug delivery , and tissue engineering . [SEP]
[CLS] collagen mimetic peptides B-material ( cmps ) are a family of synthetic peptides B-material that mimic the basic structural motif of natural collagen - the triple helix . [SEP]
[CLS] they are made of the gly - xaa - yaa triplet repeating sequence , where xaa and yaa are largely populated by proline and 4 ( r ) hydroxylproline , respectively . [SEP]
[CLS] cmps have a strong propensity to self - assemble into a triple helix structure , and have been used as synthetic models to study the structure and folding behaviors of collagens . [SEP]
[CLS] previously , our research group found that the cmps with the sequence ( gpo ) n ( n = 6 - 10 , o = hydroxylproline ) have a strong propensity to bind to denatured collagens ( also known as gelatin B-material ) by forming triple helical hybrids . [SEP]
[CLS] this hybridization process is similar to small dna fragments binding B-event to I-event dna I-event strands having complementary base pairs . [SEP]
[CLS] we also reported that the gold B-material nps B-nanoparticle conjugated with cmps are colloidally stable in aqueous solution with a wide range of ph ( ph 0 - 14 ) and even in high salt B-material concentration ( 5 m nacl ) . [SEP]
[CLS] it is believed that the polar , nonionic character , and the extended conformation of the peptides B-material prevent aggregation of the cmp conjugated nps B-nanoparticle in aqueous solution . [SEP]
[CLS] except for hybridization to gelatin B-material strands , the peptide B-material has low nonspecific binding affinity to other biomolecules . [SEP]
[CLS] therefore , cmp conjugated nps B-nanoparticle ( broadly designated as cmp - nps B-nanoparticle ) are ideal materials for biological assembly and targeting studies . [SEP]
[CLS] there are a number of potential applications for cmp - nps B-nanoparticle . [SEP]
[CLS] since cmp is known to hybridize with denatured collagen strands , cmp - np B-nanoparticle can directly be used as a contrast B-technique agent I-technique ( in tem or ct ) to detect the location of collagen damaged by either proteases or by injury . [SEP]
[CLS] the aggregation behavior of cmp - nps B-nanoparticle can be affected by the presence of collagen fragments , which could be exploited for detection of diseases associated with high collagen remodelling activity . [SEP]
[CLS] cmp - np B-nanoparticle can also be used to remove denatured collagen from protein B-material mixtures , since gelatin B-material is a common additive in protein B-material drug formulation that can cause negative host response . [SEP]
[CLS] in this study , we present the self - assembly and gelatin B-material binding properties of cmp - nps B-nanoparticle . [SEP]
[CLS] here , we show that the triple helical folding between cmps of neighboring nps B-nanoparticle can control the large - scale np B-nanoparticle assembly when the peptides B-material are displayed with correct orientation ( figure 1 ) . [SEP]
[CLS] we also show that the cmp - nps B-nanoparticle can bind to denatured collagen strands with high specificity , and demonstrate their use in selective removal of gelatin B-material from protein B-material mixtures . [SEP]
[CLS] the results suggest that the cmp - nps B-nanoparticle can potentially be used to detect diseases that produce high levels of denatured collagen ( e . g . , arthritis ) , and to remove gelatin B-material from commercial protein B-material drug formulations for reduced side effects . [SEP]
[CLS] all chemicals were purchased from sigma - aldrich ( st . louis , mo , usa ) and used without further purification . [SEP]
[CLS] citrate - capped aunp B-nanoparticle solution was purchased from ted pella , inc . ( redding , ca ) . [SEP]
[CLS] for peptide B-material synthesis , fmoc - gly - oh , fmoc - pro - oh , fmoc - ahx - oh , and 1 - hydroxy - 6 - chloro - benzotriazole ( cl - hobt ) were purchased from advanced chemtech ( louisville , ky ) . [SEP]
[CLS] o - ( benzo - triazol - 1 - yl ) - n , n , n ′ , n ′ - tetramethyluronium hexafluorophosphate ( hbtu ) was purchased from aapptec ( louisville , ky ) , n , ndiisopropylethylamine ( dipea ) from acros ( geel , belgium ) , and dimethylformamide ( dmf ) , n - methyl - 2 - pyrrolidone ( nmp ) , and trifluoroacetic acid ( tfa ) were purchased from fisher ( pittsburgh , pa ) and used without further purification . [SEP]
[CLS] fmoc - hyp ( tbu ) - oh and fmoc - cys ( trt ) - oh were purchased from emd millipore ( temecula , ca ) . [SEP]
[CLS] tentagel r ram resin was purchased from peptides B-material international ( louisville , ky ) . [SEP]
[CLS] fitc labeled gelatin B-material was purchased from life technology ( grand island , ny ) . [SEP]
[CLS] cmps were synthesized on the tentagel r ram resin via standard f - moc chemistry using focus xc peptide B-material synthesizer ( aapptec , louisville , ky ) with coupling cycles based on hbtu / diea - mediated ( adv chemtech , louisville , ky ) activation . [SEP]
[CLS] 5 - fold molar excess of the amino B-material acids I-material and coupling reagents were used in a typical coupling reaction . [SEP]
[CLS] the peptides B-material were cleaved from the resins by treatment with water B-material / 1 , 2 - ethanedithiol / thioanisole / trifluoroacetic acid ( 2 . 5 / 2 . 5 / 1 / 94 ) for at least 2 h . [SEP]
[CLS] peptides B-material were purified by reverse phase high performance liquid B-technique chromatography I-technique ( rp - hplc ) ( agilent , santa clara , ca ) on a c18 column with a gradient of water B-material - acetonitrile containing 0 . 1 % trifluoroacetic acid ( tfa ) . [SEP]
[CLS] the mass and purity of the cmps were analyzed by matrix - assisted laser desorption / ionization B-property time - of - flight mass spectrometry ( maldi - tof ms ) ( ultraflextreme , bruker daltonics , billerica , ma ) and hplc ( agilent , santa clara , ca ) . [SEP]
[CLS] the concentration of peptide B-material solution was calculated from the weight of the dry peptide B-material powder . [SEP]
[CLS] we also prepared tyrosine containing cmps , cyg ( gpo ) 9 , and ( gpo ) 9 gyc to quantify and calibrate the concentration of cmps by measuring their uv - vis absorbance at 280 nm ( extinction coefficient of 1280 m −1 cm −1 ) using a spectramax m - 2 microplate reader ( molecular devices , sunnyvale , ca ) . [SEP]
[CLS] 5 ( 6 ) - carboxyfluorescein ( cf ) conjugated cmps were synthesized as reported before . [SEP]
[CLS] cmps with a single cysteine B-material residue ( cys - cmps ) at n terminus ( compound 1 , table 1 ) was conjugated to 10 nm gold B-material np B-nanoparticle to produce cmp conjugated np B-nanoparticle designated as np B-nanoparticle - 1 . [SEP]
[CLS] similarly , cmp with a single cysteine B-material residue at c terminus ( compound 2 , table 1 ) was conjugated to 20 nm gold B-material np B-nanoparticle to produce np B-nanoparticle - 2 . [SEP]
[CLS] in a typical experiment , purified cmp was preheated at 80 °c for 5 min to melt the triple helices and added to a citrate - stabilized gold B-material np B-nanoparticle solution to give final concentrations of 25 μm of cmp and 5 nm of nps B-nanoparticle , followed by incubation B-technique at room temperature overnight . [SEP]
[CLS] the resulting reaction mixture was heated again at 80 °c for 5 min , and excess free cmps were removed by repeated centrifugation ( 20 , 000 rcf ) and washing in di water B-material . [SEP]
[CLS] the concentrations of cmp - nps B-nanoparticle were determined using optical absorbance at 520 nm . [SEP]
[CLS] for np B-nanoparticle assembly , solutions of np B-nanoparticle - 1 ( 50 μl , 20 nm ) and np B-nanoparticle - 2 ( 50 μl , 2 nm ) were mixed to yield a final concentration of 10 nm and 1 nm ( ratio 10 : 1 ) , respectively . [SEP]
[CLS] the solution mixture was left at 4 °c overnight . [SEP]
[CLS] for the disassociation experiment , free ( gpo ) 9 ( 12 . 5 μl , 4 mm ) was preheated to 80 °c for 5 min to melt the triple helix , and immediately added to the above np B-nanoparticle - 1 and np B-nanoparticle - 2 mixture solution followed by incubation B-technique at 4 °c overnight . [SEP]
[CLS] cf - ( gpo ) 6 and np B-nanoparticle - 1 were mixed together to yield a final concentration of 10 μm and 10 nm , respectively . [SEP]
[CLS] the mixture was first heated at 80 °c for 5 min to melt all the triple helices , followed by incubation B-technique at 4 °c overnight . [SEP]
[CLS] the cf - ( gpo ) 6 that did not bind to np B-nanoparticle - 1 were removed by first centrifugation of the np B-nanoparticle solution to pellet out the np B-nanoparticle - 1 , followed by decanting cf - ( gpo ) 6 containing supernatant . [SEP]
[CLS] spectramax m - 2 microplate reader was used to measure the fluorescence B-property ( ex : 489 nm , em : 533 nm ) at predetermined temperature . [SEP]
[CLS] each binding experiment was done in triplicate . [SEP]
[CLS] tem was performed on fei tecnai t12 microscope ( fei , hillsboro , or ) operated at 120 kv . tem samples were prepared by applying a drop of np B-nanoparticle containing solutions onto a copper B-material grid covered with a thin carbon B-material film ( ems , hatfield , pa ) followed by overnight drying at room temperature . [SEP]
[CLS] to test the binding of denatured collagen , a drop ( 10 μl ) of a suspension of type i collagen fibers ( 0 . 5 mg / ml in phosphate buffer solution ) which was denatured by heating to 80 °c for 5 min and cooled to room temperature , was added to a tem grid . [SEP]
[CLS] after 1 min , the excess solution was wicked away with a blotting paper , and 10 μl of np B-nanoparticle - 1 ( 10 nm ) containing 1 % bsa was applied . [SEP]
[CLS] the grid was incubated B-technique for 10 min , and washed 3 times with di water B-material . [SEP]
[CLS] the final sample was stained with 2 % ( weight / volume ) uranyl acetate by applying 10 μl staining solution onto the grid , and after 1 min , the excess solution was wicked away with a blotting paper . [SEP]
[CLS] the grid was dried at room temperature overnight . [SEP]
[CLS] the images were processed using gatan digital micrograph software ( gatan , pleasanton , ca ) and imagej ( national institutes of health , bethesda , md ) . [SEP]
[CLS] dls measurements were performed using malvern zetasizer nano s ( malvern , worcestershire , uk ) , equipped with a 50 mw laser beam and a standard 633 nm laser filter . [SEP]
[CLS] fifty microliters of sample solution in a disposable micro volume polystyrene cuvette ( malvern , worcestershire , uk ) was used for all the measurements . [SEP]
[CLS] all samples were subjected to thorough pipetting prior to measurement to ensure that nps B-nanoparticle were dispersed in solution . [SEP]
[CLS] cd spectra were recorded on a jasco j - 1500 cd ( jasco , tokyo ) in 0 . 10 mm quartz cells B-material . [SEP]
[CLS] all cmp samples were prepared in water B-material and incubated B-technique at 4 °c for at least 24 h prior to cd measurement . [SEP]
[CLS] spectra were recorded from 190 to 300 nm at a scanning rate of 100 nm / min at 0 . 5 nm increment . [SEP]
[CLS] cd melting experiments were performed in the temperature range from 20 to 90 °c at a heating rate of 1 °c / min . [SEP]
[CLS] the intensity of the cd signal at 225 nm was monitored as a function of temperature . [SEP]
[CLS] melting temperatures were determined from the maximum of the first derivative of the melting curves . [SEP]
[CLS] a 100 nm gold B-material np B-nanoparticle was conjugated with 1 as described above to produce large size cmp - np B-nanoparticle ( designated as cmp - np100 ) . [SEP]
[CLS] cmp - np100 and fitc labeled gelatin B-material were mixed to a final concentration of 5 pm and 25 μg / ml , respectively . [SEP]
[CLS] this mixture was incubated B-technique at room temperature for 2 h , followed by centrifuging at 2300 rcf for 5 min to separate the gelatinbound cmp - np100 . [SEP]
[CLS] the gelatin B-material remaining in solution was quantified by measuring their fluorescent B-property intensity using spectramax m - 2 microplate reader ( ex : 489 nm , em : 533 nm ) . [SEP]
[CLS] to prepare samples for uv - vis and sds - page characterization , a protein B-material mixture containing conalbumin ( 75 kda ) , bovine albumin serum ( bsa , 66 kda ) , carbonic anhydrase ( 29 kda ) , and ribonuclease a ( 13 . 7 kda ) , with or without gelatin B-material was mixed with cmp - np100 to yield final concentrations of 50 μg / ml of each protein B-material and 5 pm of np B-nanoparticle . [SEP]
[CLS] this mixture was incubated B-technique at room temperature for at least 2 h prior to uv - vis measurement , and centrifuged at 2 , 300 rcf for sds - page . [SEP]
[CLS] protein B-material mixture was replaced with mouse serum , and the gelatin B-material removal procedure was performed under the same condition : 25 μl of mouse serum was mixed with 75 μl of cmp - np100 solution to yield a final concentration of 5 pm cmp - np100 . [SEP]
[CLS] to prepare gelatin B-material solution , a rat tail tendon ( type i collagen ) in acidic solution was first neutralized using 1× pbs buffer ( ph 7 . 4 ) , followed by heating at 80 °c for 5 min to induce permanent protein B-material denaturation . [SEP]
[CLS] one of the main goals of this work was to develop a np B-nanoparticle system that could detect the presence of denatured collagen or collagen - like molecules by changes in the physical properties or aggregation behavior of the nps B-nanoparticle . [SEP]
[CLS] to study the behavior of np B-nanoparticle assembly resulting from the orientation of cmp display ( which could either inhibit or promote np B-nanoparticle assembly ) , we synthesized two different types of cysteine containing cmps ( cys - cmps ) , one having a single cysteine B-material residue at the n terminus designated as peptide B-material 1 ( table 1 ) , and the other having a single cysteine B-material residue at the c terminus , designated as peptide B-material 2 . [SEP]
[CLS] as a control , we also prepared a peptide B-material ( 3 ) which had a scrambled sequence containing nine residues of g , p , and o . [SEP]
[CLS] cys - cmps were used to functionalize the gold B-material np B-nanoparticle surfaces via the ligand exchange reaction as reported previously . [SEP]
[CLS] the 10 nm particles were conjugated with 1 ( designated as np B-nanoparticle - 1 , figure 1 ) , and the 20 nm particles were conjugated with 2 ( designated as np B-nanoparticle - 2 , figure 1 ) . [SEP]
[CLS] the cys - cmps were first heated in order to melt the triple helix before reacting with the nps B-nanoparticle to ensure that the cmps were conjugated to the np B-nanoparticle in a single strand form . [SEP]
[CLS] because of the opposite location of cys in the two peptides B-material , np B-nanoparticle - 1 displays cmps with its c termini pointing outward , while np B-nanoparticle - 2 displays cmps with its n termini pointing outward . [SEP]
[CLS] the size and optical properties of these particles after cys - cmps conjugation were assessed by dynamic B-technique light I-technique scattering I-technique ( dls ) , transmission B-technique electron I-technique microscopy I-technique ( tem ) and uv - vis spectroscopy B-technique ( figure 2 ) . [SEP]
[CLS] the uv - vis spectra were red - shifted by approximately 5 nm after cys - cmp conjugation for both np B-nanoparticle - 1 and np B-nanoparticle - 2 ( figure 2c ) . [SEP]
[CLS] the dls indicated hydrodynamic sizes of 31 . 0 and 38 . 3 nm respectively for np B-nanoparticle - 1 and np B-nanoparticle - 2 ( figure 2a and b , lower panels ) , which correspond to a peptide B-material layer thickness of 10 . 5 and 9 . 2 nm , respectively . [SEP]
[CLS] these values are comparable to the estimated value of 8 . 7 nm calculated for cys - cmp assuming a polyproline - ii helix as previously reported . [SEP]
[CLS] these results indicate that irrespective of cmp attachment direction , the peptides B-material are extended outward from the np B-nanoparticle surface . [SEP]
[CLS] pseudohexagonal lattice arrangement of nps B-nanoparticle in the tem ( figure 2a and b ) suggests that the nps B-nanoparticle are behaving similar to hard spheres due to densely passivated cys - cmp layers , which prevent np B-nanoparticle interactions . [SEP]
[CLS] to first demonstrate that the cmps on np B-nanoparticle surface are able to hybridize with other cmps , we investigated the binding and release characteristics of cf labeled cmp [ cf - cmp : cf - ( gpo ) 6 ] for np B-nanoparticle - 1 . [SEP]
[CLS] a mixed solution of cf - cmp and np B-nanoparticle - 1 was heated to 80 °c for 5 min , followed by incubation B-technique at 4 °c overnight . [SEP]
[CLS] np B-nanoparticle - 1 purified from unbound cf - cmps by centrifugation showed negligible fluorescence B-property intensity at 25 °c ; however when heated to 40 °c , the intensity increased by over 3 orders of magnitude ( figure 3 ) . [SEP]
[CLS] at 25 °c , cf - cmp is bound to the np B-nanoparticle - 1 by triple helical folding which puts cf in close proximity to the np B-nanoparticle surface resulting in fluorescence B-property quenching . [SEP]
[CLS] at 40 °c , however , the triple helices melt ( figure s1 ) releasing the cf - cmps from the np B-nanoparticle surface which is seen by a marked increase in fluorescence B-property intensity ( figure 3 ) . [SEP]
[CLS] under the same condition , the cmp with a scrambled sequence ( cf - g 9 p 9 o 9 ) did not hybridize with np B-nanoparticle - 1 as evidenced by negligible change in fluorescence B-property before and after the heating ( figure 3 ) . [SEP]
[CLS] the results confirm that the np B-nanoparticle - 1 is able to interact with cmps in solution via triple helical folding . [SEP]
[CLS] we were unable to determine either the cd signature or the cd melting behavior of the triple helix , because light scattering from the nps B-nanoparticle impeded the cd measurements . [SEP]
[CLS] although the two types of nps B-nanoparticle repel on their own , when mixed together , they exhibited strong tendency to aggregate ( figure 4a ) . [SEP]
[CLS] when np B-nanoparticle - 1 and np B-nanoparticle - 2 were mixed at a ratio of 10 : 1 , respectively , the initial pink color of the mixture faded after overnight incubation B-technique at 4 °c . [SEP]
[CLS] this was corroborated by the uv - vis spectra , which showed a red - shift ( from 524 to 550 nm ) and significant broadening of the peak ( figure 4b ) . [SEP]
[CLS] dls indicated formation of large clusters , which were about 6 times the size of np B-nanoparticle - 1 ( figure 4c ) . [SEP]
[CLS] under tem , the nps B-nanoparticle exhibited a well - defined assembly pattern , where nearly all 20 nm nps B-nanoparticle ( np B-nanoparticle - 2 ) were fully surrounded by 7 to 8 of the 10 nm nps B-nanoparticle ( np B-nanoparticle - 1 ) with 5 . 0 ± 1 . 0 nm spacing ( figure 4a ) . [SEP]
[CLS] this spacing is approximately half the interparticle distance between single type of nps B-nanoparticle ( 11 . 7 ± 1 . 0 nm ) , which suggests that np B-nanoparticle - 1 and np B-nanoparticle - 2 are assembled by interdigitation of cmps . [SEP]
[CLS] since tem is recorded under dry condition which intensifies np B-nanoparticle aggregation , it does not reflect the true aggregation behavior of nps B-nanoparticle in solution . [SEP]
[CLS] however , in this case where the interparticle distance is directly related to the length of the cmp , it is reasonable to infer the interparticle distance in solution from the tem , particularly knowing that there is only about 1 . 3 % length difference in collagen triple helix between dry and wet conditions . [SEP]
[CLS] the assembly of np B-nanoparticle - 1 and np B-nanoparticle - 2 into large clusters is a result of two key characteristics of cmps as they are attached to the np B-nanoparticle surfaces . [SEP]
[CLS] first , the cmps conjugated on np B-nanoparticle surfaces do not readily form stable triple helix among the neighboring cmps . [SEP]
[CLS] anchoring the ends of the peptide B-material chains to the surface of the nps B-nanoparticle without a flexible linker appears to prohibit the single amino B-material acid I-material axial shift between cmp chains , which is needed for the triple - helical folding . [SEP]
[CLS] therefore , the triple helix of full length cmp cannot be assembled on the np B-nanoparticle surface , although it is possible that a shorter triple helix of much lower stability could be assembled . inability to form a stable triple helix for cmps anchored on nps B-nanoparticle was discussed in our previous paper . [SEP]
[CLS] second , the cmps can trimerize only when they are in a parallel orientation . [SEP]
[CLS] cmps on cmp - nps B-nanoparticle repel each other when two identical particles come in contact , since cmps are in an antiparallel orientation . [SEP]
[CLS] only when two different nps B-nanoparticle , each with opposite cmp polarity ( as is the case with np B-nanoparticle - 1 and np B-nanoparticle - 2 ) , come in contact , the cmps are parallel and therefore capable of folding into triple helices . [SEP]
[CLS] when the 10 nm nps B-nanoparticle conjugated with the scrambled sequence ( np B-nanoparticle - 3 ) were mixed with np B-nanoparticle - 2 , there was neither a spectral shift ( figure 4b ) nor the formation of particle assembly ( figure 4d ) . [SEP]
[CLS] these results support the process of triple helical folding as the main mechanism of np B-nanoparticle - 1 and np B-nanoparticle - 2 assembly . [SEP]
[CLS] we questioned if the assembly between np B-nanoparticle - 1 and np B-nanoparticle - 2 can be disrupted by addition of free cmps . [SEP]
[CLS] such disruption would lead to a change in color which can be exploited for detection of collagen fragments associated with arthritis and osteoporosis . [SEP]
[CLS] when cmps with the sequence of ( gpo ) 9 , preheated to 80 °c , were added to the solution mixture containing assemblies of np B-nanoparticle - 1 and np B-nanoparticle - 2 , as can be seen in figure 4e , the large - scale assemblies broke down into small clusters . [SEP]
[CLS] although , the well - defined flower - like structures were not seen , the formation of small extended aggregates was reminiscent of the larger particles . [SEP]
[CLS] we expected that the free ( gpo ) 9 were able to disrupt np B-nanoparticle - 1 / np B-nanoparticle - 2 assembly by invading the triple helical connection by strand exchange ; however , this did not completely eliminate the large - scale assembly , possibly because the particles are held tightly together by strong ( gpo ) 9 folding . [SEP]
[CLS] preincubating the np B-nanoparticle - 1 and np B-nanoparticle - 2 with free ( gpo ) 9 separately , prior to mixing , resulted in complete separation and random dispersion of np B-nanoparticle - 1 and np B-nanoparticle - 2 , as evidenced by dls and tem ( figure s2 ) . [SEP]
[CLS] we hypothesized that the cmp ' s structural orientation on the np B-nanoparticle surface could have an effect on the assembly behavior of the nps B-nanoparticle . [SEP]
[CLS] specifically , we thought that when cmp is conjugated to the np B-nanoparticle surface via cys which is close to the middle of the peptide B-material sequence , the cmp ' s orientation could be more parallel to the np B-nanoparticle surface ( compared to end anchored cmp - np B-nanoparticle ) and that it would result in formation of a less dense cmp layer . [SEP]
[CLS] the less dense cmp layer would lead to a more favorable particle assembly because the two cmp layers can penetrate each other more readily . [SEP]
[CLS] to test this idea , we synthesized ( gpo ) 2 gco - ( gpo ) 6 ( 4 , table 1 ) , which has cys at the 1 / 3 position of the cmp . [SEP]
[CLS] due to the shorter consecutive gpo length , the melting temperature of 4 was 55 °c , which is approximately 20 °c below that of the c ( gpo ) 9 ( figure s3 ) . [SEP]
[CLS] in contrast to np B-nanoparticle - 1 and np B-nanoparticle - 2 , the 10 nm nps B-nanoparticle conjugated with 4 ( np B-nanoparticle - 4 ) had a propensity to self - assemble into large aggregates with a particleparticle distance of 3 . 2 ± 0 . 6 nm ( figure 5a ) . [SEP]
[CLS] the 20 nm nps B-nanoparticle conjugated with 4 exhibited the same behavior ( figure s4 ) . [SEP]
[CLS] we believe that the np B-nanoparticle - 4s self - assemble because the peptide B-material layer is less dense than the np B-nanoparticle - 1 and np B-nanoparticle - 2 as we had expected . [SEP]
[CLS] ( figure 5e ) [SEP]
[CLS] we also believe that the peptides B-material are unorganized and are in random direction on the np B-nanoparticle surface , all of which favor the peptide B-material interactions and aggregation of the same type of nps B-nanoparticle by triple helical association of parallely orientated cmps , particularly via the ( gpo ) 6 portion of the peptides B-material . [SEP]
[CLS] we determined the number of peptides B-material on np B-nanoparticle surface using a titration method ( figure s5 ) : 13 np B-nanoparticle - 4 had 371 ± 8 of peptides B-material , while the same size 10 nm np B-nanoparticle - 1 had 517 ± 38 peptides B-material per np B-nanoparticle . [SEP]
[CLS] these results indicate that the individual peptides B-material on np B-nanoparticle - 4 occupy a larger surface area , which results in significant reduction in the number of peptides B-material immobilized on the surface as compared to that of the np B-nanoparticle - 1 . [SEP]
[CLS] the less dense peptide B-material layer with more open area seems to offer favorable conditions for interparticle interactions . [SEP]
[CLS] when np B-nanoparticle - 4 was mixed with 20 nm au - c ( gpo ) 9 at a molar ratio of 10 : 1 ( small vs large particles ) , large aggregates were formed as the np B-nanoparticle - 4s were able to self - assemble , and also assemble with the 20 nm au - c ( gpo ) 9 ( figure 5b and 5f ) . [SEP]
[CLS] no aggregation was observed when the 20 nm np B-nanoparticle conjugated with the scrambled peptide B-material sequence ( 3 ) was mixed with np B-nanoparticle - 4 ( figure 5c ) . [SEP]
[CLS] these results indicate that the triple helical folding is dictating the aggregation behavior of the two particles in a manner similar to np B-nanoparticle - 1 / np B-nanoparticle - 2 assembly . [SEP]
[CLS] in contrast to np B-nanoparticle - 1 / np B-nanoparticle - 2 assembly , however , addition of excess cmps directly to the aggregates turned large aggregates into smaller clusters ( figure 5d ) . [SEP]
[CLS] among the combinations of nps B-nanoparticle tested , this produced the greatest change in the size of the aggregates , which we believe to be the result of nonoptimal packing of the cmps , allowing free cmps to readily exchange with the cmps on the np B-nanoparticle surface and disrupt particle aggregation . [SEP]
[CLS] we expect that these np B-nanoparticle systems , which can be induced to change their aggregation state by triple helical hybridization , could potentially be used for detecting collagen fragments commonly found in diseases associated with high extracellular matrix degradation , such as arthritis and osteoporosis . [SEP]
[CLS] since cmp has been reported to bind to denatured collagens by forming a triple - helical hybrid with the denatured collagen strands , we anticipated that cmps conjugated to gold B-material nps B-nanoparticle could also target denatured collagen with high specificity . [SEP]
[CLS] to investigate this , a denatured type i collagen solution was prepared by heating the solution to 80 °c for 5 min , followed by treatment with np B-nanoparticle - 1 . [SEP]
[CLS] tem showed that np B-nanoparticle - 1 bound to denatured collagen fibers ( figure 6a and b ) in high density , whereas , low nonspecific binding was observed for intact collagen fibers treated with np B-nanoparticle - 1 ( figure 6c ) . [SEP]
[CLS] cmp - nps B-nanoparticle were previously used to image unstable domains of the collagen triple helix in intact collagen fibers ; however , this is the first time that its specific affinity to fully denatured collagen strands is presented . [SEP]
[CLS] the results indicate that cmp - nps B-nanoparticle can specifically bind to denatured collagen strands , which can be exploited for detection or even isolation of gelatin B-material from protein B-material mixtures . [SEP]
[CLS] in addition to applications in medical device coatings B-material , gelatin B-material is widely used as stabilizers for protein - based pharmaceuticals , such as vaccines , antibodies B-material , and therapeutic proteins B-material [SEP]
[CLS] gelatin B-material in these formulations can interfere with further chemical modification , for example , in antibody conjugation chemistry . [SEP]
[CLS] moreover , gelatin B-material - containing vaccines have been found to cause severe , although rare reactions , especially in vaccinating children . [SEP]
[CLS] for these reasons , we investigated the possibility of using cmp - nps B-nanoparticle for selective removal of gelatin B-material from protein B-material mixtures . [SEP]
[CLS] we conjugated c ( gpo ) 9 to a large 100 nm gold B-material np B-nanoparticle ( designed as cmp - np100 ) which can be removed by low speed ( 2300 rcf ) centrifugation , and investigated its specific binding to gelatin B-material as well as its potential to remove gelatin B-material from a protein B-material mixture . [SEP]
[CLS] the average number of peptides B-material immobilized on the np100 surface was 52 818 ( figure s5 ) , corresponding to an average surface area of 59 a 2 for a single strand cmp which is comparable to np B-nanoparticle - 1 ( 61 a 2 ) and np B-nanoparticle - 2 ( 62 a 2 ) . [SEP]
[CLS] tem revealed that cmp - np100 bound to gelatin B-material fiber networks with high affinity ( figure 7a ) . [SEP]
[CLS] therefore , we prepared a pull - down assay using fluorescently B-property labeled gelatin B-material ( figure 7b ) . [SEP]
[CLS] the results showed that approximately 90 % of the gelatin B-material in solution was removed by cmp - np100 ( 5 pm ) , while the same np B-nanoparticle conjugated with a scrambled sequence was ineffective at gelatin B-material removal with 90 % of gelatin B-material remaining in the solution ( figure 7b ) . [SEP]
[CLS] addition of excess amounts of free single strand ( gpo ) 9 to the cmp - np100 solution prior to mixing with gelatin B-material solution drastically reduced the gelatin B-material removal capacity , to less than 40 % ( figure 7b ) . [SEP]
[CLS] these results indicate that gelatin B-material can be isolated from solution by the triple helical hybridization between gelatin B-material strands and cmps displayed on the np B-nanoparticle surface . [SEP]
[CLS] to demonstrate gelatin B-material binding specificity , we prepared a protein B-material solution composed of several common proteins B-material [ i . e . , conalbumin , bovine serum albumin ( bsa ) , carbonic B-material anhydrase , and rebonuclease a ] , into which cmp - np100 were added and incubated B-technique . [SEP]
[CLS] even in the presence of a protein B-material mixture , the surface plasmon resonance ( spr ) of the cmp - np100 remained nearly identical to the control nps B-nanoparticle in a blank solution ( figure 7c ) . [SEP]
[CLS] in contrast , a significant red - shift ( from 578 to 700 nm ) was observed in the spr signal for nps B-nanoparticle added to the solution containing the same amount of gelatin B-material . [SEP]
[CLS] there was no change in spr signal when nps B-nanoparticle conjugated with the scrambled sequence were used ( figure 7c ) under identical conditions . [SEP]
[CLS] spr is one of the most sensitive techniques available to detect molecular binding in solution . [SEP]
[CLS] we were able to detect as low as 0 . 5 ng / ml of gelatin B-material ( figure 7d and figure s6 ) . [SEP]
[CLS] the fact that there was almost no change in spr signal in the common protein B-material solutions but there was over a 100 nm shift in the gelatin B-material solution attests to the np B-nanoparticle ' s high binding specificity for denatured collagen as well as the potential for sensing collagen denaturation . [SEP]
[CLS] cmp - np100 was incubated B-technique with the protein B-material mixture solution containing gelatin B-material , and a pulldown assay was performed to remove the cmp - np100 by centrifugation . [SEP]
[CLS] only gelatin B-material was observed in the pellet , together with the nps B-nanoparticle , while all other proteins B-material remained in the supernatant , as evidenced by sds - page ( figure 7e ) . [SEP]
[CLS] similar results were obtained when the same experiment was performed with a mouse serum ( figure s7 ) . [SEP]
[CLS] the results show that the cmp conjugated gold B-material nps B-nanoparticle can selectively bind to gelatin B-material with little background binding B-event to I-event other I-event proteins I-event , and that the nps B-nanoparticle can be used to selectively remove gelatin B-material from protein B-material mixtures . [SEP]
[CLS] although cmp - nps B-nanoparticle were able to detect as low as 0 . 5 ng / ml of gelatin B-material using spr method ( figure 7d ) , the gelatin B-material removal capacity via gelatin B-material binding and centrifugation of cmp - np B-nanoparticle was determined to be 20 μg / ml of gelatin B-material removal per 1 pm of cmp - np100 . [SEP]
[CLS] vaccine and antibody B-material formulations typically contain milligrams of gelatin B-material . [SEP]
[CLS] therefore , gelatin B-material removal capacity of current system is too low for practical use ; however , with the employment of other purification methods , such as affinity column chromatography B-technique , we believe that the gelatin B-material removal capacity can be improved significantly . [SEP]
[CLS] cmp conjugated gold B-material nps B-nanoparticle can assemble into large aggregates when cmps displayed on different nps B-nanoparticle can combine in a parallel fashion to form triple helices . [SEP]
[CLS] this np B-nanoparticle assembly can be disrupted by addition of free cmps via strand exchange reaction , suggesting that the resulting shift in uv - vis signal can be used to detect the presence of cmps and possibly other degradation products derived from collagen . [SEP]
[CLS] cmp is rigid , highly resistant to degradation , and has little affinity to other biomolecules . [SEP]
[CLS] similar to dna - based np B-nanoparticle assembly explored by numerous research groups , we see great potential in cmp - nps B-nanoparticle in establishing stable np B-nanoparticle assembly with precisely designed structure and interparticle distance . [SEP]
[CLS] we demonstrated that , similar to cmp itself , the cmp - conjugated nps B-nanoparticle can bind specifically to denatured collagen strands . [SEP]
[CLS] previously , we reported that the cmp - nps B-nanoparticle are able to bind to periodic positions on intact type i collagen fibrils . [SEP]
[CLS] we speculated that this periodic binding occurs at locations of partially unfolded domains in intact collagen molecules , because it occurred at a relatively narrow temperature window , and also because of the disappearance of periodicity at higher binding temperature . [SEP]
[CLS] in this work , instead of elevating the np B-nanoparticle binding temperature , collagen fibers were heated to induce permanent protein B-material denaturation . [SEP]
[CLS] as we had expected , the denatured collagen attracted high number of cmp - nps B-nanoparticle while only low levels of nonspecifically bound nps B-nanoparticle were seen on intact collagen fibers . [SEP]
[CLS] in addition , by employing a large size cmp - np B-nanoparticle , gelatin B-material could be isolated from protein B-material mixtures by centrifugation . [SEP]
[CLS] what is most remarkable about the cmp - np B-nanoparticle is its inertness toward nonspecific binding . [SEP]
[CLS] the particle is highly stable in water B-material and exhibits almost no affinity to common proteins B-material such as albumin , carbonic B-material anhydrase , and rnase . [SEP]
[CLS] such low nonspecific binding and selective gelatin B-material affinity is mainly due to the unique binding interaction of cmp - nps B-nanoparticle which are mediated by formation of triple helical supersecondary protein B-material structures involving hydrogen B-material bonding of the protein B-material backbones . [SEP]
[CLS] the same binding mechanism will work for other types of collagen , such as type ii and iv , as we have previously demonstrated . [SEP]
[CLS] since gelatin B-material is commonly used as a stabilizer / additive for protein B-material reagents and therapeutics , the cmp - nps B-nanoparticle could be used for removal of gelatin B-material from such formulations to minimize known side effects of gelatin B-material . [SEP]
[CLS] ( 2 ) , colored in yellow , was conjugated to 20 nm nps B-nanoparticle ( np B-nanoparticle - 2 ) , to make two different size nps B-nanoparticle with opposite peptide B-material polarity . [SEP]
[CLS] on their own , both nps B-nanoparticle exhibit high colloidal stability with no sign of aggregation at a ph range 0 - 14 and high ionic strengths . [SEP]
[CLS] when the two nps B-nanoparticle are mixed together , they assemble by triple helical folding of parallelly aligned cmps . [SEP]
[CLS] remaining in the solution compared with the control solution after centrifugation . [SEP]
[CLS] a fitclabeled gelatin B-material was used for quantifying the gelatin B-material remaining in solution . [SEP]
[CLS] ( c ) uv - vis spectra of the cmp - np100 ( 5 pm ) solution ( black line ) , the same solution mixed with 50 μg / ml protein B-material mixture ( dark gray line ) or with 50 μg / ml gelatin B-material ( gray line ) , and 100 nm gold B-material np B-nanoparticle conjugated with scrambled sequence cg 9 p 9 o 9 ( 5 pm ) mixed with 50 μg / ml gelatin B-material ( dashed line ) . [SEP]
[CLS] protein B-material mixture ( pm ) : conalbumin ( 75 kda ) , bovine albumin serum ( bsa , 66 kda ) , carbonic anhydrase ( 29 kda ) , ribonuclease a ( 13 . 7 kda ) . [SEP]
[CLS] each protein B-material concentration was 50 μg / ml . [SEP]
[CLS] inset shows the compressed spectrum of cmp - np100 with 50 μg / ml gelatin B-material ( gray line ) . [SEP]
[CLS] ( d ) shift in spr peak maximum for cmp - np100 mixed with varying concentrations of gelatin B-material . [SEP]
[CLS] the same solution mixed with 50 mg / ml bsa was used as a baseline spectrum . [SEP]
[CLS] the inset shows the zoom - in data points from 0 . 0005 μg / ml to 0 . 5 μg / ml of gelatin B-material . [SEP]
[CLS] ( e ) sds - page for the study of cmp - np100 mediated gelatin B-material removal from a solution mixture . [SEP]
[CLS] mixture solution contained cmp - np100 ( 5 pm ) , protein B-material mixture ( 25 μg / ml of each protein B-material ) , and gelatin B-material ( 25 μg / ml ) . [SEP]
[CLS] lane 1 : molecular weight standards ; lane 2 : protein B-material mixture ( 25 μg / ml of each protein B-material ) + gelatin B-material ( 25 μg / ml ) ; lane 3 : supernatant of mixture solution after centrifugation ; lane 4 : pellet of mixture solution after centrifugation . [SEP]
[CLS] 1 . schematic of cmp - nps B-nanoparticle assembly . [SEP]
[CLS] two types of gold B-material nps B-nanoparticle ( 10 and 20 nm ) conjugated with cmp of opposite polarity assemble into well ordered aggregates by forming triple helical structures . [SEP]
[CLS] c ( gpo ) 9 ( 1 ) , colored in green , was conjugated to 10 nm nps B-nanoparticle ( np B-nanoparticle - 1 ) ; ( gpo ) 9 c ( 2 ) , colored in yellow , was conjugated to 20 nm nps B-nanoparticle ( np B-nanoparticle - 2 ) , to make two different size nps B-nanoparticle with opposite peptide B-material polarity . [SEP]
[CLS] on their own , both nps B-nanoparticle exhibit high colloidal stability with no sign of aggregation at a ph range 0 - 14 and high ionic strengths . [SEP]
[CLS] when the two nps B-nanoparticle are mixed together , they assemble by triple helical folding of parallelly aligned cmps . [SEP]
[CLS] transmission B-technique electron I-technique microscopy I-technique ( tem ) , dynamic B-technique light I-technique scattering I-technique ( dls ) , and uv - vis spectra of 10 nm au - c ( gpo ) 9 ( np B-nanoparticle - 1 ) and ( gpo ) 9 c - au 20 nm nps B-nanoparticle ( np B-nanoparticle - 2 ) : ( a ) 10 nm au - c ( gpo ) 9 and ( b ) ( gpo ) 9 c - au 20 nm . [SEP]
[CLS] ( c ) uv - vis spectra of unconjugated and cmp conjugated gold B-material nps B-nanoparticle . [SEP]
[CLS] the bottom panel in each tem corresponds to the dls trace with the number indicating the average size of the nps B-nanoparticle in solution . [SEP]
[CLS] scale bar represents 50 nm . [SEP]
[CLS] hybridization of cf - ( gpo ) 6 with cmp conjugated gold B-material np B-nanoparticle ( np B-nanoparticle - 1 ) . [SEP]
[CLS] the fluorescent B-property intensity of np B-nanoparticle - 1 treated with cf - ( gpo ) 6 or scrambled sequence cmp , cf - g 9 p 9 o 9 is shown for two different temperatures , 25 and 40 °c . [SEP]
[CLS] cf - g 9 p 9 o 9 is unable to fold into triple helix , and did not hybridize with np B-nanoparticle - 1 , resulting in negligible fluorescence B-property at two temperatures . [SEP]
[CLS] schematic of np B-nanoparticle - 1 hybridizing and releasing cf - ( gpo ) 6 is also shown . [SEP]
[CLS] 4 . tem , dls and uv - vis spectra of 10 nm au - c ( gpo ) 9 ( np B-nanoparticle - 1 ) and ( gpo ) 9 c - au 20 nm nps B-nanoparticle ( np B-nanoparticle - 2 ) assembly . [SEP]
[CLS] ( a ) structured aggregates formed by mixing np B-nanoparticle - 1 and np B-nanoparticle - 2 . [SEP]
[CLS] ( b ) uv - vis spectra of cmp conjugated gold B-material np B-nanoparticle assembly . [SEP]
[CLS] the black line shows the spectrum of np B-nanoparticle - 1 and np B-nanoparticle - 2 assembly . [SEP]
[CLS] the gray line shows the mixture of 10 nm au - cg 9 p 9 o 9 ( scramble sequence ) ( np B-nanoparticle - 3 ) and np B-nanoparticle - 2 . [SEP]
[CLS] ( c ) dls of np B-nanoparticle - 1 and np B-nanoparticle - 2 assembly . [SEP]
[CLS] ( d ) mixture of np B-nanoparticle - 3 and np B-nanoparticle - 2 . [SEP]
[CLS] ( e ) np B-nanoparticle assembly of np B-nanoparticle - 1 and np B-nanoparticle - 2 after the addition of 500 μm of single strand ( gpo ) 9 . [SEP]
[CLS] the bottom panels in d and e are the corresponding dls traces with the numbers indicating the average size of the nps B-nanoparticle in solution . [SEP]
[CLS] 5 . tem and corresponding dls of np B-nanoparticle systems comprised of 10 nm np B-nanoparticle conjugated with ( gpo ) 2 gco ( gpo ) 6 ( np B-nanoparticle - 4 ) and 20 nm np B-nanoparticle conjugated with c ( gpo ) 9 . [SEP]
[CLS] ( a ) tem and dls of np B-nanoparticle - 4 self - assembled into clusters ( circled ) . [SEP]
[CLS] ( b ) mixing np B-nanoparticle - 4 and 20 nm au - c ( gpo ) 9 produced large aggregates . [SEP]
[CLS] ( c ) mixing of np B-nanoparticle - 4 and 20 nm au - cg 9 p 9 o 9 ( scrambled cmp sequence ) produced no aggregate . [SEP]
[CLS] ( d ) aggregates in ( b ) are readily disrupted by addition of 500 μm of single strand ( gpo ) 9 . [SEP]
[CLS] ( e ) schematic of np B-nanoparticle - 4 self - assembly . [SEP]
[CLS] ( f ) schematic of np B-nanoparticle - 4 and 20 nm au - c ( gpo ) 9 assembly . [SEP]
[CLS] each scale bar represents 50 nm . [SEP]
[CLS] 6 . tem of cmp - nps B-nanoparticle binding to denatured type i collagens . [SEP]
[CLS] ( a ) binding of np B-nanoparticle - 1 to denatured collagen . [SEP]
[CLS] ( b ) magnification of the selected area in ( a ) , black arrows indicate nps B-nanoparticle binding along the denatured collagen fibers . [SEP]
[CLS] ( c ) binding of np B-nanoparticle - 1 to intact type i collagen fibrils . [SEP]
[CLS] white arrows indicate a few nps B-nanoparticle nonspecifically attached to the intact collagen fibrils . [SEP]
[CLS] 7 . gelatin B-material binding and removal mediated by cmp - nps B-nanoparticle . [SEP]
[CLS] ( a ) tem of 100 nm gold B-material np B-nanoparticle conjugated with c ( gpo ) 9 ( cmp - np100 ) bound to gelatin B-material . [SEP]
[CLS] scale bar represents 200 nm . [SEP]
[CLS] ( b ) gelatin B-material removal assay . [SEP]
[CLS] the percentage of gelatin B-material removal from cmp - np B-nanoparticle and gelatin B-material solution [ cmp - np100 ( 5 pm ) + gelatin B-material ( 25 μg / ml ) ] was determined by the gelatin B-material [SEP]
[CLS] in addition to tunable emission wavelengths , qds exhibit longer fluorescence B-property lifetimes ( > 10 ns ) compared to organic fluorophores ( 1 - 5 ns ) . [SEP]
[CLS] this property not only enables temporal imaging that is often limited by the short lifetime of organic dyes , but also results in a significant improvement in signal - to - noise ratios in biological applications . [SEP]
[CLS] although autofluorescence of tissues and cells B-material often contributes to high background signal , the autofluorescent species present in biological samples have shorter lifetimes . [SEP]
[CLS] thus , timegated fluorescence B-property measurements may be used to image qds by separating autofluorescence background from positive qd signal . [SEP]
[CLS] this property is especially significant in the application of qds for enhancing the sensitivity of detecting cancer biomarkers B-property , cells B-material , and tissues , which may be in low abundance at the early stages of the disease . [SEP]
[CLS] polymer B-material dots ( pds ) are a class of fluorescent B-property semi - conducting polymer B-material nanoparticles B-nanoparticle ranging from 5 to 30 nm in size that exhibit broad absorption spectra with narrow emission profiles . [SEP]
[CLS] 17 pds offer high fluorescence B-property quantum yields ( 50 - 60 % ) , which results in bright fluorescence B-property intensity , nearly 3 orders of magnitude higher than that of organic dyes . [SEP]
[CLS] in addition , altering the composition of pds results in tunable emission wavelength , 44 which is particularly useful for both in vitro assays and multiphoton in vivo imaging . [SEP]
[CLS] [SEP]
[CLS] although pds have a broader emission spectra than qds , pds are brighter in the visible and the near - uv range , are nontoxic , are highly photostable , do not blink , and are therefore utilized in diagnostic and theranostic applications . [SEP]
[CLS] upconversion nanoparticles B-nanoparticle ( ucnps ) are composed of a rare earth element crystalline host with lanthanide B-material ion B-material ( ln 3 + ) dopants . [SEP]
[CLS] most commonly , nayf 4 or nagdf 4 is used as the host lattice , with yb 3 + , tm 3 + , and er 3 + doped in varying amounts and combinations ( figure 4a ) . [SEP]
[CLS] in these structures , the ln 3 + ions B-material possess 4f n inner shell electron configurations , which gives rise to fluorescence B-property via intra - 4f and 4f - 5d electron transitions . [SEP]
[CLS] [SEP]
[CLS] varying the amounts and types of ln 3 + dopants tunes the emission wavelength 53 ( figure 4c , d ) , which is useful in multicolor imaging applications . [SEP]
[CLS] unlike organic fluorophores and qds , ucnps exhibit an anti - stokes shift , emitting a photon of higher energy than the absorbed photon . [SEP]
[CLS] this occurs through multiphoton excitation processes ( figure 4b ) , which results in the ability to excite ucnps with near - infrared ( nir ) light . [SEP]
[CLS] this is particularly useful in biological applications due to the minimization of autofluorescence from cells B-material and tissues , as well as enabling deeper tissue penetration through excitation in the tissue - transparent nir window . [SEP]
[CLS] gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) have also been used in a variety of fluorescent B-property assays for cancer detection . aunps B-nanoparticle exhibit size - dependent absorption in the ultraviolet - visible range due to a size - and shape - related property known as the surface plasmon resonance ( spr ) . [SEP]
[CLS] [SEP]
[CLS] incident light on metal B-material atoms B-material in a nanoparticle B-nanoparticle causes plasmon oscillations in the conduction band of electrons . [SEP]
[CLS] these collective oscillations result in a strong absorption of light ( on the order of 10 9 m −1 cm −1 for a 40 nm aunp B-nanoparticle ) and fast electronic relaxation . [SEP]
[CLS] based on their strong absorption , aunps B-nanoparticle are also efficient fluorescence B-property quenchers I-property and thus have been employed in many " off - on " fluorescence B-property probes . [SEP]
[CLS] compared to organic quenchers , aunps B-nanoparticle are more efficient due to surface energy transfer processes . [SEP]
[CLS] [SEP]
[CLS] cancer is the second most common cause of death in the united states , trailing only heart disease in incidence . [SEP]
[CLS] despite significant worldwide investment in research , cancer remains responsible for 1 in 4 deaths in developed countries . [SEP]
[CLS] globally , over 14 million cancer diagnoses were reported in 2012 , a figure expected to increase to over 22 million cases per annum in the next two decades . [SEP]
[CLS] estimated to kill over 1 / 2 million u . s . citizens , and with over 1 . 6 million new cases predicted to be diagnosed this year , cancer continues to present a major , yet unmet challenge to healthcare both globally and in the united states . [SEP]
[CLS] cancer emerges from our own tissues , complicating both detection and treatment methods due to the similarities between the diseased tissue and healthy tissue . [SEP]
[CLS] despite this fact , the mortality rate from cancer is often greatly reduced by early detection of the disease . [SEP]
[CLS] for example , non - small - cell B-material lung cancer is responsible for the most cancer related deaths worldwide , with patients in the advanced stages of the disease having only 5 - 15 % and < 2 % 5 - year survival rates for stage iii and iv patients , respectively . [SEP]
[CLS] in contrast , patients who start therapy in the early stages of the disease ( stage i ) have markedly improved survival rates , with an 80 % overall 5 - year survival rate . [SEP]
[CLS] consequently , early diagnosis is essential to improving cancer patient prognosis . [SEP]
[CLS] at present , clinical detection of cancer primarily relies on imaging B-technique techniques I-technique or the morphological analysis of cells B-material that are suspected to be diseased ( cytology ) or tissues ( histopathology ) . [SEP]
[CLS] imaging B-technique techniques I-technique applied to cancer detection , including x - ray , mammography , computed B-technique tomography I-technique ( ct ) , magnetic B-property resonance imaging ( mri ) , endoscopy , and ultrasound , have low sensitivity and are limited in their ability to differentiate between benign and malignant lesions . [SEP]
[CLS] while cytology , such as testing for cervical cancer via a pap smear or occult blood detection , may be used to distinguish between healthy and diseased cells B-material or tissues , it is not effective at detecting cancer at early stages . [SEP]
[CLS] similarly , histopathology , which generally relies on taking a biopsy of a suspected tumor B-material , is typically used to probe the malignancy of tissues that are identified through alternative imaging B-technique techniques I-technique , such as ct or mri , and may not be used alone to detect cancer in its early stages . [SEP]
[CLS] as such , the development of assays and methods for early detection of cancer , before the disease becomes symptomatic , presents a major challenge . [SEP]
[CLS] recent research within the field of nanotechnology has focused on addressing the limitations of the currently available methods for cancer diagnosis . [SEP]
[CLS] certain nanoparticle B-nanoparticle probes possess several unique properties that are advantageous for use in the detection of cancer at the early stages . [SEP]
[CLS] in this review , we will discuss the advances in the development of nanoparticle - based methods for the detection of cancer by fluorescence B-technique spectroscopy I-technique . [SEP]
[CLS] we will divide this topic into three categories : techniques that are designed for ( 1 ) the detection of extracellular cancer biomarkers B-property , ( 2 ) the detection of cancer cells B-material , and ( 3 ) the detection of cancerous tissues in vivo . [SEP]
[CLS] we will discuss these strategies within the context of the nanoparticle B-nanoparticle probe used as well as the recognition moieties applied in each approach . [SEP]
[CLS] ultimately , the translation of these methods from the laboratory to the clinic may enable earlier detection of cancer and could extend patient survival through the ability to administer therapeutic treatment in the early stages of the disease . [SEP]
[CLS] while this review provides a comprehensive overview of the nanoparticle B-nanoparticle probes that are used to detect cancer in vitro and in vivo through fluorescence B-property , there are several other relevant reviews that may be of interest to our readers , who may refer to the references for more generalized reviews of nanomaterials B-material used for diagnostics and therapy , or more detailed insight into the specific types of nanoparticle B-nanoparticle probes ( i . e . , quantum B-nanoparticle dots I-nanoparticle , gold B-nanoparticle nanoparticles I-nanoparticle , upconversion nanoparticles , polymer B-material dots , silica B-nanoparticle nanoparticles I-nanoparticle , polymeric B-nanoparticle nanoparticles I-nanoparticle , etc . ) for cancer diagnosis . [SEP]
[CLS] fluorescence B-property is an optical phenomenon where the absorption of photons at one wavelength results in emission at another , usually longer , wavelength . [SEP]
[CLS] the loss in energy between the absorbed and emitted photons is the result of vibrational relaxation , and this difference is referred to as a stokes shift ( figure 1b ) . [SEP]
[CLS] a typical jablonski diagram can be used to describe the process of fluorescence B-property ( figure 1a ) . [SEP]
[CLS] in the first phase , known as excitation , absorption of light results in the promotion of an electron from the ground state to the excited state . [SEP]
[CLS] once excited , release of the absorbed energy may occur through several photophysical events , including both radiative and nonradiative emission . [SEP]
[CLS] vibrational relaxation is often the first route to energy dissipation , and may be followed by internal conversion , intersystem crossing ( from a singlet to a triplet state ) , and subsequent phosphorescence B-property , or fluorescence B-property when the excited electron returns to the ground state and emits energy through the release of a photon . [SEP]
[CLS] fluorescence B-technique spectroscopy I-technique is a useful technique for the detection of biomolecules , and it is widely used in biological and biomedical applications due to its high spatial and temporal resolution . [SEP]
[CLS] assays utilizing a fluorescent B-property output for detection can employ several methods of analysis , including fluorescence B-technique spectroscopy I-technique for solution - based assays , microscopy B-technique for imaging of cells B-material and arrays used in sandwich assays , flow B-technique cytometry I-technique for high - throughput imaging of single cells B-material , and in vivo imaging . [SEP]
[CLS] the use of fluorescence B-property as a detection method depends upon the photophysical properties of the fluorophore used : photostability , quantum yield , stokes shift , and fluorescence B-property lifetime . [SEP]
[CLS] these properties , with respect to the advantages that fluorescent B-nanoparticle nanoparticles I-nanoparticle offer over organic fluorophores , will be further discussed in the context of the nanoparticle B-nanoparticle probes that are used in cancer diagnostic applications . [SEP]
[CLS] additionally , forster resonance energy transfer ( fret ) , a phenomenon that occurs when an excited donor chromophore transfers energy to an acceptor chromophore via nonradiative dipole - dipole coupling when in close proximity ( a few nanometers ) to one another , is often used in the design of nanoparticle B-nanoparticle probes for cancer diagnosis . [SEP]
[CLS] the efficiency of energy transfer is dependent upon several factors : ( 1 ) spectral overlap between the absorbance spectra of the acceptor fluorophore and the emission spectra of the donor fluorophore ( figure 2 ) , ( 2 ) their relative orientations , and ( 3 ) their proximity ( efficiency is inversely proportional to the distance between the acceptor and the donor to the sixth power ) . [SEP]
[CLS] the use of fret probes to detect cancer biomarkers B-property and cells B-material is powerful due to its ability to provide real - time spatial measurements between the donor and acceptor fluorophores , thus allowing the design of more sensitive bioassays B-technique for cancer biomarker B-property and cell B-material detection . [SEP]
[CLS] the application of nanotechnology to cancer diagnosis holds tremendous promise in enhancing the sensitivity and versatility of fluorescence - based methods of detection . [SEP]
[CLS] in particular , there are several structure - defining traits of nanoparticles B-nanoparticle that enable the development of novel cancer detection assays : size , shape , high surface area , and unique optical properties . [SEP]
[CLS] we will review these material B-material - dependent characteristics of nanoparticles B-nanoparticle with respect to their utility in cancer diagnosis through fluorescence B-property detection , focusing on optical properties and tunable surface functionality . [SEP]
[CLS] the optical properties of semiconductor and metallic B-nanoparticle nanoparticles I-nanoparticle are highly dependent on nanoparticle B-nanoparticle size , shape , and composition . [SEP]
[CLS] in particular , the optical properties most relevant in the design of fluorescence - based biosensors for cancer diagnostics , the intensity and stability of fluorescence B-property emission as well as the effectiveness of fluorescence B-property quenching in " off - on " probes , determine , in part , the sensitivity and dynamic range of a particular assay . [SEP]
[CLS] these material B-material - dependent optical properties will be further discussed in the context of the nanoparticle B-nanoparticle probes most widely used in cancer diagnostic applications : quantum B-nanoparticle dots I-nanoparticle ( qds ) , polymer B-material dots ( pds ) , upconversion nanoparticles B-nanoparticle ( ucnps ) , and gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) . [SEP]
[CLS] single crystal semiconductor nanocrystals , or qds , represent a class of inherently fluorescent B-nanoparticle nanoparticles I-nanoparticle with a range of properties that are desirable for biological imaging applications and for the development of novel cancer diagnostics . [SEP]
[CLS] semiconducting qds absorb photons of energy greater than their band gap , resulting in the promotion of electrons from their valence band to their conduction band , generating an electron - hole pair ( or exciton ) . [SEP]
[CLS] photons are then emitted from discrete bands upon the recombination of the exciton , which generates a narrow emission profile due to their quantum confined properties , which dictate that nanocrystals smaller than the bohr exciton radius of the material B-material exhibit quantized energy states , with energy levels correlating to qd size . [SEP]
[CLS] this size dependence of qd absorption and emission enables the tunable design of qds ( figure 3 ) with a range of imaging applications , especially in multicolor labeling for the simultaneous detection of multiple targets . [SEP]
[CLS] qd absorption , unlike that of organic dyes , is broad , with large molar absorption coefficients ( 100 000 - 1 000 000 m −1 cm −1 ) compared to organic dyes ( 25 000 - 250 000 m −1 cm −1 ) . [SEP]
[CLS] qds exhibit an absorption peak corresponding to the lowest energy level excited state , with absorption increasing at shorter wavelengths due to an increased probability of absorption and the presence of multiple higher energy levels ( figure 3b ) . [SEP]
[CLS] this broad absorption allows one to choose the excitation wavelength used , resulting in the ability to excite multiple qd types with a single wavelength of light . [SEP]
[CLS] in addition to their use as fluorescence B-property quenchers I-property , aunps B-nanoparticle also have been used as fluorophore labels in imaging applications . [SEP]
[CLS] small aunps B-nanoparticle or " au nanoclusters " ( auncs ) are structures typically less than 3 nm that are composed of a precise number of au atoms B-material , and unlike larger aunps B-nanoparticle , they do not exhibit spr absorption in the visible range . [SEP]
[CLS] auncs do , however , exhibit fluorescence B-property in the visible to near - infrared region with low quantum yields ( < 1 % ) . [SEP]
[CLS] despite this , auncs have been employed in the design of cancer diagnostic assays , due to their remarkable photostability and resistance to photobleaching . [SEP]
[CLS] a key advantage to the use of nanoparticles B-nanoparticle in cancer detection is their large surface area to volume ratio compared to that of bulk materials . [SEP]
[CLS] in particular , this property enables dense coverage of the nanoparticle B-nanoparticle surface with moieties that bind and recognize molecules indicative of cancer . [SEP]
[CLS] presentation of multiple binding ligands to a cancer cell B-material , for example , often enables multivalent effects that can enhance an assay ' s sensitivity . [SEP]
[CLS] for example , spherical nucleic B-material acid I-material nanoparticles B-nanoparticle derive their unique properties , including higher binding constants for their complements than free oligonucleotides of the same sequence , from the arrangement of nucleic B-material acids I-material in a dense , highly oriented fashion . [SEP]
[CLS] in addition , since surface atoms B-material contribute more significantly to determining the properties of a nanoparticle B-nanoparticle , functionalization with a variety of ligands significantly contributes to the collective properties of such structures . [SEP]
[CLS] finally , surface curvature can accommodate arrangements of ligands not possible with bulk substrates , leading to unusual and tailorable multivalent effects . [SEP]
[CLS] minor variations in nanoparticle B-nanoparticle surface functionality , ligands , size , and shape can lead to a wealth of properties advantageous for sensing and imaging applications . [SEP]
[CLS] a multitude of targeting moieties can also be attached to nanoparticles B-nanoparticle for use in the diagnosis of cancer , including peptides B-material , antibodies B-material , aptamers , and small molecules , which enable highly specific binding of nanoparticle B-nanoparticle probes to targets of interest . [SEP]
[CLS] the targeting moieties relevant to the detection of cancer will be discussed in detail . [SEP]
[CLS] peptides B-material are often used to label cancerous cells B-material based on recognition of their transmembrane proteins B-material . [SEP]
[CLS] the most commonly used peptide B-material is arginylglycylaspartic acid ( rgd ) , composed of l - arginine B-material , glycine B-material , and l - aspartic B-material acid . [SEP]
[CLS] rgd was first isolated from the cellbinding domain of fibronectin , a glycoprotein that binds to integrins , and is involved in cellcell and cell - extracellular matrix ( ecm ) attachment and signaling by binding collagen , fibrin , and proteoglycans . [SEP]
[CLS] rgd peptides B-material have the highest affinity for a type of cell B-material surface integrins , α v β 3 , which are highly expressed in tumoral endothelial cells B-material , but not in normal endothelial cells B-material . [SEP]
[CLS] in addition , the up - regulation of these integrins in breast cancer , glioblastoma , pancreatic tumors B-material , and prostate carcinoma is correlated with increased cell B-material motility and metastasis B-event . [SEP]
[CLS] since rgd peptides B-material are effective in targeting cancer cells B-material ( in tissue culture models as well as in mice ) through binding and recognition of α v β 3 integrins , they serve as promising tools for targeting nanoparticle B-nanoparticle probes to cancer cells B-material . [SEP]
[CLS] one protein B-material that is widely used to detect cancer cells B-material is transferrin , a glycoprotein that binds iron B-material ( fe 3 + ) in the blood with high affinity ( 10 −23 m ) . [SEP]
[CLS] after binding two fe 3 + ions B-material , transferrin is recognized by transferrin receptors , which mediates its uptake into cells B-material via receptor - I-event mediated endocytosis B-event . [SEP]
[CLS] transferrin receptors are usually expressed in the basal epidermis , pancreas , hepatocytes , kupffer cells B-material , testis , and the pituitary gland . [SEP]
[CLS] however , these levels are elevated in various cancers ( breast , stomach , colon , kidney , ovarian , lung , pancreas , lymphoma , skin , and bladder ) possibly due to the need for increased iron B-material uptake that is usually associated with proliferating cells B-material . [SEP]
[CLS] in addition , transferrin is an endogenous protein B-material ( 2 . 5 g / ml in normal human serum ) , and is therefore nontoxic and nonimmunogenic when used to bind and detect cancer cells B-material . [SEP]
[CLS] antibodies B-material are widely used in cancer diagnostics in vitro and in vivo since they have been commercialized and are easily procured , have high specificity to their target of interest ( both free in solution and on cells B-material ) , and bind their target with a high affinity ( the antigen B-event - I-event binding I-event affinity , k d , of most antibodies B-material lies in the range 10 −6 - 10 −9 m ) . [SEP]
[CLS] in addition , antibodies B-material can be easily conjugated to fluorescent B-property dyes and nanoparticles B-nanoparticle ( aunps B-nanoparticle , qds 84 ) , making them perfect candidates for cancer biomarker B-property and cell B-material immunoassays . [SEP]
[CLS] antibodies B-material are also widely used for in vivo cancer cell B-material detection due to their relatively low immunogenicity B-property . [SEP]
[CLS] to minimize immunogenicity B-property , smaller fragments consisting of either a single - chain variable fragment ( scfv , a fusion protein B-material of the heavy and light chains ) or the fragment - antigen binding ( fab ) region are used since antibodies B-material containing a nonhuman crystallizable ( fc ) region could result in complement system activation . [SEP]
[CLS] other types of nonimmunogenic antibodies B-material include chimera antibodies B-material ( produced by joining a mouse fab region with a human fc region ) and human antibodies B-material ( generated using transgenic mice that have human immunoglobulin B-material genes or from phage display ) . [SEP]
[CLS] although antibodies B-material play an important role as targeting moieties for nanoparticles B-nanoparticle to bind various cancerous biomarkers B-property , cells B-material , or tissues , they also act as effective therapeutics against cancer . [SEP]
[CLS] in fact , there are over 40 fda - approved antibody B-material drugs available in the united states , and examples of theranostic nanoparticle B-nanoparticle probes that simultaneously detect and treat cancer in vivo through the use of antibodies B-material will be further discussed in section 6 . 2 . 2 . [SEP]
[CLS] nucleic B-material acid I-material aptamers , oligonucleotides that bind specific targets of interest ( i . e . , proteins B-material , small molecules , cells B-material , or other nucleic B-material acids I-material ) , can also be used as targeting moieties . [SEP]
[CLS] in many cases , aptamers are discovered using a process called " systematic evolution of ligands by exponential enrichment " ( selex ) . [SEP]
[CLS] in this process , large oligonucleotide libraries ( typically 1 × 10 14 unique sequences ) are created using a single stranded dna ( ssdna ) template consisting of defined 5 ′ and 3 ′ ends and a randomized region , typically between 40 and 80 nucleotides in length , within which specific binding motifs can be evolved against proteins B-material and other biomolecules of interest . [SEP]
[CLS] after generating the library using automated dna synthesis , a double stranded dna ( dsdna ) library can be generated through polymerase chain reaction ( pcr ) and used as a starting point for either a dna or rna ( via transcription B-event with t7 rna polymerase ) in vitro selection process . [SEP]
[CLS] first the sequences are introduced to a biological target of interest and isolated based on their ability to bind the target through several rounds of in vitro selection . [SEP]
[CLS] bound sequences are typically isolated through size exclusion chromatography B-technique and other analogous techniques , and then pcr amplified to identify the sequence that binds the target of interest . [SEP]
[CLS] the bound sequences , along with permutations of new sequences , then go through this cycle 10 - 15 times , with each cycle yielding more sequences that bind the target of interest with higher specificity . [SEP]
[CLS] this method generates aptamers that have a high specificity and affinity for their target ( k d of approximately 10 −8 m ) , and may be used to develop aptamers that bind a wide range of targets , including cells B-material ( cell - selex ) . [SEP]
[CLS] this makes aptamers ideal candidates for targeting cancer biomarkers B-property and cells B-material for which antibodies B-material may not be available . [SEP]
[CLS] one promising approach in the early detection of cancer is to identify and detect substances in the blood or other bodily fluids that are correlated with the presence of cancer . [SEP]
[CLS] these substances , known as biomarkers B-property , may be proteins B-material ( either cell B-material surface glycoproteins or secreted proteins B-material ) , carbohydrates B-material , or nucleic B-material acids I-material ( i . e . , genome sequences or rna transcripts B-event ) that are associated with cancerous cells B-material . [SEP]
[CLS] measuring the levels of particular cancer biomarkers B-property from a patient ' s blood , urine , feces , or saliva could enable the detection of cancer at the early stages of the disease , identification of tumor B-material recurrence , prediction of a patient ' s risk to a new or existing cancer , and the ability to monitor a therapy ' s efficacy during treatment . [SEP]
[CLS] however , some primary challenges of early cancer detection include low abundance of biomarkers B-property in plasma at the early stages of the disease , heterogeneity in the timing and abundance of these biomarkers B-property among patients , and difficulties in executing prospective studies ( those that are serial in nature , including the collection and storage of prediagnostic samples ) . [SEP]
[CLS] research toward the identification of biomarkers B-property that are indicators B-property of cancer has generated thousands of biomarker B-property candidates , but relatively few have been granted fda clearance ( table 1 ) . [SEP]
[CLS] of the fda - cleared biomarkers B-property , the majority are utilized for monitoring the progression of cancer , rather than enabling its early detection . [SEP]
[CLS] however , despite the importance of early detection and the considerable research efforts directed toward it , the use of these assays for early diagnosis of cancer is limited . [SEP]
[CLS] in fact , no early cancer biomarker B-property assay has been fda - approved or - cleared , which highlights the challenges of developing sensors for cancer biomarkers B-property . [SEP]
[CLS] although cancer biomarker B-property assay development has faced both fundamental and technical issues , researchers are uniquely positioned to use nanotechnology to address these concerns , as the unique properties of certain classes of nanoparticle B-nanoparticle probes offer the potential to produce rapid , inexpensive , tailorable , high - throughput assays with high sensitivity and selectivity . [SEP]
[CLS] below , we will discuss nanoparticle - mediated techniques for the detection of biomarkers B-property separated by the method of analyte detection . [SEP]
[CLS] biomarkers B-property targeting bladder , breast , bone , cervical , colorectal , gastric , hepatocellular , lung , pancreas , prostate , ovarian , and thyroid cancer , will be covered in this section . [SEP]
[CLS] qds are especially promising for the detection of cancer biomarkers B-property by fluorescence B-property due to their high quantum yields , large molar extinction coefficients , and tunable emission maxima , all of which are advantageous for reducing an assay ' s limit of detection . [SEP]
[CLS] recent examples of qd - based biosensors for cancer biomarker B-property detection are reviewed below . [SEP]
[CLS] the most common design motif for the detection of biomarkers B-property is a sandwich - type assay , which consists of several components : substrate , capture antibody B-material , analyte of interest ( biomarker B-property ) , a second capture antibody B-material , and a secondary antibody , usually tagged with a fluorescent B-property probe . [SEP]
[CLS] with such an assay , the immobilized monoclonal B-material primary I-material antibody I-material binds to the biomarker B-property . [SEP]
[CLS] next , a second capture antibody B-material , specific for the biomarker B-property , is introduced and sandwiches the target . [SEP]
[CLS] a secondary antibody B-material , usually fluorophore - labeled , binds to the second primary antibody B-material , thus generating a fluorescent B-property signal that is detected using microscopy B-technique or a fluorescence B-property spectrophotometer . [SEP]
[CLS] these sandwich - type assays have high specificity due to the high affinity and selectivity a primary antibody B-material has for its analyte , and high sensitivity when qds are used to fluorophore - label the secondary antibody B-material due to the qd ' s - conjugated antibodies B-material ' intense signal . [SEP]
[CLS] sandwich - type assays can occur both on a substrate and free in solution . [SEP]
[CLS] suspension assays are advantageous because they exhibit faster kinetics in solution compared to assays performed on a substrate , allowing for faster readouts and higher sample throughput . [SEP]
[CLS] these homologous assays can be easily tailored for detecting various analytes , and do not require optimization for immobilizing antibodies B-material or antigens or extensive wash steps to separate bound versus unbound moieties . [SEP]
[CLS] one example of a homogeneous in - solution assay was reported by li et al . , who used qdconjugated antibodies B-material to detect two biomarkers B-property : carcinoembryonic antigen ( cea , one of the most widely studied cancer biomarkers B-property to monitor anticancer B-property treatments and predict tumor B-material recurrence postsurgical resection in patients with late - stage cancer ) and neuron specific enolase ( nse , an enzyme that catalyzes the conversion of 2 - phosphoglycerate to phosphoenolpyruvate and is associated with small cell lung carcinoma , carcinoids , islet cell tumors B-material upon secretion at a concentration over 15 ng / ml ) , each with a limit of detection of 1 . 0 ng / ml ( figure 5 ) . [SEP]
[CLS] since the levels of these biomarkers B-property are relatively low ( on the order of several nanograms per milliliter ) , it is important to develop assays with an even lower limit of detection to accurately monitor these biomarkers B-property in patients . [SEP]
[CLS] cao and coworkers improved upon this fluoroimmunoassay by immobilizing capture antibodies B-material on polystyrene microspheres instead of streptavidin beads to reduce the limit of detection of each of the two biomarkers B-property to 0 . 625 ng / ml . another type of qd - based immunosensor consists of either an analyte or a capture antibody B-material immobilized onto a surface commonly composed of glass , silicon B-material , or gold B-material . [SEP]
[CLS] unlike homogeneous in - solution immunoassays , these heterogeneous immunosensors require small amounts of patient samples and can provide rapid and high - throughput detection of analytes of interest . [SEP]
[CLS] in one study , kerman and co - workers developed an immunosensor that could detect total prostate specific antigen ( tpsa ) in human serum samples with a detection limit of 0 . 25 ng / ml ( figure 6 ) . [SEP]
[CLS] prostate specific antigen ( psa ) , one of the most widely tested biomarkers B-property for prostate cancer , is a glycoprotein produced by the prostate gland that is part of the kallikrein - related peptidase family ( serine B-material proteases ) and exists in multiple forms in the body ( e . g . , free and bound to serum proteins B-material ) . [SEP]
[CLS] since high psa concentrations are not always indicative of prostate cancer due to biodiversity in the patient population , psa velocity , the rate of increase in psa levels over time , can be monitored since a significant increase is indicative of prostate cancer . [SEP]
[CLS] while neither types of psa monitoring can serve as long - term predictors of prostate cancer , they serve as valuable and definitive indicators B-property in monitoring changes over time to detect cancer recurrence or treatment efficacy . [SEP]
[CLS] in order to analyze biomarkers B-property in a high - throughput fashion , gokarna and co - workers developed qd - based protein B-material microand nanoarrays B-nanoparticle for the detection of psa . [SEP]
[CLS] the psa microand nanoarrays B-nanoparticle were fabricated using dip pen nanolithography ( dpn ) and introduced to pegylated qds functionalized with an anti - psa antibody B-material . [SEP]
[CLS] the presence of psa ( marked by qd fluorescence B-property ) was evaluated using a microarray scanner . [SEP]
[CLS] since these micro - and nanoarrays B-nanoparticle allow for rapid screening of many compounds , they may enable highthroughput multiplexed screens for clinical use . [SEP]
[CLS] in order to improve the sensitivity ( to less than 0 . 5 pm or 0 . 1 ng / ml ) and speed ( to 15 min ) of biomarker B-property detection , mukundan et al . used a waveguide based biosensor to detect cea . [SEP]
[CLS] first , anti - cea monoclonal antibodies B-material were immobilized onto the sensor surface and bound to cea present in human serum patient samples . [SEP]
[CLS] next , cdse / zns core / shell qd - conjugated anti - cea monoclonal antibodies B-material sandwiched the immobilized biomarker B-property - antibody B-material complex , generating a fluorescent B-property signal as quantified by a fiber optic spectrometer . [SEP]
[CLS] microfluidic devices are often used in clinical assays to reduce the amount of patient sample needed for the detection of cancer biomarkers B-property based upon the ability to precisely control fluids on a small scale . [SEP]
[CLS] these " lab - on - a - chip " technologies operate under the premise that large laboratory experiments can be conducted on a small milli - or centimeter size scale , allowing for cost - effective and faster diagnoses . [SEP]
[CLS] for example , hu et al . used a polydime - thylsiloxane ( pdms ) microfluidic device to detect alpha fetoprotein ( afp , a plasma protein B-material found mostly in the fetus that is the most widely researched biomarker B-property for the post - treatment prognosis of hepatocellular carcinoma ) and cea at a limit of detection of 0 . 25 nm for each analyte ( both individually and in combination ) . [SEP]
[CLS] to do so , capture antibodies B-material for cea or afp were immobilized on the device surface , before the analyte was introduced . [SEP]
[CLS] next , anti - cea or anti - afp primary antibodies B-material and qdfunctionalized secondary antibodies B-material bound to the newly immobilized biomarker B-property . [SEP]
[CLS] finally , the biochip was imaged using fluorescence B-technique microscopy I-technique . [SEP]
[CLS] to improve upon the detection limit of cancer biomarkers B-property , jokerst and co - workers developed a microfluidic microporous agarose bead array to detect cea both individually and in combination with cancer antigen 125 ( ca 125 , a cell B-material surface glycoprotein that is overexpressed in patients with ovarian cancer and is used as a late - stage prognosis and monitoring tool ) in patient blood and saliva samples [SEP]
[CLS] to do this , capture antibodies B-material are immobilized onto agarose beads . [SEP]
[CLS] samples are then introduced , and the target antigen is captured by the immobilized antibodies B-material . [SEP]
[CLS] detection antibodies B-material conjugated with qds then sandwiches the antigen and provides a means for measuring its presence . [SEP]
[CLS] the fluorescence B-property intensity of photomicrographs of the fluorophore - labeled beads corresponds to the analyte concentration , and the limit of detection of cea was determined to be 20 pg / ml or 0 . 11 pm . [SEP]
[CLS] hu et al . are able to achieve an even lower limit of detection for cea ( 50 fm ) in an analogous assay . [SEP]
[CLS] one type of microfluidic device for cancer biomarker B-property detection is an immunochromatography test strip ( icts ) , which takes advantage of capillary action to carry sample through the strip . [SEP]
[CLS] these test strips are usually composed of either paper or membrane , further reducing the cost of production and analysis , as they do not require extra machinery ( i . e . , pumps are normally required for microfluidic devices ) or expensive imaging equipment ( homemade test strip readers are available ) . [SEP]
[CLS] the setup of an icts usually includes a sample pad ( where sample is loaded ) , a conjugation pad ( antibodies B-material bind analyte of interest ) , a test line ( where fluorophore - tagged antibody - analyte sandwiches are immobilized ) , and a control line ( a positive control to confirm the fluorescence B-property of fluorophore - tagged antibodies B-material ) . [SEP]
[CLS] in one study , yang and co - workers developed an icts to detect afp with a limit of detection of 1 ng / ml ( figure 7 ) . [SEP]
[CLS] another icts assay was developed by cheng et al . to detect c - reactive protein B-material ( crp , a protein secreted from the liver as a marker of inflammation that is associated with colorectal and lung cancer in addition to diabetes and cardiovascular disease ) with a limit of detection of 0 . 63 u / ml . fret - based biosensors take advantage of fret , a phenomenon in which energy is transferred from a donor fluorophore to an acceptor fluorophore ( as a result of their spectral overlap , their spatial proximity , and their relative orientation ) , due to its high sensitivity of detection and high signal - to - noise ratio . there are two types of fret - based sensors : those that turn " on " in the presence of analyte ( if ( 1 ) both chromophores are fluorescent B-property , but when they bind the analyte , fret occurs , resulting in energy transfer to the acceptor chromophore and a measurable increase in fluorescence B-property at the acceptor ' s emission wavelength or if ( 2 ) a quencher absorbs the excitation energy of a donor chromophore , but is released upon binding of analyte , resulting in fret between the donor and acceptor chromophore ) and those that turn " off " in the presence of analyte ( a biomolecule binds and fret no longer occurs , resulting in a decrease in fluorescence B-property emission by the acceptor ) . [SEP]
[CLS] qds are ideal for fret due to their tunable emission , broad absorption , and long fluorescence B-property lifetime , such that more than one dye could act as an acceptor fluorophore with one excitation event . [SEP]
[CLS] wei and co - workers developed an in - solution sandwich fluoroimmunoassay to detect estrogen receptor B-material beta ( er - β antigen , a tumor B-material suppressor that is down - regulated in the later stages of various cancers and can be used to monitor treatment efficacy ) utilizing fret . [SEP]
[CLS] er - β antigen is incubated B-technique with qd - labeled anti - er - β monoclonal B-material antibody I-material and alexa fluor labeled anti - er - β polyclonal antibody B-material , forming a sandwich . [SEP]
[CLS] the proximity of the qd 565 ( donor ) and the alexa fluor dye ( acceptor ) enables fret , and results in an increase in alexa fluor fluorescence B-property , which may be measured using confocal microscopy B-technique . [SEP]
[CLS] this assay is rapid ( only a 30 min incubation B-technique time ) , simple , and sensitive ( limit of detection was 0 . 05 nm or 2 . 65 ng / ml ) . [SEP]
[CLS] another in - solution fluoroimmunoassay was developed by chen and co - workers to detect afp with a limit of detection of 0 . 4 ng / ml using a luminescent B-property terbium chelate ( ltc ) - qd fret pair ( figure 8 ) . [SEP]
[CLS] in a similar setup , wegner et al . designed an immunoassay that also utilized a terbium - qd fret pair to detect cancer biomarkers B-property . [SEP]
[CLS] in this case , six different primary antibodies B-material were used to bind a model biomarker B-property ( psa ) , enabling psa detection at concentrations as low as 1 . 6 ng / ml . kim and co - workers developed a qd - based sandwich immunoassay on a glass substrate consisting of a vertical zinc B-material oxide I-material ( zno ) nanowire B-nanoparticle array for the detection of cea . [SEP]
[CLS] the zno nanowire B-nanoparticle substrate provides a large surface area with many binding sites for capture antibodies B-material to bind the analyte of interest . [SEP]
[CLS] fret occurs between zno nanowires B-nanoparticle and qd labeled detection antibodies B-material upon sandwich formation due to the presence of cea . [SEP]
[CLS] the fluorescence B-property enhancement is quantified using fluorescence B-technique microscopy I-technique , with a large dynamic range for detecting cea ( from 0 . 001 to 100ng / ml ) . [SEP]
[CLS] one multimodal biosensor that utilizes electron transfer to a quencher in the absence of an analyte was developed by jou et al . , who takes advantage of both fret and chemiluminesence resonance energy transfer ( cret ) to enhance the sensitivity of detecting micro - rna - 141 ( mir - 141 ) , a nucleic B-material acid I-material biomarker B-property that is found in the blood and is indicative of prostate , ovarian , and gastric cancers . [SEP]
[CLS] in this assay , a fret quencher is covalently bound to nucleic acid functionalized cdse / zns qds and is released upon mir - 141 binding , resulting in an increase in fluorescence B-property that enables the detection of mir - 141 with a limit of detection of 1 pm . to further increase the sensitivity of analyte detection , the qds are introduced to g - quadruplex - forming telomerase and dntps . [SEP]
[CLS] next , hemin is added to intercalate into the g - quadruplexes on the qd , which catalyzes the oxidation of luminol by h 2 o 2 , resulting in cret and reducing the limit of detection of mir - 141 to 0 . 28 pm . [SEP]
[CLS] ge and co - workers developed a paper - based immunosensor device to detect afp , ca 125 , ca 15 - 3 ( a biomarker B-property also derived from mucins that is used to detect tumor B-material recurrence in breast cancer patients ) , and cea with detection limits of 0 . 3 pg / ml , 6 . 1 × 10 −5 u / ml , 2 . 9 × 10 −4 u / ml , and 1 . 4 pg / ml , respectively , through the use of a qd - fret system . [SEP]
[CLS] first , antibodies B-material immobilized onto the paper sensor and copper B-material oxide I-material ( cuo ) nanoparticle B-nanoparticle - antibody B-material conjugates sandwich the analyte . [SEP]
[CLS] next , dithizone ( dz ) - quenched cdte qds are added to the sensor surface . [SEP]
[CLS] finally , the sensors are treated with hcl , which releases cu 2 + from the cuo nps B-nanoparticle in the presence of analyte and results in the recovery of qd fluorescence B-property . [SEP]
[CLS] qds can also be used as an oxidizing B-property agent I-property for the detection of cancer biomarkers B-property using a sandwich assay . [SEP]
[CLS] in one such study , zhu et al . developed an immunosensor to detect psa by using cus qds to oxidize a small molecule ( o - phenylenediamine , opd ) to generate a fluorescent B-property signal ( figure 9 ) . [SEP]
[CLS] the fluorescence B-property of the oxidized small molecule 2 , 3diaminophenazine ( opdox ) was measured using fluorescence B-technique spectroscopy I-technique to detect psa with a limit of detection of 0 . 1 pg / ml . [SEP]
[CLS] high specificity for psa was obtained even in the presence of 10 ng / ml of interfering substances , such as serum proteins B-material . [SEP]
[CLS] this cus - based fluorescent B-property immunosensor compares favorably to nonfluorescence based immunosensors ( electrochemical , colorimetric , electrochemiluminescence , and elisa based ) . [SEP]
[CLS] many biosensors utilize aunps B-nanoparticle as fluorescence B-property quenchers I-property due to their strong absorbance , which can yield sensors with lower background signal than those utilizing organic fluorophore quenchers . [SEP]
[CLS] for example , you et al . developed a " turn - on " nanoparticle B-nanoparticle probe for detecting cancer biomarkers B-property based on nonspecific electrostatic adsorption of protein B-material biomarkers B-property to a library of fluorophore - tagged aunps B-nanoparticle . [SEP]
[CLS] this system was used to detect two biomarkers B-property : acid phosphatase and alkaline phosphatase , both of which are isoenzymes found in various organs that are indicative of cancer when up - regulated . [SEP]
[CLS] ( figure 10 ) [SEP]
[CLS] though this assay is capable of distinguishing between multiple protein B-material biomarkers B-property , it is not possible to design the system to detect any arbitrary target without screening large aunp B-nanoparticle libraries for specificity to the desired target . [SEP]
[CLS] in order to develop a detection system for platelet derived growth factor ( pdgf , a growth factor that is associated with various cancers when up - regulated ) with high specificity , huang et al . used aptamers in a similar " off - on " solution - based assay . [SEP]
[CLS] in this case , the fluorescence B-property of a small molecule intercalator , n , n ′ - dimethyl - 2 , 7 - diazapyrenium dication ( dmdap ) , is quenched in the absence of analyte by the aunp B-nanoparticle , and it is restored upon introduction of pdgf , which releases dmdap . [SEP]
[CLS] the limit of detection for this assay was 8 pm . in a similar study , cheng and co - workers used the analyte of interest hyaluronidase ( haase , an endoglycosidase that degrades hyaluronic acid , and is indicative of high - grade tumors B-material in bladder cancer patients when elevated to " turn on " fluorescence B-property from the nanoparticle B-nanoparticle probe . [SEP]
[CLS] the limit of detection was 0 . 625 u / ml from urine samples . [SEP]
[CLS] rather than using aunps B-nanoparticle as fluorescence B-property quenchers I-property , cho and co - workers took advantage of surface enhanced fluorescence B-property between aunps B-nanoparticle and cyanine 3b ( cy3b ) , an organic fluorophore , to detect vascular endothelial growth factor ( vegf , another growth factor that is implicated in various cancers when elevated ) in an " on - off " nanoparticle B-nanoparticle probe . [SEP]
[CLS] specifically , cy3b - labeled aptamers targeting vegf were electrostatically associated onto 80 nm aunps B-nanoparticle using positively charged poly - l - lysine . [SEP]
[CLS] upon vegf addition , the aptamer dissociated from the nanoparticle B-nanoparticle surface to bind vegf , causing a decrease in fluorescence B-property intensity as measured by a fluorescence B-property microscope . [SEP]
[CLS] fluorophore - labeled nanoparticles B-nanoparticle can also be used to detect cancer biomarkers B-property . [SEP]
[CLS] for example , khazanov et al . fabricated a microarray biosensor to detect alpha 1 - antitrypsin precursor ( aiat , a protease inhibitor that is a biomarker B-property for gastric cancer ) using boradiazaindacene ( bodipy , 660 - 680 ) labeled nanoparticles B-nanoparticle . [SEP]
[CLS] trypsin was grafted onto a glass surface to capture aiat . [SEP]
[CLS] next , bodipy nps B-nanoparticle conjugated to anti - aiat antibodies B-material bound to the analyte , resulting in a measurable increase in fluorescence B-property as monitored by confocal B-technique laser I-technique - I-technique scanning I-technique microscopy I-technique . [SEP]
[CLS] the limit of detection for this assay was 10 μg / ml . [SEP]
[CLS] early detection of cancer enables timelier treatment , and significantly improves patient outcomes . [SEP]
[CLS] as cancer develops , metastasis B-event may occur , which can drastically diminish cancer patient prognosis . [SEP]
[CLS] current clinical methods to diagnose cancer metastasis B-event , such as imaging via computerized tomography B-technique , rely on the detection of secondary tumors B-material . [SEP]
[CLS] unfortunately , this presents a significant limitation in the current state of cancer patient care , since treatments administered after metastasis B-event has already occurred are less effective and are associated with poorer survival rates . [SEP]
[CLS] as a result , the need to develop novel tools to identify cancer metastasis B-event before the formation of secondary tumors B-material is strong . [SEP]
[CLS] metastatic tumor B-material formation occurs through the spread of cancer cells B-material from the primary tumor B-material site to the bloodstream and lymphatic system , and to new sites at distal organs and tissues to form secondary tumors B-material . [SEP]
[CLS] circulating tumor B-material cells B-material ( ctcs ) are key in this process , and detecting their presence in the blood early provides a means to predict metastatic potential before the formation of secondary tumors B-material . [SEP]
[CLS] studies have indicated that the presence of ctcs before cancer treatment begins correlates with poorer survival rates in patients with metastatic cancer , and that the presence of ctcs after treatment suggests a higher likelihood of tumor B-material relapse . [SEP]
[CLS] based on this trend , the ability to isolate and detect cancerous cells B-material in the bloodstream would enable the assessment of metastatic risk and may provide the opportunity to begin treatment prior to the development of a secondary tumor B-material to improve cancer patient outcomes . [SEP]
[CLS] to achieve this , however , significant effort must be afforded to probe the correlation of ctc presence with cancer patient prognosis . [SEP]
[CLS] additionally , quantification of ctcs from individual patients throughout their treatment plan may serve as a means to monitor the patient ' s response through a simple blood test . [SEP]
[CLS] the identification of cells B-material as cancerous is challenging , since cancer emerges from abnormal growth and differentiation of healthy tissue , and is a highly heterogeneous disease that may be manifested in significant differences in gene expression from cell B-material to cell . [SEP]
[CLS] nonetheless , in general , cancer cells B-material do indeed differ from healthy cells B-material , and there are many known biomarkers B-property that may be used to differentiate between the two populations . [SEP]
[CLS] existing technology to detect ctcs relies on the immunomagnetic separation of cells B-material based upon their expression of cell surface antigens , such as epithelial cell B-event adhesion I-event molecule ( epcam ) , which is a transmembrane protein B-material involved in cell B-material signaling , migration , and proliferation that is implicated in cancer progression . [SEP]
[CLS] while this approach has been fda - cleared and commercialized through cellsearch technology ( janssen diagnostics ) , it is limited by the need to recognize cell surface antigens and can result in the inability to distinguish ctc populations whose protein B-material expression level is not sufficient for isolation , and it does not enable the quantification of gene expression . [SEP]
[CLS] beyond the recognition of cell - surface markers , monitoring the intracellular expression levels of certain genes may also be used to distinguish cancerous cells B-material from healthy cells . [SEP]
[CLS] cancer cells B-material tend to share high expression levels of classes of genes ( oncogenes ) involved in survival , proliferation , and differentiation . [SEP]
[CLS] over the past several years , researchers have identified several crucial genetic changes that cause cancer cells B-material to become more metastatic in nature . [SEP]
[CLS] recent studies have indicated that cancer cells B-material undergo the epithelial to mesenchymal transition ( emt ) during metastasis B-event and that this process results in the loss of epithelial cellular markers and the development of mesenchymal cellular markers . [SEP]
[CLS] this process typically occurs in development and in wound healing , but cancer cells B-material use this process to enable their extravasation from tumor B-material tissue into the bloodstream . [SEP]
[CLS] expression of cell B-event adhesion I-event proteins B-material , such as e - cadherin , is suppressed to facilitate the metastatic cancer cell B-material ' s dissociation from neighboring tumor B-material cells B-material . [SEP]
[CLS] meanwhile , the expression of cell B-material migration proteins B-material such as n - cadherin , vimentin , and fibronectin is up - regulated , causing the cell B-material to invade the bloodstream and begin the metastatic process . [SEP]
[CLS] based on the known differences in the genetic expression of metastatic cancer cells B-material compared to healthy cells B-material , quantification of the intracellular expression profiles of emt markers enables the identification of cells B-material that present a risk of cancer metastasis B-event . [SEP]
[CLS] many of the current methods available to study cancer cell B-material populations , such as quantitative real time pcr ( qrt - pcr ) and the enzyme - linked immunosorbent assay ( elisa ) , interrogate bulk cell B-material samples , providing information on the population average . [SEP]
[CLS] therefore , these techniques are not capable of monitoring sample heterogeneity . [SEP]
[CLS] this is a serious limitation in the study of cancer cell B-material populations , which inherently vary significantly in gene expression . [SEP]
[CLS] in particular , a small subset of circulating tumor B-material cell B-material populations , known as cancer stem cells B-material ( cscs ) , is thought to initiate cancer metastasis B-event and increase the risk of recurrence . [SEP]
[CLS] this subset of cells B-material differs in gene expression from the majority of tumor B-material cells B-material , and they may be unidentifiable using conventionally available techniques such as qrt - pcr and elisa that provide population averages and do not offer single - cell resolution . [SEP]
[CLS] certain classes of nanoparticles B-nanoparticle are uniquely suited for the design of probes that address the challenges traditional methods face in detecting small populations of cancer cells B-material via the generation of a fluorescent B-property output . [SEP]
[CLS] for example , qds and ucnps conjugated to moieties that bind and recognize cancer cell B-material surface markers offer several important advantages over organic dyes , including high quantum yields , size - dependent emission tunability , and enhanced photostability . [SEP]
[CLS] in addition , noble metal B-nanoparticle nanoparticles I-nanoparticle and aunps B-nanoparticle , in particular , are commonly used in combination with organic fluorophores to design " off - on " probes for intracellular gene expression analysis . [SEP]
[CLS] these probes exhibit lower background signal than traditional molecular approaches due to the efficient quenching capabilities of aunps B-nanoparticle . [SEP]
[CLS] based upon their fluorescent B-property output , the systems highlighted in this section may utilize a variety of techniques for analysis , including microscopy B-technique and flow B-technique cytometry I-technique . [SEP]
[CLS] importantly , flow B-technique cytometry I-technique provides the unique capability of interrogating single cells B-material , while surveying large samples . [SEP]
[CLS] this ability is critical in the study of cancer cells B-material , which are quite heterogeneous , and improves upon the currently available technologies that may be unable to distinguish cscs from a diverse tumor B-material cell B-material population or resolve relatively scarce populations of cancer cells B-material from large populations of healthy cells . [SEP]
[CLS] in addition , the use of fluorescence activated cell B-material sorting ( facs ) enables isolation of cancer cells B-material detected with a fluorescent B-property readout , providing the opportunity for further downstream analysis . [SEP]
[CLS] notably , biocompatible B-property nanoparticle B-nanoparticle probes maintain the health of suspected tumor B-material cells B-material , enabling additional studies that may be used to develop effective treatment plans for individual cancer patients based upon the gene expression profile of the tumor B-material cell B-material population . [SEP]
[CLS] the recent developments in the use of nanoparticle B-nanoparticle probes for cancer cell B-material detection significantly expand upon the existing approaches to detect cancer cells B-material , and represent promising advances in the early detection of metastatic cancer with potential impacts for cancer patient prognosis in the clinic . [SEP]
[CLS] many groups have explored the use of nanoparticles B-nanoparticle for the detection of cancer cells B-material through cell surface marker recognition using fluorescence B-property . [SEP]
[CLS] targeting moieties , such as antibodies B-material , aptamers , and small molecules , can be conjugated to the surface of the nanoparticle B-nanoparticle probe to induce selective binding to or uptake by cancer cells B-material , fluorophore - labeling them for easy identification over noncancerous cells B-material . [SEP]
[CLS] in these approaches , a variety of fluorescent B-nanoparticle nanoparticles I-nanoparticle ranging from ucnps and qds to silica B-nanoparticle nanoparticles I-nanoparticle with fluorescent B-property dyes encapsulated are used . [SEP]
[CLS] we will divide recent advances in this field first by the core B-material composition of the nanoparticle B-nanoparticle probe and then by the moieties that are used to recognize and bind cancer cells B-material . [SEP]
[CLS] to fluorophore - label cancer cells B-material - as previously described , qds exhibit unique optical properties that make them useful for detecting cancer cells B-material . [SEP]
[CLS] in particular , the detection of cancer cell B-material populations that are present in low abundances greatly benefits from the use of qds due to their high quantum yields . [SEP]
[CLS] wu et al . first reported the use of qds in the detection of breast cancer cells B-material . [SEP]
[CLS] in their approach , antibody - conjugated qds were used to label her2 in fixed sk - br - 3 breast cancer cells B-material ( figure 11 ) . [SEP]
[CLS] in addition to labeling cancer cells B-material , the qds were also used to detect her2expressing cells B-material in fixed mammary tissue isolated from transgenic mice , demonstrating the potential use of qds in the pathological and histological diagnosis of cancer . [SEP]
[CLS] since this work , other groups have also used qds in cancer cell B-material detection , expanding the platform to target alternate cancer cell B-material types . [SEP]
[CLS] conjugated polymer B-material nanoparticles B-nanoparticle ( cpns ) are sometimes useful for the detection of cancer cells B-material due to their optical properties , which may be tuned by altering the conductive polymer B-material composition , as well as their tailorable surface chemistry , which may be modulated to present various moieties that bind cancer cells B-material . [SEP]
[CLS] however , many water - soluble conductive polymer B-material nanoparticle B-nanoparticle probes suffer from low fluorescence B-property quantum yields . [SEP]
[CLS] to overcome this , the mcneil group developed pds from a variety of conductive hydrophobic B-property polymers B-material to yield nanoparticles B-nanoparticle that have high quantum yields , are photostable , and are nontoxic . [SEP]
[CLS] as a result , pds are great candidates for detecting circulating tumor B-material cells B-material . [SEP]
[CLS] in one study , wu et al . demonstrated the use of pds to target and fluorophore - label mcf - 7 breast cancer cells B-material through antibody B-material recognition . [SEP]
[CLS] pd - labeled mcf - 7 cells B-material exhibited 25 times higher fluorescence B-property than qd - labeled cells B-material , and 18 times higher fluorescence B-property than alexa fluor labeled cells B-material , as analyzed through flow B-technique cytometry I-technique . [SEP]
[CLS] in another example , semiconducting fluorescent B-nanoparticle polymer I-nanoparticle nanoparticles I-nanoparticle were encapsulated in plga ( 230 - 260 nm in size ) and functionalized with a her2 antibody B-material to preferentially identify sk - br - 3 ( high her2 expressing ) cells B-material over mcf - 7 and nih / 3t3 ( low her2 expressing ) cells B-material both on a substrate and free in solution . [SEP]
[CLS] rare earth element doped fluorescent B-property ucnp probes have also been used for cancer cell B-material detection . [SEP]
[CLS] in these examples , ucnps are often chosen for fluorescent B-property labeling based upon the ability to excite ucnps with near - infrared ( nir ) to infrared ( ir ) light to generate fluorescent B-property emission in the visible region ( through multiphoton mechanisms ) , resulting in minimization of background noise that is often observed due to cellular autofluorescence . [SEP]
[CLS] this property is especially advantageous in the detection of cancer cells B-material , which often depends upon the ability to distinguish between a small subset of malignant cells B-material among a larger population of healthy cells B-material . [SEP]
[CLS] in addition , the tunable emission profiles of ucnps enable simultaneous multicolor imaging to detect cancer cells B-material . [SEP]
[CLS] wang et al . reported tuning naybf 4 ucnp composition to generate ucnps that emit orange , yellow , green , cyan , blue , or pink fluorescence B-property upon excitation with 980 nm nir light ( figure 12 ) . [SEP]
[CLS] these ucnps were used to label hela cervical cancer cells B-material based on cea antibody B-material recognition . using the tunable emission profiles of ucnps , it is possible to simultaneously label several cancer cell B-material surface proteins B-material to detect malignant cancer cells B-material based upon the presence of multiple cancer markers . [SEP]
[CLS] biocompatible B-property silica B-nanoparticle nanoparticle I-nanoparticle probes have also been used for fluorescence - based detection of cancer cells B-material . [SEP]
[CLS] in most cases , silica B-nanoparticle nanoparticles I-nanoparticle are doped with a fluorophore and coated with a targeting moiety to bind a cancer cell specific biomarker B-property . [SEP]
[CLS] though these approaches may not offer the enhanced photostability and high quantum yields that qd and ucnp probes do , the surface functionalization of silica B-nanoparticle nanoparticles I-nanoparticle enables multivalent interactions with target receptors that tag cancer cells B-material with multiple fluorophores to generate brighter signals . [SEP]
[CLS] tao et al . reported the use of either rhodamine 6g - doped or tris ( 2 , 2 ′ - bipyridyl ) - dichlororuthenium ( ii ) ( rubpy ) - doped mesoporous silica B-nanoparticle nanoparticles I-nanoparticle for the detection of 7721 liver cancer cells B-material based upon cd155 antibody B-material recognition . [SEP]
[CLS] similarly , huang et al . reported the detection of ovarian cancer cells B-material ( skov - 3 ) using tetramethylrhodamine - doped silica B-nanoparticle nanoparticles I-nanoparticle through antibody B-material targeting . [SEP]
[CLS] deng and co - workers expanded upon the use of dye - doped silica B-nanoparticle nanoparticles I-nanoparticle for the detection of cancer cells B-material by monitoring the change in fluorescence B-property anisotropy ( using fluorescence B-property polarization anisotropy ( fpa ) ) upon binding of the nanoparticle B-nanoparticle probe to cancer cells B-material . [SEP]
[CLS] in their approach , methylene - blue - encapsulating silica B-nanoparticle nanoparticles I-nanoparticle were used to detect t - cell B-material acute lymphoblastic leukemia cells B-material . [SEP]
[CLS] since anisotropy changes with respect to the rotational time constant of the fluorophore , binding of the dye - doped silica particles to a comparatively large cancer cell B-material results in a measurable change in anisotropy . [SEP]
[CLS] target t - cell acute lymphoblastic leukemia cells B-material were spiked into healthy blood samples , and the technique was shown to have a linear range of detection from 4000 to 70 000 cells B-material / ml of whole blood . [SEP]
[CLS] though the use of fpa may enable more sensitive detection , it requires more sophisticated instrumentation than standard fluorescence B-technique spectroscopy I-technique , limiting its potential for use in clinical cancer diagnosis . [SEP]
[CLS] in addition , both single - walled and multiwalled carbon B-nanoparticle nanotubes I-nanoparticle ( scnts and mcnts ) have been used for detecting cancer cells B-material by fluorescence B-property . [SEP]
[CLS] though the quantum yield of scnts is low ( 3 - 8 % ) , their nir fluorescence B-property emission is strong enough to enable selective in vitro labeling and fluorescent B-property imaging of cancer cells B-material . [SEP]
[CLS] in a report by welsher and co - workers , antibodies B-material against a cancer cell specific marker , cd20 , were conjugated to scnts . [SEP]
[CLS] the system was used to distinguish between t cell and b cell lymphoma cells B-material based on the selective recognition of the antibody - labeled scnts by b cell lymphoma cells B-material due to their overexpression of the cd20 cell B-material surface marker . [SEP]
[CLS] beyond using nanoparticle B-nanoparticle probes to fluorophore - label cancer cells B-material , nanoparticles B-nanoparticle can be used for simultaneous fluorescent B-property and magnetic B-property resonance imaging of cancer cells B-material ( refs 244 , 262 , 268 , 269 , 275 , 280 , 286 , 293 , 295 , 304 - 307 ) . [SEP]
[CLS] typically , iron B-nanoparticle oxide I-nanoparticle core I-nanoparticle nanoparticles I-nanoparticle are modified with fluorescent B-property dye molecules either covalently or noncovalently to provide two parallel methods of tracking nanoparticle B-nanoparticle localization . [SEP]
[CLS] in one such example , wang et al . reported a layer - by - layer assembly method to coat B-material fe 3 o 4 nanoparticles B-nanoparticle with poly ( amidoamine ) dendrimers B-nanoparticle conjugated to fluorescein B-material isothiocyanate I-material ( fitc ) and folic B-material acid I-material . [SEP]
[CLS] in their system , the iron B-nanoparticle oxide I-nanoparticle nanoparticle I-nanoparticle core B-material was used in parallel with the fitc label to determine their specificity in targeting kb cells B-material ( a hela contaminant papillary carcinoma cell B-material line that overexpresses folate B-material receptors I-material ) through magnetic B-property resonance and fluorescence B-technique imaging I-technique . [SEP]
[CLS] in addition to providing a secondary method of tracking nanoparticle B-nanoparticle localization , magnetofluorescent nanoparticles B-nanoparticle enable the simultaneous detection and isolation of cancer cells B-material through magnetic B-property separation . [SEP]
[CLS] a 2011 study by song et al . described the use of fluorescent B-property - magneticbiotargeting - multifunctional nanobioprobes ( fmbmns ) in detecting and isolating leukemia ( jurkat t ) and prostate cancer ( lncap ) cells B-material ( figure 13 ) . [SEP]
[CLS] fmbmns targeting either prostate specific membrane antigen ( overexpressed by lncap cells B-material ) or cd3 ( overexpressed by jurkat t cells B-material ) were able to detect and isolate target cancer cells B-material through magnetic B-property separation , even with only 0 . 01 % target cells B-material present in a mixture including noncancerous cells B-material . [SEP]
[CLS] in some cases , nanoparticle B-nanoparticle probes are used in the design of " off - on " fluorescent B-property sensors for the detection of cancer cells B-material . [SEP]
[CLS] aunps B-nanoparticle , in particular , are commonly used due to their ability to efficiently quench fluorescence B-property and decrease background signal in the " off state " . [SEP]
[CLS] generally , a fluorophore is held in close proximity to the aunp B-nanoparticle surface such that the fluorescence B-property is quenched in an " off state " . [SEP]
[CLS] upon selective interaction with cancer cell B-material surface moieties , the fluorophore is released , resulting in a measurable fluorescent B-property response . [SEP]
[CLS] in one such example reported by lee and co - workers , aunps B-nanoparticle were functionalized with fluorophore - labeled heparin , which is a major component of the extracellular matrix that is degraded by the overexpression of heparanase and heparinase in metastatic cancer cells B-material . [SEP]
[CLS] while conjugated to the aunps B-nanoparticle , the fluorophore label is quenched , but upon degradation by metastatic cancer cells B-material , the heparin fragments are released and generate a fluorescence B-property enhancement . [SEP]
[CLS] this method was used to selectively detect cancer cell B-material lines that express high levels of heparanase ( hela cervical cancer cells B-material ) over cancer cells B-material with low expression levels ( mcf7 breast cancer cells B-material ) , and noncancerous cells B-material ( nih - 3t3 fibroblasts ) . [SEP]
[CLS] in another approach , bajaj et al . reported an aunp B-nanoparticle - based " chemical nose " to differentiate cell B-material types and cancer states using aunps B-nanoparticle that are capped with ligands of varying hydrophobicity B-property and are coated with green fluorescent B-property protein B-material ( gfp ) . [SEP]
[CLS] based on the differences in chemical structure of the capping ligands used , each aunp B-nanoparticle - gfp complex associates with cancer cells B-material to varying extents due to the differences in cell B-material membrane composition . [SEP]
[CLS] when the aunp B-nanoparticle - gfp construct interacts with a cell B-material , the gfp is displaced , generating a fluorescence B-property enhancement . [SEP]
[CLS] this method required as few as 5000 cells B-material for detection and was able to differentiate between breast cancer cells B-material ( mcf7 ) , hepatocellular carcinoma cells B-material ( hepg2 ) , cervical cancer cells B-material ( hela ) , and testicular cancer cells B-material ( nt2 ) . [SEP]
[CLS] though this capability may be useful in the study of cancer cell B-material membrane heterogeneity and the identification of cancer cells B-material , it is limited by the inability to rationally design a nanoparticle B-nanoparticle probe to target a specific cell B-material of interest . [SEP]
[CLS] silver B-material nanoclusters ( agncs ) have also been used for cancer cell B-material detection in " off - on " fluorescent B-property systems . [SEP]
[CLS] agncs consisting of 2 - 30 ag atoms B-material may be synthesized using single stranded dna templates to yield a fluorescent B-property probe that exhibits lower cytotoxicity B-property , exhibits brighter fluorescence B-property , and is more photostable than organic dyes . [SEP]
[CLS] in addition , recent research has found that agncs may be turned " on " and " off , " or tuned in emission wavelength , based on their chemical environment . [SEP]
[CLS] this advantage has been exploited to design fluorescent B-property biosensors for dna , rna , and protein B-material detection . [SEP]
[CLS] in 2010 , yeh et al . reported that agncs exhibit a 500 - fold enhancement in red fluorescence B-property when in close proximity to guanine - rich dna . [SEP]
[CLS] the extent of fluorescence B-property turn - on was determined to be dependent upon the number of guanine bases in proximity to agncs , and it was originally hypothesized that this effect was due to charge transfer between the guanine residues and the agncs . [SEP]
[CLS] however , more recent studies have indicated that this may not be the mechanism of agnc fluorescence B-property enhancement , and the underlying cause of guanine - proximity - induced fluorescence B-property of agncs is still under investigation . [SEP]
[CLS] following the recent advances in the use of agncs for biosensing , yin et al . designed a twocomponent dna / agnc probe to detect as few as 1000 ccrf - cem acute leukemia cancer cells B-material ( figure 14 ) . [SEP]
[CLS] unlike many of the other methods reported to fluorophore - label cell B-material surface markers for cancer cell B-material detection , this approach provides a switchable fluorescent B-property output , which may lead to reduced background signal from nonspecific binding of nanoparticle B-nanoparticle probes to noncancerous cells B-material . [SEP]
[CLS] recognition - a range of moieties is used to recognize and bind cell B-material surface markers for nanoparticle - mediated fluorescence B-property detection of cancer cells B-material . [SEP]
[CLS] in some cases , proteins B-material are conjugated to fluorescent B-nanoparticle nanoparticles I-nanoparticle to generate probes that bind to cancer cell B-material surfaces through recognition by cell B-material surface I-material receptors I-material . [SEP]
[CLS] for example , mi et al . have conjugated transferrin glycoprotein to ucnps and achieved fluorescent B-property imaging and detection of hela cervical cancer cells B-material based upon recognition by transferrin receptors , which are up - regulated in many types of cancers . [SEP]
[CLS] similarly , short peptides B-material may be conjugated to nanoparticles B-nanoparticle for targeted cancer cell B-material imaging . [SEP]
[CLS] in particular , fluorescent B-nanoparticle nanoparticles I-nanoparticle labeled with rgd peptide B-material are recognized by integrin α v β 3 , a cell B-material surface I-material receptor I-material that is implicated in cancer angiogenesis B-event and metastasis B-event , and have therefore been used to detect cancer cells B-material . [SEP]
[CLS] hong et al . demonstrated the ability of fluorescent B-property zinc B-material oxide I-material nanowires B-nanoparticle coated with rgd peptide B-material to selectively label integrin α v β 3 positive human glioblastoma cells B-material ( u87mg ) over integrin α v β 3 negative human breast cancer cells B-material ( mcf - 7 ) by fluorescence B-technique microscopy I-technique , highlighting the ability to differentiate between cancer types ( figure 15 ) . [SEP]
[CLS] antibodies B-material are frequently used to recognize cancer cell B-material surface markers based on their ability to specifically bind target cell B-material surface I-material receptors with high affinity . [SEP]
[CLS] her2 , which is overexpressed in breast cancer , is frequently used for detection through antibodies B-material conjugated to nanoparticle B-nanoparticle probes for cancer cell B-material detection . [SEP]
[CLS] additionally , epidermal growth B-material factor I-material receptor I-material ( egfr ) is often targeted for selective fluorophore labeling of cancer cells B-material due to its overexpression in a range of cancer cell B-material types . [SEP]
[CLS] in 2010 , haun et al . expanded upon the use of antibody - coated nanoparticles B-nanoparticle for detecting cancer cells B-material by developing a bio - orthogonal nanoparticle B-nanoparticle detection ( bond , figure 16 ) . [SEP]
[CLS] cycloaddition of trans - cycloocetene ( tco ) - modified antibodies B-material and tetrazine - coated magnetofluorescent nanoparticles B-nanoparticle occurs in biological media , including cell B-material culture medium and serum . [SEP]
[CLS] this method was shown to detect cancer cells B-material through either her2 , egfr , epcam , mucin 1 , or cd45 receptor B-material recognition , demonstrating the tailorability of the platform to analyze multiple targets in parallel and profile cancer cell B-material populations based on the expression of various surface markers in complex biological media . [SEP]
[CLS] another method of targeting cancer cells B-material utilizes oligonucleotide aptamers , which may be designed to fold and bind with high selectivity and affinity to any target of interest . [SEP]
[CLS] prostatespecific membrane antigen ( psma ) is a common target used for aptamer - based recognition of prostate cancer cells B-material . [SEP]
[CLS] bagalkot and co - workers demonstrated that qds coated with aptamers against psma could be used to selectively deliver doxorubicin B-material to prostate cancer cells B-material ( lncap ) for combined cancer cell B-material detection and treatment ( figure 17 ) . [SEP]
[CLS] prior to cellular entry , the fluorescence B-property of both the doxorubicin B-material and the qds is quenched through bi - forster energy transfer processes . [SEP]
[CLS] upon cellular entry , doxorubicin B-material is released , generating an increase in fluorescence B-property from both the doxorubicin B-material and the qd . [SEP]
[CLS] this method provides an avenue for the design of theranostic nanomaterials B-material to simultaneously detect and treat cancer cells B-material . [SEP]
[CLS] a development in utilizing aptamers for cancer cell B-material targeting is the use of cell based systematic evolution of ligands by exponential enrichment ( cell - selex ) to design aptamers that can recognize complex targets , including whole cells B-material based upon interactions between the aptamer and cell B-material membrane components . [SEP]
[CLS] aptamers that were designed through cell - selex to target cancer cells B-material of interest have been conjugated to nanoparticles B-nanoparticle to enable their detection through fluorescence B-property . [SEP]
[CLS] in one example , an 88 - mer dna aptamer designed to bind acute leukemia ccrf - cem cells B-material with high affinity ( k d = 5 nm ) was appended to tris ( 2 , 2 ′ - bipyridyl ) dichlororuthenium ( ii ) ( rubpy ) dye doped polymeric B-nanoparticle nanoparticles I-nanoparticle and utilized for the detection of ccrf - cem cells B-material through fluorescence B-property . [SEP]
[CLS] though the specific binding interactions of aptamers designed to target cancer cells B-material are not well understood , cell - selex is a generalizable method that may be used to target cell B-material populations for which antibodies B-material are not available . [SEP]
[CLS] an alternative approach to detect cancer cells B-material is to use small molecules that bind specific cancer cell B-material receptors . [SEP]
[CLS] several groups have demonstrated that folic B-material acid I-material may be conjugated to fluorescent B-nanoparticle nanoparticles I-nanoparticle for the detection of cancerous cells B-material , which often overexpress folate B-material receptors I-material . [SEP]
[CLS] in one example , rosenholm and co - workers conjugated folic B-material acid I-material to hybrid mesoporous silica B-nanoparticle nanoparticles I-nanoparticle for hela cervical cancer cell B-material targeting . [SEP]
[CLS] human embryonic kidney epithelial cells B-material ( hek - 293 ) with low folate B-material receptor I-material expression exhibited low uptake of the hybrid particles , suggesting their ability to selectively target cancerous cells B-material . [SEP]
[CLS] an advantage of this approach over others is that some small molecules are relatively inexpensive compared to proteins B-material and antibodies B-material . [SEP]
[CLS] when small molecules bind cell B-material surface I-material receptors I-material that are not known for a cancer type of interest , large - scale screens of small - molecule - functionalized nanoparticles B-nanoparticle may be conducted to uncover specific interactions of certain molecules with target cells B-material . [SEP]
[CLS] though this approach is not based on rational design , it is a powerful high - throughput approach to uncover cancer cell B-material interactions with small molecules for diagnostic applications . [SEP]
[CLS] in 2005 , weissleder et al . synthesized a library of nanoparticle B-nanoparticle constructs conjugated to one of 146 small molecules , and screened their ability to discriminate between cell B-material populations of interest . [SEP]
[CLS] specifically , the cellular uptake of these constructs into human pancreatic ductal adenocarcinoma cells B-material ( paca - 2 ) compared to human umbilical vein endothelial cells B-material ( huvec ) and human macrophages ( u937 ) was studied . [SEP]
[CLS] of the 146 nanoparticles B-nanoparticle tested , two exhibited selective uptake ( those functionalized with either 5 - chloro - isatoic anhydride or isatoic anhydride ) into paca - 2 cells B-material . [SEP]
[CLS] though this observed difference in uptake may be used to fluorophore - label and identify pancreatic cancer cells B-material , the underlying mechanism is not known and the application of this approach for the detection of additional cancer types would require additional screens . [SEP]
[CLS] nanoflares for intracellular mrna detection - in 2007 , the mirkin group introduced the nanoflare , a spherical nucleic B-material acid I-material ( sna ) aunp B-nanoparticle - based platform , which is shown to be useful for detecting and knocking down intracellular mrna ( figure 18a ) . [SEP]
[CLS] based on the highly oriented , dense oligonucleotide coating B-material , nanoflares enter cells B-material efficiently without the use of cytotoxic B-property transfection agents . [SEP]
[CLS] in addition , the density of the oligonucleotides leads to enhanced stability against degradation , making the nanoflare less susceptible to background fluorescence B-property based upon the degradation of dna compared to traditional molecular beacon and fluorescence B-property in situ hybridization ( fish ) probes . [SEP]
[CLS] unlike fish probes , which require fixation of cell B-material samples prior to analysis , no observable cytotoxicity B-property is seen following treatment of cells B-material with nanoflares , which enables one to detect genetic and small molecule content in live cells B-material . [SEP]
[CLS] in initial studies , the nanoflare was used for the fluorescent B-property detection of the mrna transcript B-event of the oncogene survivin . [SEP]
[CLS] nanoflares were used to distinguish between breast cancer cells B-material with high survivin expression ( sk - br - 3 ) and non - cancerous mouse endothelial cells B-material ( c166 ) . importantly , this construct has enabled the profiling of cancer cells B-material based on intracellular genetic content . [SEP]
[CLS] this is significant , since relying on extracellular protein B-material markers may result in the inability to identify subpopulations of cancerous cells B-material that do not express the surface marker of interest . [SEP]
[CLS] in 2009 , the nanoflare was reported as a singleentity agent for the simultaneous detection and regulation of target mrna . [SEP]
[CLS] based on the design of nanoflares , binding of target mrna to the recognition stand may also be used in antisense gene regulation . [SEP]
[CLS] this provides an opportunity to combine targeted mrna detection and gene regulation in a single construct . [SEP]
[CLS] as such , the nanoflare was shown to be useful for detecting and knocking down expression of survivin transcripts B-event . [SEP]
[CLS] additionally , the mirkin and tang groups have expanded upon the nanoflare construct with the development of multiplexed nanoflares . [SEP]
[CLS] in these studies , functionalization of aunps B-nanoparticle with two or three dna recognition strands and subsequent hybridization of their short complement reporter strands yielded nanoflares that are capable of simultaneous intracellular detection of multiple mrna transcripts B-event . [SEP]
[CLS] in particular , the use of multiplexed nanoflares in detecting survivin in addition to actin , a housekeeping gene , was studied as a means to normalize nanoflare fluorescence B-property to account for differences in cellular uptake and to make the technique comparable with qrt - pcr in quantifying intracellular mrna , but at the single live cell level . [SEP]
[CLS] notably , the tan group has developed a " lab - on - ananoparticle " system by incorporating multiple dna recognition stands onto the nanoflare construct in order to perform intracellular dna logic gating to simultaneously monitor the presence of multiple small molecules . [SEP]
[CLS] in 2014 , the nanoflare platform was used for the detection and isolation of live circulating tumor B-material cells B-material from whole blood . [SEP]
[CLS] nanoflares targeting known markers of the emt were designed , including vimentin and fibronectin . [SEP]
[CLS] the nanoflares were used to detect metastatic breast cancer cells B-material ( mcherry labeled mda - mb - 231 ) , and were doped into healthy human blood samples . [SEP]
[CLS] samples treated with nano - flares were analyzed by flow B-technique cytometry I-technique , and the average recovery of cells B-material was determined to be 68 ± 14 % , enabling the detection of as few as 500 cells B-material / ml of blood . [SEP]
[CLS] in addition , mcherry mda - mb - 231 cells B-material were isolated from the blood of xenografted mice following tumor B-material metastases B-event ( figure 18b ) . [SEP]
[CLS] blood samples were treated with nanoflares and processed following the same procedure used for human blood experiments . [SEP]
[CLS] it was noted that 87 - 90 % of the mcherry labeled cells B-material recovered were detected by nanoflare fluorescence B-property . [SEP]
[CLS] recently , the mirkin group further expanded upon the nanoflare platform to enable both intracellular rna quantification and spatiotemporal localization in living cells B-material . [SEP]
[CLS] sticky - flares were designed to target β - actin mrna and u1 short nuclear rna such that , upon target binding to a recognition sequence on the sticky - flare , a fluorophore is transferred to the rna transcript B-event , allowing for expression quantification and tagging it for intracellular tracking through fluorescence B-technique microscopy I-technique . this construct was utilized to visualize the realtime transport of β - actin mrna in live mouse embryonic fibroblasts using confocal microscopy B-technique . notably , it is difficult , if not impossible , to analyze the dynamics of rna transport with fish probes , since they require cell B-material samples to be fixed and permeabilized prior to analysis . [SEP]
[CLS] smart - flares may be applied to the study of gene expression in cancer cells B-material to our further understanding of rna expression , localization , and transport within cancerous and metastatic cells B-material . [SEP]
[CLS] since their development , nanoflares have been commercialized by emd millipore and sold under the trade name smartflare in over 230 countries . [SEP]
[CLS] at present , there are over 1700 versions of these constructs that can be used to target different mrna sequences in a variety of flow B-technique cytometry I-technique and imaging experiments . [SEP]
[CLS] in addition to providing the only way of measuring genetic content in single live cells B-material , these structures change the paradigm of cell B-material sorting based upon extracellular protein B-material markers to intracellular genetic and small molecule markers . [SEP]
[CLS] several groups have since utilized the smartflare to study a range of biological processes including pluripotency , the inflammatory response to infection with dna viruses , and the distribution of mrna within purkinje neurons . [SEP]
[CLS] further , the hendrix group has utilized smartflares to study melanoma tumor B-material cell B-material heterogeneity . [SEP]
[CLS] in their work , nodal , an embryonic morphogen that is typically silent following early development , was chosen as a target since it is reexpressed in aggressive melanoma cells B-material . [SEP]
[CLS] tumor B-material cell B-material heterogeneity was examined by sorting smartflaretreated melanoma cells B-material based upon their expression level of nodal using fluorescence activated cell B-material sorting . [SEP]
[CLS] the metastatic potential of the subpopulations was further studied , enabling the correlation of gene expression levels to tumor B-material cell B-material phenotypes . [SEP]
[CLS] this work highlights the unique capability of the smartflare to quantify genomic expression at the single - cell level as well as the immediate implications in expanding our knowledge of cancer and metastasis B-event through the use of smartflares . [SEP]
[CLS] ultimately , the continued study of cancer cell B-material heterogeneity is essential in the development of more efficacious chemotherapeutics as well as the identification of novel markers for early cancer diagnosis . [SEP]
[CLS] aunps B-nanoparticle modified with molecular beacon dna have also been used for the fluorescent B-property detection of cancer cells B-material . [SEP]
[CLS] traditional molecular beacons consist of hairpin dna that targets mrna of interest with a quencher and fluorophore pair conjugated to each end of the dna strand . [SEP]
[CLS] in the absence of target , the proximity of the quencher to the fluorophore results in low fluorescence B-property , but when target mrna binds , the hairpin opens , increasing the distance between the quencher and fluorophore to generate fluorescence B-property " turn - on " . [SEP]
[CLS] in the aunp B-nanoparticle - based systems , traditional molecular beacons are improved upon through the enhanced fluorescence B-property quenching efficiency of aunps B-nanoparticle compared to organic dyes . [SEP]
[CLS] the tang group has expanded the aunp B-nanoparticle - molecular beacon platform by designing systems that can detect two to four mrna targets . [SEP]
[CLS] in addition , this system has been expanded to enable the simultaneous release of oligonucleotide and the chemotherapeutic drug doxorubicin B-material in a theranostic application . [SEP]
[CLS] in these designs , doxorubicin B-material is intercalated into the molecular beacon structures in their " off state , " and is released upon target mrna binding . [SEP]
[CLS] this design was shown to detect skbr - 3 cells B-material based upon the expression of target cyclin d1 mrna . [SEP]
[CLS] in addition , doxorubicin B-material - induced cell B-event death I-event of skbr - 3 cells B-material was selective , and the viability B-property of I-property mcf I-property - I-property 10a cells I-property , which express low levels of cyclin d1 , was not affected by treatment with the doxorubicin - loaded molecular beacon - aunps B-nanoparticle . [SEP]
[CLS] the detection of cancer , metastasis B-event and tumor B-material growth in particular , is essential for designing effective treatment courses . [SEP]
[CLS] beyond the ability to diagnose cancer through ex vivo analysis based upon the detection of cancer biomarkers B-property and cancer cells B-material from patient samples of blood , feces , or urine , the ability to identify cancerous tissues and tumors B-material in the body presents several advantages in the detection and treatment of cancer . [SEP]
[CLS] the development of nanoparticle B-nanoparticle probes has enabled opportunities for both prognostics ( understanding the effect of a treatment on tumor B-material growth and metastasis B-event ) and treatment ( image guided surgery and theranostics ) for improving patient outcomes . [SEP]
[CLS] though research in this field has resulted in several nanoparticle B-nanoparticle probes that may be used in the diagnosis of cancer , several challenges must be overcome before such technologies are viable for use in clinical settings . [SEP]
[CLS] for example , instruments capable of fluorescence B-technique imaging I-technique on human patient scale are limited to narrow fields of view that are primarily useful for image - guided surgery . [SEP]
[CLS] thus , the development of instrumentation capable of generating fluorescence B-property images of patients is essential for the advancement of these technologies into the clinic . [SEP]
[CLS] ultimately , translation of these nanoparticle B-nanoparticle probes toward clinical use may result in sensitive imaging platforms for the detection of tumors B-material and metastases B-event at the earlier stages of their development , which may be confirmed through histopathological evaluation . [SEP]
[CLS] in addition to fluorescence - based imaging , several alternative modalities have been developed for the diagnosis of cancer through in vivo imaging : computed B-technique tomography I-technique ( ct ) , magnetic B-property resonance imaging ( mri ) , positron B-technique emission I-technique tomography I-technique ( pet ) , single photon emission computed B-technique tomography I-technique ( spect ) , ultrasound , and photoacoustic imaging , each with their own strengths and limitations ( table 2 ) . [SEP]
[CLS] besides spect , which is relatively high - cost and low - throughput , fluorescence B-property is the only other imaging modality available for simultaneous detection of multiple probes . [SEP]
[CLS] notably , this feature is advantageous in the diagnosis of cancer where the ability to distinguish malignant cell B-material populations from benign populations is enhanced by monitoring the presence of multiple cancer markers . [SEP]
[CLS] qds and ucnps are particularly useful in this respect , due to their emission profiles that are easily tuned through changes in composition and size . [SEP]
[CLS] though fluorescence B-technique imaging I-technique is somewhat limited by lower tissue penetration depths and resolution than alternative methods , including mri , fluorescence B-property offers high sensitivity of detection at a lower cost and is less time - consuming . [SEP]
[CLS] additionally , fluorescence B-property emission may be tuned to optimize tissue penetration depths , based upon the relative transparency of tissue absorbance in the nir window ( figure 19 ) . [SEP]
[CLS] qds and ucnps are especially well - suited to address these challenges due to their tunable nir emission profiles , as well as their large stokes and anti - stokes shifts , respectively , which minimize background signal due to tissue autofluorescence . [SEP]
[CLS] in this section , we will discuss the advances that have been made toward the use of nanoparticles B-nanoparticle in vivo , as well as the development of methods to image and , in some cases , treat cancer in animal models . [SEP]
[CLS] research within this field primarily focuses on the use of fluorescent B-nanoparticle nanoparticle I-nanoparticle probes to image and diagnose cancer in vivo based upon preferential accumulation of nanoparticles B-nanoparticle in tumor B-material tissue through either passive or active targeting . [SEP]
[CLS] to achieve the goal of diagnosing cancer through the use of nanoparticle B-nanoparticle probes for fluorescent B-property imaging in patients , significant research in understanding the behavior of nanoparticles B-nanoparticle in vivo must be done . [SEP]
[CLS] while in vitro assays are useful for characterizing nanoparticle B-nanoparticle interactions on the cellular level , these models do not always accurately represent cancer development and metastasis B-event in vivo . [SEP]
[CLS] as such , in order to design a nanoparticle B-nanoparticle for biomedical applications , and cancer diagnosis in particular , it is necessary to use animal models of cancer to understand nanoparticle B-nanoparticle probe behavior in vivo . [SEP]
[CLS] an ideal nanoparticle B-nanoparticle probe for cancer tissue detection should have a long circulation time with specificity to the tumor B-material tissue and low toxicity B-property to surrounding healthy tissue . [SEP]
[CLS] the fundamental design rules for developing such a probe are an active area of ongoing research . [SEP]
[CLS] we will discuss these recent advances , focusing on the properties that affect nanoparticle B-nanoparticle circulation times and biodistribution ( especially tumor B-material accumulation ) , and potential toxicity B-property . [SEP]
[CLS] tumor B-material tissue - nanoparticle B-nanoparticle size , shape , surface charge , and surface modification each play important roles in determining in vivo behavior and contribute to the complex interactions between nanomaterials B-material and biological systems . [SEP]
[CLS] here , we will discuss these interactions within the context of nanoparticle B-nanoparticle association with blood proteins B-material , the uptake and clearance of nanoparticles B-nanoparticle by the reticuloendothelial system ( res ) organs , penetration into solid tumors B-material , and the optimization of active ( versus passive ) targeting for cancer diagnosis . [SEP]
[CLS] passive targeting refers to the ability of nanoparticles B-nanoparticle with diameters 10 - 150 nm to preferentially extravasate from the bloodstream into tumor B-material tissue . [SEP]
[CLS] the tumor B-material ' s rapid growth initiates local angiogenesis B-event , the process of forming new blood vessels , to supply cancer cells B-material with nutrients . [SEP]
[CLS] as a result , the tight junctions between endothelial cells B-material do not form properly , generating " leaky " blood vessels . [SEP]
[CLS] in terms of blood supply , oxygen B-material , and nutrient delivery , these newly formed vessels are inefficient , necessitating the formation of additional blood vessels . [SEP]
[CLS] in vivo delivery of diagnostic and therapeutic nanoparticles B-nanoparticle can take advantage of the abundance of vasculature at tumor B-material sites in addition to the poor formation of tight junctions ( which are larger than the usual 8 nm size in tumor B-material tissue ) for preferential accumulation of nanoparticles B-nanoparticle in tumor B-material tissue . [SEP]
[CLS] in addition , large tumors B-material tend to have poor lymphatic drainage , leading to long retention times of extravasated nanoparticles B-nanoparticle in the tumor B-material tissue . [SEP]
[CLS] this form of passive nanoparticle B-nanoparticle entry into the tumor B-material microenvironment is termed the enhanced permeability and retention ( epr ) effect ( figure 20 ) , which was observed nearly 30 years ago with the transport of macromolecules into tumor B-material tissue . despite the ability to passively target nanoparticles B-nanoparticle to tumor B-material tissue via the epr effect , there are several challenges associated with this approach , including heterogeneity within and between tumor B-material types , which may compromise the utility of passive targeting in clinical settings . [SEP]
[CLS] thus , active tumor B-material targeting , in which a moiety that specifically binds a cell B-material surface marker that is associated with cancer , is frequently used to enhance the delivery of nanoparticles B-nanoparticle to tumor B-material tissue for cancer imaging applications . [SEP]
[CLS] though the epr effect allows nanoparticles B-nanoparticle to passively extravasate from circulation and accumulate within tumor B-material tissue , it is well understood that the adsorption of serum proteins B-material onto a nanoparticle B-nanoparticle surface ( opsonization ) significantly alters in vivo nanoparticle B-nanoparticle trafficking , uptake , and clearance . [SEP]
[CLS] the formation of a nanoparticle B-nanoparticle protein B-material corona I-material enables the recognition of nanoparticles B-nanoparticle by cell B-material - surface I-material receptors I-material on macrophages , resulting in rapid clearance rates . [SEP]
[CLS] it is widely known that nanoparticle B-nanoparticle composition , shape , size , and surface charge can dictate the types of proteins B-material that adsorb to the nanoparticle B-nanoparticle surface and can significantly alter in vivo pharmacokinetics and biodistribution . [SEP]
[CLS] this must be taken into consideration when designing nanoparticle B-nanoparticle probes for in vivo cancer diagnosis to enhance tumor B-material accumulation and improve signal . [SEP]
[CLS] one common method to reduce nonspecific adsorption of serum proteins B-material while also enhancing nanoparticle B-nanoparticle circulation time is to use poly ( ethylene glycol ) ( peg ) to passivate the surface of nanoparticles B-nanoparticle . [SEP]
[CLS] peg is widely accepted as a noncytotoxic molecule that imparts decreased sequestration by resident tissue macrophages in the res organs by minimizing the formation of a protein B-material corona I-material . [SEP]
[CLS] experimentally , pegylation of a range of nanoparticles B-nanoparticle , including aunps B-nanoparticle and qds , results in an enhancement in nanoparticle B-nanoparticle circulation time and slower accumulation into the liver and spleen when administered by intravenous ( iv ) injection into the tail vein of mice . [SEP]
[CLS] since circulation time and uptake into various organs can be tuned by altering peg length and packing density on the nanoparticle B-nanoparticle surface , pegylation provides an additional avenue for designing in vivo nanoparticle B-nanoparticle probes for cancer diagnosis . [SEP]
[CLS] it is important to note , however , that the accelerated blood clearance ( abc ) phenomenon may result in the rapid clearance of pegylated B-nanoparticle nanoparticles I-nanoparticle following repeat intravenous injections . [SEP]
[CLS] due to this challenge , the design of alternatives to enhance the blood residence time of pharmaceuticals remains an active field of research . [SEP]
[CLS] in addition to the epr effect and the formation of a protein B-material corona I-material , nanoparticle B-nanoparticle size and shape are also important considerations in the design of nanoparticle B-nanoparticle probes that exhibit high tumor B-material accumulation . [SEP]
[CLS] nanoparticles B-nanoparticle smaller than 10 nm are rapidly cleared by the renal system , minimizing their ability to localize in tumor B-material tissue . [SEP]
[CLS] additionally , the microenvironment surrounding tumor B-material tissue is complex and extremely heterogeneous : there are areas of hypoxia and necrosis , varying degrees of hyperpermeability in blood vessels , differences in the proliferative capacity between cells B-material at the edge of the tumor B-material and the core B-material , and dense extracellular matrix surrounding the solid tumor B-material . [SEP]
[CLS] these heterogeneities can lead to uneven distribution of diagnostic nanoparticle B-nanoparticle probes within tumor B-material tissue and afford smaller nanoparticles B-nanoparticle with the ability to permeate tumor B-material tissue more deeply . [SEP]
[CLS] nanoparticle B-nanoparticle shape also affects in vivo behavior , and must be considered when designing nanoparticle B-nanoparticle probes for the detection of tumor B-material tissue . [SEP]
[CLS] generally speaking , anisotropic particles exhibit longer circulation times in the blood , which is most likely due to the decreased probability of anisotropic nanoparticles B-nanoparticle permeating the endothelial gaps found in the fenestrations of the liver that range from hundreds of nanometers to tens of micrometers . [SEP]
[CLS] due to this , flexible nanoparticles B-nanoparticle with high aspect ratios may enable accumulation in tissues that are more difficult to access ( such as the brain ) , and carry a larger number of active targeting or drug molecules compared to a spherical B-nanoparticle nanoparticle I-nanoparticle of the same diameter . [SEP]
[CLS] such effects may alter nanoparticle B-nanoparticle accumulation in tumor B-material tissue , and should be considered in the design of diagnostic nanoparticles B-nanoparticle for in vivo imaging . [SEP]
[CLS] safety of systemic nanoparticle B-nanoparticle administration - in order to effectivelydiagnose and treat cancer in vivo through the use of novel nanoparticle B-nanoparticle probes , it is necessary to assess any possible toxicity B-property effects caused by their systemic administration . [SEP]
[CLS] while some nanoparticles B-nanoparticle , such as those derived from silica and certain polymers B-material , are generally regarded as safe due to their biocompatibility B-property and biodegradability B-property , the long - term safety of systemic injection of metal B-material and semiconductor nanoparticles B-nanoparticle is still under investigation . [SEP]
[CLS] ultimately , the specific toxicity B-property concerns associated with each nanoparticle B-nanoparticle probe must be thoroughly addressed on a case - by - case basis as the developing technologies are translated to clinical use . [SEP]
[CLS] mesoporous silica B-nanoparticle nanoparticles I-nanoparticle are often employed as carriers of drugs and other small molecules into cells B-material . [SEP]
[CLS] the ability to modify both the surface chemistry and internal pore size of these nanoparticles B-nanoparticle can increase the loading capacity to greater than the solubility B-property limit of many drugs . [SEP]
[CLS] this is a feature unique to mesoporous silica B-nanoparticle nanoparticles I-nanoparticle , and one that is highly desirable for tumor B-material targeting and treatment . [SEP]
[CLS] furthermore , it is generally accepted that mesoporous silica B-nanoparticle nanoparticles I-nanoparticle are biocompatible B-property and that any observed toxicity B-property in vivo is due to altered surface chemistry , specifically the surface silanol groups . [SEP]
[CLS] polymeric B-nanoparticle nanoparticles I-nanoparticle , including micelles B-material , hydrogels , and polymer - drug conjugates , are often useful for in vivo diagnostics and therapeutics due to their biodegradability B-property , biocompatibility B-property , and near complete clearance of degradation products . [SEP]
[CLS] some polymers B-material , such as poly ( glycolic acid ) and poly ( lactic acid I-material ) , have been approved by the fda for medical applications , making the use of such polymers B-material highly attractive for the rapid development of novel nanoparticles B-nanoparticle for cancer diagnostics in vivo . [SEP]
[CLS] the use of aunps B-nanoparticle for biomedical applications , and cancer diagnostics in particular , is rapidly gaining interest due to their ease of surface modification and attractive optical properties . [SEP]
[CLS] aunps B-nanoparticle have long been considered to be both bioinert and biocompatible B-property , and they have not been shown to cause cytotoxicity B-property in human cell B-material lines in vitro . [SEP]
[CLS] however , these cell B-material culture conditions do not replicate the complexity of in vivo conditions , and in vivo experimentation is necessary to better understand the potential side effects of aunp B-nanoparticle accumulation in organs and tissues over time . [SEP]
[CLS] though several studies have aimed to understand the role of aunp B-nanoparticle size , shape , dose , and route of administration [SEP]
[CLS] on biocompatibility B-property [SEP]
[CLS] , the results are often conflicting and difficult to compare based on variations in study design and nanoparticle B-nanoparticle synthesis . [SEP]
[CLS] the safety of administering ucnps and qds is also controversial , due to the concern that toxic B-property metals B-material ( cd , in particular , for qds ) may leach from the nanoparticle B-nanoparticle core B-material , causing adverse effects in vivo . [SEP]
[CLS] though few reports characterize the in vivo toxicity B-property of ucnps , some studies have used caenorhabditis elegans ( c . elegans ) to investigate biocompatibility B-property and have observed minimal toxicity B-property . [SEP]
[CLS] furthermore , a few studies have claimed that the injection of mice with ucnps does not cause any overt toxic B-property effects . [SEP]
[CLS] meanwhile , qds have consistently been shown cytotoxicity B-property in vitro , but translation of these results to systemic toxicity B-property is not always straightforward . [SEP]
[CLS] recent reports have demonstrated contrasting accounts of safety and toxicity B-property of qd administration in various small animal models , which is attributed to differences in administration dosage , qd synthesis strategies , and surface ligands . [SEP]
[CLS] ye et al . reported that when a certain class of qds was injected into nonhuman primates , no adverse effects on body weight , daily behavior , immune response , kidney and liver function , or blood chemistry were observed for 90 days post injection ( figure 21 ) . [SEP]
[CLS] though this study , which was the first to use an animal model with high genetic similarity to humans , supports the notion that qds may be safe for use in humans , qds have not yet been approved for human application , and many in the community are skeptical about their potential for in vivo clinical use . [SEP]
[CLS] many groups have investigated the use of fluorescent B-nanoparticle nanoparticle I-nanoparticle probes for imaging B-technique tumor I-technique tissue in vivo . [SEP]
[CLS] current research in this field utilizes animal models ( usually mouse models ) to study the accumulation of fluorescent B-nanoparticle nanoparticle I-nanoparticle probes in tumor B-material tissue for cancer diagnosis . [SEP]
[CLS] in these examples , a nanoparticle B-nanoparticle is typically injected systemically and preferentially localized to tumor B-material tissue either passively via the epr effect or actively through conjugation of a surface moiety that binds and recognizes the cancer cells B-material found within tumors B-material . [SEP]
[CLS] we will discuss these approaches within the context of the nanoparticle B-nanoparticle probe used to generate the fluorescent B-property signal and the recognition moiety used to bind and tag cancer cells B-material within the animal model . [SEP]
[CLS] imaging - many groups have utilized the epr effect to passively target fluorescent B-nanoparticle nanoparticles I-nanoparticle to tumor B-material tissue for in vivo imaging and diagnostics . [SEP]
[CLS] due to their unusual properties , including remarkable photostability , tunable emission , and high quantum yield , qds have been used in the fluorescent B-property imaging B-technique of I-technique tumor I-technique tissue by passive accumulation via the epr effect . [SEP]
[CLS] in 2012 , hong et al . reported the use of silver B-material sulfide ( ag 2 s ) qds for in vivo imaging B-technique of I-technique tumor I-technique tissues in a xenograft mouse model of 4t1 metastatic breast cancer cells B-material . [SEP]
[CLS] in another example , popovic and colleagues reported the use of qds coated with a silica B-material shell I-material of varying thickness to probe the role of nanoparticle B-nanoparticle size in determining tumor B-material tissue accumulation for cancer diagnostic applications . [SEP]
[CLS] by varying the composition of the qd cores B-material , the researchers found that each of the resulting nanoparticles B-nanoparticle , with diameters ranging from 12 to 120 nm , exhibited distinct emission colors . [SEP]
[CLS] a mixture of 12 , 60 , and 120 nm silica - coated qds was injected intravenously into a xenografted mu89 human melanoma mouse model . [SEP]
[CLS] due to the unique emission wavelength of each qd used , real - time fluorescence B-technique imaging I-technique of qd extravasation and tumor B-material penetration was gathered simultaneously for each nanoparticle B-nanoparticle size . [SEP]
[CLS] the results indicated that the 12 nm qds penetrated the tumor B-material tissue with minimal hindrance , while the 60 nm qds extravasated but remained within 10 μm from the blood vessels . [SEP]
[CLS] in contrast , the 120 nm qds did not extravasate to an appreciable extent . [SEP]
[CLS] this data supports the use of nanoparticles B-nanoparticle for the detection of cancer in vivo through fluorescence B-property based upon passive accumulation in tumor B-material tissue , and highlights the notion that a nanoparticle B-nanoparticle probe ' s size must be appropriately designed to enhance tumor B-material targeting and penetration . [SEP]
[CLS] additionally , there have been recent advances in the use of qds for in vivo cancer diagnosis based upon sentinel lymph node imaging . [SEP]
[CLS] sentinel lymph nodes are the first lymph nodes that a cancer will metastasize to , and evidence of metastasis B-event in the sentinel lymph nodes serves as an important prognostic for the progression of cancer [SEP]
[CLS] the ability to locate and surgically remove the sentinel lymph node provides an avenue to understanding the development of cancer : the absence of cancerous cells B-material in the sentinel lymph node indicates that the cancer is likely unable to have advanced through the formation of secondary tumors B-material , while the presence of cancerous cells B-material suggests that the cancer has begun the metastatic process . [SEP]
[CLS] kim et al . first reported the use of near - infrared qds for in vivo mapping of sentinel lymph nodes in both a mouse and pig model . [SEP]
[CLS] to demonstrate the utility of qds in performing diagnostic surgeries to excise sentinel lymph nodes , qds were injected into pigs . [SEP]
[CLS] localization of the qds in the sentinel lymph nodes highlighted their location , and the fluorescence B-property signal was used to direct surgery to remove them with penetration depths on the centimeter length scale . [SEP]
[CLS] the use of qds enabled deeper penetration depths compared to organic dyes used for sentinel lymph node mapping . [SEP]
[CLS] in 2007 , ballou et al . expanded upon the use of qds to map sentinel lymph nodes in tumor B-material - bearing animals . [SEP]
[CLS] mice with subcutaneous m21 human melanoma tumors B-material were injected with pegylated qds intratumorally . [SEP]
[CLS] the qds rapidly transferred from the tumor B-material tissue to the adjacent lymph nodes , and the fluorescence B-property emission was visible through the skin of the animal almost immediately . [SEP]
[CLS] conducting polymer B-material nanoparticles B-nanoparticle ( cpns ) have also been used in vivo for sentinel lymph node imaging . [SEP]
[CLS] kim et al . reported the use of cyanovinylene - backbone cpns ( 60 nm ) to map sentinel lymph nodes in real time in the nir wavelength range . [SEP]
[CLS] cpns were injected intradermally in the paws of mice , and could be tracked by the naked eye ( due to their high fluorescence B-property , at 365 nm ) as they drained to the lymph nodes . [SEP]
[CLS] ucnps are also used to detect tumor B-material tissue in vivo through fluorescence B-property . [SEP]
[CLS] cheng and co - workers demonstrated the tunability of ucnps for in vivo multicolor imaging applications . [SEP]
[CLS] nayf 4 nanocrystals doped with either er 3 + / yb 3 + or er 3 + / tm 3 + were synthesized and modified by adsorbing B-property one of three organic fluorophores on the nanoparticle B-nanoparticle surface : rhodamine b , rhodamine 6g , and tide quencher 1 . [SEP]
[CLS] five sets of ucnps were synthesized , each exhibiting distinct emission profiles . [SEP]
[CLS] each ucnp was subcutaneously injected into the backs of nude mice and imaged to demonstrate the potential use of ucnps in imaging and diagnostic applications ( figure 22 ) . [SEP]
[CLS] beyond multicolor imaging , ucnps have been used in the design of probes to diagnose cancer through the ability to detect hypoxia in vivo , since tumor B-material tissues are often low in oxygen B-material due to their abnormal vasculature . [SEP]
[CLS] liu et al . designed composite nanoparticles B-nanoparticle consisting of a ucnp core B-material and a mesoporous silica B-material shell I-material containing tris ( 4 , 7 - diphenyl - 1 , 10phenanthroline ) ruthenium ( ii ) dichloride ( [ ru ( dpp ) 3 ] 2 + cl 2 ) . [SEP]
[CLS] in the presence of oxygen B-material , the [ ru ( dpp ) 3 ] 2 + is quenched due to reversible photochemical B-property oxidation processes . [SEP]
[CLS] in the absence of oxygen B-material , the [ ru ( dpp ) 3 ] 2 + may be excited through energy transfer from the ucnps , providing a fluorescent B-property output to detect hypoxia in tumor B-material tissues . [SEP]
[CLS] this system was tested in a zebrafish embryo model of hypoxia ( figure 23 ) , and could be expanded upon for the detection of hypoxic cancerous tissues in vivo . [SEP]
[CLS] auncs have also been used in fluorescent B-property in vivo tumor B-technique imaging I-technique applications based on passive targeting . [SEP]
[CLS] in one report , wu et al . described the use of auncs , about 2 . 7 nm in diameter , for fluorescent B-property tumor B-technique imaging I-technique in vivo in cervical cancer ( hela ) and breast cancer ( mda - mb - 45 ) xenograft mouse models . [SEP]
[CLS] as previously described , small auncs of this size exhibit inherent fluorescence B-property . [SEP]
[CLS] the ultrasmall auncs were injected systemically into balb / c nude mice with subcutaneous hela or mda - mb - 45 tumors B-material . [SEP]
[CLS] within 6 h post injection , fluorescence B-property signal from the tumors B-material increased , demonstrating the potential use of aunps B-nanoparticle for in vivo cancer diagnostics . [SEP]
[CLS] for in vivo fluorescence B-technique tumor I-technique imaging I-technique using larger aunps B-nanoparticle , organic fluorophores are typically conjugated to the nanoparticle B-nanoparticle surface . [SEP]
[CLS] for example , chou and chan used fluorophore - labeled aunps B-nanoparticle of varying sizes to study the effect of nanoparticle B-nanoparticle size on tumor B-material accumulation for diagnostic and therapeutic applications . [SEP]
[CLS] aunps B-nanoparticle from 15 to 100 nm were synthesized and functionalized with alexa fluor 750 . [SEP]
[CLS] smaller , 15 nm aunps B-nanoparticle dispersed faster into the tumor B-material tissue to provide fluorescence B-property contrast of the cancerous lesions . [SEP]
[CLS] this approach may be used for tumor B-material tissue detection through passive accumulation , but also aids in our understanding of the size dependence of the epr effect . [SEP]
[CLS] perrault and chan later expanded upon the use of aunps B-nanoparticle in cancer tissue imaging through the use of aunps B-nanoparticle as an " anchor " in a xenograft breast cancer ( mda - md - 435 ) mouse model ( figure 24 ) . [SEP]
[CLS] this approach combines passive targeting of the anchor aunps B-nanoparticle to tumor B-material tissue with active targeting of a fluorophore to label the aunps B-nanoparticle in vivo . [SEP]
[CLS] this method resulted in a 200 times faster rate of fluorophore accumulation in tumor B-material tissue compared to fluorophore - labeled aunps B-nanoparticle of the same size , based on the active targeting of the fluorophore to the biotinylated aunps B-nanoparticle . [SEP]
[CLS] this method ' s use of actively targeting biotinylated aunps B-nanoparticle within the tumor B-material tissue may be beneficial for imaging cancer tissues when the cell B-material surface markers unique to the cancer cells B-material are not well understood . [SEP]
[CLS] in 2011 , von maltzahn et al . expanded upon this approach by utilizing photothermal nanoparticles B-nanoparticle to passively target tumor B-material tissue and locally induce a coagulation cascade that was then actively targeted with fluorescent B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] gold B-material nanorods B-nanoparticle ( aunrs ) were intravenously injected into mda - mb - 435 breast cancer tumor B-material - bearing mice . mice were irradiated to locally heat tumor B-material tissue based on passive aunr accumulation , resulting in local blood vessel disruption and coagulation . [SEP]
[CLS] in a labeling step , magnetofluorescent iron B-material oxide I-material nanoworms coated with a peptide B-material substrate for the coagulation transglutaminase fxiii were administered intravenously to the mice for the detection of tumor B-material tissue through fluorescence B-property . [SEP]
[CLS] in addition to metal B-material and semiconducting nanoparticles B-nanoparticle , polymeric B-nanoparticle nanoparticles I-nanoparticle encapsulating organic fluorophores have also been used for in vivo tumor B-technique imaging I-technique . [SEP]
[CLS] while these approaches do not utilize the unique optical properties of inorganic B-nanoparticle nanoparticles I-nanoparticle , such as qds and ucnps , the nanoparticles B-nanoparticle ' size enables passive tumor B-material targeting via the epr effect . [SEP]
[CLS] in one such report , fortin et al . describe the use of liposomal B-nanoparticle nanoparticles B-nanoparticle loaded with a near - infrared organic fluorophore , 1 , 1 ′ - dioctadecyl - 3 , 3 , 3 ′ , 3 ′ tetramethylindotricarbocyanine iodide B-material ( dir ) , to image brain tumors B-material in a mouse model by fluorescence B-property . [SEP]
[CLS] in a similar study , schadlich et al . demonstrated the use of polymeric B-nanoparticle nanoparticles I-nanoparticle for tumor B-technique imaging I-technique in a xenograft mouse model of colorectal cancer ( dsred2 labeled colorectal adenocarcinoma ht29 cells B-material ) . [SEP]
[CLS] tumor B-material accumulation of the nanoparticles B-nanoparticle was evaluated in vivo and ex vivo following excision of the tumor B-material tissues ( figure 25 ) . [SEP]
[CLS] colocalization of the dsred2 fluorescence B-property from the tumor B-material tissue and the dir dye from the peg - pla nanoparticles B-nanoparticle increased from 10 min to 24 h post injection as a result of enhanced retention of the nanoparticles B-nanoparticle over time . [SEP]
[CLS] in addition to tumor B-technique imaging I-technique based I-technique upon nanoparticle B-nanoparticle accumulation through passive targeting via the epr effect , extensive work in the use of nanoparticles B-nanoparticle that actively target tumor B-material tissue by recognizing cell B-material surface I-material receptors I-material on cancer cells B-material has been studied . [SEP]
[CLS] often , these approaches enhance the sensitivity of in vivo tumor B-material detection by increasing the amount of nanoparticles B-nanoparticle that are delivered to tumor B-material tissue per unit time . [SEP]
[CLS] we will discuss these approaches , dividing them by the recognition moiety used to target cancer cells B-material in vivo . [SEP]
[CLS] peptides B-material are frequently used to actively target cancerous tissues in vivo . [SEP]
[CLS] in particular , the rgd peptide B-material , which is recognized by a cell B-material surface I-material receptor I-material that is implicated in cancer angiogenesis B-event and metastasis B-event ( integrin α v β 3 ) , has been used to target tumor B-material tissue in vivo for imaging and diagnostic applications . [SEP]
[CLS] in some cases , polymeric B-nanoparticle nanoparticles I-nanoparticle conjugated to organic fluorophores are modified with rgd to target tumors B-material . [SEP]
[CLS] more commonly , inherently fluorescent B-nanoparticle nanoparticles I-nanoparticle , such as ucnps , are used to target integrin α v β 3 through presentation of the rgd peptide B-material for in vivo imaging B-technique of I-technique tumors I-technique . [SEP]
[CLS] in one such example , xiong et al . used pegylated ucnps conjugated to rgd to actively target tumors B-material in u87mg glioma xenografted mice . [SEP]
[CLS] to evaluate the effect of rgd active targeting , mice were injected with two xenograft tumors B-material : u87mg glioma , which has high integrin α v β 3 expression , and mcf7 breast cancer , which has low integrin α v β 3 expression . [SEP]
[CLS] the rgdconjugated ucnps were injected intravenously , and the fluorescence B-property intensity of the two tumors B-material was compared ( figure 26 ) . [SEP]
[CLS] ex vivo analysis of the ucnp content in organs by inductively coupled plasma atomic emission B-technique spectroscopy I-technique ( based on y 3 + content ) revealed that ucnp content in the u87mg tumor B-material was roughly 30 times higher than that of the mcf7 tumor B-material . [SEP]
[CLS] in another example , wu and co - workers utilized polymer B-material dots ( pds ) modified with chlorotoxin , a tumor B-material targeting peptide B-material , to target medullo - blastoma tumors B-material in nd2 : smoa1 mice . [SEP]
[CLS] high fluorescence B-property intensity of the pds was detected in the brain tumor B-material regions ( 2 . 3fold increase over wild type mice , compared to a 1 . 2 - fold increase in nontargeting pds ) following intravenous injection via the tail vein . [SEP]
[CLS] these works highlight the benefit of active tumor B-material targeting compared to passive targeting via the epr effect for cancer diagnostics , when high contrast enables the detection of tumors B-material at earlier stages . [SEP]
[CLS] conjugation of antibodies B-material that bind cancer cell B-material surface I-material receptors I-material to fluorescent B-nanoparticle nanoparticles I-nanoparticle has also been used for in vivo tumor B-technique imaging I-technique through active nanoparticle B-nanoparticle probe targeting . [SEP]
[CLS] kolitz - domb and co - workers used polymeric B-nanoparticle fluorescent I-nanoparticle nanoparticles I-nanoparticle for in vivo imaging of colon cancer through antibody B-material recognition of carcinoembryonic antigen ( cea ) . [SEP]
[CLS] indocyanine green , a near - infrared fluorophore , was encapsulated into proteinoidpoly ( lactic acid ) nanoparticles B-nanoparticle , which were used to image colon cancer tumors B-material in a ls174t colorectal cancer orthotopic mouse model . [SEP]
[CLS] the nanoparticles B-nanoparticle were administered to mice through the anus , and the animals were sacrificed 4 h postdelivery for ex vivo imaging B-technique of I-technique nanoparticle I-technique tumor I-technique accumulation in the colon . [SEP]
[CLS] based on the overexpression of cea by ls174t cells B-material , the nanoparticles B-nanoparticle localized to tumor B-material tissue and generated fluorescent B-property signal at the tumor B-material sites . [SEP]
[CLS] in comparison , control nanoparticles B-nanoparticle without anti - cea antibody B-material produced no appreciable fluorescence B-property signal , indicating the enhancement of tumor B-material targeting achieved through the use of antibodies B-material . [SEP]
[CLS] aptamers have also been used in the development of nanoparticle B-nanoparticle probes for in vivo fluorescent B-property imaging B-technique of I-technique tumor I-technique tissue through active targeting . [SEP]
[CLS] tong et al . reported the use of polymeric B-nanoparticle nanoparticles I-nanoparticle incorporating cy5 to target psma using a dna aptamer conjugated to the nanoparticle B-nanoparticle surface . [SEP]
[CLS] in vitro experiments demonstrated that nanoparticle B-nanoparticle labeling of prostate cancer cells B-material was specific to lncap cells B-material , which have high psma expression compared to pc3 cells B-material . [SEP]
[CLS] though the nanoparticles B-nanoparticle were not used to image prostate cancer in vivo , they exhibited no observable adverse effects in mice . [SEP]
[CLS] in 2015 , ding and co - workers expanded upon this and used an aptamer designed to bind scg7901 gastric cancer cells B-material through cell - selex for in vivo imaging B-technique of I-technique tumors I-technique in a mouse model . [SEP]
[CLS] in addition to the use of peptides B-material , antibodies B-material , and aptamers , small molecules also are used to actively target nanoparticle B-nanoparticle probes to tumor B-material tissue for cancer diagnostics . [SEP]
[CLS] in particular , folate is frequently attached to fluorescent B-nanoparticle nanoparticles I-nanoparticle to target folate B-material receptors I-material , which are overexpressed by many cancer cells B-material . [SEP]
[CLS] in one such example , ma et al . synthesized polymeric B-nanoparticle nanoparticles I-nanoparticle loaded with indocyanine green and conjugated to folate for in vivo imaging of xenograft mda - mb - 231 breast cancer tumors B-material in a mouse model . [SEP]
[CLS] similarly , xiong and co - workers reported the conjugation of folate to nayf 4 : yb , er ucnps for active tumor B-material targeting in cancer diagnostic B-technique imaging I-technique . [SEP]
[CLS] folate - tagged ucnps resulted in an observable fluorescence B-property signal from cervical cancer hela tumors B-material in mice 24 h following intravenous injection compared to no observable signal from hela tumors B-material in mice injected with control ucnps lacking the folate tag . [SEP]
[CLS] to quantify the difference in uptake , inductively coupled plasma atomic emission B-technique spectroscopy I-technique was used to measure ucnp accumulation in the tumor B-material tissue based on y 3 + content . [SEP]
[CLS] approximately 6 times more folate - receptor - targeting ucnps per gram of tumor B-material tissue than ucnps without folate was observed , indicating the fold enhancement in nanoparticle B-nanoparticle accumulation that may be achieved by actively targeting tumor B-material tissue . [SEP]
[CLS] such enhancements can also improve fluorescence B-property signal from smaller tumors B-material and enable the early detection of tumors B-material . [SEP]
[CLS] considerable effort has been devoted to the development of nanoparticle B-nanoparticle probes for simultaneous imaging through both fluorescence B-property and additional techniques , such as mri , computed B-technique tomography I-technique ( ct ) , and x - ray imaging . [SEP]
[CLS] these approaches typically seek to combine the benefits of fluorescence B-technique imaging I-technique with a complementary imaging modality to expand upon the use of nanoparticle B-nanoparticle probes for the detection of cancerous tissues in vivo . [SEP]
[CLS] while the design of multimodal nanoparticle B-nanoparticle probes has been an active area of research , the infrastructure required to support the use of such technologies , and the availability of instrumentation to conduct fluorescence B-technique imaging I-technique while simultaneously imaging via an alternative modality , has yet to be developed . [SEP]
[CLS] in one such example , yi et al . reported the design of ucnps for in vivo imaging through simultaneous upconversion fluorescence B-property and x - ray imaging . [SEP]
[CLS] similarly , shen et al . described the synthesis of a ucnp sandwiched structure containing a nagdf 4 : yb / tm core B-material , a naluf 4 : yb / tm middle layer , and a nayf 4 top layer for simultaneous in vivo upconversion fluorescence B-property and ct imaging . [SEP]
[CLS] zhang et al . described the encapsulation of aunps B-nanoparticle and the organic fluorophore , bis ( 4 - ( n - ( 2 - naphthyl ) phenylamino ) phenyl ) fumaronitrile ( npapf ) , in pegylated micelles B-material for in vivo imaging of colon carcinoma ( ct26 ) tumors B-material through both fluorescence B-property and through ct . [SEP]
[CLS] combining fluorescence B-technique imaging I-technique capabilities with either ct or x - ray imaging may be especially useful in the translation of nanoparticle B-nanoparticle probes from animal models to clinical patient use since the relatively deeper tissue penetration afforded by these methods may be used in parallel with the high sensitivity of fluorescence B-technique imaging I-technique . [SEP]
[CLS] some approaches to image tumor B-material tissue in vivo focus on the use of magnetofluorescent nanoparticles B-nanoparticle for simultaneous fluorescent B-property and magnetic B-property resonance imaging . [SEP]
[CLS] one common approach in the design of magentofluorescent nanoparticles B-nanoparticle is to synthesize self - assembling fluorophore - labeled polymeric B-nanoparticle nanoparticles I-nanoparticle that encapsulate smaller magnetic B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] in some cases , chitosan B-nanoparticle nanoparticles I-nanoparticle containing magnetic B-property and fluorescent B-property components are used for in vivo imaging B-technique of I-technique tumor I-technique tissue through passive targeting by the epr effect . [SEP]
[CLS] since chitosan B-material is a biodegradable B-property polysaccharide B-material , it is regarded as highly biocompatible B-property with low toxicity B-property . [SEP]
[CLS] in 2010 , nam et al . used self - assembling chitosan B-nanoparticle nanoparticles I-nanoparticle encapsulating a gadolinium B-material ( gd ) mri contrast B-technique agent I-technique and labeled with the near - infrared dye cyanine 5 . 5 ( cy5 . 5 ) for squamous carcinoma tumor B-material detection in mice . [SEP]
[CLS] similarly , lee et al . reported the use of chitosan B-nanoparticle nanoparticles I-nanoparticle for multimodal fluorescence B-property and mri imaging B-technique of I-technique tumor I-technique tissue in vivo . [SEP]
[CLS] chitosan B-nanoparticle nanoparticles I-nanoparticle encapsulating superparamagnetic B-nanoparticle iron I-nanoparticle oxide I-nanoparticle nanoparticles I-nanoparticle ( spions ) with a cy5 . 5 fluorophore label were synthesized and intravenously injected into mice bearing glioma u87mg tumors B-material ( figure 27 ) . [SEP]
[CLS] others have utilized antibodies B-material in the development of multimodal imaging platforms for in vivo cancer diagnostics through active tumor B-material targeting . [SEP]
[CLS] for example , liu et al . described the use of magnetic B-property , gd - containing ucnps for multimodal mri and fluorescence B-technique imaging B-technique of I-technique small I-technique tumors B-material in vivo . [SEP]
[CLS] in another example reported by chen et al . , mesoporous silica B-nanoparticle nanoparticles I-nanoparticle were used to actively target tumor B-material tissue for multimodal fluorescence B-property and positron B-technique emission I-technique tomography I-technique ( pet ) imaging . [SEP]
[CLS] mesoporous silica B-nanoparticle nanoparticles I-nanoparticle were labeled with cu and ir dye 800 , and conjugated to an anti - cd105 trc105 antibody B-material ( tracon pharmaceuticals inc . ) , which targets a glycoprotein receptor B-material that is overexpressed on tumor B-material vessel endothelial cells B-material and is implicated in angiogenesis B-event and tumor cell proliferation . [SEP]
[CLS] following intravenous administration , the cu pet - active label was used to quantify the percentage of the injected dose localized to tumor B-material tissue in vivo . [SEP]
[CLS] while fluorescence B-property alone may not provide this capability , multimodal imaging systems impart many advantages that may expand upon the capabilities of fluorescence B-technique imaging I-technique to provide complementary information in cancer diagnosis . [SEP]
[CLS] finally , folate is also used in actively targeting multifunctional nanoparticles B-nanoparticle to tumor B-material tissue through recognition of folate by folate B-material receptors I-material , as reported by rolfe et al . in 2014 . [SEP]
[CLS] specifically , a hyperbranched polymer B-material scaffold labeled with rhodamine b and f was assembled into nanoparticles B-nanoparticle and conjugated to folate to generate magnetofluorescent nanoparticles B-nanoparticle for multimodal imaging B-technique of I-technique b16 I-technique melanoma tumors B-material in vivo . [SEP]
[CLS] mri and fluorescence B-technique imaging I-technique indicated an enhanced tumor B-material accumulation of magnetofluorescent nanoparticles B-nanoparticle tagged with folate compared to nanoparticles B-nanoparticle without folate by combining the high resolution of mri with the sensitivity of fluorescence B-technique imaging I-technique within a single nanoparticle B-nanoparticle construct . [SEP]
[CLS] probes - theranostic nanoparticle - based applications take advantage of the preferential accumulation of nanoparticles B-nanoparticle in tumor B-material tissue to simultaneously detect and treat cancer , where simply incorporating a chemotherapeutic drug into a fluorescent B-nanoparticle nanoparticle I-nanoparticle used for tumor B-technique imaging I-technique may also enable therapy . [SEP]
[CLS] additionally , many reports describe the use of plasmonically active nanoparticles B-nanoparticle , such as nanoshells B-nanoparticle , for tumor B-material detection and light - triggered local heating of cancerous cells B-material , causing death via photothermal therapy . [SEP]
[CLS] these methods will be discussed in further detail below . [SEP]
[CLS] many reports describe the incorporation of chemotherapeutics into fluorescent B-nanoparticle nanoparticles I-nanoparticle for use in theranostic applications . [SEP]
[CLS] these nanoparticles B-nanoparticle may be passively or actively targeted to the tumor B-material tissue , and are used to treat cancer based upon preferential nanoparticle B-nanoparticle accumulation in the tumor B-material . [SEP]
[CLS] a variety of nanoparticles B-nanoparticle ( i . e . , polymeric and inorganic ) are used in these approaches . [SEP]
[CLS] the kwon group , for example , has reported the use of dye - labeled chitosan B-nanoparticle nanoparticles I-nanoparticle for the simultaneous detection and treatment of cancer through delivery of paclitaxel B-material to tumor B-material tissue through passive nanoparticle B-nanoparticle accumulation . [SEP]
[CLS] dye - doped polymeric B-nanoparticle nanoparticles I-nanoparticle have also been used to deliver camptothecin and irinotecan chemotherapeutics for theranostic applications . [SEP]
[CLS] additionally , chen et al . reported the use of magnetofluorescent ucnps to deliver doxorubicin B-material to tumor B-material tissue via passive targeting for simultaneous cancer imaging and therapy . [SEP]
[CLS] in order to improve nanoparticle B-nanoparticle ' s selectivity for cancer cells B-material , active nanoparticle B-nanoparticle targeting is often used to deliver theranostic nanoparticle B-nanoparticle probes to tumor B-material tissue . [SEP]
[CLS] in some cases , delivery of the targeting moiety to tumor B-material tissue alone may result in a therapeutic response . [SEP]
[CLS] corsi et al . reported the suppression of her2 , a cell B-material surface I-material receptor I-material that promotes cancer cell B-material growth , through active targeting via antibody B-material conjugation to magnetofluorescent ucnps for cancer imaging applications . [SEP]
[CLS] clinical treatment of her2 - positive cancers typically utilizes the monoclonal B-material antibody I-material trastuzumab to target her2 and subsequently inhibit cancer cell B-material proliferation . [SEP]
[CLS] in their work , corsi and co - workers demonstrated that conjugation of trastuzumab to ucnps for in vivo tumor B-technique imaging I-technique in a breast cancer xenograft mouse model ( mcf7 ) through active targeting also results in a significant decrease in her2 expression on the tumor B-material cell B-material surface for up to 1 week following intravenous administration ( figure 28 ) . [SEP]
[CLS] this may be further developed into a theranostic system that can both diagnose and treat her2 - positive cancers . [SEP]
[CLS] in another example of active tumor B-material targeting for theranostic applications , santra et al . reported the synthesis of dendrimer B-nanoparticle polymeric B-nanoparticle nanoparticles I-nanoparticle encapsulating cytochrome c , a protein B-material known to initiate apoptosis B-event , and the fluorophore indocyanine green . [SEP]
[CLS] the nanoparticles B-nanoparticle were tagged with folate for targeted theranostic detection and treatment of a549 lung cancer cells B-material , a cell line that overexpresses folate B-material receptors I-material . [SEP]
[CLS] selectivity in cellular uptake and death for a549 cells B-material over mcf7 cells B-material , which have low folate B-material receptor I-material expression , was demonstrated in vitro ; this approach may later be applied to in vivo theranostic applications . [SEP]
[CLS] in addition to chemotherapeutics , photoactive materials may also be delivered to tumor B-material tissue for theranostic imaging and treatment . [SEP]
[CLS] in these approaches , materials with strong absorbance in the near - infrared region are used to convert optical energy into heat to locally kill cancer cells B-material . [SEP]
[CLS] this method , termed photothermal therapy , typically utilizes nir light , which does not cause damage to cells B-material , to activate photothermal nanoparticles B-nanoparticle localized in tumor B-material tissue throughout the body . [SEP]
[CLS] one concern with this approach , however , is that nanoparticles B-nanoparticle accumulated in the liver and spleen may damage healthy tissue upon irradiation . [SEP]
[CLS] this disadvantage hinders the applicability of photothermal therapies to treat cancer once metastasis B-event has occurred , and limits its potential use to local treatment of tumors B-material . [SEP]
[CLS] nanoparticles B-nanoparticle that allow for both imaging and photothermal therapy have demonstrated passive targeting to tumor B-material tissue via the epr effect . [SEP]
[CLS] in some cases , silica or polymeric B-nanoparticle nanoparticles I-nanoparticle are designed to encapsulate photothermal - sensitizing dyes , such as protoporphyrin ix or naphthalocyanine [SEP]
[CLS] in one such example , chen et al . reported the use of a conductive polymer - based nanoparticle B-nanoparticle for photothermal therapy of 4t1 breast cancer tumors B-material in mice . [SEP]
[CLS] poly ( 3 , 4 - ethylenedioxythiophene ) : poly ( 4 - styrenesulfonte ) ( pedot : pss ) nanoparticles B-nanoparticle were synthesized via a layer - by - layer assembly process and labeled with cy5 fluorophores . [SEP]
[CLS] the resulting nanoparticles B-nanoparticle were intravenously administered to tumor bearing mice for fluorescent B-property detection of tumors B-material in vivo . [SEP]
[CLS] at 48 h post injection , mice were irradiated with a 808 nm laser for 5 min , raising the temperature of the tumor B-material tissue to about 50 °c ( figure 29 ) . [SEP]
[CLS] one day following laser treatment , the tumors B-material were eliminated , while control groups ( no treatment , nanoparticle B-nanoparticle only , or laser only ) , showed no change in tumor B-material growth . [SEP]
[CLS] active targeting of nanoparticles B-nanoparticle for combined imaging and photothermal therapy can enhance the selectivity of nanoparticle B-nanoparticle accumulation in tumor B-material tissue . [SEP]
[CLS] in some examples , polymeric B-nanoparticle nanoparticles I-nanoparticle are used to actively target tumor B-material tissue for theranostic applications . the cai group encapsulated two photosensitizing B-property dyes , indocyanine green and ir - 780 in polymer B-material nanoparticles B-nanoparticle to target folate B-material receptor I-material in a breast cancer mouse model . forty - eight hours following intravenous injection with folate - coated indocyanine green encapsulating nanoparticles B-nanoparticle , mice were irradiated with a near - infrared laser for 5 min , resulting in an increase in intratumoral temperature to 50 °c . [SEP]
[CLS] this localized heating resulted in significant cancer cell B-event death I-event , as indicated by histological analysis , and complete tumor B-material ablation . [SEP]
[CLS] bardhan et al . reported the use of her2 antibody - conjugated nanoshells B-nanoparticle for combined imaging and therapy of breast cancer . [SEP]
[CLS] indocyanine green and anti - her2 antibody B-material were conjugated to gold B-material nanoshells B-nanoparticle and used to selectively image bt474az breast cancer tumors B-material with high her2 expression over mda - mb - 231 breast cancer tumors B-material with low her2 expression in mouse models . [SEP]
[CLS] within 4 h following intravenous injection , strong fluorescence B-property was observed from the tumors B-material of bt474az xenografted mice , which was about 2 times the intensity of tumors B-material in mda - mb - 231 xenografted mice . [SEP]
[CLS] though the photothermal treatment of tumors B-material was not examined in vivo , nanoshells B-nanoparticle have been reported for effective cancer cell B-material ablation , and the anti - her2 nanoshells B-nanoparticle may be further explored for theranostics in vivo . [SEP]
[CLS] we have reviewed many of the recently reported nanoparticle - mediated methods for the detection of cancer biomarkers B-property , cells B-material , and tissues through fluorescence B-property . [SEP]
[CLS] nanotechnology - based assays for cancer detection are an increasingly relevant alternative to traditional techniques . [SEP]
[CLS] the characteristics of certain nanoparticle B-nanoparticle probes , such as high surface - area - tovolume ratio and unique optical properties , allow them to overcome some of the limitations of currently available methods of cancer detection . [SEP]
[CLS] in particular , the four main nanoparticle B-nanoparticle types used in the design of probes for the detection of cancer biomarkers B-property , cells B-material , and tissues through fluorescence B-property are ( 1 ) qds , which exhibit tunable absorption and high quantum yields ; ( 2 ) pds , which have high fluorescence B-property quantum yields and show no cytotoxicity B-property ; ( 3 ) ucnps , which exhibit anti - stokes shifts and are excitable in the tissue - transparent nir window ; ( 4 ) aunps B-nanoparticle , which are excellent fluorescence B-property quenchers I-property and are thus commonly used in " off - on " probes ; and ( 5 ) fluorophore - encapsulating polymeric or mesoporous silica B-nanoparticle nanoparticles I-nanoparticle , which are useful in theranostic applications that simultaneously deliver chemotherapeutics or photosensitizing B-property agents to image and treat cancer cells B-material . [SEP]
[CLS] beyond the nanoparticle B-nanoparticle probe ' s core B-material composition , surface functionalization with a range of moieties ( i . e . , antibodies B-material , oligonucleotides , aptamers , peptides B-material , and small molecules ) can enable the detection of cancer biomarkers B-property , cells B-material , and tissues by imparting high binding affinity and specificity to a target . [SEP]
[CLS] the high surface - area - to - volume ratio of nanoparticles B-nanoparticle can enable dense functionalization , and provides tailorable and sometimes multivalent binding of nanoparticle B-nanoparticle probes to target proteins B-material , cell B-material surface markers , and oligonucleotides that may be indicative of cancer . [SEP]
[CLS] this property allows one to detect cancer markers with low lods and high specificity . [SEP]
[CLS] as discussed in this review , the combination of the optical properties of certain classes of nanoparticles B-nanoparticle ( qds , ucnps , and aunps B-nanoparticle in particular ) with a variety of tailorable surface chemistries has been utilized in the design of nanoparticle B-nanoparticle probes for the detection of cancer . [SEP]
[CLS] the desirable properties for each nanoparticle B-nanoparticle probe are often dependent upon the application . [SEP]
[CLS] most notably , the design considerations for nanoparticle B-nanoparticle probes used in the detection of secreted biomarkers B-property do not require the same cellular and systemic biocompatibility B-property that is required for the detection of cancerous tissues in vivo . [SEP]
[CLS] for example , although though qds and ucnp exhibit enhanced photostability and are efficiently excited in the tissue - penetrating nir window , their long - term safety and toxicity B-property have not yet been fully evaluated . [SEP]
[CLS] thus , these types of nanoparticles B-nanoparticle may be more rapidly translated to clinical use for ex vivo cancer biomarker B-property detection after further in - depth studies regarding the safety of systemic qd and ucnp administration are completed . [SEP]
[CLS] while a variety of nanoparticle B-nanoparticle probes have been designed for the detection of cancer biomarkers B-property through fluorescence B-property , no specific assay has been selected as the " gold B-material standard " for clinical use . [SEP]
[CLS] perhaps this is due to difficulties in comparing results across reports with differing experimental conditions . [SEP]
[CLS] notably , there is a significant difference between lods that are obtained by measuring pure target compared to those obtained measuring target in the presence of complex mixtures that more accurately represent patient - derived samples , including serum , for example . [SEP]
[CLS] in general , however , sandwich - type immunoassays which utilize qds to generate fluorescence B-property in the presence of a target biomarker B-property have continued to demonstrate low lods , which are attainable due to the high quantum yield of qds . [SEP]
[CLS] there are two main classes of nanoparticle - based fluorescent B-property assays for the detection of cancer cells B-material : those that rely of the binding of nanoparticle B-nanoparticle probes to cancer cell B-material surface markers , and those that enter cells B-material and detect genetic content . [SEP]
[CLS] most of the nanoparticle B-nanoparticle probes reported operate solely through binding to cell surface markers . [SEP]
[CLS] these approaches often improve upon the currently available system for detection of ctcs ( i . e . , cellsearch ) , due to the enhanced fluorescence B-property properties ( i . e . , higher photostability , longer fluorescence B-property lifetimes , and high quantum yields ) of certain nanoparticles B-nanoparticle including qds and ucnps . [SEP]
[CLS] however , they are limited in their ability to detect malignant cells B-material that may not express sufficient quantities of the surface marker . [SEP]
[CLS] the nanoflare or smartflare ( commercialized by emd millipore ) , however , overcomes this challenge by providing a method to detect and quantify intracellular genetic content in live cell B-material samples . [SEP]
[CLS] this approach has been successfully used to detect , isolate , and culture metastatic breast cancer cells B-material from a murine model as well as from human blood samples , and represents a paradigm shift in the ability to detect cancer cell B-material populations based upon genetic markers . [SEP]
[CLS] additionally , multiplexed nanoflares provide the opportunity to distinguish truly malignant cancer cells B-material based upon the expression levels of multiple oncogenes . [SEP]
[CLS] in addition to the detection of cancer biomarkers B-property and cells B-material , nanoparticle B-nanoparticle probes for the detection of cancerous tissues in vivo have also been discussed in this review . [SEP]
[CLS] one remarkable property of certain nanoparticles B-nanoparticle , which enables their utility in cancer tissue detection , is their in vivo behavior . [SEP]
[CLS] specifically , the preferential uptake of fluorophorelabeled nanoparticle B-nanoparticle probes into tumor B-material tissues via the epr effect or through active tumor B-material targeting offers a powerful tool for tumor B-material detection , image - guided therapy , and theranostics . [SEP]
[CLS] methods to detect tumor B-material tissue in vivo are enhanced through active targeting based upon the recognition of cancer cell B-material surface markers by providing higher image contrast in less time . [SEP]
[CLS] this effect is due to the selective and enhanced accumulation of nanoparticles B-nanoparticle in cancerous tissue . [SEP]
[CLS] compared to alternative imaging modalities , fluorescence B-property offers relatively high sensitivity and tailorable excitation and emission maxima to optimize tissue penetration depths for in vivo imaging . [SEP]
[CLS] qds , pds , and ucnps , in particular , are ideal for in vivo use due to their tunable nir profiles and large stokes and anti - stokes shifts , respectively , which minimize background signal caused by tissue autofluorescence to enhance image contrast . [SEP]
[CLS] though initial studies on the safety of qds , pds , and ucnps in vivo are promising , showing little to no toxicity B-property in mouse and nonhuman primate models , translation of the use of such nanoparticle B-nanoparticle probes to the clinic will require significant research on the long - term safety of qd and ucnp administration . [SEP]
[CLS] overall , significant effort has been afforded toward the design of nanoparticle B-nanoparticle probes for the detection of cancer biomarkers B-property , cells B-material , and tissues through fluorescence B-property . [SEP]
[CLS] a wide range of assays have been developed , and many of them improve upon the currently available detection methods either through enhanced sensitivity and selectivity , or by offering entirely new and unique capabilities that are not attainable with conventional methods . [SEP]
[CLS] in addition to their potential use in cancer diagnosis and prognosis , these technologies may also be used to further study the progression of cancer and may aid in our understanding of the disease . [SEP]
[CLS] ultimately , translation of nanoparticle B-nanoparticle probes to clinical cancer diagnosis will require further knowledge of the correlation between levels of cancer biomarkers B-property , cells B-material , or tissues present in a patient with the stage of their disease to enhance prognostic capabilities . [SEP]
[CLS] this ability will improve cancer patient care by enabling early detection to improve survival outcomes and by providing a means for monitoring the progress of the disease in response to treatment . [SEP]
[CLS] the latter will lead to the design of better treatment strategies for an individual patient . [SEP]
[CLS] with an understanding of the strides made in using nanoparticle B-nanoparticle probes to detect cancer biomarkers B-property , cells B-material , and tissues , as well as the challenges associated with this type of research , researchers in this field are poised to move nanoparticle B-nanoparticle probes for cancer diagnosis , prognosis , and therapeutics rapidly into the clinic . [SEP]
[CLS] emission and excitation spectra of cyp and yfp , a commonly used fret pair , with the spectral overlap shown in gray [SEP]
[CLS] schematic representation of silver B-material nanoclusters ( agncs ) used in the detection of ccrf - cem acute leukemia cells B-material . [SEP]
[CLS] in the " off state " , the system consists of two dna strands : the " signal probe " which is tethered to agncs that are not fluorescent B-property and a linker region that is complementary to the arm segment of the " recognition probe " . [SEP]
[CLS] upon binding of the scg8c aptamer region of the recognition probe to ccrf - cem cells B-material , the arm segment is exposed , enabling the hybridization of the signal probe and the recognition probe . [SEP]
[CLS] this brings the agncs in close proximity of the g - rich segment of the recognition probe , leading to enhanced agnc fluorescence B-property in the " on " state . [SEP]
[CLS] adapted from ref 320 . [SEP]
[CLS] copyright 2013 american chemical society [SEP]
[CLS] ( a ) jablonski diagram including typical time scales of photophysical processes for organic molecules . [SEP]
[CLS] ( b ) molecular fluorescence B-property spectrum illustrating the broadening of the spectral lines due to the presence of vibrational energy levels , and the stokes shift between the excitation and emission maxima . [SEP]
[CLS] adapted from ref 22 . [SEP]
[CLS] copyright 2010 american chemical society [SEP]
[CLS] ( a ) increasing qd size results in a red shift in qd emission : zns - capped cdse qds of varying size with emission maxima ranging from 443 to 655 nm . [SEP]
[CLS] samples were excited with a near - uv lamp . [SEP]
[CLS] ( b ) representative cdse qd absorption ( represented by lines ) and emission profiles ( represented by circles ) . [SEP]
[CLS] qd size increases from left to right , resulting in red - shifted emission . [SEP]
[CLS] note the broad absorption . [SEP]
[CLS] ( c ) fluorescence B-property lifetime of cds / zns qd compared to organic dyes nile red and cy5 . [SEP]
[CLS] ( a ) reprinted with permission from ref 31 . [SEP]
[CLS] copyright 2001 macmillan publishers ltd . ( b and c ) reprinted with permission from ref 32 . [SEP]
[CLS] copyright 2008 macmillan publishers ltd . [SEP]
[CLS] ( a ) schematic representation of rare earth element crystalline host with ln 3 + dopant ( red ) . [SEP]
[CLS] ( b ) two - photon excitation mechanisms common in ucnps result in the release of a photon of higher energy and an anti - stokes shift . [SEP]
[CLS] ( c ) emission spectra of nayf 4 : yb / tm compared to nayf 4 : yb / er demonstrates the composition - dependent emission profiles of ucnps . [SEP]
[CLS] ( d ) luminescent B-property photos showing colloidal solutions of ucnps doped with varying ratios of yb , tm , and er are excited at 980 nm with a 600 mw diode laser . [SEP]
[CLS] the different colors represent changes in the emission spectra . [SEP]
[CLS] ( a and b ) reprinted with permission from ref 49 . [SEP]
[CLS] copyright 2010 the royal society of chemistry . [SEP]
[CLS] ( c and d ) adapted from ref 51 . [SEP]
[CLS] copyright 2008 american chemical society [SEP]
[CLS] 5 . homogenous in - solution sandwich assay for detecting cea and nse . [SEP]
[CLS] ( 1 , 2 ) biotinylated capture antibodies B-material and qd - functionalized detection antibodies B-material against each biomarker B-property bind the analyte of interest to form a sandwich . [SEP]
[CLS] ( 3 ) streptavidin beads are then used to capture the in - solution sandwich constructs . [SEP]
[CLS] ( 5 , 6 ) finally , qds are freed from the sandwich , and detected using a plate reader ( fluorophore excitation at 355 nm ) . [SEP]
[CLS] adapted with permission from ref 144 . [SEP]
[CLS] copyright 2011 the royal society of chemistry . [SEP]
[CLS] 6 . scheme for a qd immunosensor utilized to detect tpsa . [SEP]
[CLS] ( a ) protein B-material a and an anti - tpsa antibody B-material are immobilized onto a screen - printed carbon B-material substrate . [SEP]
[CLS] ( b ) upon introduction to the sensor , the analyte binds to the capture antibody B-material and ( c ) a biotinylated a second antibody B-material . [SEP]
[CLS] ( d ) streptavidin functionalized qds sandwich the analyte onto the sensor , and produce fluorescence B-property with an emission at 525 nm . [SEP]
[CLS] adapted with permission from ref 150 . [SEP]
[CLS] copyright 2007 elsevier [SEP]
[CLS] ( a ) immunochromatography test strip for afp detection . [SEP]
[CLS] ( b ) afp - containing sample is loaded onto the sample pad , and binds qd antibodies B-material ( qd - ab1 conjugates ) on the conjugation pad . [SEP]
[CLS] ( c ) next , afp - qd - ab1 travels to the test line and binds to immobilized anti - afp antibodies B-material . [SEP]
[CLS] ( d ) unbound qd - ab1 conjugates bind to a secondary antibody B-material . [SEP]
[CLS] fluorescence B-property along the test and control line is quantified using a fluorescence B-property reader . [SEP]
[CLS] adapted with permission from ref 191 . [SEP]
[CLS] copyright 2011 elsevier [SEP]
[CLS] in - solution fret fluoroimmunoassay to detect afp . [SEP]
[CLS] qds are incorporated onto the surface of polymeric microparticles ( qps ) that are conjugated to an anti - afp antibody B-material . [SEP]
[CLS] luminescent B-property terbium chelates ( ltc ) are also conjugated to an anti - afp antibody B-material , and in the presence of afp , the target is sandwiched , and brings the qds and ltc in close proximity to each other , initiating fret . [SEP]
[CLS] adapted with permission from ref 198 . [SEP]
[CLS] copyright 2012 elsevier [SEP]
[CLS] 9 . multiwalled carbon B-nanoparticle nanotube I-nanoparticle ( mcnt ) immunosensor for psa detection . [SEP]
[CLS] ( a ) first , cus qds are functionalized with an anti - psa antibody B-material while ( b ) ( a - c ) ito substrates are functionalized with carbon B-nanoparticle nanotubes I-nanoparticle and poly ( diallyldimethylammonium chloride B-material ) ( pdda ) before anti - psa capture antibodies B-material are immobilized to the surface . [SEP]
[CLS] ( b ) ( d , e ) next , the anti - psa capture antibodies B-material and the cus qds sandwich psa , resulting in the oxidation of o - phenylenediamine ( opd ) to 2 , 3 - diaminophenazine ( opdox ) , thus producing a fluorescence B-property signal . [SEP]
[CLS] adapted with permission from ref 214 . [SEP]
[CLS] copyright 2014 the royal society of chemistry . [SEP]
[CLS] 10 . schematic representation of a nanoparticle B-nanoparticle probe for the detection of various cancer biomarkers B-property . [SEP]
[CLS] ( a ) this in - solution assay uses a library of aunps B-nanoparticle capped with various cationic B-material functional groups that is then coated with an electrostatically associated fluorophore - labeled polymer B-material ( poly ( p - phenyleneethynylene ) or ppe ) . [SEP]
[CLS] in the absence of target , the aunp B-nanoparticle quenches the fluorescence B-property of the fluorophore , and the sensor is in an " off state . [SEP]
[CLS] " binding B-event of I-event various I-event proteins I-event to the aunp B-nanoparticle can trigger the dissociation of ppe from the nanoparticle B-nanoparticle , resulting in a fluorescence B-property enhancement and the transition of the nanoparticle B-nanoparticle probe to an " on state " . [SEP]
[CLS] ( b ) in a typical experiment , one aunp B-nanoparticle from a library is contained within each of the wells of a microplate , and a protein B-material sample is added to each well . [SEP]
[CLS] due to the differences in the surface charge of various proteins B-material , the biomarkers B-property that are analyzed bind to each of the aunps B-nanoparticle in the library to varying extents , thereby generating a unique " fingerprint " that enables their differentiation through fluorescence B-technique spectroscopy I-technique . [SEP]
[CLS] reprinted with permission from ref 222 . [SEP]
[CLS] copyright 2007 macmillan publishers ltd . [SEP]
[CLS] 11 . [SEP]
[CLS] ( a , c ) fixed breast cancer sk - br - 3 cells B-material labeled with anti - her2 535 - nm - emitting qds and anti - her2 630 - nm - emitting qds . [SEP]
[CLS] ( b , d ) sk - br - 3 cells B-material treated with igg coated 535 - nmemitting qds and 630 - nm - emitting qds were not specifically labeled . [SEP]
[CLS] cell B-material nuclei were stained with hoechst 33342 ( blue ) , and the scale bar represents 10 μm . [SEP]
[CLS] adapted with permission from ref 274 . [SEP]
[CLS] copyright 2003 macmillan publishers ltd . [SEP]
[CLS] 12 . photographs of 1 wt % colloidal solutions of naybf 4 : er / tm / ho upconversion nanoparticles B-nanoparticle of ( a ) naybf 4 : 2 % er , ( b ) naybf 4 : 2 % tm , ( c ) naybf 4 : 2 % ho , ( d ) naybf 4 : 1 % tm , 1 % ho , ( e ) naybf 4 : 1 % er , 1 % ho , and ( f ) naybf 4 : 1 % er , 1 % tm excited with 980 nm nearinfrared light . [SEP]
[CLS] despite their differing emission profiles ( as seen by the different colors in the photographs ) , all samples can be excited with the same wavelength of near - infrared light . [SEP]
[CLS] adapted from ref 272 . [SEP]
[CLS] copyright 2009 american chemical society . [SEP]
[CLS] 13 . fluorescent B-property magnetic I-nanoparticle bifunctional I-nanoparticle nanoparticles I-nanoparticle ( fmbns ) used in the simultaneous fluorescence B-property detection and magnetic B-property isolation of target cancer cells B-material . [SEP]
[CLS] ( a ) fmbns consist of cdse / zns core / shell qds and fe 2 o 3 nanoparticles B-nanoparticle that are encapsulated in a copolymer nanosphere B-nanoparticle . [SEP]
[CLS] biotinylated monoclonal antibodies B-material against a target protein B-material are recognized by the avidin - conjugated fmbns through avidin - biotin interactions . [SEP]
[CLS] ( b ) upon binding of the fmbns to target cells B-material , magnetic B-property separation may be performed to isolate the cancer cells B-material that express the cell surface marker of interest . [SEP]
[CLS] adapted from ref 268 . [SEP]
[CLS] copyright 2011 american chemical society [SEP]
[CLS] 14 . [SEP]
[CLS] rgd peptide functionalized zno nanowires B-nanoparticle ( nw - peg - rgd ) were used to selectively label integrin α v β 3 in u87mg glioblastoma cells B-material . [SEP]
[CLS] cells B-material were treated with either pegylated zno nanowires B-nanoparticle ( nw - peg ) or nw - peg - rgd . [SEP]
[CLS] as a control , integrin α v β 3 was also blocked on u87mg cells B-material by pretreating with cyclic rgdyk peptide B-material , resulting in decreased labeling of the u87mg cells B-material . [SEP]
[CLS] note that images were taken under 200× magnification . [SEP]
[CLS] adapted from ref 323 . [SEP]
[CLS] copyright 2011 american chemical society [SEP]
[CLS] 16 . [SEP]
[CLS] ( a ) schematic representation of a tetrazine - labeled magnetofluorescent nanoparticle B-nanoparticle ( mfnp ) and a trans - cyclooctene ( tco ) - functionalized antibody B-material used in the detection of cancer cells B-material . [SEP]
[CLS] ( b ) cells B-material are first treated with the tco - ab and then treated with mfnps , which react in cell B-material culture conditions to fluorophore - label cells B-material expressing the target of interest . [SEP]
[CLS] adapted with permission from ref 262 . [SEP]
[CLS] copyright 2010 macmillan publishers ltd . [SEP]
[CLS] 17 . schematic representation of aptamer - functionalized qds for combined cancer cell B-material imaging and therapy . [SEP]
[CLS] ( a ) doxorubicin B-material ( dox ) is intercalated into dna aptamers bound to the qd probe . [SEP]
[CLS] ( b ) aptamers recognize cell B-material surface markers of a target cancer cell B-material , and once the construct is internalized , dox is released . [SEP]
[CLS] adapted from ref 277 . [SEP]
[CLS] copyright 2007 american chemical society [SEP]
[CLS] 18 . [SEP]
[CLS] ( a ) nanoflares are used in the fluorescence - based detection of intracellular mrna and consist of thiolated " recognition " antisense dna adsorbed onto the surface of a spherical aunp B-nanoparticle . [SEP]
[CLS] the " reporter flare , " a shorter complementary dna with a cyanine 5 ( cy5 ) fluorophore , is hybridized to the recognition strand , resulting in the quenching of the cy5 fluorophore . [SEP]
[CLS] upon target binding , the reporter flare is released generating a measurable fluorescence B-property signal . [SEP]
[CLS] ( b ) nanoflares have been shown to enable the detection and isolation of circulating tumor B-material cells B-material from a murine model of triple negative breast cancer . [SEP]
[CLS] blood samples from mice with xenografted mcherry labeled mda - mb - 231 tumors B-material were treated with vimentin - targeting nanoflares , and mda - mb - 231 cells B-material were retrieved based on nanoflare fluorescence B-property . [SEP]
[CLS] representative scatter plots ( n = 1 mouse per scatter plot ) are shown for a mouse that was not injected with mcherry mda - mb - 231 breast cancer cells B-material ( control ) and mice that were injected with mcherry mda - mb - 231 breast cancer cells B-material and developed widespread metastases B-event ( experimental 1 and 2 ) . [SEP]
[CLS] cancerous cells B-material are shown in red , and noncancerous cells B-material are shown in black ( b ) . [SEP]
[CLS] adapted with permission from ref 343 . [SEP]
[CLS] copyright 2014 national academy of sciences of the united states of america . [SEP]
[CLS] 19 . nir [SEP]
[CLS] figure 19 . window is optimal for in vivo imaging due to minimal light absorption by hemoglobin ( hb ) , oxyhemoglobin ( hbo 2 ) , and water B-material in tissues from 650 to 900 nm . [SEP]
[CLS] adapted with permission from ref 406 . [SEP]
[CLS] copyright macmillan publishers ltd . [SEP]
[CLS] 20 . [SEP]
[CLS] diagram depicting the enhanced permeability and retention effect displayed by tumor B-material tissue . [SEP]
[CLS] due to the leaky tumor B-material vasculature as a result of poor lymphatic drainage , nanoparticles B-nanoparticle will escape the blood stream and preferentially localize in tumor B-material tissue , also known as passive targeting . [SEP]
[CLS] reprinted with permission from ref 414 . [SEP]
[CLS] copyright 2007 macmillan publishers ltd . [SEP]
[CLS] 21 . [SEP]
[CLS] histological ( hematoxylin and eosin staining ) analysis of the major organs of nonhuman primates 90 days after injection of phospholipid micelle - encapsulated qds . [SEP]
[CLS] tissues were collected from control ( left image ) and treated ( right image ) animals . [SEP]
[CLS] tissue analysis shows no significant differences in the ( a ) brain , ( b ) heart , ( c ) liver , ( d ) spleen , ( e ) kidneys , or ( f ) lymph nodes . [SEP]
[CLS] reprinted with permission from ref 525 . [SEP]
[CLS] copyright 2012 macmillan publishers ltd . [SEP]
[CLS] 22 . [SEP]
[CLS] upconversion nanoparticles B-nanoparticle with varying emission profiles for multicolor in vivo imaging . [SEP]
[CLS] ( a ) nayf 4 : yb , er , ( b ) nayf 4 : yb , er - rhodium B-material b , ( c ) nayf 4 : yb , er - rhodium B-material 6g , ( d ) nayf 4 : yb , er - tide quencher 1 , and ( e ) nayf 4 : ertm upconversion nanoparticles B-nanoparticle subcutaneously injected into the back of a nude mouse . [SEP]
[CLS] ( f ) fluorescence B-property merge of the upconversion nanoparticles B-nanoparticle and ( g ) white light image . [SEP]
[CLS] adapted from ref 365 . [SEP]
[CLS] copyright 2011 american chemical society [SEP]
[CLS] 23 . [SEP]
[CLS] zebrafish embryo induced with cerebral hypoxia imaged using an ucnp sensor for low oxygen B-material detection . [SEP]
[CLS] zebrafish embryos were injected with ucnp sensors via intracerebral microinjection prior to treatment with 2 , 3 - butanedione monoxime ( bdm ) to induce cerebral hypoxia . [SEP]
[CLS] the increase in fluorescence B-property intensity from the cranium 0 - 7 min post bdm treatment indicates the decrease in oxygen B-material . [SEP]
[CLS] adapted from ref 378 . [SEP]
[CLS] copyright 2014 american chemical society [SEP]
[CLS] 24 . schematic representation of aunp B-nanoparticle " anchors " for in vivo tumor B-technique imaging I-technique . [SEP]
[CLS] biotinylated aunps B-nanoparticle are injected into mice and passively accumulate in target tumor B-material tissue before streptavidin - fluorophore is injected to fluorophore - label the aunps B-nanoparticle in vivo . [SEP]
[CLS] reprinted with permission from ref 381 . [SEP]
[CLS] copyright 2010 national academy of sciences of the united states of america . [SEP]
[CLS] 25 . [SEP]
[CLS] ( a ) image of xenografted mouse 10 min post injection with dir - encapsulating peg - pla nanoparticles B-nanoparticle ( red ) illustrates location of dsred2 labeled ht29 tumors B-material ( green ) . [SEP]
[CLS] ( b ) time course of dir fluorescence B-property from 10 min to 48 h post injection of peg - pla nanoparticles B-nanoparticle . [SEP]
[CLS] ( c ) ex vivo imaging B-technique of I-technique excised I-technique tumor I-technique demonstrates colocalization of dsred2 labeled ht29 tumors B-material and dir from peg - pla nanoparticles B-nanoparticle . [SEP]
[CLS] adapted from ref 383 . [SEP]
[CLS] copyright 2011 american chemical society [SEP]
[CLS] 26 . nude mouse with subcutaneous u87mg xenograft tumor B-material ( left hind leg , short arrow ) and mcf7 xenograft tumor B-material ( right hind leg , long arrow ) imaged 1 ( top ) and 4 ( bottom ) h post intravenous injection with rgd labeled ucnps . [SEP]
[CLS] ucnp accumulation is higher in the u87mg tumor B-material as compared to the mcf7 tumor B-material , due to active targeting of integrin α v β 3 . [SEP]
[CLS] adapted from ref 531 . [SEP]
[CLS] copyright 2009 american chemical society [SEP]
[CLS] 27 . [SEP]
[CLS] ( a ) schematic representation of the synthesis of superparamagnetic B-nanoparticle iron I-nanoparticle oxide I-nanoparticle nanoparticle I-nanoparticle ( spion ) encapsulated in chitosan B-nanoparticle nanoparticles I-nanoparticle labeled with cy5 . 5 . ( b ) u87mg tumorbearing mice ( b ) preinjection , and ( c ) 1 , ( d ) 3 , and ( e ) 5 h post injection of spion - loaded cy5 . 5 - labeled chitosan B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] adapted from ref 376 . [SEP]
[CLS] copyright 2011 american chemical society [SEP]
[CLS] 28 . [SEP]
[CLS] theranostic trastuzumab - conjugated ucnps are targeted to tumor B-material tissue via active her2 recognition . [SEP]
[CLS] the magnetofluorescence of the ucnps allows for diagnostic B-technique imaging I-technique , while trastuzumab results in therapeutic down - regulation of her2 . [SEP]
[CLS] immunohistochemical analysis of her2 expression in tumor B-material sections from mcf7 xenografted mice ( a ) prior to intravenous injection with trastuzumab - conjugated ucnps , ( b ) 5 h post injection , ( c ) 24 h post injection , and ( d ) 1 week post injection . [SEP]
[CLS] note that brown staining corresponds to her2 while blue represents cell B-material nuclei , and that the images were taken under 40× magnification . [SEP]
[CLS] adapted from ref 395 . [SEP]
[CLS] copyright 2011 american chemical society [SEP]
[CLS] 29 . [SEP]
[CLS] cy5 labeled pedot : pss passively target 4t1 tumors B-material . [SEP]
[CLS] ( a ) fluorescence B-property images 1 - 48 h post intravenous administration . [SEP]
[CLS] ( b ) tissue temperature with 10 s to 5 min of laser irradiation . [SEP]
[CLS] tumor B-material location is indicated by the arrow . [SEP]
[CLS] adapted from ref 568 . [SEP]
[CLS] copyright 2012 american chemical society [SEP]
[CLS] fda - approved or - cleared cancer biomarkers B-property a [SEP]
[CLS] chemical bonds are a key determinant of the structure and properties of a material B-material . [SEP]
[CLS] thus , rationally designing arbitrary materials requires complete control over the bond . [SEP]
[CLS] while atomic bonding is dictated by the identity of the atoms B-material , nanoparticle B-nanoparticle superlattice engineering , where nanoparticle B-nanoparticle " atoms B-material " are held together by dna " bonds " , offers a route to design crystal lattices in a way that nature cannot : through altering the oligonucleotide bond . [SEP]
[CLS] herein , the use of rna , as opposed to dna , is explored by synthesizing superlattices in which nanoparticles B-nanoparticle are bonded by dna / dna , rna / rna , and dna / rna duplexes . [SEP]
[CLS] by moving beyond nanoparticle B-nanoparticle superlattices assembled only with dna , a new degree of freedom is introduced , providing programmed responsiveness to enzymes and greater bond versatility . [SEP]
[CLS] therefore , the oligonucleotide bond can have programmable function beyond dictating the structure of the material B-material and moves nanoparticle B-nanoparticle superlattices closer to naturally occurring biomaterials , where the line between structural and functional elements is blurred . [SEP]
[CLS] dna is a powerful ligand for programming the assembly of nanoparticles B-nanoparticle into superlattices with a vast number of crystallographic symmetries . [SEP]
[CLS] this can be achieved by using a programmable atom B-material equivalent ( pae ) , which consists of a nanoparticle B-nanoparticle core B-material densely functionalized with geometrically defined oligonucleotides , where dna mediates interactions between nanoparticles B-nanoparticle . [SEP]
[CLS] the oligonucleotide density and rigid nanoparticle B-nanoparticle core B-material impose a radial orientation of the dna and valency to the nanoparticles B-nanoparticle . [SEP]
[CLS] initially , spherical gold B-material nano - particles ( aunps B-nanoparticle ) were studied as pae cores B-material , but subsequent work has found that this approach is core B-material generalizable , as other inorganic 2a , b and organic 1f cores I-material , anisotropic cores , 3a , b as well as biological materials , such as proteins B-material , can be assembled using the same design rules . [SEP]
[CLS] the unifying element of all these studies is the dna " bond " that programs nanoparticle B-nanoparticle interactions and drives their assembly into ordered crystalline structures . [SEP]
[CLS] while recent work has been dedicated to understanding the function of these materials including emergent plasmonic 2a , 5 and catalytic properties , these properties are predominantly derived from the nanoparticle B-nanoparticle core B-material . studies of how the bond contributes to the functional properties of the crystalline superlattice are absent . [SEP]
[CLS] when considering materials that could in principle be used as a programmable ligand to assemble nanoparticles B-nanoparticle , dna is not the only candidate . [SEP]
[CLS] specifically , the incorporation of rna into nanoparticle B-nanoparticle superlattices would enable new classes of functional and stimuliresponsive superstructures that are not achievable with dna or solely by engineering the pae building block core B-material . [SEP]
[CLS] though rna is chemically similar to dna ( the primary difference is the presence of a 2 ′ - hydroxyl ( 2 ′ - oh ) group in rna ) , it has a vast chemical , structural , and functional design space that exceeds that of dna . [SEP]
[CLS] for example , in cells B-material , while dna is often found in the form of long double helices , rna is generally composed of short helices surrounded by loops and bulges . [SEP]
[CLS] notable forms of biofunctional rna include small interfering rna ( sirna ) that can regulate gene expression , 10 ribozymes ( ribonucleic B-material acid I-material enzymes ) which are catalytic rna molecules , and riboswitches which are structures formed in mrna that can regulate gene expression in bacteria and even act as stimuli responsive sensors . [SEP]
[CLS] while the vast chemical and biological space that rna occupies may appear to make it an ideal ligand for endowing paes with additional functionalities , its instability and vulnerability to nuclease - catalyzed hydrolysis provides a substantial barrier B-property to realizing biomaterials based upon rna . [SEP]
[CLS] research on synthesizing rna biomaterials has focused on the analogy to dna hybridization , where rigidity is imposed by the dna hybridization events , which leads to rigid structures and therefore valency . [SEP]
[CLS] this approach , based purely on dna hybridization , has been extended to rna for the synthesis of micrometer scale rna filaments , molecular jigsaw puzzles , and square - shaped rna particles [SEP]
[CLS] in order for these syntheses to work for rna , however , a hierarchical multistep process is required , whereas dna structures can typically be made in a " one pot " synthesis . [SEP]
[CLS] in addition , the dna and rna - based hybridization approaches require the use of both simulation and experiment to rationally design the 3d rna architectures through initial computer modeling . [SEP]
[CLS] this strategy is conceptually related but different from the method discussed herein for forming nanoparticle - based templated bonds , where the rigid nanoparticle B-nanoparticle core B-material leads to a radial upright orientation of the densely packed dna , leading to valency imposed by the core B-material . [SEP]
[CLS] therefore , the well - understood nature of dna programmable assembly , through the established design rules for the rational construction of dna nanoparticle superlattices , 1d provides the perfect platform for exploring the degree to which non - dna oligonucleotides can serve as programmable " bonds " . [SEP]
[CLS] here , the conventional design space for dnaprogrammable assembly is transformed by introducing oligonucleotide identity ( i . e . , paes held together by dna / dna , rna / rna , or dna / rna duplexes ) as an important design parameter . [SEP]
[CLS] similar to conventional dna - based assembly , the programmable nature of the oligonucleotide bond is the driving force , and is independent of the oligonucleotide identity such that dna / dna , rna / rna , and dna / rna duplexes are all suitable programmable ligands [SEP]
[CLS] however , the ability to tune the bond identity enables the rational design of responsive materials , whereby the oligonucleotide bond identity and interparticle distance dictate the response to enzymes . [SEP]
[CLS] design rules for the synthesis of nanoparticle B-nanoparticle superlattices with a variety of crystallographic symmetries have been established , which allow one to independently adjust each of the relevant crystallographic parameters , including particle size , periodicity , and interparticle distance . [SEP]
[CLS] because these design rules are based upon explorations of dna as the programmable ligand , one must first explore how the oligonucleotide bond identity affects the programmable assembly of nanoparticle B-nanoparticle superlattices . [SEP]
[CLS] we hypothesize that since rna / rna and rna / dna B-event binding I-event proceeds in a similar fashion to dna B-event / I-event dna B-event binding I-event , the use of dna , rna , or a dna / rna heteroduplex will not significantly change the resulting nanoparticle B-nanoparticle superlattice crystal structure . [SEP]
[CLS] as an initial proof - of - concept study , a twocomponent system which is expected to yield superlattices with a body - centered cubic ( bcc ) crystallographic symmetry , was evaluated . [SEP]
[CLS] four binary sets of particles were functionalized with dna or rna with non - self - complementary sticky ends , such that particle a can only bind to particle b and vice versa ( figure 1a ) . [SEP]
[CLS] the dna and rna design ( table s1 and figure s1 ) contains short overhang regions on the 3 ′ end of the linkers , which facilitate the interactions between nanoparticles B-nanoparticle . [SEP]
[CLS] for each sample , particle a and particle b were mixed in a 1 : 1 ratio so that each sample was allowed to form aggregates . [SEP]
[CLS] the possible permutations of oligonucleotide bonds are a - dna / b - dna ( red ) , a - rna / b - rna ( blue ) , a - dna / b - rna ( dark purple ) , and a - rna / b - dna ( light purple ) . [SEP]
[CLS] it is well - known that the density of dna affects the cooperative melting transition and crystallization of paes . [SEP]
[CLS] in order to explore new oligonucleotide identities as bonding ligands , a method to functionalize particles with rna at a density similar to that attainable with dna must be developed ( figure s2 ) . [SEP]
[CLS] in previous reports , rna immobilized on gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) has been exclusively in the form of double stranded rna , 22a , b where a backfill molecule was added to passivate the remaining gold B-material surface to account for the lower loading of rna compared to what is observed with dna . [SEP]
[CLS] but herein , rna particles a and b need to be synthesized with single - stranded rna without backfill molecules . [SEP]
[CLS] therefore , rna particles a and b were synthesized using methods analogous to their dna counterparts ( figure s2 ) . [SEP]
[CLS] this was found to significantly increase the density of thiolated rna on particles a and b from 25 - 60 pmol / cm 2 to 50 - 75 pmol / cm 2 , thus allowing rna particle a and particle b to be analogous to their dna counterparts in terms of oligonucleotide density . [SEP]
[CLS] once this has been accomplished , the only parameters that need to be optimized to synthesize nanoparticle B-nanoparticle superlattices with different bond compositions are the strength and length of the sticky end , the spacer B-material unit between the nanoparticle B-nanoparticle surface , and the oligonucleotide recognition sequence ( figure s1 ) . [SEP]
[CLS] it has been widely observed that paes exhibit cooperative and sharp melting transitions ( transition widths of 2 - 8 °c ) compared to linear nucleic B-material acids I-material , which exhibit broad melting transitions ( transition width ~ 20 °c ) . [SEP]
[CLS] a typical melting experiment involves monitoring the optical extinction at 260 and 520 nm , which is dampened for room temperature assembled pae aggregates , but increases as the temperature is raised and the nanoparticles B-nanoparticle begin to dissociate . [SEP]
[CLS] all aforementioned pae - oligonucleotide combinations ( figure 1a ) exhibited sharp and cooperative melting transitions ( figure 1b ) . [SEP]
[CLS] notably , the characteristic melting transition ( t m ) for the rna - pae aggregates occurred about 13 °c higher than analogous dna - pae aggregates ( full width at half - maximum ( fwhm ) = 2 . 7 °c for both ) , where the only difference is the identity of the oligonucleotide . [SEP]
[CLS] a similar stabilization effect exists in molecular duplexes of rna , which exhibit greater thermal stability and higher melting temperatures than their dna counterparts . [SEP]
[CLS] additionally , the two pae aggregates held together by heteroduplexes exhibited two distinct melting transitions ( t m = 39 °c , fwhm = 3 °c for aggregates with rna - pae a ; t m = 50 °c , fwhm = 1 . 3 °c for aggregates with dna - pae a ) . [SEP]
[CLS] while this result may appear surprising given the similarity of rna and dna , the position of the melting transitions for the two heteroduplexes can be understood by examining the molecular counterparts for their sticky ends , where the same trend in melting temperatures was observed for molecular duplexes of similar sequences as the sticky ends . [SEP]
[CLS] 24a , b more specifically , the stability of homopurinehomopyrimidine oligomer duplexes mirrors the trend in superlattice melting temperatures when looking at sticky end identity , 25a - c thus demonstrating that the characteristics of hybrid molecular duplexes are maintained when they are used as programmable ligands . [SEP]
[CLS] additionally , adenine dna / uracil rna heteroduplexes are known to be exceptionally unstable . [SEP]
[CLS] this hetero - duplex is analogous to the sticky end interaction in a - rna / b - dna and thus explains the lower melting temperature . [SEP]
[CLS] finally , this trend in melting temperatures was found to persist as the sticky end was increasingly moved away from the particle surface by utilizing longer linker oligonucleotides ( figure s3 ) . [SEP]
[CLS] recent work has demonstrated that slowly cooling paes through their melting transition is an effective method for synthesizing micron - scale single crystals . [SEP]
[CLS] to test whether rnaprogrammable assembly could also be used to form such large scale crystals , pae aggregates were slowly cooled ( 0 . 01 °c / min ) from 5 to 10 °c above their melting temperature down to room temperature . [SEP]
[CLS] small - angle x - ray scattering ( saxs ) was used to confirm the bcc crystallographic symmetry 1d ( figure 2a ) across three different oligonucleotide linker length scales and four different oligonucleotide bond compositions ( figure 2b ) . [SEP]
[CLS] despite differences at the molecular level , both dna and rna can be used interchangeably with the same crystal design principles , as evidenced by saxs and sem . [SEP]
[CLS] for example , the macroscopic crystallites formed by this slow cooling process were examined by sem and determined to be rhombic dodecahedra ( figure 2c ) , as observed in pure dna systems . [SEP]
[CLS] though the translated sequences used for assembly are identical ( i . e . , the dna and rna used on all a type particles had the same sequence just a different oligonucleotide identity ) , the a - dna / b - dna superlattices exhibited the largest interparticle distance , while the a - rna / b - rna superlattices consistently exhibited the shortest interparticle distances ( table 1 ; si eq 1 ) . [SEP]
[CLS] these data can be understood by looking at the typical characteristics of the molecular duplexes and specifically the 0 . 275 nm rise per base pair for rna ( a - form ) as compared with 0 . 34 nm for dna ( b - form ) . [SEP]
[CLS] dna / rna heteroduplexes are typically intermediate B-property in pitch , however , it is difficult to predict the properties of a dna - rna heteroduplex compared to its homoduplex counterpart , as they are known to be highly sequence specific . [SEP]
[CLS] use of the scherrer equation ( si eq 2 ) allows one to calculate the mean crystallite size for a given sample , defined as the average diameter of a single crystalline domain . [SEP]
[CLS] these calculations show that the grain sizes are all very similar regardless of bond type ( table 1 ) , thus demonstrating the power of programmable assembly for generating crystals of similar size but with different oligonucleotide constituents . [SEP]
[CLS] together , these data demonstrate that tuning the oligonucleotide bond is an important new handle for on - demand materials properties including melting temperature and interparticle distance in crystalline nanoparticle B-nanoparticle materials . [SEP]
[CLS] finally , saxs patterns of analogous dna and rna superlattices stored at 25 °c were obtained throughout the course of 100 days ( figure s4 ) . [SEP]
[CLS] these data revealed that the superlattices remain well ordered , with the interparticle distance changing < 1 nm and crystalline domain size changing < 20 nm over this time period ( table s2 ) . [SEP]
[CLS] this demonstrates that the rna stability is adequate for its use as a programmable ligand in nanoparticle B-nanoparticle superlattices . [SEP]
[CLS] having shown that dna / dna , rna / rna , and dna / rna duplexes can all serve as programmable ligands to synthesize nanoparticle B-nanoparticle superlattices , it was explored whether the oligonucleotide bond could play a significant role in dictating the properties of the material B-material , rather than acting as a passive " glue " . [SEP]
[CLS] to this end , time - dependent saxs measurements were performed to probe the interaction of nanoparticle B-nanoparticle superlattices with ribonuclease ( rnase ) a ( figure 3 ) , an enzyme that is known to recognize and degrade both single and double stranded rna duplexes . [SEP]
[CLS] we hypothesized that nanoparticle B-nanoparticle superlattices would become more accessible to the enzyme as interparticle distances increased due to larger pores for diffusion . [SEP]
[CLS] 30a - c to eliminate a purely diffusion - based interaction , a flow - cell setup was utilized , where the enzyme and superlattice were in constant oscillation , as described in si materials and methods . [SEP]
[CLS] for the rna superlattices , as the linker length between the nanoparticles B-nanoparticle was increased , the time span over which the superlattices retained their structure decreased dramatically ( 6 min for short and medium linkers and 0 . 25 min for long linkers ; figure 3a and s5 ) . [SEP]
[CLS] while pure dna superlattices were stable in the presence of ribonuclease , one might expect that dna / rna superlattices would still be able to respond to enzymes due to the presence of rna . [SEP]
[CLS] indeed , the ribonuclease can degrade the dna / rna superlattices , though they retain their order over a longer period of time than the rna superlattices ( 13 min for the short linker , 8 . 5 min for the medium linker , and 9 min for the long linker ; figure 3b , s5c ) . [SEP]
[CLS] this process is also concentration dependent ( figure s5 ) . [SEP]
[CLS] taken together , these data show that the rna - containing bonds in the nanoparticle B-nanoparticle superlattices are responsive to enzymes and such responses are dependent on both oligonucleotide identity and length . [SEP]
[CLS] in order to better understand the structural changes that occur during enzymatic degradation , several attributes of the time - dependent saxs data were studied . [SEP]
[CLS] by examining the breadth and position of the first - order scattering peak ( q 0 ) , one can begin to quantify how the bond length and identity affects the superlattice ' s response to enzymes . [SEP]
[CLS] the position of q 0 is used to calculate the interparticle distance . [SEP]
[CLS] the first analysis involved monitoring changes in the position of q 0 as the lattice is degraded by the enzyme , which is manifested by shifts in peak positions to lower values of q and thus larger interparticle distances . [SEP]
[CLS] the greatest overall change in the position of q 0 ( 0 . 002 a - 1 ) is observed for rna superlattices over the course of 6 . 5 min , whereas for the hybrid superlattices , a smaller change in q 0 ( 0 . 0017 a −1 ) is observed over a longer period of time ( 13 min ; figure 4a ) . [SEP]
[CLS] variations in the breadth of the q 0 peak , indicating changes in domain size and relative crystal quality , are characterized by the fwhm , where a smaller value of the fwhm indicates a larger domain and higher quality crystal . [SEP]
[CLS] for rna superlattices , the fwhm increases over 8 min ( 0 . 0014 a −1 ) before the structure falls apart , which is in stark contrast to the hybrid superlattices , where almost no change in fwhm is observed ( 0 . 0003 a −1 ; figure 4b ) . [SEP]
[CLS] similar trends are observed for the medium and long linkers ( figure s6 ) . [SEP]
[CLS] for rna superlattices with short linkers , these data indicate that rna connections are lost as enzyme incubation B-technique time increases . [SEP]
[CLS] this degradation reduces the number of connections holding the rna superlattice together , thus allowing more conformational degrees of freedom for each nanoparticle B-nanoparticle . [SEP]
[CLS] this manifests as an increase in fwhm ( figure 4a , b ) . [SEP]
[CLS] specifically , the structure is able to retain long - range order for 5 min before sufficient rna - rna interconnects are lost and the structure rapidly becomes disordered over the next 3 min . [SEP]
[CLS] in contrast , while a small change in q 0 is observed , a minimal change in fwhm is seen for the hybrid superlattices , thus indicating that some dna / rna connections may be lost but not enough to result in a change in overall crystal quality . [SEP]
[CLS] this suggests that having one component of dna allows the crystal to retain grain size and relative ordering as the interparticle distance is increased . [SEP]
[CLS] this is because rnase a is only known to cleave single and double stranded rna , 29a , b and thus is only able to recognize and degrade rna originating from particle b , and not the dna on particle a or the hybrid sticky end . [SEP]
[CLS] almost no change is seen in q 0 position or fwhm for the dna superlattices ( figure s6 ) , which further confirms that the enzyme recognition is oligonucleotide bond specific . [SEP]
[CLS] for both the rna and hybrid superlattices , it was observed that crystals with shorter interparticle distances are better able to withstand enzymatic degradation . [SEP]
[CLS] taken together , these data suggest that the introduction of rna into nanoparticle B-nanoparticle superlattices leads to bonds that are selectively addressable . [SEP]
[CLS] thus , it has been demonstrated that the bond in nanoparticle B-nanoparticle superlattices is responsive to enzymes and structural changes in the nanoparticle B-nanoparticle superlattices can be monitored by timedependent saxs . [SEP]
[CLS] this transition also can be monitored using uv - visible ( uv - vis ) spectroscopy B-technique , where rnase a was added to a solution of rna superlattices and an increase in extinction over time was observed , much like what is observed in a melting experiment ( figure 4c ) . [SEP]
[CLS] in this case , the strong extinction by the nanoparticles B-nanoparticle at 520 nm provides a spectroscopic and colorimetric handle for tracking this process . [SEP]
[CLS] again , it is observed that the enzymatic degradation is concentration - and oligonucleotide - bond - dependent , as almost no change in extinction was observed for the hybrid and dna superlattices . [SEP]
[CLS] thus , the strong aunp B-nanoparticle absorption can be used as a spectroscopic handle to monitor the function of the oligonucleotide bond quickly on the benchtop , without the need for a synchrotron light source ( figure 4d ) . [SEP]
[CLS] the data presented herein show that design rules for nanoparticle B-nanoparticle superlattices 1d still hold true when using rna as opposed to dna as a programmable ligand , and that the identities of the oligonucleotide " bonds " in nanoparticle B-nanoparticle superlattices can be independently changed without changing the " atoms B-material " . [SEP]
[CLS] this novel capability provides a pathway for deliberately tailoring superlattice properties , something not possible with conventional atomic and molecular systems . [SEP]
[CLS] indeed , the realization of responsive oligonucleotide bonds within such structures dramatically increases the breadth and sophistication of the design space for these materials and creates several challenges for the field moving forward . [SEP]
[CLS] these challenges include : ( 1 ) the study of other specialty oligonucleotides , such as peptide B-material nucleic B-material acids I-material and locked nucleic B-material acids I-material , which could allow one to further tailor the charge , stability , and function of oligonucleotide bonds , ( 2 ) the creation of mixed superlattice systems with different bonds that can be addressed and modified independently and selectively with enzymes , and ( 3 ) the creation of bonds that move beyond linear struts , such as structures that contain catalytic loops . [SEP]
[CLS] taken together , the development of a programmable system to utilize oligonucleotides other than dna to direct the assembly of nanoparticles B-nanoparticle into threedimensional crystals shows promise in developing nanoparticle B-nanoparticle superlattices with dynamic and functional bonds for many areas , including catalysis and sensing . [SEP]
[CLS] conclusiona the interparticle distance is defined as the distance from the center - to - center of each nanoparticle B-nanoparticle on the bcc diagonal and is therefore the length of the oligonucleotide between the nanoparticles B-nanoparticle plus the sum of the nanoparticle radii ( n = 2 ) . [SEP]
[CLS] color scheme is as follows : a - dna / b - dna ( red ) , a - rna / b - dna ( light purple ) , a - dna / b - rna ( dark purple ) , a - rna / b - rna ( blue ) . [SEP]
[CLS] of the aps was supported by the u . s . doe , office of science , office of basic energy sciences , under contract de - ac02 - 06ch11357 . [SEP]
[CLS] nanoparticle B-nanoparticle superlattices synthesized with modular oligonucleotide bonds . [SEP]
[CLS] ( a ) twocomponent system where particles a and b are linked by non - self - complementary sticky ends . [SEP]
[CLS] four types of oligonucleotide bonds are explored : dna / dna ( red ) , rna / rna ( blue ) , dna / rna ( dark purple ) , and rna / dna ( light purple ) . [SEP]
[CLS] ( b ) uv - vis melts of aggregates linked with four different oligonucleotide bond compositions at short linker lengths ( color scheme is the same as in ( a ) ) . [SEP]
[CLS] tunable melting transitions ( t m ) emerge based on oligonucleotide bond composition . [SEP]
[CLS] body - centered cubic nanoparticle B-nanoparticle superlattices . [SEP]
[CLS] ( a ) depictions of body - centered cubic ( bcc ) nanoparticle B-nanoparticle superlattices of four different oligonucleotide bond compositions ( to - scale ) . [SEP]
[CLS] gold B-nanoparticle nanoparticles I-nanoparticle are shown in yellow and the oligonucleotide bond in red , blue , or purple . [SEP]
[CLS] ( b ) small - angle x - ray scattering ( saxs ) of nanoparticle B-nanoparticle superlattices with four different " bond " compositions and three different interparticle distances ( from bottom to top : short ( 46 - base pair ( - bp ) ) , medium ( 67 - bp ) , and long ( 128 - bp ) linkers ) . [SEP]
[CLS] ( c ) scanning electron B-technique microscopy I-technique ( sem ) images of nanoparticle B-nanoparticle super - lattices in the solid state . [SEP]
[CLS] in all cases , single crystal rhombic dodecahedra are observed . [SEP]
[CLS] scale bars = 100 nm . [SEP]
[CLS] functional oligonucleotide bonds in nanoparticle B-nanoparticle superlattices . [SEP]
[CLS] ( a ) time - dependent saxs scattering patterns for rna superlattices with short and long linkers upon addition of 1 μg ribonuclease ( rnase ) a . ( b ) time - dependent saxs scattering patterns for dna - rna superlattices with short and long linkers upon addition of 1 μg rnase a . [ aunp B-nanoparticle ] ≈ 45 nm . [SEP]
[CLS] 4 . measurement of the superlattice response to enzymes . ( a ) [SEP]
[CLS] changes in position of the firstorder scattering peak ( δq 0 ) versus time extracted from the time - dependent saxs data for rna ( blue circles ) and hybrid ( purple squares ) superlattices with short linkers . [SEP]
[CLS] lines are not fits but rather guides for the eye . [SEP]
[CLS] the zero point is represented at 0 . 01 min . [SEP]
[CLS] ( b ) changes in grain size and relative crystal quality indicated by changes in the full width at half - maximum ( δfwhm ) during incubation B-technique with enzymes extracted from the time - resolved saxs data . [SEP]
[CLS] analysis of the rna superlattice stopped at 6 . 5 min , after which long - range order was no longer maintained . [SEP]
[CLS] lines are not fits but rather guides for the eye . [SEP]
[CLS] the zero point is represented at 0 . 01 min . [SEP]
[CLS] ( c ) uv - vis kinetics demonstrating changes in the localized surface plasmon resonance ( lspr ) upon incubation B-technique with rnase a for superlattices of rna ( blue ) , a - dna / b - rna ( purple ) and dna ( red ) . [SEP]
[CLS] open circles represent 1 μg rnase a and closed circles represent 5 μg rnase a . [SEP]
[CLS] [ aunp B-nanoparticle ] ≈ 1 . 5 nm . [SEP]
[CLS] ( d ) optical images showing the change in rna nanoparticle B-nanoparticle superlattices before ( left ) and after ( right ) incubation B-technique with rnase a . [SEP]
[CLS] the effect of serum protein B-material adsorption on the biological fate of spherical nucleic B-material acids I-material ( snas ) is investigated . [SEP]
[CLS] through a proteomic analysis , it is shown that g - quadruplexes templated on the surface of a gold B-nanoparticle nanoparticle I-nanoparticle in the form of snas mediate the formation of a protein B-material corona I-material that is rich in complement proteins B-material relative to snas composed of poly - thymine ( poly - t ) dna . [SEP]
[CLS] cellular uptake studies show that complement receptors on macrophage cells B-material recognize the sna protein B-material corona I-material , facilitating their internalization , and causing g - rich snas to accumulate in the [SEP]
[CLS] spherical nucleic B-material acids I-material ( snas ) are a unique class of nanoparticles B-nanoparticle ( nps B-nanoparticle ) that are comprised of a dense and highly oriented nucleic B-material acid I-material shell B-material . [SEP]
[CLS] this architecture imparts several novel properties to snas that are fundamentally different from those of their linear counterparts . [SEP]
[CLS] for example , unlike linear nucleic B-material acids I-material , snas readily enter most cell B-material types without the use of additional transfection reagents . [SEP]
[CLS] this process is often facilitated by the recognition of the 3d architecture of snas by class a scavenger receptors , which have high affinities for snas but not the particle - free sequences . [SEP]
[CLS] these structure - dependent properties make the sna a promising single - entity agent for probing and regulating nucleic acid - dependent cellular B-event processes I-event . [SEP]
[CLS] though elucidating the mechanism of cellular uptake of snas has been an active area of research , little is known about the effect of serum protein B-material adsorption on this process . [SEP]
[CLS] it is conceivable that the formation of a sna protein B-material corona I-material could impact cellular uptake by altering the surface chemistry that is presented to cells B-material and tissues . [SEP]
[CLS] in particular , the adsorption of proteins B-material could enable snas to be internalized by macrophage cells B-material and cause them to accumulate in the liver and spleen following intravenous administration . [SEP]
[CLS] while previous work has shown that the identity and types of proteins B-material that comprise the sna protein B-material corona I-material can impact their uptake by macrophage cells B-material , no data have been collected to probe how macrophage cells B-material internalize protein - coated snas or the effects of this process on the biodistribution of snas in mice . [SEP]
[CLS] herein , we utilize two snas , one composed of guanine - rich ( g - rich ) oligonucleotides that form g - quadruplexes and one composed of poly - thymine ( poly - t ) oligonucleotides ( scheme 1 ) , to study the effect of nucleic B-material acid I-material sequence on the formation of a np B-nanoparticle protein B-material corona I-material and the subsequent effects on their uptake by macrophage cells B-material . [SEP]
[CLS] we find that nucleic B-material acid I-material sequence can dictate the chemical composition of the sna protein B-material corona I-material and alter their recognition by macrophage cells B-material and their accumulation in macrophage - rich tissues in vivo . [SEP]
[CLS] our results show that sna surface ligand architecture , and the formation of gquadruplexes in particular , can alter the biological fate of snas . [SEP]
[CLS] while the biological fate of nps B-nanoparticle is known to be dependent upon both size and surface charge , it is increasingly apparent that the chemical composition and architecture of surface ligands also plays a significant role in facilitating the interactions of nanomaterials B-material with biological entities , including serum and cell B-material surface proteins B-material . [SEP]
[CLS] previous work has shown that snas composed of g - rich oligonucleotide sequences facilitate interactions with both cell - surface scavenger receptors and a variety of serum proteins B-material due to the enhanced formation of g - quadruplexes , which is mediated by the dense layer of oligonucleotides that brings preoriented dna strands in close proximity to one another on the sna surface . [SEP]
[CLS] based upon the enhanced interaction of g - quadruplexforming , g - rich snas with serum proteins B-material , we hypothesized that the protein B-material corona I-material formed on g - rich snas would facilitate their uptake into phagocytic macrophage cells B-material , and that this would lead to subsequent accumulation in the macrophage - rich liver and spleen in vivo . [SEP]
[CLS] to probe the effect of secondary oligonucleotide structure on sna protein B-material corona I-material formation and subsequent biodistribution in vivo , we designed two oligonucleotide sequences and synthesized g - rich snas and poly - t snas with a gold B-material np B-nanoparticle ( aunp B-nanoparticle ) core B-material . [SEP]
[CLS] we studied the amounts and types of human serum proteins B-material that bind to g - rich and poly - t snas , and their effect on sna uptake into phagocytic cells B-material and subsequent sequestration by the liver and spleen in mice . [SEP]
[CLS] to study the effect of g - quadruplex formation on the sna protein B-material corona I-material , we incubated B-technique grich snas and poly - t snas in 10 % human serum ( hs ) for 15 - 90 min and studied the quantity and identity of serum proteins B-material that adsorb to snas . [SEP]
[CLS] protein - coated snas were isolated from unbound serum proteins B-material by centrifugation and analyzed by dynamic B-technique light I-technique scattering I-technique ( dls ) to determine the relative increase in the hydrodynamic diameter of the snas upon protein B-material corona I-material formation ( figure 1a ) . [SEP]
[CLS] the hydrodynamic diameter of proteincoated g - rich snas increased from 49 . 2 ± 0 . 6 nm prior to incubation B-technique with hs to 195 . 2 ± 9 . 9 nm in a linear fashion over the 90 min incubation B-technique period . [SEP]
[CLS] meanwhile , the hydrodynamic diameter of poly - t snas increased from 52 . 4 ± 2 . 3 nm prior to incubation B-technique with hs to 128 . 2 ± 7 . 4 nm after 90 min . [SEP]
[CLS] importantly , the hydrodynamic diameter of g - rich snas is consistently higher than that of poly - t snas throughout the 90 min incubations B-technique with hs , most likely due to the increase in protein - sna interactions that is caused by the enhanced formation of g - quadruplexes on the sna surface . [SEP]
[CLS] in addition , the increase in sna size is likely due to two factors : ( 1 ) the adsorption of protein B-material to the nanoparticle B-nanoparticle surface , and ( 2 ) nanoparticle B-nanoparticle clustering facilitated by protein - protein interactions . [SEP]
[CLS] to quantify the amount of protein B-material bound to g - rich and poly - t snas , we performed a bicinchoninic acid ( bca ) assay ( figure 1b ) . [SEP]
[CLS] protein - coated snas were incubated B-technique with tween - 20 and heated to isolate the tightly bound proteins B-material . [SEP]
[CLS] the amount of protein B-material bound to g - rich snas increased from 4 . 09 × 10 −12 ±1 . 8 × 10 −13 μg per sna following a 15 min incubation B-technique in hs to 6 . 27 × 10 −12 ± 3 . 9 × 10 −13 μg per sna following a 90 min incubation B-technique in hs . [SEP]
[CLS] in contrast , the amount of protein B-material isolated from poly - t snas increased from 3 . 60 × 10 −12 ± 6 . 9 × 10 −13 μg per sna following a 15 min incubation B-technique in hs to 4 . 50 × 10 −12 ± 2 . 5 × 10 −13 μg per sna following a 90 min incubation B-technique in hs . [SEP]
[CLS] these results indicate that g - rich snas bind more total protein B-material than poly - t snas , and that the amount of protein B-material bound to both g - rich and poly - t snas increases over time , which is consistent with the increase in hydrodynamic diameter observed by dls . [SEP]
[CLS] in addition to quantifying the amount of protein B-material bound to the nanoparticle B-nanoparticle , the identity of the specific proteins B-material that adsorb to g - rich and poly - t snas with increasing incubation B-technique time in hs was determined . [SEP]
[CLS] to study corona composition , we isolated proteins B-material from snas incubated B-technique in 10 % hs for 15 - 90 min and then separated them by sodium B-material dodecyl sulfate - polyacrylamide B-technique gel I-technique electrophoresis I-technique ( sds - page , figure 1c ) . [SEP]
[CLS] the gel indicates that at each time point , more types and amount of protein B-material are bound to g - rich snas than poly - t snas . [SEP]
[CLS] to identify the specific proteins B-material that bind to g - rich and poly - t snas over time , mass spectrometry of the trypsin - digested isolated proteins B-material was performed ( tables s3 - s10 , supporting information ) . [SEP]
[CLS] the number of distinct proteins B-material identified by mass spectrometry ( figures 2a and s1 ) increases for g - rich snas from 59 to 86 following 15 - 90 min incubations B-technique in hs . [SEP]
[CLS] for poly - t snas , 62 distinct proteins B-material were isolated after a 15 min incubation B-technique in hs . [SEP]
[CLS] after 30 - 90 min incubations B-technique in hs , this levels off to around 50 distinct proteins B-material . [SEP]
[CLS] this suggests that the protein B-material corona I-material composition for snas changes over time , which correlates well with observations that the specific types of proteins B-material that form a nanoparticle B-nanoparticle protein B-material corona I-material changes over time , a phenomenon known as the vroman effect . [SEP]
[CLS] in addition , a comparison of the distinct proteins B-material isolated from g - rich and poly - t snas indicates that about 45 proteins B-material bind to both types of snas , regardless of incubation B-technique time in hs ( figure 2b ) . [SEP]
[CLS] importantly , the number of proteins B-material that bind exclusively to g - rich snas increases over time , while those that bind to poly - t snas decreases initially before leveling off . [SEP]
[CLS] these results further enforce the dynamic interaction of serum proteins B-material with snas over time and demonstrate the sequence - specific nature of this process . [SEP]
[CLS] western blotting of proteins B-material isolated from g - rich and poly - t snas ( 3 pmol ) was used to quantify the relative abundance of proteins B-material bound to g - rich and poly - t snas over time : apolipoprotein b100 ( apo b100 ) , complement factor h , complement c3b , and serum albumin ( figure 2c ) . [SEP]
[CLS] the relative amount of human serum albumin ( hsa ) , the highest abundant serum protein B-material studied , decreased for g - rich snas over time . [SEP]
[CLS] for poly - t snas , hsa initially increases from 15 to 30 min of incubation B-technique in hs before decreasing in relative abundance . [SEP]
[CLS] the band intensities for apo b100 , factor h , and c3b are low after a 15 min incubation B-technique , but increases and plateaus for both g - rich and poly - t snas over time . [SEP]
[CLS] in addition , an exchange of higher abundance proteins B-material ( hsa ) for lower abundance proteins B-material ( apo b100 , complement factor h , and complement c3b ) is observed , indicating that the composition of the sna protein B-material corona I-material changes over time , which is consistent with the vroman effect . [SEP]
[CLS] these results corroborate the dls and bca assay experiments , suggesting that g - rich snas bind more types and amount of protein B-material following incubation B-technique in hs than their poly - t sna counterparts . [SEP]
[CLS] in particular , more complement c3b adsorbs B-property onto g - rich than poly - t snas , which is a protein B-material that is recognized by complement receptors on the macrophage cell B-material surface , and can lead to np B-nanoparticle phagocytosis B-event . [SEP]
[CLS] based on the observed differences in sna protein B-material corona I-material composition over time , we hypothesized that g - rich snas would be recognized by macrophages , resulting in higher uptake compared to poly - t snas . [SEP]
[CLS] to test this hypothesis , phorbol 12 - myristate 13 - acetate ( pma ) induced human macrophages differentiated from thp - 1 monocytes were treated with g - rich and poly - t snas with and without 10 % hs for 15 - 90 min . [SEP]
[CLS] cell B-material uptake and association were then quantified by inductively coupled plasma mass spectrometry ( icp - ms , figure 3 a ) . [SEP]
[CLS] the results reveal that g - rich snas in 10 % hs exhibit higher cellular uptake and association than g - rich snas without hs and poly - t snas with or without hs . [SEP]
[CLS] at initial times , uptake and association of g - rich snas in 10 % hs is about three times higher than uptake and association of g - rich snas without hs and poly - t snas with or without hs . [SEP]
[CLS] over time , uptake and association of g - rich snas in hs increases substantially , resulting in a fourfold higher number of aunps B-nanoparticle / cell B-material . [SEP]
[CLS] though a protein B-material corona I-material is formed on both poly - t and g - rich snas , no significant increase in the uptake of poly - t snas by macrophages is observed in the presence of serum proteins B-material . [SEP]
[CLS] this suggests that beyond the presence of adsorbed protein B-material , the identity and types of proteins B-material that comprise the sna protein B-material corona I-material impact their recognition by macrophage cells B-material . [SEP]
[CLS] one possible explanation for this increase in the uptake of g - rich snas transfected in hs is the presence of opsonin proteins B-material , which may bind to g - rich snas and flag them for phagocytosis B-event . [SEP]
[CLS] two main classes of cell B-material surface I-material receptors I-material that bind opsonin proteins B-material are complement receptors and fc receptors , which recognize complement proteins B-material and igg proteins , respectively . [SEP]
[CLS] these classes of proteins B-material have been identified to bind to snas by proteomic mass spectrometry analysis , and it was hypothesized that their presence in the sna protein B-material corona I-material may be responsible for recognition and uptake by macrophages ( tables s3 - s10 , supporting information ) . [SEP]
[CLS] to probe the role of these receptors in facilitating sna uptake by macrophages , chemical blockers were used to inhibit recognition of differentiated thp - 1 cells B-material by class a scavenger receptors ( fucoidan ) , complement receptors ( trypan blue ) , and fc receptors ( fc block ) . [SEP]
[CLS] cells B-material pretreated with blocker were treated with g - rich and poly - t snas with and without hs for 90 min , and cell B-material uptake and association was quantified by icp - ms ( figure 3b ) . [SEP]
[CLS] although class a scavenger receptors have been shown to facilitate uptake of snas in serum - free conditions , the uptake of g - rich and poly - t snas was not substantially decreased by blocking class a scavenger receptors , suggesting that cellular uptake of snas by macrophages in the presence of serum may occur primarily by an alternate mechanism . [SEP]
[CLS] blocking fc receptors on thp - 1 macrophages did not result in a significant change in uptake and association of g - rich and poly - t snas transfected with or without hs . [SEP]
[CLS] in contrast , blocking complement receptors resulted in about a fourfold decrease in the uptake of g - rich snas in the presence of hs . [SEP]
[CLS] this appears to inhibit the increase in uptake that is observed in the presence of hs , resulting in uptake that is comparable to g - rich snas in the absence of hs . [SEP]
[CLS] this suggests that complement receptors recognize complement proteins B-material on the sna protein B-material corona I-material , such as complement c3 , and facilitate phagocytosis B-event . [SEP]
[CLS] this result supports the observed increase in macrophage uptake of g - rich snas in the presence of hs compared to poly - t snas in the absence of hs , since g - rich snas adsorb B-property more complement c3b than poly - t snas ( figure 2c ) . [SEP]
[CLS] in order to probe the relative abundance of complement receptors , fc receptors , and class a scavenger receptors on thp - 1 macrophages compared to hacat cells B-material , which primarily uptake snas via class a scavenger receptor - I-event mediated I-event endocytosis B-event , we treated cells B-material with fluorophore - tagged antibodies B-material targeting these three classes of receptors and analyzed them using flow B-technique cytometry I-technique ( figure 4 ) . [SEP]
[CLS] our results indicate that differentiated thp - 1 macrophages express ≈11 times more complement receptors and ≈26 times more fc receptors than hacat cells B-material and that hacat cells B-material express about approximately two times more scavenger receptors than thp - 1 macrophages . [SEP]
[CLS] taken together , these results suggest that the difference in cell B-material surface I-material receptor I-material expression in macrophage cells B-material , in addition to the formation of the sna protein B-material corona I-material , may account for the difference in uptake mechanism observed in thp - 1 macrophage cells B-material compared to hacat cells . [SEP]
[CLS] based upon the difference in uptake of g - rich and poly - t snas by macrophages in cell B-material culture , we hypothesized that in vivo , g - rich snas would be sequestered by macrophagerich tissues , such as the liver and spleen , to a greater extent than poly - t snas . [SEP]
[CLS] to probe this hypothesis , c57bl / 6 mice were injected with either g - rich snas or poly - t snas via the tail vein . [SEP]
[CLS] mice were sacrificed , perfused , and the organs were collected for analysis of aunp B-nanoparticle content via icp - ms 8 - 672 h following injection ( figure 5 ) . [SEP]
[CLS] more than 80 % of the injected dose of g - rich snas is found in the liver , compared to nearly 50 % of the injected dose of poly - t snas , 8 h postinjection . [SEP]
[CLS] over time , snas are cleared from the liver , and at 672 h postinjection , the difference in the percentage of the injected dose of g - rich versus poly - t snas in the liver is statistically insignificant . [SEP]
[CLS] similar trends are observed for the accumulation of snas in the spleen , though the percent of the injected dose is less than 10 % , due to the relatively smaller size of the spleen compared to the liver . [SEP]
[CLS] these results confirm our hypothesis that secondary oligonucleotide structure formation on snas , which influences the np B-nanoparticle protein B-material corona I-material , can significantly alter the biodistribution profile of snas in vivo . [SEP]
[CLS] we have shown that the presence of g - quadruplexes on the sna surface increases protein B-event binding I-event over time , where more proteins B-material , both in number and in type , bind to g - rich snas than poly - t snas . [SEP]
[CLS] this difference in the protein B-material corona I-material alters the interaction of g - rich and poly - t snas with macrophage cells B-material in vitro , where g - rich snas exhibit higher uptake via complement receptors . [SEP]
[CLS] indeed , in vivo experiments show that g - rich snas accumulate in macrophage - rich tissues , a consequence of the recognition of certain proteins B-material on the corona by cell B-material surface I-material receptors I-material . [SEP]
[CLS] this work highlights the importance of nucleic B-material acid I-material sequence in designing therapeutic snas , since the formation of nucleic B-material acid I-material secondary structures can result in markedly different biodistribution profiles . [SEP]
[CLS] in addition , some of the observations herein likely apply to other nucleic acid - modified nanostructures , and should be taken into consideration for therapeutic design . [SEP]
[CLS] citrate capped , 13 nm gold B-nanoparticle nanoparticles I-nanoparticle were synthesized using the frens method . [SEP]
[CLS] dna was synthesized using a mm38 oligonucleotide synthesizer ( bioautomation ) using standard solid - phase phosphoramidite coupling chemistry . [SEP]
[CLS] snas were synthesized by functionalizing aunps B-nanoparticle with 3 × 10 −9 mole thiolated dna per ml of aunps B-nanoparticle ( at 7 . 5 × 10 −9 m ) . [SEP]
[CLS] snas were stabilized with 0 . 2 % tween - 20 and salted slowly over time ( 0 . 5 and 1 m final nacl concentrations for g - rich and poly - t snas , respectively ) . [SEP]
[CLS] to ensure the snas were consistent in dna loading , 1 × 10 −9 m of each sna was dissolved using 40 × 10 −3 m kcn , and the number of dna strands per particle was determined using the oligreen assay ( life technologies , table s1 , supporting information ) . [SEP]
[CLS] snas ( 5 × 10 −9 m ) were incubated B-technique in 10 % type ab male hs ( sigma ) for 15 - 90 min at 37 °c with shaking . [SEP]
[CLS] unbound protein B-material was removed by centrifugation ( 3x , for 30 min at 13 000 rpm ) . [SEP]
[CLS] the concentration of purified snas with proteins B-material bound was determined , and adjusted to 120 × 10 −9 m by dilution with phosphate buffered saline ( pbs ) . [SEP]
[CLS] tween - 20 was added to the protein - coated snas at a final concentration of 1 % , and heated for 5 min at 95 °c to release the specifically bound proteins B-material . [SEP]
[CLS] the mixture was then centrifuged for 30 min at 13 000 rpm , and the supernatant was collected to analyze the specifically bound proteins B-material . [SEP]
[CLS] snas were collected from the pellet and further analyzed . [SEP]
[CLS] dls measurements were taken using a malvern zetasizer nanozs using the refractive B-property index I-property of gold B-material . [SEP]
[CLS] samples were prepared in pbs at a concentration of 1 × 10 −9 m sna and measurements were taken at 25 °c . [SEP]
[CLS] for diameter , the average and standard deviation of five measurements are reported and for zeta B-property - I-property potential I-property , the average of three measurements is reported . [SEP]
[CLS] the bca assay was used to quantify protein bound to snas following incubation B-technique in human serum . [SEP]
[CLS] protein B-material samples were isolated from 1 pmol sna following the procedure described previously , and were analyzed using the bca protein B-material assay kit ( pierce ) , and using bovine serum albumin to generate a standard curve from 0 to 250 μg protein B-material . [SEP]
[CLS] proteins B-material harvested from 3 pmol sna were analyzed by sds - page using precast mini protean tgx 4 % - 15 % polyacrylamide gels ( biorad ) . [SEP]
[CLS] to compare the amount of protein B-material from each type of sna , the protein B-material samples loaded were isolated from the same volume and concentration of snas . [SEP]
[CLS] spectra multicolor broad range protein B-material ladder was used as a molecular weight marker ( pierce ) and the gels were run for 1 h at 100 v in tris - glycine - sds buffer . [SEP]
[CLS] gels were then stained using ir blue protein B-material stain and imaged on an odyssey imager at 700 nm ( li - cor biosciences ) . [SEP]
[CLS] protein B-material samples isolated as previously described were prepared for mass spectrometry analysis by using acetone precipitation to remove sds , denatured in 8 m urea , reduced using dithiothreitol ( dtt ) , and alkylated using iodoacetamide . [SEP]
[CLS] the proteins B-material were then digested with sequencing grade trypsin in 1 m urea at 37 °c overnight . [SEP]
[CLS] the samples were desalted before they were loaded into a 10 cm long , 75 × 10 −6 m reverse phase capillary column ( proteopep ii c18 , 300a , 5 μm size , new objective ) and separated using a 100 min gradient from 5 % to 100 % acetonitrile on a proxeon easy n - lc ii ( thermo scientific ) . [SEP]
[CLS] next , the peptides B-material were eluted into an ltq orbitrap velos mass spectrometer ( thermo scientific ) with electrospray ionization B-property at 350 nl min −1 flow rate . [SEP]
[CLS] the mass spectrometer was run in a data - dependent mode , where 10 of the most intense ions B-material from each ms1 precursor ion B-material scan were selected from fragmentation by collision - induced dissociation . [SEP]
[CLS] the parameters for mass spectrometry were as follows : the resolution of ms1 was set at 60 000 , normalized collision energy was set to 35 % , activation time was 10 ms , isolate width was 1 . 5 , and the + 1 and + 4 and higher charge states were rejected . [SEP]
[CLS] the results were processed using proteome discoverer ( thermo scientific ) and searched using an in - house mascot server . [SEP]
[CLS] the results were also searched against the swiss - prot database , with the following database search filters : homo sapiens , trypsin for enzyme specificity , cysteine B-material carbamidomethylation for fixed modification , methionine B-material oxidation , and n - terminal acetylation for variable modification , precursor mass tolerance of ±10 ppm , and a fragment ion mass tolerance of ±0 . 8 da . [SEP]
[CLS] all spectra were searched against target / decoy databases , and the mascot significance threshold was chosen to achieve a targeted false discovery rate of 1 % . [SEP]
[CLS] the peptide B-material identification was considered valid if its corresponding mascot score was equal or less than the threshold . [SEP]
[CLS] protein B-material grouping was enabled in proteome discoverer , and were grouped to satisfy the rule of parsimony . [SEP]
[CLS] finally , proteins B-material with less than three unique peptides B-material were not considered to eliminate false discovery . [SEP]
[CLS] proteins B-material isolated from 3 pmol of snas were run on a 4 % - 15 % mini - protein B-material tgx gel ( bio - rad ) , transferred onto a nitrocellulose membrane ( thermo scientific ) using a trans - blot sd semi - dry transfer cell B-material ( bio - rad ) . [SEP]
[CLS] odyssey blocking buffer ( li - cor ) was used to block the membranes , and the following proteins B-material were probed with an antibody B-material : apolipoprotein b100 using a primary goat antibody B-material ( 1 : 1000 , abcam ab98132 ) , factor h using a primary goat antibody ( 1 : 2000 , abcam ab36134 ) , complement c3 using a primary rabbit antibody B-material ( 1 : 500 , abcam ab97462 ) , and serum albumin using a primary goat antibody B-material ( 1 : 1000 , abcam ab19194 ) . [SEP]
[CLS] the bands were labeled with the following secondary antibodies B-material : anti - goat ( 1 : 10 000 ) igg irdye 800 ( li - cor ) and anti - rabbit ( 1 : 10 000 ) igg irdye 680 ( li - cor ) , and the protein B-material bands were visualized using the odyssey clx infrared imaging system ( li - cor ) . [SEP]
[CLS] cells B-material were cultured in a 5 % co 2 incubator B-technique following atcc ' s recommended instructions . [SEP]
[CLS] thp - 1 cells B-material were cultured in rpmi containing 10 % fetal bovine serum ( fbs ) and 1 % penicillin / streptomycin supplemented with 0 . 05 × 10 −3 m 2 - mercaptoethanol . [SEP]
[CLS] hacat cells B-material were cultured in dmem containing 10 % fbs and 1 % penicillin / streptomycin and subcultured using trypsin . [SEP]
[CLS] one million thp - 1 cells B-material per well were seeded in a 12 - well plate , and were differentiated using 100 × 10 −9 m pma in rpmi containing 20 % fbs for 72 h . [SEP]
[CLS] to quantify cellular uptake , differentiated thp - 1 cells B-material were treated with 1 × 10 −9 m sna in optimem with and without 10 % hs for 15 , 30 , 60 , and 90 min . [SEP]
[CLS] for receptor B-material blocking studies , 50 μg ml −1 fucoidan ( sigma - aldrich ) or 5 μg ml −1 fc block ( bd biosciences ) in optimem was incubated B-technique with cells B-material for 30 min prior to transfection with snas , and 1 × 10 −3 m trypan blue ( life technologies ) was incubated B-technique with cells B-material for 60 min . [SEP]
[CLS] before snas were added to the cells B-material , the wells were washed twice with optimem to remove blockers . [SEP]
[CLS] thp - 1 cells B-material were then treated with snas in optimem with or without 10 % hs for 90 min . [SEP]
[CLS] following transfection , cells B-material from both experiments were washed with optimem three times before they were counted ( countess cell B-material counter , invitrogen ) and digested using 3 % hcl in 97 % hno 3 at 55 °c for 1 h . [SEP]
[CLS] acid - treated samples were resuspended in 2 % hcl and 2 % hno 3 in water B-material and analyzed using an x series ii icp - ms ( thermofisher ) . [SEP]
[CLS] the data presented represent the average and standard deviation from n = 3 for each sample group . [SEP]
[CLS] thp - 1 monocytes were differentiated using 100 × 10 −9 m pma in rpmi containing 20 % fbs for 48 h . 300 000 thp - 1 monocytes , thp - 1 differentiated macrophages , and hacat cells B-material were stained with fcγrii antibody B-material ( cd32 , bd biosciences 550586 , diluted to 1 : 50 ) , class a scavenger receptor B-material antibody B-material ( sr - ai , r & d systems fab2708 , diluted 1 : 100 ) , and complement receptor 3 antibody B-material ( cd11b , bd biosciences 564518 , diluted 1 : 200 ) for 30 min at 4 °c . [SEP]
[CLS] samples were then analyzed on an lsrii flow cytometer . [SEP]
[CLS] the mean fluorescence B-property intensity of each sample was plotted following dead cell B-material and doublet exclusion . [SEP]
[CLS] the values reported represent the mean and standard deviation of triplicate experiments with values normalized to the fluorescence B-property associated with hacat cells B-material for each receptor B-material type studied . [SEP]
[CLS] all experiments were conducted under an approved protocol of the institutional animal care and use committee of northwestern university . [SEP]
[CLS] - 8 week old male cd57bl / 6 mice were injected with 200 μ l of 100 × 10 −9 m g - rich sna , poly - t sna , or 1x pbs as a control intravenously via the tail vein . [SEP]
[CLS] blood samples were collected 1 , 5 , 10 , 15 , 30 , 45 , 60 , 120 , 240 , 480 , 720 , 960 , 1440 , and 2880 min following injection via retro - orbital draw . [SEP]
[CLS] blood samples were weighed , dried , and digested with acid for quantification of gold B-material content using icp - ms . [SEP]
[CLS] to determine sna accumulation in the liver and spleen , mice were sacrificed and perfused 8 h , 24 h , and 28 d postinjection , and tissues were collected for analysis by icp - ms . [SEP]
[CLS] detailed procedures for the preparation of samples for icp - ms can be found in the supporting information . [SEP]
[CLS] the data presented represent the average and standard deviation from n = 4 for each experimental group ( g - rich or poly - t snas ) . [SEP]
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[CLS] a . b . c . and c . m . g . both acknowledge a national defense science and engineering graduate fellowship . [SEP]
[CLS] analysis and identification of proteins B-material isolated from snas postincubation in hs . [SEP]
[CLS] a ) the number of distinct proteins B-material isolated from g - rich snas increases with longer incubations B-technique in hs . [SEP]
[CLS] b ) a comparison of the proteins B-material that bind g - rich and poly - t snas indicates that there are about 45 proteins B-material that bind to both particle types , while only the number of proteins B-material bound uniquely to g - rich snas increases with longer incubations B-technique in hs . [SEP]
[CLS] c ) western B-technique blot I-technique analysis I-technique of the relative amounts of apolipoprotein b100 , complement factor h , complement c3 , and human serum albumin isolated from g - rich and poly - t snas after incubation B-technique in hs from 15 to 90 min . [SEP]
[CLS] effects of sna human serum protein B-material corona I-material formation on macrophage uptake . [SEP]
[CLS] a ) the uptake and association of g - rich and poly - t snas in the presence and absence of hs following 15 - 90 min incubations B-technique with thp - 1 cells B-material was quantified by icp - ms . [SEP]
[CLS] g - rich snas exhibit significantly higher uptake in the presence of hs compared to poly - t snas with or without hs present . [SEP]
[CLS] b ) the uptake of snas into thp - 1 macrophages following treatment with chemical blockers to inhibit binding B-event to I-event cell I-event surface I-event receptors I-event was quantified . [SEP]
[CLS] fucoidan was used to block class a scavenger receptors , fc block was used to block fc receptors , and trypan blue was used to block complement receptors . [SEP]
[CLS] the results indicate that the macrophage uptake and association of snas in the presence of hs was significantly inhibited by trypan blue and suggests that complement receptor - mediated phagocytosis B-event plays a large role in the uptake of protein - coated snas into thp - 1 macrophage cells B-material . [SEP]
[CLS] * p < 0 . 05 . [SEP]
[CLS] g - rich snas adsorb more types of proteins B-material and more total protein B-material from serum than poly - t snas . [SEP]
[CLS] characterization of protein - coated snas and quantification of protein bound to snas . [SEP]
[CLS] a ) the hydrodynamic diameter of g - rich and poly - t snas incubated B-technique in hs from 15 to 90 min increases . [SEP]
[CLS] b ) quantitative analysis of the amount of protein B-material bound to g - rich and poly - t snas after incubation B-technique in hs . [SEP]
[CLS] c ) sds - page gel of proteins B-material isolated from g - rich and poly - t snas after incubation B-technique in hs from 15 to 90 min indicates that more proteins B-material , both in number and type , bind to g - rich snas than to poly - t snas . [SEP]
[CLS] note that the heavy band in hs corresponds to human serum albumin ( hsa ) . [SEP]
[CLS] comparison of cell B-material surface I-material receptor I-material expression between thp - 1 macrophages and hacat cells B-material . [SEP]
[CLS] relative cell - associated fluorescence B-property of thp - 1 macrophages and hacat cells B-material treated with fluorophore - labeled antibodies B-material targeting complement receptors , fc receptors , and class a scavenger receptors . [SEP]
[CLS] quantification of sna accumulation in the a ) liver and b ) spleen of mice injected with either g - rich or poly - t snas 8 , 24 , and 672 h postinjection . [SEP]
[CLS] * p < 0 . 05 [SEP]
[CLS] complementary tetrahedral small molecule - dna hybrid ( smdh ) building blocks have been combined to form nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle without the need for an underlying template or scaffold . [SEP]
[CLS] the sizes of these particles can be tailored in a facile fashion by adjusting assembly conditions such as smdh concentration , assembly time , and nacl concentration . [SEP]
[CLS] notably , these novel particles can be stabilized and transformed into functionalized spherical nucleic B-material acid I-material ( sna ) structures through the incorporation of capping dna strands conjugated with functional groups . [SEP]
[CLS] these results demonstrate a systematic , efficient strategy for the construction and surface functionalization of well - defined , size - tunable nucleic B-material acid I-material particles from readily accessible molecular building blocks . [SEP]
[CLS] furthermore , because these nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle exhibited enhanced cellular internalization and resistance to dnase i compared to free synthetic nucleic B-material acids I-material , they should have a plethora of applications in diagnostics and therapeutics . [SEP]
[CLS] over the last few decades , dna has emerged as a key building block for molecular assemblies and nanostructures due to the versatile programmability encoded into the base - pairing interactions ( a - t and g - c ) between complementary strands . [SEP]
[CLS] such high specificity and predictability at the molecular level can be readily translated into an exquisite control of assembly interactions at the nanoscale , enabling the formation of a variety of topologically distinct dna - based constructs such as rings , boxes , tubes , and macroscopic crystals . [SEP]
[CLS] recently , well - defined small molecule - dna hybrids ( smdhs ) , comprising multiple dna strands covalently attached to small - molecule cores B-material , have quickly gained popularity as dna - containing building blocks for nanomaterials B-material with valency that can be tuned through the number and orientation of the strands . [SEP]
[CLS] smdhs greatly increase the scope and versatility of the design and construction of dna - based nanostructures because branched building blocks can be made efficiently from relatively short oligonucleotides and a broad range of organic B-material cores I-material . [SEP]
[CLS] early work in smdh - based assembly has often focused on the careful selection of an organic B-material core I-material with three or fewer dna strands , along with a predesigned sequence and strand orientation of the dna , to afford thermodynamically stable discrete 2d or 3d supramolecular structures . [SEP]
[CLS] however , when smdhs with more than three strands are hybridized with their complementary smdhs , a broad range of illdefined products can result , decreasing the yield of the desired discrete product . [SEP]
[CLS] indeed , only macroscopic aggregates were observed in the assembly of self - complementary tetrahedral smdh building blocks ( smdh 4 ) possessing as few as two base pairs per dna arm . [SEP]
[CLS] herein , we report that the assembly of two complementary smdh 4 building blocks , with four dna strands around relatively rigid tetrahedral cores B-material , can be readily controlled to yield well - defined , narrowly dispersed spherical B-nanoparticle nanoparticles I-nanoparticle ( figure 1 ) . [SEP]
[CLS] these small particles are almost entirely nucleic B-material acids I-material in composition and their sizes can be tuned easily by controlling nucleic B-material acid I-material concentration , assembly time , and nacl concentration . [SEP]
[CLS] notably , they can be stabilized , and transformed into structures similar to functionalized spherical nucleic B-material acids I-material ( sna ) , through the incorporation of capping dna strands conjugated with functional groups . [SEP]
[CLS] these nanoparticles B-nanoparticle exhibit efficient cellular uptake compared to the free dna arms of the smdh 4 building blocks , and show enhanced resistance to a dnase i enzyme when capped with a peg - functionalized strand . [SEP]
[CLS] such characteristics make them highly attractive for applications in both diagnostics and therapeutics . [SEP]
[CLS] we and others have previously shown that the hybridization of smdhs containing more than three dna strands with their complementary smdhs at high dna concentrations ( 40 - 150 μm ) can result in ill - defined networks and visually observable large aggregates . [SEP]
[CLS] however , this outcome can be modulated either with an additive or by changing the flexibility of the spacer B-material between the core B-material and the dna arms . [SEP]
[CLS] for example , sleiman and coworkers have employed a ru ( bpy ) 3 guest template to preferentially induce the formation of fibers over ill - defined networks in the assembly of complementary rigid smdh 3 s . [SEP]
[CLS] subsequently , we demonstrated that the yields of small discrete cage - like smdh 3 dimers can be greatly increased over those of higher - order aggregates when unhybridized oligo ( deoxythymidine ) spacers B-material are included between the relatively rigid organic B-material core I-material and the hybridizing dna arms to provide more flexibility for the assembly to " anneal " into more thermodynamically stable discrete structures . [SEP]
[CLS] results and discussionhis spacer B-material flexibility is particularly important when the hybridizing dna arms are long or when the total nucleic B-material acid I-material concentration of the assembly is high ( > 5 μm ) . [SEP]
[CLS] parallel to the aforementioned developments , recent work in the growth of colloidal nanoparticles B-nanoparticle has shown that tuning the assembly conditions - such as concentrations of the components that are participating in the assembly , temperature , and assembly rate - can significantly affect the morphological outcome of an assembly process . [SEP]
[CLS] our own work in the assembly of complementary smdh 3 s has also indicated that the total concentration of dna in solution , the annealing rate , and the salt B-material concentrations are very important parameters that dictate the proportions of well - defined dimers vs higher - order structures . [SEP]
[CLS] these results prompted us to hypothesize that discrete nanostructures may be obtained readily from tetrahedral smdh 4 building blocks at low - to - moderate nucleic B-material acid I-material concentrations ( 2 - 15 μm ) and through a careful control of the assembly conditions . [SEP]
[CLS] to test the aforementioned hypothesis , we selected smdh 4 building blocks with a tetraphenylmethane core B-material and 18 - base dna arms that are linked together through ( ch 2 ) 4triazole spacers B-material . [SEP]
[CLS] from previous studies , we suspected that this design would afford the right combination of stability and flexibility needed for optimizing the formation of discrete nanostructures . [SEP]
[CLS] to our advantage , these building blocks can be obtained readily in largeenough quantities for our study through a solid - state click coupling strategy that was previously shown to favor multiarm products ( see experimental section ) . [SEP]
[CLS] in an initial screen , equimolar mixtures of the smdh 4 and its complementary partner were combined in tris - buffered saline ( tbs , 20 mm tris • hcl and 150 mm nacl buffer solution , ph 7 . 4 ) at dna concentrations in the 2 - 100 μm range and annealed following established dna hybridization protocols ( see experimental section ) . [SEP]
[CLS] in a typical annealing procedure , the assembly mixture is heated to 90 °c in a heating block and kept there for 5 - 10 min to eliminate all initial dna interactions ; turning off the heating block then allows the mixture to slowly cool to rt over 3 h and for dna - hybridized colloidal aggregates to form . [SEP]
[CLS] these aggregates were then examined by dynamic B-technique light I-technique scattering I-technique ( dls ) , which reports the hydrodynamic diameter ( d h ) of the particles in solution , and scanning B-technique transmission I-technique electron I-technique microscopy I-technique ( stem ) , which reflects the sizes and shapes for the particles in a vacuum . [SEP]
[CLS] while the assemblies at high smdh concentrations ( total [ dna ] = 100 μm ) yielded only macroscopic aggregates ( supporting information ( si ) , figure s3 ) , as reported previously ; 15 , 16 those at lower concentrations ( total [ dna ] = 2 - 15 μm ) afforded welldefined , narrowly dispersed particles ( figure 2 ) . [SEP]
[CLS] at 15 μm total nucleic B-material acid I-material concentration , the average d h of the assembled particles is 360 ± 60 nm , while that at 2 μm concentration is a remarkably small 24 ± 6 nm . [SEP]
[CLS] together with these dls data ( figure 2a ) , agarose B-technique gel I-technique electrophoresis I-technique analysis ( si , figure s4 ) confirms that these small particles are completely different from the species formed by hybridizing one of our smdh 4 building blocks with the complementary single - stranded ( ss ) dna . [SEP]
[CLS] the stem images ( figure 2b ) of the assembled products support the dls results and show all particles are spherical in shape . [SEP]
[CLS] in addition , the particles prepared at high concentration ( total [ dna ] = 15 μm ) cannot be converted to smaller particles when diluted to similarly low concentration ( total [ dna ] = 2 μm ; si , figure s5 ) , suggesting that the individual smdh components in the assembled particles are strongly connected to each other . [SEP]
[CLS] when the assemblies were carried out at temperatures well above 57 °c , the characteristic melting temperature ( t m ) of the hybridized products between smdh 4 and its complementary partner ( si , figure s6 ) , the hybridization interactions between the arms are not stable and both smdhs remain apart in solution . [SEP]
[CLS] as the temperature of the assembly mixture is decreased toward t m , duplex formation between the dna arms on adjacent molecules begins to link them together , forming oligomers and small polymeric B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] if the total dna concentration in the initial mixture is high , hybridization occurs in a fast , unoptimized manner throughout the mixture , which will result in large aggregates . [SEP]
[CLS] however , if the total dna concentration in the initial mixture is low , the smdh 4 concentration around the nucleation sites can be quickly depleted and the probability for forming large aggregates decreases , just as in condensation polymerization . [SEP]
[CLS] this represents an opportunity for the nanoscale smdh 4 particles to be " trapped " in a kinetically stable state . [SEP]
[CLS] if this process is further controlled in a way that does not allow neighboring nanoparticles B-nanoparticle to undergo aggregation , they would remain as discrete entities . [SEP]
[CLS] within this kinetically stable regime , the size of the nanoparticles B-nanoparticle should be easily tuned by varying the initial dna concentration and the cooling rate . [SEP]
[CLS] expanding upon the aforementioned idea of controllable kinetic trapping , fast cooling ( short assembly time ) near t m should decrease the number of collisions between neighboring particles , resulting in a stable population of smaller particles . [SEP]
[CLS] in contrast , prolonged cooling ( long assembly time ) near t m should correspond to more collisions and give rise to larger particles . [SEP]
[CLS] to evaluate this effect , an equimolar mixture of the two smdh 4 components ( total [ dna ] = 10 μm ) was prepared in tbs . [SEP]
[CLS] after being heated at 90 °c for 10 min in a thermal cycler , this mixture was cooled down to room temperature at different rates ( 5 . 5 , 1 . 0 , 0 . 34 , and 0 . 17 °c / min ) and monitored using dls . [SEP]
[CLS] as shown in figure 3a , the particles obtained in the fast - cooled ( 5 . 5 °c / min ) solution are much smaller ( d h = 23 ± 7 nm ) than those obtained from the slow - cooled ( 0 . 17 °c / min ) solution ( d h = 300 ± 80 nm ) . [SEP]
[CLS] as the cooling rate decreased from 5 . 5 °c / min , bimodal populations of larger and larger nanoparticles B-nanoparticle were observed . [SEP]
[CLS] correspondingly , the stem images of all mixtures show spherically shaped nanoparticles B-nanoparticle . [SEP]
[CLS] not only do these results confirm that the size of the nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle can indeed be modulated by the assembly time , they also suggest that the small particles that were previously observed only at low concentrations ( total [ dna ] ≤ 5 μm ) could now be successfully synthesized at moderate concentrations ( total [ dna ] up to 15 μm ; see si , figure s7 ) with fast cooling . [SEP]
[CLS] in addition to the total dna concentration and the assembly time , the concentration of na + ions B-material in the assembly solutions also has significant effects on the average size of the assembled particles . [SEP]
[CLS] as shown in figure 4a , higher [ nacl ] leads to larger particles : the d h of the particles is 650 ± 100 nm at 300 mm nacl , and 25 ± 12 nm at 25 mm nacl . [SEP]
[CLS] this is not surprising when one considers that na + ions B-material can increase the rate of duplex formation , as well as the stability of the final duplex , by reducing the repulsive force between the negatively charged dna strands . [SEP]
[CLS] interestingly , the magnitude of the effect of [ nacl ] depends significantly on the concentration of the smdhs . [SEP]
[CLS] at low smdh concentration ( total [ dna ] = 2 μm ) , the average diameters of the particles at high ( 300 mm ) and low ( 50 mm ) [ nacl ] are not significantly different ( cf . the top dls profile in figure 4b and the top dls profile in figure s9 in the si ) . [SEP]
[CLS] in contrast , the particles prepared at > 2 μm total [ dna ] show significant variations in size and population distributions when the [ nacl ] varies ( cf . the lower dls profiles in figure 4b and the lower dls profiles in figures s9 in the si ) . [SEP]
[CLS] these results suggest that while increasing the [ nacl ] can increase the size of nucleic acidbased polymeric B-nanoparticle nanoparticles I-nanoparticle , through either aggregation or ostwald ripening , there is a minimal smdh concentration required before this effect becomes significant . [SEP]
[CLS] because our nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle were derived from two complementary components , their surfaces have an approximately equal population of two types of complementary dna strands that do not hybridize with each other due to steric constraints . [SEP]
[CLS] however , as expected for kinetically stabilized species , the as - prepared particles continue to aggregate over the next 4 weeks at room temperature through hybridization events between unhybridized strands on the surfaces of neighboring particles ( figure 5 , top path ) . [SEP]
[CLS] this propensity to aggregate can be minimized by capping one of these two types of unhybridized strands with complementary ssdna strands , leading to particles that are fully stabilized for over 4 weeks ( figure 5 , bottom path ) . [SEP]
[CLS] this advance not only is important for stabilization purposes , but also transforms these nucleic B-material acid I-material materials into novel forms of spherical nucleic B-material acids I-material ( snas ) , which have become extremely important as building blocks in materials science , labels in in vitro and intracellular diagnostics , and delivery agents in therapeutic applications . [SEP]
[CLS] the availability of two different types of complementary dna strands on the surface of our nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle also makes it an excellent platform for orthogonal functionalization . [SEP]
[CLS] one of these strands can be capped with a functionalized ssdna and the remaining uncapped dna strand can subsequently be modified with a second set of functionalized capping strands ; the whole platform would then bear two different functional groups . [SEP]
[CLS] as a proof - of - concept experiment , an alexa fluor 647 ( af647 ) conjugated capping dna strand ( 5 μm ) that hybridizes with the remaining unbound strands of smdh 4 on the surface of the particles was added to a solution of as - prepared particles ( equimolar amounts of smdh 4 and its complementary partner ; total [ dna ] = 10 μm ) and incubated B-technique at room temperature for 8 h ( figure 6a ) . [SEP]
[CLS] examining the resulting solutions by confocal B-technique laser I-technique scanning I-technique microscopy I-technique ( clsm ) at room temperature clearly shows bright fluorescent B-property dots against a darker background ( figure 6a ) , suggesting the presence of individual spherical particles that concentrated the af647 fluorophores . [SEP]
[CLS] this is in clear contrast to the uniform , nondistinct image of a control solution of only af647 capping strands ( si , figure s10 ) . [SEP]
[CLS] as expected , these af647 - capped particles exhibit brownian motion in solution as observed by continuous clsm monitoring ( see the video that accompanies this manuscript as part of the si ) . [SEP]
[CLS] while these af647 - capped particles are now stabilized , they still have unbound dna strands from the complementary partner of smdh 4 on their surfaces that can be used for orthogonal labeling with cy3 , another dye molecule . [SEP]
[CLS] after the as - prepared af647 - capped particles were mixed with the appropriate cy3conjugated dna strands ( 5 μm ) and incubated B-technique at room temperature for 8 h , their clsm images ( figure 6b , right ) clearly show fluorescence B-property signals from both af647 and cy3 . [SEP]
[CLS] nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle can serve as efficient vehicles B-material for transporting synthetic nucleic B-material acids I-material into cells B-material . [SEP]
[CLS] synthetic nucleic B-material acids I-material such as antisense dna and small interfering rna have long been used for both gene - regulation study and gene therapy . [SEP]
[CLS] however , they are not readily uptaken by cells B-material : their high molecular weights and polyanionic backbones sterically and electrostatically impede them from passing through the negatively charged cellular membranes . [SEP]
[CLS] in addition , they also suffer from a high susceptibility to enzymatic degradation when administered in isolated molecular form . [SEP]
[CLS] thus , appropriate delivery systems that can encapsulate the nucleic B-material acids I-material are often used to carry them into cells B-material and protect them from degradation prior to reaching the therapeutic targets inside the cells B-material ( e . g . , mrna ) . [SEP]
[CLS] among the emerging cellular delivery systems , nanoparticles B-nanoparticle are predicted to have the optimal endocytotic cellular uptake potentials when they are in the 25 - 30 nm size regimes . [SEP]
[CLS] indeed , for synthetic nucleic B-material acid I-material delivery , snas in this size regime have shown excellent promises in their ability to easily enter into cells B-material through an endocytotic pathway . [SEP]
[CLS] given these precedents , we hypothesize that our nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle in the 25 - 30 nm size regimes could also be efficiently internalized into cells B-material . [SEP]
[CLS] in addition , because the synthetic nucleic B-material acids I-material in our nanoparticle B-nanoparticle are packed throughout the whole volume in a high - density ( > 98 wt % nucleic B-material acid I-material composition ) fashion , capping them , as shown in the previous section , with nuclease - resistant agents may confer good protection of the whole cargo against enzymatic degradation compared to free synthetic nucleic B-material acids I-material . [SEP]
[CLS] to evaluate the cellular uptake efficiency of our nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle , we treated skov - 3 human carcinoma ovarian cells B-material with either a sample that has been capped with an af647 - conjugated ssdna ( total [ dna ] = 15 μm ; capping [ dna ] = 75 nm ; d h = 24 ± 5 nm ) or free af647 - conjugated ssdna ( 75 nm ) for 24 h , and analyzed their cellular uptake by clsm and flow B-technique cytometry I-technique . [SEP]
[CLS] clsm images ( figure 7a ) of the cells B-material that were exposed to the af647 - capped nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle clearly shows the localization of af647 - conjugated ssdna ( red - colored regions ) inside the cells B-material , indicating successful cellular internalization . [SEP]
[CLS] in stark contrast , no red region is visually observed in the control sample of cells B-material that were exposed to the free af647 - conjugated ssdna ( figure 7b ) . [SEP]
[CLS] quantitative analysis by flow B-technique cytometry I-technique ( figure 7c ) further confirmed the clsm observation by showing that the average af647 - based fluorescence B-property intensity of the cells B-material that were exposed to the nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle is about ten times higher than that of the control ( i . e . , exposed to free af647 - conjugated ssdna ) . [SEP]
[CLS] such enhanced cellular uptake may prove to be advantageous in gene delivery applications where increases in the amounts of internalized synthetic nucleic B-material acids I-material can lead to excellent transfection B-property efficiency I-property . [SEP]
[CLS] before evaluating the hypothesis that capping our nanoparticles B-nanoparticle with nuclease - resistant agents can lead to enhanced protection of the smdh 4 building blocks against attack by nucleases , we first examined the size - dependent resistance of our nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle against dnase i , an endonuclease that is capable of degrading both single - and double - stranded dna as well as chromatin . [SEP]
[CLS] samples of large ( d h = 340 ± 50 nm ) and small ( d h = 24 ± 5 nm ) nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle , along with a control smdh 4 that has been hybridized with complementary free dna strands , were exposed to a solution of dnase i ( 5 units / ml ) at 37 °c for 1 h and then denatured and analyzed by polyacrylamide B-technique gel I-technique electrophoresis I-technique ( page ) . [SEP]
[CLS] as shown in figure 8a , significant amounts of the smdh 4 remain intact in both nanoparticle B-nanoparticle samples , suggesting that they are better protected against dnase i than the smdh 4 control , which completely degraded into smaller fragments . [SEP]
[CLS] as an additional control , the page gel image of denatured samples of both types of nanoparticles B-nanoparticle showed that virtually all of the smdh 4 building blocks remain intact during the particle formation process ( figure 8b ) . [SEP]
[CLS] that the smdh 4 control qualitatively degraded faster than the small nanoparticles B-nanoparticle , which in turn degraded faster than the larger ones , can be partially attributed to two size - dependent factors . [SEP]
[CLS] first , there is a higher probability for the smdh 4 building blocks to encounter the enzyme when existing as individual units ( instead of being " packaged " inside the nanoparticles B-nanoparticle ) . [SEP]
[CLS] that is , at the same dna concentration , the assembly of smdh 4 into nanoparticles B-nanoparticle ensures that there is a lower number of available dna - containing entities ( either nanoparticles B-nanoparticle or free smdh 4 units ) with which the enzyme can react . [SEP]
[CLS] second , the synthetic nucleic B-material acids I-material near the center of the smaller nanoparticle B-nanoparticle will be exposed to the enzyme faster than those near the center of the larger nanoparticles B-nanoparticle . [SEP]
[CLS] we note in passing that the high concentration of salts B-material ( ion B-material cloud ) surrounding the nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle , which repels the binding of the enzyme , can also contribute to the overall enhanced stability of the smdh 4 building blocks inside the nanoparticles B-nanoparticle . [SEP]
[CLS] this potential third factor has recently been reported for snas , and is currently being investigated by us ; these results will be reported in due course . [SEP]
[CLS] to test the hypothesis that capping our nanoparticles B-nanoparticle with nuclease - resistant agents , such as poly ( ethylene glycol ) ( peg ) , can lead to enhanced protection of the smdh 4 building blocks against attack by nucleases , we employed two types of peg - conjugated capping ssdna strands , each with a sequence that is complementary to the two smdh strands comprising the nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] two samples of each particle diameter , i . e . , large ( d h = 340 ± 50 nm ) and small ( d h = 24 ± 5 nm ) , were then capped with one or both of these peg - conjugated capping ssdna strands using the aforementioned orthogonal functionalization method ( si , section s3 ) . [SEP]
[CLS] all four samples were then exposed to the same dnase i treatment described previously in this section ( 5 units / ml at 37 °c for 1 h ) , and then denatured and analyzed by polyacrylamide B-technique gel I-technique electrophoresis I-technique ( page ) to evaluate the effect of relative peg density . [SEP]
[CLS] as shown in figure 9a , most of the dually peg - capped nanoparticles B-nanoparticle with large diameters were not degraded and even the dually peg - capped small nanoparticles B-nanoparticle were also highly stabilized against the enzyme . [SEP]
[CLS] having a higher density of peg - capping is clearly more advantageous : the singly peg - capped samples appear to degrade slightly more under the same dnase i treatment regime ( figure 9b ) . [SEP]
[CLS] these results again highlight the modular characteristic of our nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle : higher levels of protection of the smdh 4 content can be engineered into their surfaces with higher peg density . [SEP]
[CLS] in addition , because peg chains with two different functional endgroups are readily available , the incorporation of nuclease resistance can be readily incorporated without sacrificing the capacity for orthogonal surface functionalization . [SEP]
[CLS] in summary , we have shown that tetravalent molecular cores I-material can be used to create tetravalent dna constructs for rapidly preparing nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] the size and distributions of these novel nanomaterials B-material can be modulated by tuning the smdh concentration , the assembly time / temperature , and the nacl concentration , demonstrating a systematic strategy for constructing nucleic acid nanoparticles B-nanoparticle from purely organic building blocks without the need for a template or scaffold . [SEP]
[CLS] these nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle exhibit enhanced cellular internalization and can be functionalized to have highly enhanced resistance against dnase i compared to the smdh 4 building blocks . [SEP]
[CLS] together with the ability to transform these particles into snas , while still maintaining active surface B-nanoparticle groups I-nanoparticle for further functionalization , these novel materials have the potential to become useful in areas spanning both diagnostics and therapeutics . [SEP]
[CLS] see si , section s1 . s1 for sequence descriptions ) [SEP]
[CLS] at the end of the synthesis , the beads were dried overnight under a high vacuum , removed from the column , and kept in a tightly capped vial that is filled with dry nitrogen B-material gas . [SEP]
[CLS] the solid - phase coupling reactions with the tetraazide core B-material were then carried out using these dry cpg beads . [SEP]
[CLS] ( b ) solid - phase synthesis of smdh 4 - the as - prepared dry cpg beads containing alkyne - modified dna ( 1 μmol ) were placed in a 1 . 5 ml eppendorf tube . [SEP]
[CLS] to the tube were sequentially added the tetraazide core B-material ( 5 - 7 μmol ) in dmf ( 0 . 75 ml ) , tris ( 3hydroxypropyltriazolylmethyl ) amine ( 7 μmol ) in dmf ( 0 . 2 ml ) , cuso 4 • 5h 2 o ( 5 μmol ) in dmf ( 0 . 05 ml ) , and l - ascorbic acid ( 10 μmol ) in dmf ( 0 . 1 ml ) . [SEP]
[CLS] the reaction tube was then filled with dry nitrogen B-material gas before being capped and shaken for 18 h at 25 °c in an thermomixer r ( eppendorf , hauppauge , ny ) instrument at 1200 rpm ( note : the cpg beads should be properly agitated in dmf solution and not allowed to settle at the bottom of the tube during the reaction ) . [SEP]
[CLS] the resulting cpg beads were filtered using a one - side - fritted 1 μmol expedite dna synthesis column ( glen research , # 20 - 0021 - 01 ) , washed with dmf ( 10 × 1 ml ) and acetone ( 10 × 1 ml ) , and dried using a stream of dry nitrogen B-material . [SEP]
[CLS] the cpg beads containing the products were placed in a vial containing 1 ml of ama ( 1 : 1 v / v 30 % ammonium hydroxide B-material solution : methylamine solution ; caution ! only freshly made ama solutions should be used ) , and the vial was capped and heated at 65 °c for 15 min to cleave the smdhs from the solid supports . [SEP]
[CLS] the ammonia and methylamine byproducts were then removed by passing a stream of dry nitrogen B-material gas over the content of the vial until the characteristic ammonia smell disappears . [SEP]
[CLS] the remaining liquid , which contains the crude smdhs , was collected by pipet and the remaining beads were further extracted with ultrapure deionized B-material water I-material ( 3 × 200 μl ) . [SEP]
[CLS] these extracts were combined with the initial solution of crude smdhs ( affording a total volume of 0 . 8 ml at the end ) and filtered through 0 . 45 μm nylon syringe filter ( acrodisc 13 mm syringe filter # pn 4426t ) . [SEP]
[CLS] ( c ) purification and characterization of smdh 4 s - see si , section s2 . [SEP]
[CLS] equimolar mixtures of the as - prepared smdh 4 and its complementary partner were added into 0 . 5 ml eppendorf tubes . [SEP]
[CLS] enough tris • hcl buffer solution ( 20 mm tris • hcl with appropriate concentrations of nacl ; ph = 7 . 4 ) was then added to make solutions with the desired total concentration of dna ( 2 , 5 , 10 , 15 , and 100 μm ) . [SEP]
[CLS] the resulting solutions were heated to 90 °c in a thermomixer r 5355 instrument without shaking and kept there for 10 min to remove all initial dna interactions . [SEP]
[CLS] the power to the heating block was then turned off to allow the solution to slowly cool to room temperature over 3 h ( for a typical cooling profile of this equipment , please see figure s16 in the si of the reported publication ) . [SEP]
[CLS] the resulting nucleic acid - based polymeric particles were analyzed by dls and stem ( see si , section s3 ) . [SEP]
[CLS] for other programmable cooling profiles used in the cooling - rate study , a mj mini gradient thermal cycler ( bio - rad laboratories , inc . , hercules , ca ) was utilized . [SEP]
[CLS] in a typical experiment , an equimolar mixture of the as - prepared smdh 4 and its complementary partner ( total [ dna ] = 10 or 15 μm ) was placed into a 0 . 5 ml eppendorf tube . [SEP]
[CLS] enough tris - buffered saline ( tbs , 20 mm tris • hcl and 150 mm nacl buffer solution , ph 7 . 4 ) was then added to make a 0 . 1 ml solution . [SEP]
[CLS] this solution was heated to 90 °c in a thermomixer r 5355 instrument and kept there for 10 min to clear out all initial dna interactions . [SEP]
[CLS] the power to the heating block was then turned off to allow the solution to slowly cool to room temperature over 3 h . [SEP]
[CLS] the cooled - down solution of assembled nucleic acid - based polymeric particles was then mixed with capping dna strands ( 5 or 7 . 5 μm ; [SEP]
[CLS] with or without af647 dye ) [SEP]
[CLS] and [SEP]
[CLS] the resulting solution was left at rt for 8 h without shaking . [SEP]
[CLS] the resulting solution of capped particles was analyzed by dls and clsm . [SEP]
[CLS] in a typical experiment , an equimolar mixture of the as - prepared smdh 4 and its complementary partner was placed into a 0 . 5 ml eppendorf tube . [SEP]
[CLS] enough tbs was then added to make a 0 . 1 ml solution ( total [ dna ] = 10 μm ) . [SEP]
[CLS] this solution was heated to 90 °c in a thermomixer r 5355 instrument and kept there for 10 min to clear out all initial dna interactions . [SEP]
[CLS] the power to the heating block was then turned off to allow the solution to slowly cool to room temperature over 3 h . [SEP]
[CLS] the cooled - down solution of assembled nucleic acid - based polymeric particles was then mixed with af647 - conjugated capping dna strands ( final concentration = 5 μm , in stoichiometric equivalence to the total dna concentration in each of the two smdh 4 assembly partners but in excess of the concentration of unhybridized dna strands on the surface ) , and the resulting solution was left at rt for 8 h without shaking to form a solution of af647 - labeled particles . [SEP]
[CLS] next , cy3conjugated capping dna strands ( final concentration = 5 μm , in stoichiometric equivalence to the total dna concentration in each of the two smdh 4 assembly partners but in excess of the concentration of unhybridized dna strands on the surface ) were added and the resulting solution was left at rt for 8 h without shaking . [SEP]
[CLS] the resulting solution of orthogonally labeled particles ( with both af647 and cy3 fluorophores on the surface ) was analyzed by clsm . [SEP]
[CLS] see si , section s4 . [SEP]
[CLS] ( a ) assessment of cellular uptake by flow B-technique cytometry I-technique - skov - 3 cells B-material ( 100 , 000 cells ) were plated into each well of 12 - well plates and incubated B-technique for 24 h . [SEP]
[CLS] the media were then replaced with preprepared media containing either free af647 - conjugated ssdna ( 75 nm , 0 . 5 ml / well ) or nucleic - acid based polymeric B-nanoparticle nanoparticles I-nanoparticle ( total [ dna ] = 15 μm ; d h = 24 ± 5 nm ) capped with af647 - conjugated ssdna ( 75 nm , 0 . 5 ml / well ) . [SEP]
[CLS] after 24 h incubation B-technique , the cells B-material were washed with dpbs ( 3×1 ml / well ) , treated with a tryple express ( 1× ) solution ( 0 . 5 ml / well ) , harvested , transferred to 1 . 5 ml microcentrifuge tubes , and centrifuged at 1000 g for 5 min . [SEP]
[CLS] after removing the supernatant via aspiration , the resulting cell B-material pellets were washed in dpbs ( 0 . 5 ml / tube ) and resuspended in dpbs media ( 0 . 5 ml / tube ) . [SEP]
[CLS] the fluorescence B-property intensity of af647 - conjugated ssdna in collected cells B-material was measured by a flow cytometer ( bd lsrii , bd biosciences , san jose , ca ) . [SEP]
[CLS] data were analyzed using flowjo software ( tree star , inc . , ashland , or ) to obtain the average fluorescence B-property intensity of collected cells B-material . [SEP]
[CLS] ( b ) cellular uptake assessed by clsm - skov - 3 cells B-material ( 100 000 cells B-material ) were plated in 35 mm fluorodish cell B-material culture dishes ( world precision instruments , sarasota , fl ) and incubated B-technique for 24 h . [SEP]
[CLS] the media in the dishes were then replaced with preprepared media containing either free af647 - conjugated ssdna ( 75 nm , 0 . 5 ml / well ) or nucleic - acid based polymeric B-nanoparticle nanoparticles I-nanoparticle capped with af647 - conjugated ssdna ( 75 nm , 0 . 5 ml / well ) . [SEP]
[CLS] after 24 h incubation B-technique , the cells B-material were washed with dpbs ( 3×2 ml / dish ) followed by immediate clsm imaging . [SEP]
[CLS] two different sizes of nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle ( total [ dna ] = 15 μm ; d h = 24 ± 5 nm and d h = 340 ± 50 nm , respectively ) as well as a control smdh 4 ( total [ dna ] = 15 μm ) that was hybridized with a complementary free ssdna strand were prepared using the methods described above . [SEP]
[CLS] either the particles ( 25 μl ) or control smdh 4 ( 25 μl ) were then mixed with dnase i ( 2 . 5 μl of a 100 units / ml solution ) . [SEP]
[CLS] the resulting solutions were diluted to 50 μl and incubated B-technique at 37 °c for 1 h . [SEP]
[CLS] edta solution ( 5 μl of a 0 . 5 m solution ) [SEP]
[CLS] was then added to quench the dnase i reaction and the resulting mixture was immediately lyophilized . [SEP]
[CLS] the remaining dried powders were redispersed in a mixture of aqueous urea ( 5 μl of a 7 m solution ) and gel - loading dye solution ( 5 μl , life technologies division of thermo fisher scientific , inc . , grand island , ny ) , heated at 90 °c for 5 min to denature the assembly , and quickly immersed in an ice bath . [SEP]
[CLS] the resulting solutions were immediately loaded on denaturing polyacrylamide gel ( 12 wt % ) . [SEP]
[CLS] the gel experiments were carried out in 0 . 5× tbe buffer at 350 v for 1 h and stained with sybr gold B-material ( life technologies division of thermo fisher scientific , inc . , grand island , ny ) . [SEP]
[CLS] images of the gels were taken using an imagequant las 4010 gel imaging system ( ge healthcare , pittsburgh , pa ) . [SEP]
[CLS] ( a ) denaturing page - gel ( 12 % ) image of the dually peg - capped nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle after a 1 h treatment with dnase i ( 5 units / ml ) . [SEP]
[CLS] lane 1 = ssdna ladder ; lanes 2 and 3 = large ( d h = 340 ± 50 nm ) and small ( d h = 24 ± 5 nm ) nucleic acidbased polymeric B-nanoparticle nanoparticles I-nanoparticle that were dually capped with two peg - functionalized strands , respectively . [SEP]
[CLS] samples were run after particle dissolution ( i . e . , denaturing ) . [SEP]
[CLS] in a qualitative comparison to the gel image shown in figure 8a , there was much less degradation of the smdh 4 building blocks in these samples into small fragments : most of the smdh 4 in the large nanoparticle B-nanoparticle samples and a large amount of those in the small sample stay intact . [SEP]
[CLS] ( b ) denaturing page - gel ( 12 % ) image of the nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle that have been capped with only one peg - functionalized strands after a 1 h treatment with dnase i ( 5 units / ml ) . [SEP]
[CLS] lanes 1 and 2 = large ( d h = 340 ± 50 nm ) and small ( d h = 24 ± 5 nm ) nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle that were capped with only one pegfunctionalized strand , respectively . [SEP]
[CLS] samples were run after particle dissolution . [SEP]
[CLS] in a qualitative comparison to the gel images shown in figures 8a and 9a , the smdh 4 building blocks in these samples are slightly less protected than those in the dually peg - capped samples but much better protected than those in the unprotected samples . [SEP]
[CLS] synthesis of alkyne - modified ssdna on cpg beads - the alkyne - modified ssdna on cpg beads were synthesized according to a previously published procedure . [SEP]
[CLS] syntheses were carried out from the 3 ′ direction using controlled pore glass ( cpg ) beads possessing 1 μmol of either adenine ( glen research , da - cpg # 20 - 2001 - 10 , ( 1000 a , 38 μmol / g ) ) or thymine ( glen research , dt - cpg # 20 - 2031 - 10 ( 1000 a , 26 μmol / g ) ) attached to the surface . [SEP]
[CLS] the cpg beads were placed in a 1 μmol synthesis column and 3 ′ phosphoramidites ( glen research , da - ce phosphoramidite # 10 - 1000 - c5 , ac - dc - ce phosphoramidite # 10 - 1015 - c5 , dmf - dg - ce phosphoramidite # 10 - 1029 - c5 , dt - ce phosphoramidite # 10 - 1030 - c5 ) and 5 ′ - hexynyl phosphoramidite ( glen research , # 10 - 1908 - 90 ) were then added using the standard 1 μmol protocol on an expedite 8909 synthesizer to make the cpg - 3 ′ - ssdna - c 4 - alkyne ( see si , table [SEP]
[CLS] 1 . assembly of nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle from smdh 4 and its complementary partner [SEP]
[CLS] size - tunable particles can be obtained by slow ( [UNK] . 2 °c / min ; right ) or fast ( [UNK] °c / min ; left ) cooling of a " hot " assembly solution ( total [ dna ] = 15 μm , t = 90 °c ) , respectively . [SEP]
[CLS] the " diamond lattice " drawings that were embedded in the spherical cartoons of the nanoparticles B-nanoparticle are only ideal representations of the network and should not be taken literally . [SEP]
[CLS] effect of smdh concentration on the size of assembled nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle in tbs . [SEP]
[CLS] the nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle were prepared with a 1 : 1 ratio of smdh 4 and its complementary partner using an established dna hybridization method ( see experimental section ) . [SEP]
[CLS] the average sizes of the particles were determined by ( a ) dls and ( b ) stem . [SEP]
[CLS] the concentration values listed above the dls graphs and stem images are the total dna concentrations present ( 4 × [ smdh 4 ] , as there are four dna strands in each smdh 4 molecule ) in the assembly solution . [SEP]
[CLS] the " control " sample was a smdh 4 that hybridized with 4 equiv of complementary ssdna under the same assembly condition . [SEP]
[CLS] effect of the assembly time on the size of assembled nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] equimolar solutions of smdh 4 and its complementary partner ( total [ dna ] = 10 μm ) were heated at 90 °c for 10 min in a thermal cycler and then cooled down at different rates as indicated above the dls graphs ( a ) and stem images ( b ) of the samples . [SEP]
[CLS] effect of [ nacl ] on the size of assembled nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] ( a ) d h data for particles prepared by combining equimolar quantities of the smdh components ( total [ dna ] = 15 μm ) in solutions with different concentrations of na + ions B-material ( [ nacl ] = 25 , 50 , 100 , 150 , and 300 mm ) . [SEP]
[CLS] data for the particles prepared with total [ dna ] = 10 μm can be found in the si , figure s8 . [SEP]
[CLS] ( b ) d h data for particles prepared by combining equimolar quantities of the smdhs at different dna concentrations ( total [ dna ] = 2 , 5 , 10 , and 15 μm ) and at [ nacl ] = 300 mm . [SEP]
[CLS] the [ nacl ] and total [ dna ] are indicated above each graph and the d h of the particles was determined by dls . [SEP]
[CLS] ( left ) schematic illustration of the capping - stabilization experiment . [SEP]
[CLS] ( right ) comparative d h data ( determined by dls ) for nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle formed from an equimolar mixture of smdh 4 and its complementary partner ( total [ dna ] = 15 μm ) at rt without ( top ) and with ( bottom ) capping dna ( added [ dna ] = 7 . 5 μm ) . [SEP]
[CLS] the incubation B-technique times are indicated above each graph ( wk = week ) . [SEP]
[CLS] orthogonal incorporation of two dyes ( af647 and cy3 ) into nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] the particles were first prepared from an equimolar mixture of smdh 4 and its complementary partner ( total [ dna ] = 10 μm ) . [SEP]
[CLS] they were then sequentially mixed with ( a ) af647 - labeled and ( b ) cy3 - labeled capping dna strands ( added [ dna ] = 5 μm each ) at room temperature . [SEP]
[CLS] clsm images were obtained at each step as shown on the right side of each panel ( scale bars are 2 μm ) . [SEP]
[CLS] in panel b , the left and right images at the top are obtained at the fluorescent B-property emission wavelengths for af647 ( shown as red ) and cy3 ( shown as green ) dyes , respectively ; the image at the bottom is the colocalized fluorescence B-property image of these top two , suggesting effective orthogonal functionalization . [SEP]
[CLS] ( a , b ) clsm images of skov - 3 cells B-material after 24 h exposure to either nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle capped with an af647 - conjugated ssdna ( total [ dna ] = 15 μm ; capping [ dna ] = 75 nm ; d h = 24 ± 5 nm ) ( top image ) or free af647 - conjugated ssdna ( [ dna ] = 75 nm ) ( bottom image ) . [SEP]
[CLS] the visible red - color regions in the top image indicate high uptake of af647 - capped nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle into cells B-material in contrast to minimal uptake of the free af647 - conjugated ssdna in the bottom image . [SEP]
[CLS] ( c ) quantitative flow cytometric profiles showing that cellular internalization of the af647capped nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle ( red ) is ten times that of the free af647conjugated ssdna ( gray ) ( 2200 vs 230 au , respectively ) . [SEP]
[CLS] ( a ) denaturing page - gel ( 12 % ) image of the nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle and a control duplex - hybridized smdh 4 after a 1 h treatment with dnase i ( 5 units / ml ) . [SEP]
[CLS] the control is a discrete smdh 4 that was initially hybridized with a complementary free ssdna strand prior to dnase i treatment . [SEP]
[CLS] from left to right : lane 1 = ssdna ladder ; lane 2 = control duplex - hybridized smdh 4 ; lane 3 = large ( d h = 340 ± 50 nm ) nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle , and lane 4 = small ( d h = 24 ± 5 nm ) nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle [SEP]
[CLS] the samples were run after particle dissolution ( i . e . , denaturing ) , showing a significant amount of the smdh 4 building blocks remain intact in the two nanoparticlederived samples . [SEP]
[CLS] in contrast , the smdh 4 component of the control has completely degraded . [SEP]
[CLS] ( b ) denaturing page - gel ( 12 % ) image of our nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle after particle dissolution , showing that virtually all of the smdh 4 building blocks remain intact during the particle formation process . [SEP]
[CLS] from left to right : lane 1 = large ( d h = 340 ± 50 nm ) nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle and lane 2 = small ( d h = 24 ± 5 nm ) nucleic acid - based polymeric B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] small - sized ( ~ 65 nm ) doxorubicin B-material ( dox ) - loaded polymeric B-nanoparticle nanoparticles I-nanoparticle ( pnps ) were modified with oligonucleotides to form colloidally stable dox - loaded polymeric spherical nucleic B-material acid I-material ( dox - psna ) nanostructures in biological media . [SEP]
[CLS] the nucleic B-material acid I-material shell B-material facilitates the cellular uptake of dox - psna , resulting in in - vitro cytotoxicity B-property against skov3 cancer cells B-material . [SEP]
[CLS] polymeric B-nanoparticle nanoparticles I-nanoparticle ( pnps ) prepared from amphiphilic B-property romp block copolymers ( romp = ring - opening metathesis polymerization ) represent a versatile class of multifunctional nanoconstructs for biodetection , imaging , and drug delivery . [SEP]
[CLS] in particular , the ease with which these nanoparticles B-nanoparticle can be surface - functionalized with a multitude of biologically active entities has shown promise in many drug delivery applications . [SEP]
[CLS] previously , we reported that romp - based doxorubicin - loaded pnps can be modified with cationic B-material surface moieties post - assembly to improve their stability in buffered media and in vitro cellular uptake . [SEP]
[CLS] however , the large diameters of these particles ( ~ 100 - 200 nm ) may limit their cellular uptake and tumor B-material penetration when delivered in vivo . [SEP]
[CLS] thus , we set out to improve the bioavailability B-property of the doxorubicin - loaded pnp platform by reducing nanoparticle B-nanoparticle size and covalently modifying the surface with negatively charged oligonucleotides , which have been shown to promote higher cellular uptake . [SEP]
[CLS] since sub - 100 nm - sized nanoparticles B-nanoparticle have been shown to exhibit increased cellular uptake and better penetration into tumor B-material tissue , these two modifications may allow for extended stability under physiologically relevant conditions and should enhance overall efficacy for in vivo applications . [SEP]
[CLS] the high - density surface modification of nanoparticles B-nanoparticle with oligonucleotides results in a three - dimensional architecture known as a spherical nucleic B-material acid I-material ( sna ) . [SEP]
[CLS] the dense oligonucleotide shells B-material in snas have been shown to stabilize a broad range of nanoparticle B-nanoparticle cores B-material - from inorganic au , ag , and silica ones to organic structures such as ultra - small liposomes B-nanoparticle , proteins B-material , and polymers B-material - under physiological conditions via electrostatic repulsion . [SEP]
[CLS] the sna architecture also promotes superior cellular internalization for the nanoparticle B-nanoparticle cores B-material over peg - functionalized analogs and linear nucleic B-material acids I-material , given its ability to bind to cellular membrane - bound scavenger receptors , which facilitate cellular internalization via endosomal pathways [SEP]
[CLS] as a result , sna structures have been utilized in a wide variety of applications spanning biodetection , gene regulation , drug delivery , and immunomodulation B-property . [SEP]
[CLS] by covalently modifying small ( sub - 100 nm ) doxorubicin - loaded pnps with oligonucleotides ( scheme 1 ) , we show herein that the resulting doxorubicin - loaded polymeric sna ( dox - psna ) structures are stable under physiologically relevant conditions , are capable of active cellular internalization , and exhibit cytotoxicity B-property with respect to skov - 3 ovarian cancer cells B-material . [SEP]
[CLS] while the potency of these dox - psna nanoparticles B-nanoparticle is comparable to that of the free drug , they should not be prone to the renal clearance issues that plague small molecules . [SEP]
[CLS] in an inert - atmosphere glovebox , monomer B-material 2 ( 25 mg , 45 . 2 μmol ; see si , section s2 for the synthesis of monomer B-material 2 ) was dissolved in an anhydrous mixture of chcl 3 / meoh ( 9 : 1 v / v , 1 ml ) in a 20 ml scintillation vial equipped with a magnetic B-property stir bar . [SEP]
[CLS] a stock solution of grubbs ' first - generation catalyst B-property ( 5 mg ) in chcl 3 / meoh ( 9 : 1 v / v , 5 ml ) was prepared , a portion of which ( 2 . 48 ml , 3 . 01 μmol ) was added to the vial containing the solution of monomer B-material 2 under vigorous stirring . [SEP]
[CLS] the resulting reaction mixture was stirred for 45 min at room temperature , at which time an aliquot ( 100 μl ) was removed and quenched with excess ethyl vinyl ether B-material . [SEP]
[CLS] a portion of this quenched aliquot was evaporated to dryness , redissolved in cdcl 3 , and analyzed by 1 h nmr spectroscopy B-technique , which indicated complete consumption of the monomer B-material . [SEP]
[CLS] the remaining portion was evaporated to dryness , dissolved in hplc - grade thf , and subjected to gpc analysis ( m n = 9000 ( theoretical m n = 8000 ) , pdi = 1 . 13 ) . [SEP]
[CLS] immediately after aliquot removal , a solution of monomer B-material 1 ( see si , section s1 , 30 . 7 mg , 45 . 2 μmol ) in chcl 3 / meoh ( 9 : 1 v / v , 1 ml ) was added to the reaction vial and the resulting polymerization mixture was stirred for an additional 45 min before being terminated with the addition of ethyl vinyl ether B-material ( 1 ml ) . [SEP]
[CLS] the reaction mixture was added quickly into vigorously stirred cold ( −10 °c ) pentanes ( 200 ml ) , and the resulting precipitate was isolated via vacuum - filtration and washed thoroughly with fresh pentanes to afford the product copolymer quantitatively as a red solid . [SEP]
[CLS] see the si for the 1 h nmr spectrum , section s3 ) . [SEP]
[CLS] due to the lack of complete polymerization of monomer B-material 1 when being initiated as the first block , we did not attempt to synthesize the reverse block copolymer 1 15b - 2 15 . [SEP]
[CLS] to maximize the number of chemical handles that can subsequently be used for surface functionalization of the pnps , we employed a block copolymer with an equimolar ratio of monomers B-material 2 : 1 , while keeping the pnp diameter < 100 nm . [SEP]
[CLS] an aqueous suspension of the polymer B-material nanoparticles B-nanoparticle was prepared by dialysis following a modification of the published procedure . [SEP]
[CLS] an aliquot ( 2 . 5 ml ) of a stock solution of block copolymer 2 15 - b - 1 15 ( 0 . 01 wt % ) in dmso was transferred to a 4 ml scintillation vial and stirred vigorously . [SEP]
[CLS] ultrapure deionized B-material water I-material was added to this stirring copolymer solution at a rate of 1 drop ( 50 μl , 1 . 75 wt % ) every 1 s using a 20 - 100 μl micro - pipette until the mixture contained 18 wt % water B-material . [SEP]
[CLS] the resulting cloudy mixture was placed in a 3 ml dialysis cassette and dialyzed against ultrapure deionized B-material water I-material ( 500 ml ) , with the dialate changed every 2 h . [SEP]
[CLS] complete absence of dmso in the dialysate after 48 h was verified by uv - vis spectroscopy B-technique as indicated by the disappearance of the uv cut - off for dmso at 268 nm . [SEP]
[CLS] dls analysis of the final pnp aqueous suspension derived from block copolymer 2 15 - b - 1 15 revealed relatively monodisperse pnps with an average d h = 63 ± 7 nm and a pdi = 0 . 11 ( figure 1b ) . [SEP]
[CLS] tem analysis indicated a uniform size distribution for the pnps in the solid - state , with an average diameter of ~ 65 nm ( figure 1c ) that is consistent with the dls data . [SEP]
[CLS] the zeta B-property potential I-property of these pnps was determined to be −6 . 83 ± 0 . 5 mv . [SEP]
[CLS] the average concentration of pnp particles is ~ 1 × 10 10 particles / ml , as determined by nta . [SEP]
[CLS] the doxorubicin B-material loading for the pnps is 64 wt % , as calculated based on the doxorubicin B-material wt % in the 2 15 - b - 1 15 copolymer . [SEP]
[CLS] in a 1 . 5 ml safe - lock eppendorf tube , solutions of n - ( 3 - dimethylaminopropyl ) - n ′ ethylcarbodiimide hydrochloride ( edc • hcl , final concentration in reaction solution = 5 μm ) and n - hydroxysulfosuccinimide ( sulfo - nhs , final concentration in reaction solution = 7 . 5 μm ) in ultrapure deionized B-material water I-material were added to an aliquot ( 1 ml ) of the carboxyfunctionalized pnp suspension ( 1 × 10 10 particles / ml , ~ 125 , 000 carboxylate B-material groups I-material ) derived from the desired block copolymer . [SEP]
[CLS] the reaction mixture was agitated ( 1000 rpm ) on a multi - therm platform shaker ( benchmark scientific , inc . , south plainfield , nj , usa ) for 5 min at room temperature . [SEP]
[CLS] a freshly prepared aliquot of 3 ′ - amino - terminated oligonucleotides ( 100 μl of a 10 μm solution ) was then added to the reaction mixture , and the resulting mixture was covered with aluminum foil and agitated on a multi - therm platform shaker for 4 h at room temperature . [SEP]
[CLS] to increase the density of oligonucleotide packing on the pnp surface , edc and nhs solutions ( to 5 μm and 7 . 5 μm final concentrations , respectively , assuming that all the initially added reagents have been consumed / decomposed ) were added to the pnp solution , and the resulting mixture was subjected to a salt - aging process . [SEP]
[CLS] aliquots of nacl ( 8 μl of a 5 m solution to attain a final [ nacl ] = 50 mm ) were added to the reaction mixture every 30 min , and the eppendorf tube was sonicated for 15 s ( branson 2510 ultrasonic cleaner , branson ultrasonics , danbury , ct , usa ) after each salt B-material addition . [SEP]
[CLS] upon reaching a total nacl concentration of 300 mm , the reaction mixture was agitated on a multi - therm platform shaker for an additional 12 h at room temperature . [SEP]
[CLS] the resulting psnas were then purified using size - exclusion chromatography B-technique ( sepharose cl - 4b , sigma aldrich ) . [SEP]
[CLS] the average concentration of psna particles was ~ 10 9 / ml , as determined by nta . [SEP]
[CLS] dls analysis of the final dna - functionalized pnp aqueous suspension derived from block copolymer 3 15 - b - 5 15 revealed relatively monodisperse pnps with an average d h = 88 ± 3 nm and a pdi = 0 . 13 . [SEP]
[CLS] the zeta B-property potential I-property of the oligonucleotide - modified pnps was determined to be −31 . 2 ± 3 . 2 , indicating that negatively charged oligonucleotides are conjugated to the pnp surface . [SEP]
[CLS] for tem analysis , the pnps were pre - stained with uranyl acetate solution ( 10 μl , 2 % w / v ) . [SEP]
[CLS] to assess the degree of oligonucleotide surface modification , an aliquot ( 20 μl ) of the purified cy5 - oligonucleotide - modified pnps was added to dmso solution and placed on a multi - therm shaker for 2 h at 37 °c to dissolve the psnas . [SEP]
[CLS] an aliquot ( 50 μl ) of the resulting solution was further mixed with dmso ( final volume = 200 μl ) , and its fluorescence B-property at the emission wavelength of cy5 ( λ ex = 649 nm , λ em = 670 nm ) was recorded using a synergy ht multi - mode microplate reader ( biotek , winooski , vt , usa ) . [SEP]
[CLS] this fluorescence B-property was compared against a calibration curve constructed from absorbance values for solutions of the cy5 - labeled oligonucleotides in dmso ( concentrations = 0 , 0 . 1 , 0 . 25 , 0 . 5 , 1 , 2 . 5 , 5 μm ) . [SEP]
[CLS] from this analysis and the number of psna nanoparticles B-nanoparticle in solution ( obtained from nta ) , each cholesterol - containing psna particle was estimated to have ~ 1900 oligonucleotides strands and each doxorubicin - loaded psna particle was estimated to have ~ 1200 oligonucleotides strands . [SEP]
[CLS] the doxorubicin B-material loading is 63 . 8 wt % , as calculated based on the doxubicin B-material wt % in the 2 15 - b - 1 15 copolymer and the total weight of the dna , assuming an average density of 1 for the whole construct . [SEP]
[CLS] two separate aliquots of the doxorubicin B-material - containing pnps and psnas ( 500 μl ) were transferred to a 1 . 5 ml safe - lock eppendorf tube and centrifuged for 30 min at 10k rpm to a solid pellet . [SEP]
[CLS] the supernatant was removed and the pnps were re - suspended in pbs ( 500 μl , 10 mm , ph 7 . 4 , 150 mm [ nacl ] ) . [SEP]
[CLS] as expected , characterization of the pnp suspension in pbs resulted in loss of the original well - defined morphology of the parent pnps as observed by tem and dls . [SEP]
[CLS] the size of psnas was analysed periodically over a two month period using dls . [SEP]
[CLS] we note in passing that while single - stranded dna has been adsorbed to the surface of polymer B-material nanoparticles B-nanoparticle through non - covalent modification , such constructs have not been tested for stability in cell B-material culture media . [SEP]
[CLS] melt analysis experiments were carried out using a cary 5000 uv - vis spectrometer equipped with a programmable heating stage ( thermofisher scientific , carlsbad , ca , usa ) . [SEP]
[CLS] doxorubicin - containing psnas functionalized with melt a strands were prepared as described above . [SEP]
[CLS] 13 nm au - core snas functionalized with the complementary melt b strands ( table s1 ) were prepared following literature protocols . [SEP]
[CLS] the aggregates were formed by combining these two materials in a 1 : 1 ratio ( total dna concentration = 1 . 5 μm , total volume = 1 ml ) . [SEP]
[CLS] the aggregates were then subjected to a temperature ramp from 20 to 65 °c ( rate of 0 . 25 °c / min ) while the absorbance for the aggregates was continuously monitored at 260 nm . [SEP]
[CLS] skov - 3 cells B-material were purchased from american tissue culture collection ( atcc , manassas , va , usa ) and grown in mccoy ' s 5a medium ( invitrogen , carlsbad , ca , usa ) supplemented with 10 % heat - inactivated fetal bovine serum , penicillin ( 100 iu / ml ) , and streptomycin ( 50 μg / ml ) and maintained at 37 °c with 5 % co 2 as per atcc instructions . [SEP]
[CLS] for cellular studies , the cells B-material were plated at 60 % confluency 24 h prior to the treatment . [SEP]
[CLS] in a typical experiment , skov - 3 cells B-material were plated on a 35 mm fluorodish ( world precision instruments , saratosa , fl , usa ) and incubated B-technique for 24 h before being transfected with the appropriate oligonucleotide formulation ( final dna concentration = 0 . 1 μm ) . [SEP]
[CLS] the treated cells B-material were incubated B-technique for 24 h before being washed three times with 1× phosphate - buffered saline ( pbs , invitrogen ) and imaged under a zeiss lsm 510 inverted laser - scanning confocal microscope using hoechst 33342 as a nuclear stain . [SEP]
[CLS] the fluorescence B-property excitation for cy5 was set as 630 nm and emission 650 - 710 nm . [SEP]
[CLS] for z - stack images , the images were collected in different planes with a depth of 0 . 3 μm . [SEP]
[CLS] the cellular uptake of the psnas was compared to that of the free drug in skov3 cells B-material . [SEP]
[CLS] in preparation for this analysis , the cells B-material were seeded at 60 % confluency in a 96 well plate 24 h before treatment with psnas and free doxorubicin B-material . [SEP]
[CLS] the treatments were carried out at 0 . 1 , 0 . 5 , 1 , and 2 . 5 μm of doxorubicin B-material . [SEP]
[CLS] the cells B-material were then incubated B-technique with either free drug or the psnas for 24 h before washing three times with 1× pbs . [SEP]
[CLS] the cells B-material were then detached from the plate surface by exposing to trypsin ( 20 μl ) for 5 mins and fixed using a 4 % paraformaldehyde solution ( in 1× pbs ) . [SEP]
[CLS] the intracellular delivery of doxorubicin B-material was quantified via flow B-technique cytometry I-technique on a guava easycyte 8ht instrument ( millipore , billerica , ma , usa ) using the doxorubicin B-material channel and normalized based on the intensity of the untreated cells B-material . [SEP]
[CLS] skov - 3 ( ovarian cancer ) cells B-material were seeded in 96 well plates at a density of ~ 5000 cells B-material per well 24 h before the experiment . [SEP]
[CLS] cells B-material were incubated B-technique with a fixed amount ( 100 μl ) of solution containing either doxorubicin B-material or dox - psnas that were prepared at varying concentrations in optimem ( invitrogen ) at 37 °c . [SEP]
[CLS] after overnight incubation B-technique , the media was replaced with regular growth medium ( mccoy ' s 5a supplemented with 10 % heatinactivated fetal bovine serum , penicillin ( 100 iu / ml ) , and streptomycin ( 50 μg / ml ) ) , and the cells B-material were incubated B-technique at 37 °c for an additional 48 h . [SEP]
[CLS] cell B-property viability I-property was assessed using the alamarblue® cell B-technique viability I-technique assay I-technique ( thermofisher scientific , carlsbad , ca , usa ) according to the manufacturer ' s recommended protocol . [SEP]
[CLS] briefly , 10 % alamarblue® reagent in regular growth medium was added to cells B-material ( 100 μl per well ) , and the treated cell B-material plate was incubated B-technique at 37 °c for 4 h before being analyzed on a synergy h4 multimode microplate reader . [SEP]
[CLS] fluorescence B-property data at 570 nm were collected and normalized to that for an untreated control . [SEP]
[CLS] reported values represent the mean ± standard deviation of 5 replicates . [SEP]
[CLS] as a general strategy , romp - based pnps were assembled from short amphiphilic B-property block copolymers consisting of one hydrophobic B-property doxorubicin - conjugated block and a hydrophilic B-property carboxy - terminated , poly ( ethylene oxide B-material ) ( peo ) - loaded block ( figure 1 ) . [SEP]
[CLS] with this design , the doxorubicin - loaded block constitutes the core B-material of the pnps , and the peo - loaded segment forms a shell B-material with terminal carboxy groups that can be used for further surface modification . [SEP]
[CLS] the doxorubicin B-material payload was linked to the hydrophobic B-property segment through an acid - cleavable linkage B-property , which can be released in the low - ph environment of the late endosome or in the tumor B-material tissue [SEP]
[CLS] the amphiphilic B-property block copolymers needed for pnp preparation were synthesized by blockcopolymerizing the carboxy - terminated , peo - conjugated monomer B-material 2 and the doxorubicinconjugated monomer B-material 1 ( figure 1a ) . [SEP]
[CLS] a 15 : 15 molar ratio for monomers B-material 2 : 1 was chosen to limit the molecular weight of the polymer B-material < 45 kda , thus facilitating renal clearance of the polymers B-material post drug release . [SEP]
[CLS] monomer B-material 2 was successfully homo - polymerized using grubbs ' first - generation olefin metathesis ( ( pcy 3 ) 2 cl 2 ru = chph ) catalyst B-property ( 3 ) as confirmed by h nmr spectroscopy B-technique and gel - permeation chromatography B-technique ( gpc ) analyses ( m n = 9000 ( theoretical m n = 8000 ) , polydispersity index ( pdi ) = 1 . 17 ; supporting information ( si ) , section s2 ) . [SEP]
[CLS] sequential [SEP]
[CLS] results and discussionaddition of monomer B-material 1 to the homo - polymerized block 2 15 , followed by quenching with ethyl vinyl ether B-material resulted in the formation of our desired monodisperse amphiphilic B-property block copolymer 2 15 - b - 1 15 ( final polymer m n = 15000 ( theoretical m n = 14000 ) , pdi = 1 . 12 ; see materials and methods section and si , section s3 ) . [SEP]
[CLS] following our previously established strategy , dropwise addition of water B-material to a dmso solution of copolymer 2 15 - b - 1 15 , followed by exhaustive dialysis against ultrapure deionized B-material water I-material , afforded an aqueous suspension of carboxy - functionalized , doxorubicin - loaded pnps ( figure 1a ) . [SEP]
[CLS] these nanoparticles B-nanoparticle have small hydrodynamic diameters ( d h = 63 ± 7 nm , figure 1b ) and exhibit a narrow size distribution ( pdi = 0 . 11 ± 0 . 02 ) , as measured by dynamic B-technique light I-technique scattering I-technique ( dls ) . [SEP]
[CLS] transmission B-technique electron I-technique microscopy I-technique ( tem ) also confirmed their homogeneous spherical morphology ( figure 1c ) with an average d h of 65 ± 5 nm . [SEP]
[CLS] key to the realization of these small nanoparticle B-nanoparticle sizes was a fast rate of water B-material addition over a shorter time period than previously reported ( see materials and methods section ) . [SEP]
[CLS] the covalent surface modification of the carboxy - functionalized pnps with 3 ′ - amineterminated dna was achieved using edc / nhs coupling ( figure 1a ) . [SEP]
[CLS] a mixture of the two components was stirred together with the edc and nhs reagents for 4 h before being subjected to a salt - aging process to further increase the dna density on the pnp surface ( see materials and methods section ) . [SEP]
[CLS] after the total salt B-material concentration reached 0 . 3 m , this mixture was allowed to react further overnight before being purified by size - exclusion chromatography B-technique . [SEP]
[CLS] a tem image ( figure 1d ) of the isolated psna clearly shows the retention of the discrete and spherical morphology of the parent pnp ( diameter ~ 87 nm ) . [SEP]
[CLS] their dls - derived size distribution plot ( figure 1b ) shows an increase in the hydrodynamic diameter ( d h = 88 ± 3 nm and pdi = 0 . 13 ± 0 . 03 ) that can be attributed to the addition of a dense dna shell B-material on the pnp surfaces . [SEP]
[CLS] the shift in zeta B-property potential I-property ( −5 . 1 ± 1 . 5 mv for the parent pnps ) to a higher negative value ( −31 . 2 ± 3 . 2 mv for the psnas ) also corroborates the successful conjugation of dna to the pnp surface . [SEP]
[CLS] notably , the presence of the dna shell B-material on the surface of the pnp can be clearly observed upon staining of the psnas with uranyl acetate ( z - contrast and transmission modes ; figure 1d ) . [SEP]
[CLS] while not a primary focus of the current work , the thickness of the dna shell B-material can be changed by altering the length and composition of the oligonucleotide strands . [SEP]
[CLS] on average , ~ 1200 dna strands / pnp were conjugated to the surface of each pnp as determined by fluorescence B-technique spectroscopy I-technique and nanoparticle B-nanoparticle tracking analysis ( nta ) to quantify the number of psnas in a given sample via light scattering and brownian motion . [SEP]
[CLS] this dna surface density ( ~ 7 pmol / cm 2 , see materials and methods section for calculation ) is comparable to but slightly higher than the loadings obtained for our previously reported liposomal B-nanoparticle sna constructs . [SEP]
[CLS] the doxorubicin - loaded psnas show excellent stability in comparison to the parent pnps , a property that enables their use in cellular systems . [SEP]
[CLS] for example , the psnas remain colloidally stable after 2 months of incubation B-technique in phosphate - buffered saline ( 1× pbs ) at room temperature ( figure 2a ) . [SEP]
[CLS] in contrast , subjecting the unmodified pnps to the same treatment for only a week resulted in formation of polydisperse aggregates irreversibly agglomerated , presumably through interparticle fusion . [SEP]
[CLS] most relevant for physiological applications , no change was observed in the dls distribution plot for the psnas after 3 days of incubation B-technique at 37 °c in 1× pbs ( figures 2c and 2d ) . [SEP]
[CLS] we attribute this enhanced stability to the electrostatic repulsion between the negatively charged oligonucleotides that comprise the shell B-material of the psna , which inhibits particle - particle fusion under physiologically relevant conditions . [SEP]
[CLS] notably , dox - psnas remain stable in the presence of human serum while still maintaining their acid - triggered drug - release properties . [SEP]
[CLS] the doxorubicin - based fluorescence B-property profiles for dox - psnas after being incubated B-technique at 37 °c in pbs and human serum solution ( 10 % v / v ) are very similar as evidenced by minimal leakage of the drug ( si , figure s8 ) . [SEP]
[CLS] in contrast , exposing dox - psnas to 0 . 1 m hcl ( aq ) leads to a six - fold increase in drug release after one hour ( si , figure s8 ) . [SEP]
[CLS] consistent with the assigned sna structure , psnas cooperatively hybridize with complementary au core snas . [SEP]
[CLS] for example , when psnas were combined with complementary 13 nm au - core snas ( figure 3a ) , visible aggregates can be observed and isolated . [SEP]
[CLS] tem analysis ( figures 3b and 3c ) of these hybridized materials clearly shows a complete coverage of the larger psnas by the smaller au - core snas . [SEP]
[CLS] the increase in the melting temperature ( t m of the pnp - aunp B-nanoparticle hybrid = 54 . 7 °c vs t m = 52 °c ( as calculated at 150 mm nacl and 1 . 5 um oligonucleotides using idt oligoanalyzer 41 ) for the free dna duplex analog ) of these aggregates and their narrow thermal denaturation profile ( full width at half - maximum ( fwhm ) of the first derivative is [UNK] . 3 °c ) confirms cooperative binding behavior that is characteristic of snas 15 ( figure 3d ) [SEP]
[CLS] as expected , the dense oligonucleotide shell B-material of the dox - psnas greatly facilitates their entrance into cells B-material . [SEP]
[CLS] as shown by confocal microscopy B-technique , skov - 3 cells B-material ( ovarian cancer ascites ) readily uptake dox - psnas that were surface - modified with 5 ′ - cy5 - labelled oligonucleotides ( figure 4a ) . [SEP]
[CLS] this is in stark contrast to the non - functionalized pnp analog , which settles out of cell B-material culture media due to aggregation and shows low and non - uniform uptake . [SEP]
[CLS] such enhanced cellular internalization of sna structures has been attributed by mirkin and coworkers to the interaction of the oligonucleotide shell B-material with class a scavenger receptors ( sr - a ) present on the cell B-material surface , followed by caveolin - mediated endocytosis B-event . [SEP]
[CLS] because our goal was to enhance the delivery of the therapeutically active payload of dox - psnas to diseased cells B-material , a generic t 20 dna sequence was sufficient for this purpose . [SEP]
[CLS] we note in passing that although a detailed mechanistic study comprising competitive and endocytosis B-event inhibition assays can be carried out to ascertain the exact uptake mechanism , such a study is beyond the scope of the present work . [SEP]
[CLS] the cellular internalization properties of the dox - loaded psnas ( dox - psnas ) lead to invitro cytotoxicity B-property against skov3 cancer cells B-material that is comparable to that of free doxorubicin B-material ( figure 4b ) . [SEP]
[CLS] such a study could not have been properly carried out with the parent pnps due to their aggregation behavior in biological media . [SEP]
[CLS] indeed , pnps have been observed to settle out of cell B-material culture media over time , leading to low and non - uniform uptake in sknsh wildtype cells B-material . [SEP]
[CLS] the observed cytotoxicity B-property of the dox - psnas can be attributed to the acid - induced cleavage of the carbamate linkage B-property between the block copolymer backbone and the doxorubicin B-material , allowing for the release of the latter into the cytoplasm . [SEP]
[CLS] supporting this conclusion is the observation that the analogous psnas with cholesterol - loaded cores B-material does not show any cytotoxicity B-property against skov3 cells B-material ( si , section s5 ) . [SEP]
[CLS] in summary , we have demonstrated that the successful modification of doxorubicin - loaded pnps with a dense oligonucleotide shell B-material greatly increases their colloidal stability in biological media under physiological conditions . [SEP]
[CLS] the dense shell B-material of oligonucleotides allows the resulting dox - psnas to have enhanced cellular uptake , manifesting in cytotoxicity B-property that is comparable to the free drug . [SEP]
[CLS] importantly , this sna - based strategy should be generalizable to other therapeutic - loaded nanoparticles B-nanoparticle for in vivo applications in the treatment of cancer and other diseases . [SEP]
[CLS] the covalent surface modification of carboxy - functionalized pnps with amine - terminated oligonucleotides ( green ) via amide B-material - coupling chemistry . [SEP]
[CLS] 1 . a ) synthesis of block copolymer 1 15 - b - 2 15 and subsequent formation of the corresponding pnps . [SEP]
[CLS] b ) dynamic B-technique light I-technique scattering I-technique ( dls ) data for the pnps before and after covalent dna functionalization . [SEP]
[CLS] c - d ) tem image of the pnps before ( c ) and after ( d ) dna functionalization . [SEP]
[CLS] a ) doxorubicin - loaded psnas remain stable for over two months in 1× pbs as analysed by tem and dls . b ) doxorubicin - loaded pnps aggregate when incubated B-technique in 1× pbs for a week . [SEP]
[CLS] c and d ) dls profiles for dox - psnas confirming their stablity in 1× pbs at 37 °c for 3 days . [SEP]
[CLS] a ) a schematic drawing of the dox - psnas hybridized to complementary 13 nm au - core snas . [SEP]
[CLS] b - c ) tem images of the dox - psnas surrounded by complementary au - core snas conjugates . [SEP]
[CLS] scale bar is 100 nm . [SEP]
[CLS] d ) the thermal denaturation profile ( red ) of the [ dox - psnas + au - core snas ] hybridized aggregates monitored as a change in extinction at 260 nm . [SEP]
[CLS] the overlaid 1 st derivative ( blue ) of this profile shows the sharp melting transition . [SEP]
[CLS] see si , section s10 for the control experiment . [SEP]
[CLS] 4 . a ) [SEP]
[CLS] confocal B-technique laser I-technique - I-technique scanning I-technique microscopy I-technique images of skov - 3 cells B-material that have been incubated B-technique for 24 h with free doxorubicin B-material ( dox ) and doxorubicin - loaded psnas ( dox - psnas ) at 2 . 5 μm dox concentration . [SEP]
[CLS] given the qualitative nature of the confocal image ( e . g . , change in focal point between sample wells ) , the various signal intensities should not be compared across rows . [SEP]
[CLS] in addition , each panel is limited to a few cells B-material and do not represent the entire population . [SEP]
[CLS] a statistically significant flow B-technique cytometry I-technique study ( si , figures10 ) does show higher doxorubicin B-material fluorescence B-property signal for the cells B-material that have been incubated B-technique with free doxorubicin B-material . [SEP]
[CLS] b ) cytotoxicity B-property profiles for skov - 3 cells B-material that have been incubated B-technique for 48 h with dox and dox - psna formulations at 5 nm - 10 μm [ dox ] . [SEP]
[CLS] the similarity in toxicity B-property between the doxorubicin - loaded nps B-nanoparticle and free doxorubicin B-material is not surprising given previous studies from our group , and the fact that ic 50 measurements are highly dependent on many variables ( treatment time , incubation B-technique time post - treatment , release of payload from the delivery vehicle B-material , cell B-material type and confluency ) and can vary by several fold , depending on conditions . [SEP]
[CLS] here , we report dna - induced polymer B-material segregation and dna island formation in binary block copolymer assemblies . [SEP]
[CLS] a dna diblock copolymer of polymethylacrylate - block - dna ( pma - b - dna ) and a triblock copolymer of poly ( butadiene ) - block - poly ( ethylene oxide B-material ) - block - dna ( pbdb - peo - b - dna ) were synthesized and each was co - assembled with a prototypical amphiphilic B-property polymer B-material of poly ( butadiene ) - block - poly ( ethylene oxide B-material ) ( pbd - b - peo ) . [SEP]
[CLS] the binary self - assembly of pma - b - dna and pbd - b - peo resulted in giant polymersomes with dna uniformly distributed in the hydrophilic B-property peo shell B-material . [SEP]
[CLS] when giant polymersomes were connected through specific dna interactions , dna block - copolymers migrated to the junction area , forming dna islands within polymersomes . [SEP]
[CLS] these results indicate that dna hybridization can induce effective lateral polymer B-material segregation in mixed polymer B-material assemblies . [SEP]
[CLS] the polymer B-material segregation and local dna enrichment has important implications in dna melting properties , as mixed block copolymer assemblies with low dna block copolymer contents can still exhibit useful dna melting properties that are characteristic of dna nanostructures with high dna density . [SEP]
[CLS] gold B-nanoparticle nanoparticles I-nanoparticle modified with a dense layer of oligonucleotides have been extensively studied for many applications ranging from materials syntheses to diagnostics and drug delivery [SEP]
[CLS] the most attractive characteristic of dna - modified gold B-material particles is their unique dna hybridization properties such as sharp melting transitions and high binding constants . [SEP]
[CLS] these unique properties originate from the cooperative interaction of densely packed dna strands , and thus they are independent of the core B-material composition . [SEP]
[CLS] therefore , researchers have developed ways to fabricate densely packed dna nanostructures without the gold B-material core B-material . [SEP]
[CLS] among them , dna block copolymers are a particularly promising building block to fabricate such dna nanostructures . [SEP]
[CLS] assemblies of dna block copolymers are composed of the polymer B-material core B-material and a high density dna corona , and thus show similar dna melting properties as dna - modified gold B-material particles mentioned above . [SEP]
[CLS] moreover , functional molecules or nanoparticles B-nanoparticle can be readily incorporated into the polymer B-material core B-material of dna block copolymer micelles B-material . [SEP]
[CLS] capitalizing on these attributes , dna block copolymers have been actively studied for various applications including drug delivery and gene therapy . [SEP]
[CLS] thirdly , the polymer B-material strands composing the assemblies can undergo strand rearrangement and exchange , which allows for dynamic morphology changes in response to various external stimuli . [SEP]
[CLS] an intriguing possibility arising from the strand rearrangement is the phase segregation and domain formation in mixed assemblies , which has not been previously investigated for dna block copolymers . [SEP]
[CLS] phase segregation is a common phenomenon found in lipid B-material bilayers I-material composing cell B-material membranes where different membrane components are segregated to form domains . [SEP]
[CLS] there are evidences indicating that such domain formation plays a critical role in many cellular functions such as signal transduction pathways , cell B-event adhesion I-event , cell migration , and synaptic transmission . [SEP]
[CLS] here , we fabricated giant vesicles from a dna diblock copolymer of polymethylacrylateblock - dna ( pma - b - dna ) and a prototypical block copolymer of poly ( butadiene ) - blockpoly ( ethylene oxide B-material ) ( pbd - b - peo ) ( scheme 1 ) . [SEP]
[CLS] we demonstrated that the two polymers B-material composing the giant polymersomes undergo efficient polymer B-material segregation upon the introduction of polymersomes containing complementary dna . [SEP]
[CLS] while such lateral segregation has been reported for lipids B-material , efficient dna - induced segregation in polymer B-material assemblies has remained elusive . [SEP]
[CLS] phase segregation in polymer B-material assemblies is typically much slower than that in lipid B-material bilayers I-material due to the entanglement of high molecular weight polymers B-material , and thus the segregation behavior can be quite different in the two systems . [SEP]
[CLS] the dna - induced polymer B-material segregation shown here generates high density dna domains on vesicle surfaces , which allows for cooperative dna B-event binding I-event even at low dna block copolymer contents . [SEP]
[CLS] to the best of our knowledge , this report is the first to show the efficient dna - induced segregation in polymer B-material assemblies , and to demonstrate how the phase behavior influences the molecular recognition properties of dna . [SEP]
[CLS] the stability and chemical diversity of polymers B-material together with the dna segregation observed here open up new possibilities in polymeric dna nanostructures . [SEP]
[CLS] an amphiphilic B-property dna block copolymer , pma - b - dna was synthesized through the coupling of carboxylic acid terminated polymethylacrylate ( pma , mn = 7800 kg mol −1 ) to 5 ′ - aminemodified 25 base oligonucleotide strands ( dna 1 : 5 ′ - a10 - atccttatcaatatt - fam - 3 ′ ) attached on solid supports ( figure 1a ) . [SEP]
[CLS] a green fluorescent B-property dye ( 6 - fam ) was attached at the 3 ′ end of dna to monitor the presence of dna . [SEP]
[CLS] typically , pyrene acrylate dyes were incorporated into pma at a ratio of one pyrene molecule per polymer B-material chain to track the presence of pma ( supporting information ) . [SEP]
[CLS] gel B-technique electrophoresis I-technique data show that dna block copolymers were successfully synthesized and purified from the crude product of dna block copolymers ( figure 1b ) ; as dna block copolymers form nanoscale assemblies in water B-material , they remain in the loading well , while unconjugated free dna strands move along the electric field . [SEP]
[CLS] the successful conjugation was also confirmed by the coexistence of the fingerprint - like absorption peaks of pyrene and the absorption peak of fam at 494 nm as well as the dna peak at 260 nm ( figure 1c ) . [SEP]
[CLS] based on the absorbance at 494 nm of 6 - fam and 335 nm of pyrene , the molar ratio of the two dye molecules was calculated to be 0 . 88 : 1 , which is close to the predesigned 1 : 1 ratio . [SEP]
[CLS] due to the amphiphilic B-property nature , pma - b - dna spontaneously form micelles B-material in water B-material after gel purification . [SEP]
[CLS] the diameter of the polymer B-material micelles B-material was determined to be 14 nm by dynamic B-technique light I-technique scattering I-technique ( dls ) ( figure 1d ) . [SEP]
[CLS] giant dna polymersomes were prepared by the film hydration of pma - b - dna and pbd - b - peo ( scheme 1 ) . [SEP]
[CLS] pbd - b - peo diblock copolymers can self - assemble into various structures , such as spherical micelles B-material , bilayers and cylindrical micelles B-material in water B-material , depending on the relative block ratio . [SEP]
[CLS] in this study , pbd 52 - b - peo 32 with the peo weight fraction ( w peo ) of 0 . 33 was used for the binary self - assembly , as pbd 52 - b - peo 32 readily forms giant vesicles by film hydration method ( figure s6a ) . [SEP]
[CLS] in typical experiments , pma - b - dna and pbd 52 - b - peo 32 were mixed at a molar ratio of 1 : 1600 in chcl 3 / dmso mixture ( 5chcl 3 : 1dmso ) . [SEP]
[CLS] the solution ( 60 μl ) was placed on the bottom of a glass vial and dried by a stream of n 2 gas , which generated a thin film of mixed polymers B-material on the bottom of the vial . [SEP]
[CLS] the film was further dried under vacuum overnight , and then hydrated in 500 μl of 0 . 1 m phosphate buffered saline ( pbs ) solution ( 100 mm nacl , 10 mm phosphate , ph = 7 . 17 ) . [SEP]
[CLS] the 12 hour incubation B-technique in the buffer produced suspensions of giant vesicles of the two polymers B-material . [SEP]
[CLS] s6b for more images ) of giant polymersomes formed with 0 . 062 % dna block copolymer , showing well - defined giant vesicles composed of the hydrophobic B-property inner layer of pbd and pma and the hydrophilic B-property corona of peo and dna ( scheme 1 ) . [SEP]
[CLS] green fluorescence B-property from vesicles indicates that dna block copolymers are incorporated into the vesicle membranes . [SEP]
[CLS] z - stack images of giant polymersomes obtained by immobilizing them onto a micropipette ( figure 2c ) shows uniform distribution of fam - labeled dna on the polymersome surface ( figure 2d ) . [SEP]
[CLS] two sets of giant dna polymersomes ( polymersome 1 and polymersome 1 ′ ) were prepared using complementary dna strands , dna 1 and dna 1 ′ ( scheme 2 ) . [SEP]
[CLS] the two sets of giant dna polymersomes were mixed together in 0 . 1 m pbs buffer to induce the hybridization of dna 1 and dna 1 ′ and consequently the aggregation of polymersomes ( scheme 2 ) . [SEP]
[CLS] optical microscope images taken after 16 hour incubation B-technique showed polymersome aggregates as expected ( figure 3a , figure s8a , b , figure s9 ) . [SEP]
[CLS] in our control experiment where polymersomes 1 and 1 ′ were mixed in water B-material , there was no obvious aggregation ( figure s10a , b ) . [SEP]
[CLS] to further confirm the duplex formation at the junction , ethidium bromide B-material , which is a commonly used reagent to visualize dna duplexes , was introduced to polymersome aggregates . [SEP]
[CLS] the orange fluorescence B-property observed at the junction area ( figure 3b , figure s8c , d ) confirms that the polymersome aggregates were formed through specific dna interactions . [SEP]
[CLS] interestingly , fluorescent B-property microscope images reveal that fam fluorescence B-property from dna is also localized at the junction between polymersomes ( figure 3c , d , figure s8a , b ) . [SEP]
[CLS] this result indicates that polymer B-material strands in the giant vesicles are mobile B-property and dna block copolymers accumulate at the junction area , creating dna islands on polymersome surfaces . [SEP]
[CLS] fluorescence B-property recovery after photobleaching ( frap ) measurements show that dna block copolymers in mixed polymersomes have lateral diffusivity ( figure s7 ) , which is consistent with the observation of dna - rich island formation . [SEP]
[CLS] the size of dna islands should mainly depend on the number of junctions per vesicles , and the diameter of the island ranged from sub - micrometers to a few micrometers . [SEP]
[CLS] the dna - induced lateral segregation of polymers B-material in this study occurred with overnight incubation B-technique , which is relatively fast , compared to previously studied phase segregation in polymersomes . [SEP]
[CLS] phase segregation in polymersomes is known to be much slower than in lipid B-material bilayers I-material due to the entanglement of large molecular weight polymers B-material . [SEP]
[CLS] for example , discher and coworkers reported that micrometer - sized polymersomes made of pbd - b - peo and poly ( butadiene ) - block - poly ( acrylic acid ) ( pbd - b - paa ) showed fully segregated polymer B-material domains after 40 hr incubation B-technique with the addition of cross - bridging polyvalent cations B-material . [SEP]
[CLS] in another example , nanometer - sized polymersomes made of pbd - b - peo and poly ( 2 - ( diisopropylamino ) ethyl methacrylate ) - block - poly ( ( 2methacryloyloxy ) ethyl phosphorylcholine ) ( pdpa - b - pmpc ) showed the evolution of surface patterns of phase segregation over the time - scale of more than a month . [SEP]
[CLS] we attribute the relatively efficient polymer B-material separation in mixed polymersomes observed here to the driving force of forming multiple dna linkages B-property between polymersomes . [SEP]
[CLS] the dna island formation shown in figure 3a is advantageous , as it indicates that the useful dna binding properties of dna block copolymer micelles B-material 8 might occur in the mixed assemblies with low dna content . [SEP]
[CLS] to examine how the polymer B-material segregation affects dna B-event binding I-event properties , we prepared a new set of mixed assemblies from a dna triblock copolymer of poly ( butadiene ) - block - poly ( ethylene oxide B-material ) - block - dna ( pbd - b - peo - b - dna ) ( scheme 3 ) . [SEP]
[CLS] this new design allows for the formation of binary assemblies where dna strands are not buried inside the peg layer , which can potentially destabilize dna duplexes . [SEP]
[CLS] the dna triblock copolymer , pbd - b - peo - b - dna was synthesized with dna 2 ( 5 ′ - atccttatcaatatt - fam - 3 ′ ) and pbd - b - peo ( mn : 3800 kg mol −1 , w peo : 0 . 34 ) , following the same procedure used for dna diblock copolymers ( see supporting information for details ) . [SEP]
[CLS] the synthesized polymers B-material were purified by gel B-technique electrophoresis I-technique ( figure 4a ) . [SEP]
[CLS] the purified dna triblock copolymers showed distinct dna absorption peak at 260 nm and 6 - fam peak at 480 nm ( figure 4b ) . [SEP]
[CLS] to prepare binary assemblies , pbd - b - peo - b - dna was mixed with pbd 46 - b - peo 30 at varying molar ratios ( 100 mol % , 50 mol % , 10 mol % of pbd - b - peo - b - dna ) in small amount of chcl 3 / dmso mixture ( 4chcl 3 : 1dmso , 50 μl ) . [SEP]
[CLS] suspensions of binary polymer B-material assemblies were prepared following the procedure described above for diblock copolymers . [SEP]
[CLS] the polymer B-material suspensions were extruded through a polycarbonate membrane filter with 400 nm pores to obtain uniform nanoscale assemblies for dna melting studies . [SEP]
[CLS] transmission B-technique electron I-technique microscopy I-technique ( tem ) images showed that small spherical assemblies were formed by the procedure ( figure s11a , b ) . [SEP]
[CLS] the diameters of the assemblies were determined to be 62 nm , 63 nm , 65 nm for 100 % , 50 % , 10 % samples , respectively , by dls ( figure s11c - e ) . [SEP]
[CLS] another dna triblock copolymer , polystyrene - block - poly ( ethylene oxide B-material ) - block - dna ( psb - peo - b - dna , mn : 7000 kg mol −1 , w peo : 0 . 29 ) and their assemblies were also prepared by the same procedure as pbd - b - peo - b - dna to investigate the effect of the core B-material polymer B-material ( see supporting information for synthesis and characterization data , figure s4 and figure s12 ) . [SEP]
[CLS] to investigate the melting properties of binary assemblies , pbd - b - peo - b - dna triblock copolymer assemblies were mixed with gold B-nanoparticle nanoparticles I-nanoparticle modified with dna 2 ′ ( 5 ′ - a 10 - aatattgataaggat - 3 ′ ) in 0 . 1 m pbs buffer , as illustrated in figure 5a . [SEP]
[CLS] the mixture was then incubated B-technique at 50 °c for 16 hr to facilitate the polymer B-material strand migration and dna duplex formation . [SEP]
[CLS] dna hybridization connect nanoparticles B-nanoparticle and polymer B-material assemblies together into macroscopic aggregates ( figure 5b , c ) . [SEP]
[CLS] the assembly process caused expected red - shift and broadening of 520 nm ( spr ) band of gold B-nanoparticle nanoparticles I-nanoparticle and corresponding red to purple color change ( figure 5b ) . [SEP]
[CLS] melting curves were obtained by monitoring the extinction of gold B-nanoparticle nanoparticles I-nanoparticle at 520 nm ( figure 6 ) . [SEP]
[CLS] a sharp melting transition was observed for the assemblies made of 100 mol % pbd - b - peo - b - dna triblock copolymer ( fwhm : 1 . 9 °c ) , as expected , due to the cooperative interaction of densely packed dna strands ( figure 6a , figure s15a ) . [SEP]
[CLS] melting curves from binary assemblies containing 50 mol % pbd - b - peo - b - dna ( figure 6b , figure s15b ) and 10 mol % dna block copolymers ( figure 6c , figure s15c ) showed slight broadening with fwhm values of 2 . 4 °c and 4 . 1 °c , respectively . [SEP]
[CLS] however , they still remain much sharper than that of plain dsdna ( fwhm : 9 . 8 °c , figure s16a , b ) . [SEP]
[CLS] the melting curves measured without the thermal annealing process showed fwhm of 2 . 2 °c , 2 . 8 °c and 6 . 7 °c for 100 % , 50 % and 10 % samples , respectively ( figure s13 ) , showing that the annealing process causes sharpening of melting transition for low dna content assemblies . [SEP]
[CLS] to further investigate the effect of polymer B-material segregation on melting behaviors , the same set of assemblies were prepared from ps - b - peo - b - dna and ps - b - peo containing a high glass temperature ( 100 °c ) polymer B-material , ps . [SEP]
[CLS] 6d presents the melting curve of the binary assembly containing 10 % ps - b - peo - b - dna , which indeed shows broader melting transition ( fwhm : 7 . 7 °c ) and lower melting temperature ( 45 . 3 °c ) than the assemblies with pbd core B-material ( figure 6c ) ( see figure s14 for more melting data ) . [SEP]
[CLS] the fwhm values and melting temperatures of mixed assemblies are summarized in table 1 . [SEP]
[CLS] these results support our hypothesis that the polymer B-material segregation ( figure 3 ) leads to cooperative dna B-event binding I-event and sharp melting transitions of dna in mixed assemblies . [SEP]
[CLS] unlike dna affixed on nanoparticle B-nanoparticle surface by covalent bonds , dna block copolymers can diffuse laterally as long as tg of the core B-material polymer B-material is sufficiently low . [SEP]
[CLS] in the assemblies made of ps , the polymer B-material strand migration should be much slower than those made of pbd , and therefore the melting transition becomes significantly broader as the dna block copolymer content is reduced . [SEP]
[CLS] on the other hand , for nonglassy polymers B-material , dna block copolymers can form locally concentrated dna islands at the binding sites , which allows for cooperative binding even at low dna contents . where dna block copolymers are uniformly distributed in the membrane . [SEP]
[CLS] note that dna block copolymer alone typically forms small micelles B-material in water B-material due to the highly charged dna backbone . [SEP]
[CLS] mixed assembly reported here provides a way to form various types of dna block copolymer assemblies that are difficult to make on its own . [SEP]
[CLS] interestingly , when the giant polymersomes with complementary dna strands were mixed together to induce aggregation of polymersomes , dna block copolymers segregated to the binding area , forming dna islands at the junction between polymersomes . [SEP]
[CLS] the efficient accumulation of dna block copolymers at the junction observed here compared to the typical phase segregation in polymersomes was attributed to the driving force of forming multiple dna linkages B-property between polymersomes . [SEP]
[CLS] this polymer B-material segregation has important consequences in dna melting properties of mixed assemblies . [SEP]
[CLS] binary micelles B-material formed from dna triblock copolymer of pbd - b - peo - b - dna and pbd - b - peo at varying dna triblock copolymer contents showed that the unique sharp melting transition of dna block copolymer micelles B-material is maintained in micelles B-material with low dna block copolymer contents ( i . e . , 50 % , 10 % ) . [SEP]
[CLS] the type of hydrophobic B-property polymer B-material affected the melting behavior of binary assemblies . [SEP]
[CLS] the assemblies made from ps - b - peo - b - dna and ps - b - peo , which contains a glassy ps core B-material , showed broader melting transitions at low dna block copolymer contents . [SEP]
[CLS] this result supports that the segregation of dna block copolymers affect their dna melting properties . [SEP]
[CLS] note that it is advantageous to use low dna block copolymer content in forming dnadecorated polymer B-material nanostructures , as dna block copolymers are more costly to make than the matrix amphiphilic B-property polymers B-material . [SEP]
[CLS] we believe that this work is the first to demonstrate the efficient dna - induced polymer B-material segregation in mixed assemblies and to show how it affects the dna melting properties . [SEP]
[CLS] the findings of this study demonstrate that the binary assemblies of dna block copolymers and other commonly used amphiphilic B-property polymers B-material provides an opportunity to form various types of assembly structures that are difficult to make by dna block copolymer by itself without losing its excellent dna hybridization properties . [SEP]
[CLS] pma - b - dna was synthesized by coupling carboxylate - terminated pma and aminemodified oligonucleotides . [SEP]
[CLS] pma was synthesized by raft polymerization . [SEP]
[CLS] see supporting information for its complete synthetic procedure and characterization data . [SEP]
[CLS] for dna coupling , purified pma ( 0 . 264 g , 34 μmol ) was dissolved in 500 μl of anhydrous dmf . [SEP]
[CLS] then , n , n - diisopropylethylamine ( 48 μl , 280 μmol ) and 1 - [ bis ( dimethylamino ) methylene ] - 1h - 1 , 2 , 3 - triazolo [ 4 , 5 - b ] pyridinium 3 - oxid hexafluorophosphate ( hatu ) ( 13 mg , 34 μmol ) were subsequently added to the solution . [SEP]
[CLS] the mixture was vortexed for 10 min to pre - activate the coupling reaction . [SEP]
[CLS] then , 5 ′ - aminomodified dna on cpg beads solid support ( ca . 1 μmol , mmt deprotected ) was added to the solution . [SEP]
[CLS] the mixture was kept on a shaker at room temperature overnight . [SEP]
[CLS] the cpg beads were then washed with ~ 200 ml of dmf to remove unbound pma homopolymers . [SEP]
[CLS] the dna block copolymer strands were cleaved from the cpg beads by incubating B-technique them in ~ 1 ml of concentrated ammonia at 65 °c for 2 h . after 2 h reaction , ammonia was evaporated by loosening the vial cap . [SEP]
[CLS] the cpg beads were filtered and subsequently washed with about 4 ml of water B-material . [SEP]
[CLS] dna block copolymers and unbound single strand dnas were collected and separated by page gel B-technique electrophoresis I-technique . [SEP]
[CLS] giant polymersomes were prepared by the film hydration method . [SEP]
[CLS] first , 50 μl pbd 52 - b - peo 32 solution ( chcl 3 , 4 mg / ml ) was mixed with 10 μl pma - b - dna solution ( dmso , 3 μm ) . [SEP]
[CLS] the mixture was dried under the nitrogen B-material gas flow in the bottom of a glass vial and then the dried polymer B-material mixture was kept under vacuum for > 6 h to ensure that the solvents were completely removed . [SEP]
[CLS] finally , 500 μl of 0 . 1 m pbs buffer ( 100 mm nacl , 10 mm phosphate buffer ph = 7 . 17 ) was added to the film and heated at 50 °c for 12 h to form mixed giant polymersomes . [SEP]
[CLS] for optical B-technique imaging I-technique , 5 μl of the polymersome solution was placed into the glass bottom of cell B-material culture dish . [SEP]
[CLS] mixed small micelles B-material were prepared by passing the assemblies formed by the abovedescribed film hydration method through the membrane extrusion filter . [SEP]
[CLS] typically , 100 μl of pbd - b - peo - b - dna ( dmso , 4 μm ) solution was mixed with 35 μl of pbd 46 - b - peo 30 solution ( chcl 3 , 0 . 4 mg / ml ) for mixed assemblies with 10 mol % dna block copolymer content or with 38 μl of pbd 46 - b - peo 30 solution ( chcl 3 , 0 . 04 mg / ml ) for mixed assemblies with 50 mol % dna block copolymer contents . [SEP]
[CLS] for ps - b - peo - b - dna assemblies , 133 μl of ps - b - peo - b - dna ( dmso , 3 μm ) solution was mixed with 35 μl of ps 48 - b - peo 46 solution ( chcl 3 , 0 . 74 mg / ml ) or 38 μl of ps 48 - b - peo 46 ( chcl 3 , 0 . 074 mg / ml ) solution for binary assemblies containing 10 mol % or 50 % dna block copolymers , respectively . [SEP]
[CLS] the mixture was placed into a glass vial and the solvent was evaporated under vacuum for at least 6 h . [SEP]
[CLS] then , 100 μl of 0 . 1 m pbs buffer ( 100 mm nacl , 10 mm phosphate buffer ph = 7 . 17 ) was added to the polymer B-material film in the vial . [SEP]
[CLS] the solution was vortexed , frozen and thawed 5 times before the extrusion . [SEP]
[CLS] finally , the samples were extruded 38 times through whatman nucleopore tracketch membrane with the pore size of 400 nm . [SEP]
[CLS] in a typical experiment , 100 μl of each polymersome solution was placed in a microcentrifuge tube . [SEP]
[CLS] the mixture was incubated B-technique at 55 °c for 5 min , and then allowed to cool down to room temperature overnight prior to the optical B-technique imaging I-technique . [SEP]
[CLS] in summary , we have fabricated binary assemblies of dna block copolymers ( i . e . , pma - b - dna , pbd - b - peo - b - dna , ps - b - peo - b - dna ) and a prototypical block copolymer of pbd - b - peo . [SEP]
[CLS] binary self - assembly of pbd - b - peo and pma - b - dna at low dna block copolymer content adopt the morphology of pbd - b - peo and form giant polymersomes , [SEP]
[CLS] 1 . ( a ) synthetic schemes for pma - b - dna . [SEP]
[CLS] ( b ) page analyses ( lane 1 : dna 1 , lane 2 : crude product containing pma - b - dna and unbound dna strands , lane 3 : purified pma - b - dna . [SEP]
[CLS] ( c ) extinction spectrum of purified pma - b - dna in water B-material . [SEP]
[CLS] ( d ) dls data of purified pma - b - dna micelles B-material in water B-material . [SEP]
[CLS] ( a - b ) confocal laser scanning fluorescence B-property ( a ) and transmission ( b ) images of giant dna polymersomes formed from pbd 52 - b - peo 32 and pma - b - dna . [SEP]
[CLS] ( c ) a pictorial description of a dna polymersome immobilized onto a micropipette . [SEP]
[CLS] ( d ) a z - stack image of a dna giant polymersome . [SEP]
[CLS] ( a - b ) transmission ( a ) and fluorescence B-property ( b ) images of polymersome aggregates incubated B-technique with ethidium bromide B-material . [SEP]
[CLS] ( c - d ) confocal laser scanning I-technique fluorescence B-property ( c ) and transmission ( d ) images of polymersome aggregates . [SEP]
[CLS] a fluorescence B-property intensity line profile is shown in the inset of ( c ) [SEP]
[CLS] ( a ) page analysis of synthesized triblock copolymers ( lane 1 : dna 2 , lane 2 : crude product containing pbd - b - peo - b - dna and unbounded dna 2 , lane 3 : purified pbd - b - peo - b - dna ) . [SEP]
[CLS] ( b ) extinction spectrum of purified pbd - b - peo - b - dna in water B-material . [SEP]
[CLS] ( c ) dls data of pbd - b - peo - b - dna micelles B-material prepared by film hydration and subsequent membrane extrusion . [SEP]
[CLS] ( a ) schematic description of dna - induced self - assembly of dna triblock copolymer micelles B-material and dna - modified gold B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] ( b ) extinction spectra of dispersed gold B-nanoparticle nanoparticles I-nanoparticle ( red line ) and gold B-nanoparticle nanoparticle I-nanoparticle aggregates cross - linked by polymer B-material assemblies ( black line ) . [SEP]
[CLS] a picture of dispersed nanoparticles B-nanoparticle ( left ) and nanoparticle B-nanoparticle aggregates are shown in the inset . [SEP]
[CLS] ( c ) a tem image of nanoparticle B-nanoparticle networks . [SEP]
[CLS] 6 . dna melting transitions of aggregates formed from dna - modified gold B-nanoparticle nanoparticles I-nanoparticle and binary micelles B-material with ( a ) 100 mol % , ( b ) 50 mol % , and ( c ) 10 mol % dna triblock copolymer ( pbd - b - peo - b - dna ) content , and ( d ) 10 mol % ps - b - peo - b - dna , obtained by monitoring the extinction at 520 nm . [SEP]
[CLS] the insets show the first derivatives of the melting curves . [SEP]
[CLS] the black and the red curves are experimental data and fitted curves , respectively . [SEP]
[CLS] fwhm / melting temperatures for mixed assemblies with different dna block copolymer contents . [SEP]
[CLS] dna - modified particles are used extensively for applications in sensing , material B-material science , and molecular biology . [SEP]
[CLS] the performance of such dna - modified particles is greatly dependent on the degree of surface coverage , but existing methods for quantitation can only be employed for certain particle compositions and / or conjugation chemistries . [SEP]
[CLS] we have developed a simple and broadly applicable exonuclease iii ( exo iii ) digestion assay based on the cleavage of phosphodiester bonds - a universal feature of dna - modified particles - to accurately quantify dna probe surface coverage on diverse , commonly used particles of different compositions , conjugation chemistries , and sizes . [SEP]
[CLS] our assay utilizes particle - conjugated , fluorophore - labeled probes that incorporate two abasic sites ; these probes are hybridized to a complementary dna ( cdna ) strand , and quantitation is achieved via cleavage and digestion of surface - bound probe dna via exo iii ' s apurinic endonucleolytic and exonucleolytic activities . [SEP]
[CLS] the presence of the two abasic sites in the probe greatly speeds up the enzymatic reaction without altering the packing density of the probes on the particles . [SEP]
[CLS] probe digestion releases a signal - generating fluorophore and liberates the intact cdna strand to start a new cycle of hybridization and digestion , until all fluorophore tags have been released . [SEP]
[CLS] since the molar ratio of fluorophore to immobilized dna is 1 : 1 , dna surface coverage can be determined accurately based on the complete release of fluorophores . [SEP]
[CLS] our method delivers accurate , rapid , and reproducible quantitation of thiolated dna on the surface of gold B-nanoparticle nanoparticles I-nanoparticle , and also performs equally well with other conjugation chemistries , substrates , and particle sizes , and thus offers a broadly useful assay for quantitation of dna surface coverage . [SEP]
[CLS] dna - modified nano - and microparticles can specifically recognize a variety of targets , including nucleic B-material acids I-material , proteins B-material , peptides B-material , small molecules , and metal B-material ions B-material . [SEP]
[CLS] accordingly , such particles have proven extraordinarily useful for bioimaging , bioseparation , diagnostic assays , drug targeting , nanotherapeutics , and nanomaterial B-material assembly . [SEP]
[CLS] different conjugation chemistries have been used to immobilize dna strands onto various particle substrates , including metallic B-nanoparticle nanoparticles I-nanoparticle , semiconductive quantum B-nanoparticle dots I-nanoparticle ( qds ) , inorganic upconversion nanoparticles B-nanoparticle ( ucnps ) , silica microspheres , and magnetic B-property beads . [SEP]
[CLS] in particular , alkanethiol adsorption has been employed for attaching thiolated dna onto gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) . [SEP]
[CLS] the lanthanide B-material - phosphate interaction has been used to conjugate unmodified dnas onto lanthanide - doped hydrophobic B-property ucnps . [SEP]
[CLS] the streptavidin ( sa ) biotin interaction is often utilized to attach biotinylated dna onto sa - coated magnetic B-property beads , qds , and ucnps . [SEP]
[CLS] in addition , when analyte detection or sample separation is being performed at elevated temperatures , amino - modified dna has been covalently conjugated onto carboxylated magnetic B-property or silica particles . [SEP]
[CLS] such covalently bound dna probes offer greater stability for assays that employ heat - induced dehybridization , as high temperatures can disrupt sa - biotin interaction . [SEP]
[CLS] the extent of dna surface coverage can profoundly influence dna hybridization efficiency , enzyme B-property activity I-property , assay sensitivity , cellular uptake efficiency , and thermostability of dna nanoparticle - assembled superlattices [SEP]
[CLS] many studies have shown that high surface coverage is associated with steric hindrance , electrostatic repulsive interactions , and elevated surface salt B-material concentration , whereas low surface coverage can result in nonspecific binding of oligonucleotides to the particle surface . [SEP]
[CLS] both scenarios can greatly reduce dna hydridization efficiency and enzyme B-property activity I-property for surface - bound dna probes , and strategies for accurately quantifying the degree of surface coverage are critical for research applications based on dna - conjugated particles . [SEP]
[CLS] radioisotope B-material labeling has been used to determine the number of conjugated oligonucleotides on a variety of surfaces . [SEP]
[CLS] briefly , probe dnas are labeled at their 5 ′ end with a radioisotope B-material such as p with t4 polynucleotide B-material kinase before conjugation . [SEP]
[CLS] quantitation is then achieved by direct measurement of the radioactivity B-property of radiolabeled dna - modified particles using a liquid scintillation counter or a geiger counter . [SEP]
[CLS] although p can be easily incorporated into the phosphate groups of dntps to generate highly sensitive , radiolabeled probes , the short half - life of p means that the labeled probes should be used within a week of preparation , leading to increased costs . [SEP]
[CLS] meanwhile , radioactive B-property hazards are also a concern requiring personal protection and separate waste disposal procedures . [SEP]
[CLS] radioisotope B-material methods have become less popular as a variety of fluorescence - based assays have been developed for accurate quantitation of dna surface coverage on various particle surfaces . [SEP]
[CLS] fluorophore - labeled dna is strongly quenched when bound to particles such as aunps B-nanoparticle and magnetic B-property beads , but this fluorescence B-property is fully recovered once the dna is detached from the surface . [SEP]
[CLS] for thiolated , dna - modified aunps B-nanoparticle , surface coverage is typically determined by measuring the fluorescence B-property of oligonucleotides liberated via ligand displacement with mercaptoethanol or dithiothreitol ( dtt ) . [SEP]
[CLS] the standard dtt displacement protocol usually requires overnight incubation B-technique of dna - modified aunps B-nanoparticle with high concentrations ( [UNK] . 5 m ) of dtt at room temperature . [SEP]
[CLS] although time - consuming , the dtt displacement - based assay is recognized as a reliable " gold B-material standard " for quantifying surface coverage on aunps B-nanoparticle irrespective of dna length or sequence . [SEP]
[CLS] recently , several labelfree methods utilizing pcr , oligreen , and unimolecular beacon have been developed to characterize dna surface coverage . [SEP]
[CLS] however , these methods are only compatible with thiol - coupled dna - aunps B-nanoparticle . [SEP]
[CLS] fluorescent B-property assays are also available for accurate determination of surface coverage on sa - coated inorganic particles such as qds , ucnps , or magnetic B-property beads . [SEP]
[CLS] in this scenario , the interaction between biotinylated probe dnas and sa - coated beads is disrupted in a mixture of ethylenediaminetetraacetic acid ( edta ) and formamide via high - temperature treatment ( 90 °c for 10 min ) . [SEP]
[CLS] the eluted biotinylated dnas can then be fluorescently B-property measured . [SEP]
[CLS] however , this method is not applicable to other conjugation chemistries , such as covalent B-property linkage I-property of thiolated dna to aminated particles or of aminolabeled dna to carboxylated magnetic B-property or silica substrates [SEP]
[CLS] alternatively , dissolution of particle substrates can ensure full release of the attached oligonucleotides for accurate quantification of dna surface coverage . [SEP]
[CLS] this is typically achieved with fluorophore labeling rather than absorbance measurement at 260 nm , which suffers two important impediments . [SEP]
[CLS] first , such measurements are not sensitive enough to accurately quantify dna at low levels of surface coverage ( [UNK] ) . [SEP]
[CLS] second , the chemicals used for aunp B-nanoparticle dissolution , such as ferrocyanide and ferricyanide , exhibit strong absorbance at 260 nm , and this can interfere with accurate quantitation . [SEP]
[CLS] dissolution - based determination of dna surface coverage has been reported for only two substrates : aunps B-nanoparticle and metal - organic framework ( mof ) nanoparticles B-nanoparticle . [SEP]
[CLS] a mixture of kcn and k 3 fe ( cn ) 6 was used to dissolve dna - modified aunps B-nanoparticle for several minutes , after which the absorbance of the cn − - released fluorescein ( fam ) - labeled dnas was measured at 494 nm . [SEP]
[CLS] dnamodified mof nanoparticles B-nanoparticle can be dissolved by incubating B-technique in naoh for 18 h under mechanical shaking , with the absorbance of the released tamra - labeled dnas measured at 566 nm . [SEP]
[CLS] such methods are constrained to specific substrates , however , due to the limited availability of dissolution chemistries . [SEP]
[CLS] thus , there is presently no simple and fast general approach that can be employed for rapidly and accurately quantifying dna surface coverage across the large spectrum of commonly used particle compositions and conjugation chemistries . [SEP]
[CLS] to address this need , we have developed a generalizable method based on the chemical cleavage of phosphodiester bonds - a feature shared by all dna - modified particles - that consistently achieves rapid and accurate quantitation based on fluorophore release , regardless of the conjugation chemistry employed , particle substrate , or size . [SEP]
[CLS] our assay utilizes exonuclease iii ( exo iii ) to achieve cleavage and digestion at abasic sites incorporated into surface - bound , fluorophore - labeled probe dnas that are hybridized with a complementary dna ( cdna ) strand . [SEP]
[CLS] hybridization of the cdna prevents the surfacebound probes from wrapping around the particle surface , rendering them accessible to exo iii for digestion and enabling accurate quantitation . [SEP]
[CLS] the presence of the abasic sites in the probe is not strictly essential , but greatly speeds up the process of enzyme digestion , and we have experimentally demonstrated that these abasic sites do not alter the packing density of probes on particle surface relative to probes lacking these sites . [SEP]
[CLS] as an initial proof - ofconcept , we have demonstrated complete fluorophore release from aunp B-nanoparticle - conjugated , famlabeled dna using our exo iii - based assay after 60 min at room temperature . [SEP]
[CLS] the results were in very good agreement with those from an overnight dtt displacement assay for both high and low dna surface coverage conditions . [SEP]
[CLS] our assay also achieved similar sensitivity to this " gold B-material standard " dtt displacement approach . [SEP]
[CLS] more importantly , our exo iii - based assay proved equally effective for quantifying surface coverage of dna conjugated to other particles , including magnetic B-property beads ( 1 μm ) , qds ( 18 nm ) , silica microspheres ( 1 μm ) , and ucnps ( 25 or 76 nm ) . [SEP]
[CLS] we further demonstrated that our method is effective with probes conjugated via streptavidin - biotin interaction , covalent B-property linkage I-property , or electrostatic interaction . [SEP]
[CLS] in most instances , we observed far more efficient fluorophore release with our exo iii - based assay relative to existing assays . [SEP]
[CLS] our assay therefore delivers valuable new analytical capabilities for accurate dna quantitation , greatly reducing the measurement time needed for dna - conjugated aunps B-nanoparticle while also offering a general - purpose assay that can readily be applied to various conjugation chemistries , substrates , and sizes . [SEP]
[CLS] we first employed our exo iii - based assay with fam - labeled probe dna - modified aunps B-nanoparticle . [SEP]
[CLS] exo iii has 3 ′ - to - 5 ′ exonuclease activity and processes nucleic B-material acids I-material in a sequenceindependent fashion . [SEP]
[CLS] we first exploited the exonucleolytic activity of exo iii to catalyze the stepwise removal of mononucleotides from 3 ′ - hydroxyl termini of duplex dna as a means to accurately determine dna surface coverage on aunps B-nanoparticle . [SEP]
[CLS] the procedure for the exo iii - based assay is illustrated in figure 1 . [SEP]
[CLS] these aunp B-nanoparticle - conjugated , fam - labeled probes ( figure 1a ) are hybridized to a complementary dna ( cdna ) strand ( supporting information ( si ) , table s1 , cdna - 8a ) to form a perfectly matched duplex , and quantitation is achieved via exo iii digestion of surface - bound probe dnas ( figure 1b ) . [SEP]
[CLS] digestion releases a signal - generating fluorophore and liberates the intact cdna strand to start anew until all probes have been digested from the particle surface , generating an aunp B-nanoparticle aggregation - induced visible color shift from red to blue ( figure 1c ) . [SEP]
[CLS] since the molar ratio of fam to immobilized probe dna is 1 : 1 , dna surface coverage can be determined accurately based on the complete release of fluorophores after enzyme digestion . [SEP]
[CLS] we designed a 47 - nt probe dna with the following sequence : 5 ′ - poly ( t 6 ) - accacatcatccatataactgaaagccaaacagtttttttt - 3 ′ . the 5 ′ poly ( t 6 ) acts as a flexible linker to improve cdna hybridization . [SEP]
[CLS] we synthesized the probe with a 5 ′ thiol B-material group and a 3 ′ fam - label ( si , table s1 , sh probe ) and conjugated it onto aunps B-nanoparticle via thiol - gold B-material chemistry at a ratio of 300 : 1 . [SEP]
[CLS] after the modification , we found that 70 % of the dna was unconjugated and remained in the supernatant ( data not shown ) . [SEP]
[CLS] this large excess confirms that these conditions ensure saturated dna loading of the aunps B-nanoparticle , and indicate that coverage would not meaningfully increase at even higher dna : aunp B-nanoparticle ratios . [SEP]
[CLS] these dnamodified aunps B-nanoparticle are stable in reaction buffer , and the resulting solution is red in color due to the strong electrostatic repulsion of dna - modified nanoparticles B-nanoparticle . [SEP]
[CLS] we hybridized the modified aunps B-nanoparticle with cdna - 8a , and then incubated B-technique with exo iii . [SEP]
[CLS] digestion of the surfacebound probes causes the probe - free aunps B-nanoparticle to become unstable in the reaction buffer , leading to salt - induced aggregation that gives rise to a red - to - blue color change . [SEP]
[CLS] we tracked this response by performing time - dependent measurements of uv absorbance ( figure 2a ) , specifically monitoring the ratio of absorbance at 650 to 520 nm ( a 650 / a 520 ) ( figure 2b ) that has served as an indicator B-property of aunp B-nanoparticle aggregation . [SEP]
[CLS] we observed that aggregation occurred gradually , with complete digestion of surface - bound probe dnas achieved after 255 min . [SEP]
[CLS] we also measured the time - dependent release of the fluorophore from aunpconjugated sh probes . [SEP]
[CLS] we found that 75 % of the fam molecules were released in the first 60 min , and fluorescence B-property intensity subsequently achieved saturation after 180 min ( figure 2c ) . [SEP]
[CLS] as expected , the fluorophore was released before the complete degradation of immobilized probe dnas on aunp B-nanoparticle . [SEP]
[CLS] to test if our exo iii - based assay can quantify aunp B-nanoparticle - conjugated probe dna in both high and low surface - coverage scenarios , we prepared dna - modified aunps B-nanoparticle at sh probe dna : aunp B-nanoparticle ratios of 60 , 80 , 120 , 150 , 200 , and 300 and characterized their surface coverage under the conditions described above . [SEP]
[CLS] after a 255 min incubation B-technique , we separated the supernatant from the aunp B-nanoparticle precipitate and measured its fluorescence B-property . [SEP]
[CLS] we confirmed that exo iii was able to cleave the 3 ′ fam - modified nucleotide from the probe , thereby releasing it into solution . [SEP]
[CLS] the fluorescence B-property intensity increased in proportion to the amount of dna used for the modification ( figure 3a and si , figure s1 ) , and we used a standard curve generated from unconjugated fam - labeled , sh probe dna with exo iii and cdna under the same experimental conditions ( figure 3b ) to calculate dna surface coverage . [SEP]
[CLS] we determined that aunps B-nanoparticle modified with dna : aunp B-nanoparticle ratios of 60 , 80 , 120 , 150 , 200 , and 300 respectively displayed 38 ± 1 , 44 ± 1 , 53 ± 1 , 65 ± 1 , 69 ± 1 , and 79 ± 2 oligonucleotides per particle , equivalent to surface coverage of 12 . 1 ± 0 . 3 , 13 . 8 ± 0 . 2 , 16 . 7 ± 0 . 2 , 20 . 4 ± 0 . 3 , 21 . 7 ± 0 . 3 , and 24 . 7 ± 0 . 6 pmol / cm 2 , respectively ( figure 3c ) . [SEP]
[CLS] to evaluate the accuracy of our exo iii - based assay , we performed a dtt displacement assay with the same set of samples . [SEP]
[CLS] we mixed each batch of sh probe dna - modified aunps B-nanoparticle with an equal volume of 1 . 0 m dtt and incubated B-technique the mixture at room temperature for 12 h , after which we centrifuged the samples and measured the fluorescence B-property of the collected supernatant ( figure 3a , si , figure s2 ) . [SEP]
[CLS] we used the constructed calibration curve ( figure 3b ) to determine that the surface coverage measurements obtained via dtt displacement were in very good agreement with the values obtained via exo iii digestion ( figure 3c ) . [SEP]
[CLS] in addition to 3 ′ - to - 5 ′ exonuclease activity , exo iii has also been reported to have apurinic ( ap ) endonuclease activity that digests propanyl abasic sites inserted into dna strands . [SEP]
[CLS] we predicted that the enzymatic digestion in our exo iii - based assay could be profoundly accelerated by inserting propanyl abasic sites into the dna probe , thereby exploiting both activities of exo iii . [SEP]
[CLS] specifically , we utilized exo iii ' s endonuclease activity to generate internal nicks at the abasic sites ( marked as x in the probe sequence ) , with subsequent exonucleolytic digestion at the newly created nicks considerably accelerating the assay process . [SEP]
[CLS] we inserted two internal propanyl abasic sites into our sh probe dna , producing the sh - 2x probe ( si , table s1 , sh - 2x probe ) . [SEP]
[CLS] the sh - 2x probe dna was then attached onto aunps B-nanoparticle via thiol B-material linkage B-property ( figure 4a ) . [SEP]
[CLS] when the immobilized probe hybridizes to the cdna strand ( si , table s1 , cdna ) , it forms a duplex with an 8 - nt sticky end ( figure 4b ) . [SEP]
[CLS] the two duplexed abasic sites provide binding sites for exo iii , which endonucleolytically cleaves the phosphodiester bond and generates two nicks with hydroxyl B-material groups I-material at the 3 ′ end ( figure 4c ) . [SEP]
[CLS] this converts the sh - 2x probe into three nicked duplex fragments ( 12 , 11 , and 8 bp ) . [SEP]
[CLS] the fam - labeled 8 - bp strand ( t m = 21 . 9 °c ) readily dissociates from the cdna at room temperature , while the 11 - and 12 - bp fragments ( t m = 27 . 8 and 44 . 5 °c , respectively ) remain hybridized to the cdna . [SEP]
[CLS] exo iii subsequently initiates 3 ′ - to - 5 ′ exonucleolytic digestion of the sh - 2x probe dna at these newly formed 3 ′ - hydroxyl termini . [SEP]
[CLS] when the probe is degraded , liberating the fluorophore , the intact cdna is released ( figure 4d ) to start a new cycle of dna hybridization and exo iii digestion ( figure 4e ) . [SEP]
[CLS] once all of the attached probes have been sheared from the surface , we use the resulting fluorescence B-property to accurately determine dna surface coverage ( figure 4f ) . [SEP]
[CLS] we experimentally confirmed that the insertion of abasic sites accelerated dna probe digestion and that the number of abasic sites strongly influences the reaction rate . [SEP]
[CLS] we immobilized two versions of our probe dna containing either one or two abasic sites ( figure 5a and si , table s1 , sh - 1x and - 2x probes ) onto aunps B-nanoparticle with a saturating dna : aunp B-nanoparticle ratio of 300 . [SEP]
[CLS] we determined that the surface coverage for these two new probes was essentially identical to that of the original sh - probe dna ( 79 ± 2 oligonucleotides / particle ) , with surface densities of 77 ± 3 and 79 ± 3 oligonucleotides / particle for the sh - 1x and - 2x probes , respectively . [SEP]
[CLS] we then performed exo iii digestion of these three batches of modified aunps B-nanoparticle and recorded the absorbance change at 520 and 650 nm . [SEP]
[CLS] the time course of aggregation confirmed that the sh - 2x probe dna produced a much more rapid reaction ( si , figure s3a ) relative to the sh ( figure 2a ) or sh - 1x probes ( si , figure s3b ) . [SEP]
[CLS] the reaction times required for complete aggregation of the aunps B-nanoparticle were 60 , 230 , and 255 min for aunps B-nanoparticle modified with sh - 2x , - 1x probes and sh probe , respectively ( figure 5b ) . [SEP]
[CLS] clearly , the presence of two abasic sites in the 2x probe dna enables far more rapid digestion . [SEP]
[CLS] compared with the sh probe , the sh - 1x probe exhibited only moderately faster digestion , presumably because the 20 - bp 3 ′ duplexed fragment ( t m = 56 . 9 °c ) formed by cleavage at the 5 ′ abasic site is very stable , which prevents the cdna from releasing at room temperature and thereby delays further digestion steps . [SEP]
[CLS] we therefore used the sh - 2x probe dna for all subsequent experiments with our exo iii - based assay . [SEP]
[CLS] previous studies have shown that the ap activity of exo iii is marginally influenced by the presence of different bases at positions complementary to abasic sites . [SEP]
[CLS] we conducted experiments to test for this effect under our assay conditions . [SEP]
[CLS] we found that cdnas with c or t opposite the abasic site ( si , table s1 , cdna - c and - t ) contributed to more efficient digestion by exo iii relative to those with a or g opposite the abasic site ( si , table s1 , cdna - a and - g ) ( si , figure s4 ) at low enzyme concentrations ( 0 . 004 u / μl ) , but the difference became negligible and did not affect the assay ' s efficiency when 0 . 2 u / μl of exo iii was used ( si , figure s5 ) . [SEP]
[CLS] to verify that all fluorophore labels had been released from the particle surface at the point of aunp B-nanoparticle aggregation , we measured the time - dependent release of the fluorophore from aunp B-nanoparticle - conjugated sh - 2x probes by monitoring the change in fam intensity . [SEP]
[CLS] most fam molecules were released in the first 15 min of the reaction , with 75 % of the fam released into solution ( si , figure s6 ) . [SEP]
[CLS] fluorescence B-property intensity subsequently increased slowly , achieving saturation after 60 min , with no further increase after 18 h . [SEP]
[CLS] clearly , the complete release of fluorophores is consistent with the aggregation of the aunps B-nanoparticle , indicating that exo iii can achieve entire digestion of surface - bound 2x - probe within 1 h . [SEP]
[CLS] digestion of surface - bound probes requires the formation of a duplex with cdna , since exo iii is inactive on single - stranded dna . [SEP]
[CLS] when hybridized with the aunp B-nanoparticle - immobilized dna probe , the 3 ′ end of the cdna is close to the surface of the aunps B-nanoparticle . [SEP]
[CLS] we believe that the 3 ′ to - 5 ′ exonuclease activity of exo iii is greatly inhibited by steric hindrance near the particle surface . [SEP]
[CLS] we have previously shown that cdna concentrations as low as 2 nm can result in complete removal of all probe dna from aunps B-nanoparticle , resulting in aggregation , demonstrating that low concentrations of cdna can be recycled efficiently . [SEP]
[CLS] however , it is not necessary for the cdna to be recycled as long as we have sufficient cdna in the sample . [SEP]
[CLS] we further investigated the effect of cdna concentration on exo iii efficiency . [SEP]
[CLS] we observed that the initial reaction rate of exo iii increased with increasing concentrations of cdna until reaching a plateau at 200 nm ( si , figure s7 ) . [SEP]
[CLS] further increases in the cdna concentration up to 500 nm yielded only a slightly higher reaction rate . [SEP]
[CLS] we therefore used 200 nm cdna for subsequent experiments . [SEP]
[CLS] we also examined the effect of exo iii concentration , and found that increasing the amount of exo iii greatly increased the initial reaction rate ( si , figure s8 ) . [SEP]
[CLS] 0 . 2 u / μl of exo iii was used for subsequent experiments to achieve a rapid reaction . [SEP]
[CLS] we confirmed the accuracy of the exo iii - based assay utilizing sh - 2x probe modified aunps B-nanoparticle under both low and high surface coverage conditions . [SEP]
[CLS] we conjugated sh - 2x probe dna with aunps B-nanoparticle at dna : aunp B-nanoparticle ratios of 60 , 80 , 120 , 150 , 200 , and 300 under the same modification conditions described above and characterized the surface coverage of the six batches of aunps B-nanoparticle with the exo iii - based ( si , figure s9 ) and dtt displacement ( si , figure s10 ) assays . [SEP]
[CLS] we found that the measurements in high and low surface coverage scenarios obtained via both methods were in very good agreement ( si , figure s11 ) . [SEP]
[CLS] however , the exo iii - based assay can be performed in a far shorter time relative to the dtt displacement assay , especially when using abasic site containing probes ( 1 h vs 12 h ) . [SEP]
[CLS] since the surface coverage values of the sh - 2x probe ( si , figure s11 ) were essentially identical to those obtained with the sh probe ( figure 3 ) , we conclude that the inclusion of the abasic sites speeds up the enzymatic reaction without altering either the dna packing density on the particle surface or the accuracy of quantitation . [SEP]
[CLS] the exo iii - based assay also demonstrated a comparable limit of quantitation ( loq ) to the dtt displacement assay . [SEP]
[CLS] we performed exo iii digestion using sh - 2x probe dnamodified aunps B-nanoparticle with maximum surface coverage at particle concentrations ranging from 31 fm to 3 . 1 nm , equivalent to a concentration of sh - 2x probe dna ranging from 2 . 5 pm to 250 nm . [SEP]
[CLS] our results demonstrated good linearity , with a loq of 34 . 6 pm probe dna ( 8 . 3 × 10 8 oligonucleotides ) ( figure 6 ) . [SEP]
[CLS] this demonstrates the high sensitivity of the exo iii - based assay ; based on the small testing volume of our assay ( 40 μl ) , only 1 . 4 fmol dna - or a subfemtomole quantity of aunps B-nanoparticle - is required for accurate quantitation . [SEP]
[CLS] we observed a similar loq ( 26 . 3 pm , 6 . 3 × 10 8 oligonucleotides ) for the dtt displacement - based assay using the same set of samples . [SEP]
[CLS] our exo iii - based assay achieves rapid and accurate determination of dna surface coverage due to the high efficiency of exo iii and increased enzyme accessibility resulting from cdna hybridization . [SEP]
[CLS] dnase i digestion offers an alternative nuclease - based method that cleaves fluoro - phore - labeled dna from gold B-material surfaces for subsequent quantification . [SEP]
[CLS] dnase i is an endonuclease that nonspecifically cleaves single - and double - stranded dna to release di - , tri - , and oligonucleotide products with 5 ′ - phosphorylated and 3 ′ - hydroxylated ends . [SEP]
[CLS] to compare the digestion efficiency of exo iii and dnase i , we monitored the time course for digestion of sh - 2x probe dna - modified aunps B-nanoparticle with maximum surface coverage with 80 nm exo iii ( 0 . 2 u / μl ) or 80 nm dnase i ( 0 . 125 u / μl ) as reported optimized condition [SEP]
[CLS] the [SEP]
[CLS] presence [SEP]
[CLS] of 200 nm cdna [SEP]
[CLS] after 60 min , exo iii achieved 100 % probe digestion whereas dnase i digested only 42 % of the probe dna ( figure 7a ) . [SEP]
[CLS] we further compared the capacity of the dnase i - based assay to quantify dna at different levels of surface coverage relative to exo iii . [SEP]
[CLS] we mixed six batches of sh - 2x probe dna - modified aunps B-nanoparticle displaying various dna surface coverage with dnase i and incubated B-technique the mixture at room temperature for 16 h , then collected the supernatants to obtain fluorescence B-property spectra ( si , figure s12a ) . [SEP]
[CLS] we used a standard curve established with different concentrations of unconjugated fam - labeled , thiolated 2x probe dna and 0 . 125 u / μl of dnase i to calculate the dna surface coverage on these modified aunps B-nanoparticle ( si , figure s12b ) . [SEP]
[CLS] the performance of the dnase i - based assay was generally inferior to that of the exo iii - based assay , with estimates of surface coverage for all six batches of dna - modified aunps B-nanoparticle that were significantly smaller than those obtained by our exo iii - based assay ( figure 7b and si , table s2 ) . [SEP]
[CLS] this incomplete digestion by dnase i could be attributable to two reasons . [SEP]
[CLS] first , dnase i activity is highly dependent on salt B-material concentration , and digestion can be inhibited when dna surface coverage is high due to an elevated local salt B-material concentration . [SEP]
[CLS] in contrast , exo iii is robust against high local salt B-material concentrations . furthermore , the wrapping of immobilized single - stranded probe dnas around particles typically reduces the accessibility of the probes to enzymes , impairing accurate quantitation with dnase i - especially when dna surface coverage is low . [SEP]
[CLS] the persistence length of ssdna is around 8 nm ( [UNK] nucleotides ) , and this lack of stiffness makes it easy for the probe to bind to the particle surface . [SEP]
[CLS] when cdna binds with the probe to form a duplex , however , the persistence length of dsdna increases to 50 nm ( [UNK] nucleotides ) , which is much longer than our probe - cdna duplex ( 14 nm / 41 nucleotides ) . [SEP]
[CLS] thus , the duplexed probe would be predicted to not bend and wrap around the curved particle surface , and should extend perpendicularly to the aunp B-nanoparticle 59 regardless of the degree of surface coverage . [SEP]
[CLS] we predicted that duplex formation between surface - bound probe dna and cdna should minimize surface adsorption in the dnase i - based assay and thus enhance the accuracy of the measurements . [SEP]
[CLS] to confirm this , we incubated B-technique our sh - 2x probe dna - modified aunps B-nanoparticle with 0 . 125 u / μl of dnase i and 200 nm of cdna for 16 h . [SEP]
[CLS] the supernatants were collected and recorded ( si , figure s13a ) . [SEP]
[CLS] we used a standard curve ( si , figure s13b ) established with different concentrations of unconjugated sh - 2x probe dna , 200 nm of cdna , and 0 . 125 u / μl of dnase i to calculate dna surface coverage on these modified aunps B-nanoparticle . [SEP]
[CLS] as expected , the presence of the cdna improved the performance of the dnase i assay , yielding measurements that were in good agreement with our exo iii - based assay for aunps B-nanoparticle with relatively low surface coverage ( figure 7b and si , table s2 ) . [SEP]
[CLS] this suggests that the duplexed probe dnas provide more access to dnase i , thereby facilitating complete digestion . [SEP]
[CLS] however , the dnase i - based assay still produced lower measurements when dna surface coverage was greater than 56 oligonucleotides / particle ( figure 7b and si , table s2 ) , presumably due to inhibition of dnase i by high local salt B-material concentrations at the surface . [SEP]
[CLS] to demonstrate the generalizability of our exo iii - based method for determining dna surface coverage on various substrates with different conjugation chemistries and sizes , we first quantified surface coverage of biotinylated 2x probe dna ( si , table s1 , biotin - 2x probe ) on sa - coated magnetic B-property beads ( mb - sa , 1 μm diameter ) . [SEP]
[CLS] we modified the beads with equal volumes of 5 μm biotinylated dna in binding buffer , and then washed and separated the dna - modified beads from unbound dna using a magnetic B-property particle concentrator . [SEP]
[CLS] using the same exo iii digestion procedure described above , we could readily obtain complete fluorophore release after 60 min , with no measurable increase of fluorescence B-property after an overnight reaction ( figure 8a ) . [SEP]
[CLS] based on the corresponding standard curve ( si , figure s14 ) , we measured an average ( 3 . 4 ± 0 . 1 ) × 10 5 dna molecules per mb - sa , equivalent to a surface coverage of 18 . 0 ± 0 . 6 pmol / cm 2 . [SEP]
[CLS] heat - treated elution in a mixture of edta and formamide has been used to measure dna surface coverage under such conditions by disrupting sa - biotin binding . [SEP]
[CLS] using this approach , we obtained a value of 16 . 1 ± 0 . 6 pmol / cm 2 ( si , figure s15a and c ) . [SEP]
[CLS] this is 10 % less than the exo iii - based measurement , indicating that not all of the biotinylated dna was successfully dissociated under such conditions . [SEP]
[CLS] employing the same conjugation method , we attached the biotin - 2x probe dna onto sa - coated quantum B-nanoparticle dots I-nanoparticle ( qd - sa , 18 nm diameter ) and performed exo iii digestion with the dna - modified qds . [SEP]
[CLS] we used the established standard curve ( si , figure s16 ) to calculate surface coverage on the dna - modified qds after 60 min ( figure 8b ) , measuring an average 34 ± 1 oligonucleotides per qd - sa , equivalent to a surface coverage of 5 . 6 ± 0 . 1 pmol / cm 2 . [SEP]
[CLS] we obtained a comparable value using the heating - based elution assay , finding an average of 32 ± 1 oligonucleotides per qd - sa ( 5 . 3 ± 0 . 1 pmol / cm 2 ) ( si , figure s17a , b ) . [SEP]
[CLS] our method was equally successful in quantifying amino - labeled dna ( si , table s1 , nh 2 - 2x probe ) covalently conjugated onto carboxylated magnetic B-property beads ( mb - cooh , 1 μm diameter ) and silica microspheres ( sio 2 - cooh , 1 μm diameter ) . [SEP]
[CLS] we used ethyl ( dimethylaminopropyl ) carbodiimide ( edc ) to activate the carboxylic B-material acid I-material groups B-nanoparticle on I-nanoparticle the I-nanoparticle surface I-nanoparticle of mb - cooh or sio 2 - cooh in mes buffer , and incubated B-technique with nh 2 - 2x probe dna . [SEP]
[CLS] the dna - modified magnetic B-property beads were collected with a magnetic B-property particle concentrator , and the dna - modified silica microspheres were separated from unbound dna by several rounds of centrifugation and resuspension . [SEP]
[CLS] we mixed the modified beads with 0 . 2 u / μl of exo iii and 200 nm of cdna and incubated B-technique for either 1 or 16 h . [SEP]
[CLS] after centrifuging the samples , we collected the supernatants and obtained the sample fluorescence B-property . [SEP]
[CLS] based on a standard curve ( si , figure s18 ) established with different concentrations of unconjugated nh 2 - 2x probe dna , 200 nm of cdna , and 0 . 2 u / μl of exo iii , we determined that the surface coverage was ( 1 . 1 ± 0 . 1 ) × 10 5 dna strands per magnetic B-property bead ( figure 8c ) and ( 7 . 4 ± 0 . 2 ) × 10 3 dna strands per silica microsphere ( figure 8d ) , equivalent to surface coverage of 5 . 8 ± 0 . 3 and 0 . 39 ± 0 . 01 pmol / cm 2 , respectively . [SEP]
[CLS] we noted that overnight incubation B-technique was required to achieve complete digestion with covalently conjugated dna particles . [SEP]
[CLS] the reason for this is still unclear , but one possibility is that nonspecific adsorption of exo iii onto the surface of these nonprotein - coated microparticles greatly reduces the enzyme ' s ability to access the conjugated oligonucleotides . [SEP]
[CLS] this limitation aside , it is important to note that our exo iii - based assay represents the first published fluorescence B-property method capable of accurately quantifying coverage for such covalently conjugated dna probes . [SEP]
[CLS] finally , we demonstrated successful determination of dna surface coverage on ucnps . [SEP]
[CLS] dna - modified ucnps have gained considerable attention in the field of dna - based bionanotechnology because of their distinctive properties , such as exceptional photostability , suppression of autofluorescence , and low in vitro and in vivo toxicity B-property . [SEP]
[CLS] dna can be attached to ucnps through either sa - biotin binding or direct adsorption . [SEP]
[CLS] therefore , we prepared 2x probe dna - modified ucnps by attaching biotin - 2x probe dna onto sacoated ucnps ( ucnp - sa , 76 nm diameter ) or directly conjugating 5 ′ - unmodified 2x probe dna onto oleic acid - capped ucnps ( ucnp - oa , 25 nm diameter ) via the electrostatic interaction between phosphoric acid groups on the dna and lanthanide B-material ions B-material on the ucnps . [SEP]
[CLS] employing the exo iii - based assay , we readily obtained complete fluorescence B-property recovery after 60 min , with no measurable increase of fluorescence B-property after an overnight reaction ( figure 8e , f ) . [SEP]
[CLS] based on the corresponding standard curves ( si , figures s14 and s19 ) , we measured an average of 30 ± 1 dna molecules per ucnp - sa and an average of 19 ± 1 dna molecules per ucnp - oa , equivalent to a surface coverage of 0 . 27 ± 0 . 01 pmol / cm 2 and 1 . 6 ± 0 . 1 pmol / cm 2 , respectively . [SEP]
[CLS] we obtained a comparable value using the heating - based elution assay , finding an average of 25 ± 1 dna molecules per ucnp - sa , equivalent to a surface coverage of 0 . 22 ± 0 . 01 pmol / cm 2 ( si , figure s15b , c ) . [SEP]
[CLS] we have demonstrated a general - purpose method for the quantitation of dna surface coverage that targets the phosphodiester bonds of dna probes - a feature shared by all dna - modified particles . [SEP]
[CLS] our system exploits the distinct dual enzymatic activities of exo iii ( with the assistance of a hybridized cdna ) to achieve efficient probe digestion and complete fluorophore release . [SEP]
[CLS] the resulting fluorescence B-property serves as a reporter for accurately quantifying dna conjugated onto particles of diverse sizes and compositions via a variety of different chemistries . [SEP]
[CLS] using dna - modified aunps B-nanoparticle as an initial proof - of - concept , we demonstrated that our exo iii - based method can deliver accurate and reproducible quantitation for both high - and lowsurface - coverage scenarios , with results closely comparable to the " gold B-material standard " dttdisplacement assay , yet much faster . [SEP]
[CLS] importantly , our method generated essentially identical measurements from aunp B-nanoparticle conjugation experiments with probes that either contain or lack abasic sites , and both probes achieve equivalent coverage on the aunp B-nanoparticle surface after the modification . [SEP]
[CLS] however , the addition of abasic sites to the probe significantly accelerates enzymatic digestion . [SEP]
[CLS] note that we obtained equivalent surface coverage measurements for both unmodified and abasic site - containing probes , indicating that the effects of any alterations in cdna hybridization in the latter scenario are minimal in terms of assay performance . [SEP]
[CLS] additionally , our assay proved effective with other particle substrates , such as qds , ucnps , magnetic B-property beads , and silica microspheres , delivering accurate quantitation of both noncovalently and covalently bound dnas with results that were comparable to or superior to other standard assays ( si , table s3 ) . [SEP]
[CLS] although the fluorophore - labeled dna probes and excess amount of complementary dna are required to complete the characterization of dna surface coverage in the exo iii - based assay , we believe that our exo iii - based assay has the potential to provide simple and accurate measurement of surface coverage for virtually any class of dna - particle conjugates . [SEP]
[CLS] quantitation of dna surface coverage on aunps B-nanoparticle via exo iii - based assay 1 . 25 μl of sh - 2x probe dna - modified aunps B-nanoparticle ( final concentration 3 . 1 nm ) was mixed with 36 . 75 μl of reaction buffer ( 20 mm tris - ac , 50 mm kac , 25 mm nacl , 20 mm cacl 2 , 3 mm mgcl 2 , 1 × bsa , ph 7 . 9 ) , 2 μl of 4 μm cdna and 3 μl of 2 . 7 u / μl exo iii ( both diluted with reaction buffer ) . [SEP]
[CLS] after 60 min incubation B-technique at room temperature , the solution was centrifuged at 21 , 130 rcf × 10 min to remove the aunp B-nanoparticle precipitates , after which 40 μl of supernatant was transferred into a 384 - well microplate to record fluorescence B-property intensities of fluorescein label using a tecan infinite m1000 pro with λ ex = 495 nm and λ em = 520 nm . [SEP]
[CLS] to establish the standard calibration curve , 38 μl of reaction buffer containing known concentrations of sh - 2x probe dna was mixed with 2 μl of 4 μm cdna and 3 μl of exo iii ( 2 . 7 u / μl ) to obtain probe concentrations ranging from 0 to 250 nm . [SEP]
[CLS] these samples underwent the same incubation B-technique and centrifugation process as the modified aunp B-nanoparticle samples , with 40 μl of each solution used for measurement . [SEP]
[CLS] to investigate the effect of cdna concentration on the reaction time , sh - 2x probe dna - modified aunps B-nanoparticle with a surface coverage of 79 oligonucleotides / particle were mixed with various concentrations of cdna ( 0 , 10 , 20 , 50 , 100 , 200 , and 500 nm ) and exo iii ( 0 . 01 u / μl ) in 40 μl reaction buffer . [SEP]
[CLS] the mixtures were loaded into a 384 - well microplate and the timedependent fluorescence B-property changes ( λ ex = 495 nm and λ em = 520 nm ) were recorded . [SEP]
[CLS] the initial reaction rate was determined by the slope of the exponential fitting curve at 0 min . [SEP]
[CLS] 1 . 25 μl of 2x probe dna - modified particles ( final concentration 3 . 1 nm ) were mixed with 36 . 75 μl of reaction buffer , 2 μl of 4 μm cdna , and 3 μl of 2 . 7 u / μl exo iii ( both diluted with reaction buffer ) . [SEP]
[CLS] after incubation B-technique for 60 min or 16 h at room temperature , the solution was centrifuged at 21 , 130 rcf × 10 min to remove the particles , after which 40 μl of supernatant was transferred into a 384 - well microplate to record the fluorescence B-property spectra from 504 to 850 nm ( λ ex = 495 nm ) . [SEP]
[CLS] to establish the standard calibration curve , 38 μl of reaction buffer containing known concentrations of biotin - 2x probe dna ( for sa - coated magnetic B-property beads , qds or ucnps ) , nh 2 - 2x probe dna ( for carboxylated magnetic B-property or silica beads ) , or 5 ′ - unmodified - 2x probe dna ( for oleic acid - capped ucnps ) was mixed with 2 μl of 4 μm cdna and 3 μl of 2 . 7 u / μl exo iii to obtain probe solutions ranging from 0 to 250 nm . [SEP]
[CLS] these samples underwent the same incubation B-technique and centrifugation process as the dna - modified aunp B-nanoparticle samples , with 40 μl of each solution used for fluorescence B-property measurement . [SEP]
[CLS] since dna - modified qd - sa particles cannot be completely removed by centrifugation and dna - modified qd - sa particles did not significantly quench fam fluorescence B-property , we directly collected the fluorescence B-property spectra from 504 to 850 nm ( λ ex = 495 nm ) from samples of dna - modified qd - sa particles . [SEP]
[CLS] the same amount of unmodified qd - sa particles was added for calibration before exo iii digestion . [SEP]
[CLS] schematic illustration of our exo iii - based assay for quantifying the surface coverage of sh - 2x probe dna conjugated with aunps B-nanoparticle . [SEP]
[CLS] aunps B-nanoparticle are modified with dna probes containing two abasic sites ( a ) . [SEP]
[CLS] these are hybridized to cdna ( b ) and then incubated B-technique with exo iii ( c ) . [SEP]
[CLS] the enzyme introduces internal nicks at the abasic sites , and then performs exonucleolytic digestion of the remaining probe fragments ( d ) . [SEP]
[CLS] the cdna is released intact for further rounds of digestion ( e ) , until all probes have been stripped away and the released fluorophores can be quantified to assess surface coverage ( f ) . [SEP]
[CLS] figure 1 . of the exo iii - based assay for quantifying surface coverage of probe dna on aunps B-nanoparticle . [SEP]
[CLS] fluorophore - labeled , aunp B-nanoparticle - coupled dnas ( a ) are hybridized with a complementary cdna and subsequently undergo exonucleolytic digestion by exo iii ( b ) . [SEP]
[CLS] this releases fluorophores and intact cdna strands , allowing multiple rounds of digestion until all fluorophores are released ( c ) . [SEP]
[CLS] the resulting aggregation of the probe - free aunps B-nanoparticle produces a visible red - to - blue color change . [SEP]
[CLS] exo iii digestion of dna - conjugated aunps B-nanoparticle modified at a dna : aunp B-nanoparticle ratio of 300 . [SEP]
[CLS] timedependent absorbance spectra ( a ) and absorbance ratio ( a 650 / a 520 ) ( b ) and fluorescence B-property intensity ( c ) of the sh probe - modified aunps B-nanoparticle in exo iii - based assay . [SEP]
[CLS] the a 650 / a 520 of sh probe dna - modified aunps B-nanoparticle reached saturation after 255 min . [SEP]
[CLS] comparison of the exo iii - based and the dtt displacement assays with sh probe dnaconjugated aunps B-nanoparticle prepared at different dna : aunp B-nanoparticle ratios . [SEP]
[CLS] ( a ) fluorescence B-property intensities of supernatants after exo iii digestion ( black ) and dtt displacement ( red ) . [SEP]
[CLS] ( b ) standard calibration curve obtained with known concentrations of fam - labeled , thiolated probe dna after exo iii digestion ( black ) and dtt displacement ( red ) . [SEP]
[CLS] ( c ) surface coverage of sh probe dna - modified aunps B-nanoparticle as characterized by exo iii digestion ( black ) and dtt displacement ( red ) . [SEP]
[CLS] error bars show standard deviations obtained from three measurements . [SEP]
[CLS] 5 . effect of the number of abasic sites on the rate of digestion for dna - modified aunps B-nanoparticle in the exo iii - based assay . [SEP]
[CLS] ( a ) scheme of exo iii digestion for cdna duplexes formed with aunp B-nanoparticle - conjugated sh - 2x , sh - 1x , and sh probes . [SEP]
[CLS] ( b ) time - dependent absorbance changes ( a 650 / a 520 ) of aunps B-nanoparticle modified with sh - 2x , sh - 1x , and sh probe dna . [SEP]
[CLS] 6 . sensitivity of the exo iii - based ( black ) and dtt displacement ( red ) assays . [SEP]
[CLS] ( a ) fluorescence B-property intensities at 520 nm for supernatants from different dna concentrations of sh - 2x probe dna - modified aunps B-nanoparticle ( 79 oligonucleotides per particle ) after performing both assays . [SEP]
[CLS] ( b ) we determined the limit of quantitation ( loq ) for both assays based on a signal - to - noise ratio of 10 ( ref 50 ) . [SEP]
[CLS] error bars show standard deviations obtained from three measurements . [SEP]
[CLS] reaction kinetics for exo iii digestion and dnase i hydrolysis of sh - 2x probe dnamodified aunps B-nanoparticle . [SEP]
[CLS] ( a ) time - dependent fluorescence B-property changes for aunps B-nanoparticle modified with 79 oligonucleotides per particle upon addition of either 80 nm exo iii ( 0 . 2 u / μl ) or dnase i ( 0 . 125 u / μl ) in the presence of 200 nm cdna . [SEP]
[CLS] ( b ) surface coverage measurements obtained for dna - modified aunps B-nanoparticle with different amounts of sh - 2x probe dna , as characterized by exo iii digestion with 200 nm cdna ( black ) or dnase i hydrolysis with ( pink ) or without 200 nm cdna ( blue ) . [SEP]
[CLS] error bars show standard deviations from three measurements . [SEP]
[CLS] exo iii - based quantification of dna surface coverage on various particles . [SEP]
[CLS] fluorescence B-property spectra of unmodified beads ( black ) , undigested modified beads ( red ) , and supernatants from exo iii - treated , dna - modified ( a ) streptavidin - coated magnetic B-property beads ( mb - sa ) , ( b ) streptavidin - coated quantum B-nanoparticle dots I-nanoparticle ( qd - sa ) , ( c ) carboxylated magnetic B-property beads ( mb - cooh ) , ( d ) carboxylated silica microspheres ( sio 2 - cooh ) , ( e ) streptavidin - coated ucnps ( ucnp - sa ) , and ( f ) oleic acid - capped ucnps ( ucnp - oa ) . [SEP]
[CLS] assays were conducted for 1 h ( blue ) or 16 h ( magenta ) with 200 nm cdna and 0 . 2 u / μl exo iii . [SEP]
[CLS] recently , surface - enhanced raman scattering ( sers ) nanoprobes B-nanoparticle ( nps B-nanoparticle ) have shown promise in the field of cancer imaging due to their unparalleled signal specificity and high sensitivity . [SEP]
[CLS] here we report the development of a dna aptamer targeted sers np B-nanoparticle . [SEP]
[CLS] recently , aptamers are being investigated as a viable alternative to more traditional antibody B-material targeting due to their low immunogenicity B-property and low cost of production . [SEP]
[CLS] we developed a strategy to functionalize sers nps B-nanoparticle with dna aptamers , which target mucin1 ( muc1 ) in human breast cancer ( bc ) . [SEP]
[CLS] thorough in vitro characterization studies demonstrated excellent serum stability and specific binding of the targeted nps B-nanoparticle to muc1 . [SEP]
[CLS] in order to test their in vivo targeting capability , we co - injected muc1targeted sers nps B-nanoparticle , and as controls non - targeted and blocked muc1 - targeted sers nps B-nanoparticle in bc xenograft mouse models . [SEP]
[CLS] a two - tumor mouse model with differential expression of muc1 ( mda - mb - 468 and mda - mb - 453 ) was used to control for active versus passive targeting in the same animals . [SEP]
[CLS] the results showed that the targeted sers nps B-nanoparticle home to the tumors B-material via active targeting of muc1 , with low levels of passive targeting . [SEP]
[CLS] we expect this strategy to be an advantageous alternative to antibody - based targeting and useful for targeted imaging B-technique of I-technique tumor I-technique extent , progression , and therapeutic response . [SEP]
[CLS] in recent years , raman imaging has emerged as a promising modality in the field of bioimaging . [SEP]
[CLS] raman reporter coated gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) with an encapsulant layer of silica have emerged as the preferred architecture for the fabrication of bright sers nps B-nanoparticle ( with enhancement factors in the order of 10 7 - 10 ) [SEP]
[CLS] owing to the narrow , ' fingerprint ' like raman spectral signatures , sers nps B-nanoparticle have shown very high specificity of detection and multiplexing capabilities . [SEP]
[CLS] additionally , sers nps B-nanoparticle exhibit little to no susceptibility to photobleaching and have been shown to be non - toxic B-property . [SEP]
[CLS] the ultrahigh sensitivity and specificity of detection , the multiplexing capabilities , and the photostability are key advantages of the newest generations of sers nps B-nanoparticle over imaging agents based I-technique on I-technique fluorescence I-technique . [SEP]
[CLS] due to these perennial advantages , sers nps B-nanoparticle have gained a lot of attention as a highly sensitive tool for cancer imaging . [SEP]
[CLS] sers nps B-nanoparticle functionalized with polyethylene glycol ( peg ) chains exhibit relatively long blood circulation time and accumulate in cancer tissues by the virtue of the so - called enhanced permeability and retention ( epr ) effect . [SEP]
[CLS] this uptake mechanism is also known as " passive targeting " . [SEP]
[CLS] however , if one desires to detect the expression of a specific marker of interest in a tumor B-material , then " active targeting " via specific interaction of the np B-nanoparticle with a surface ligand with the tumor B-material cells B-material or cells in the tumor microenvironment is required . [SEP]
[CLS] several studies have shown promising results in achieving specific targeting of cancer biomarkers B-property in vitro and in vivo using antibodies B-material as the targeting moiety . [SEP]
[CLS] nevertheless , major disadvantages of using antibodies B-material for active sers np B-nanoparticle targeting are considerable immunogenicity B-property and high cost of production of humanized monoclonal antibodies B-material , both of which could hinder clinical translation . [SEP]
[CLS] recently , aptamers , also termed chemical antibodies B-material , have emerged as a promising candidate for targeting cancer biomarkers B-property with high selectivity and specificity . [SEP]
[CLS] aptamers possess several advantages over monoclonal antibodies B-material , such as : ( i ) low molecular weight , ( ii ) low or completely absent immunogenicity B-property , ( iii ) reproducible , large scale and inexpensive synthesis via solid phase chemistry , and ( iv ) available chemical modifications which allow different imaging agents to be attached using straight forward conjugation techniques . [SEP]
[CLS] therefore , efficient conjugation of aptamers with radioisotopes B-material , fluorophores and nanoparticles B-nanoparticle have produced several promising aptamer - targeted imaging modalities to date . [SEP]
[CLS] more encouragingly , in recent clinical trials aptamers have shown no significant immunogenicity B-property advocating the use of aptamers as a targeting moiety . [SEP]
[CLS] recently aptamer functionalized nanoparticles B-nanoparticle and sers nps B-nanoparticle gained attention for in vitro cancer targeting while in vivo targeted cancer detection still remains challenging . [SEP]
[CLS] in this work , we have developed a novel dna aptamer targeted sers nps B-nanoparticle for in vivo imaging of muc1 overexpression in bc . [SEP]
[CLS] muc1 is a transmembrane glycoprotein overexpressed in 90 % of bc , and other cancer types as well . [SEP]
[CLS] muc1 has been shown to have an important role in cancer progression and metastasis B-event and is overexpressed in early stages of triple negative breast cancer . [SEP]
[CLS] the overexpression of muc1 also alters the muc1 glycosylation pattern and exposes certain epitopes in the core B-material protein B-material , which act as an active site for specific binding . [SEP]
[CLS] recently , several monoclonal antibodies B-material have been developed for muc1 specific tumor B-technique imaging I-technique and therapy in preclinical studies . [SEP]
[CLS] however , antibody B-material targeting inherits several limitations such as long plasma half - life , immunogenicity B-property , and relatively large molecular weight . [SEP]
[CLS] to alleviate these shortcomings , nucleic acidbased aptamers are being pursued as a potential alternative to more traditional antibody B-material targeting . [SEP]
[CLS] in this work , we chose a well - studied dna aptamer that was shown to bind to muc1 with subnanomolar dissociation constant and demonstrated the active targeting capabilities of muc1 dna aptamer targeted sers nps B-nanoparticle in vivo as depicted in figure 1 . [SEP]
[CLS] we postulated that dna aptamer functionalized sers nps B-nanoparticle , a new member of the spherical nucleic B-material acid I-material ( sna ) family , will inherit low nuclease degradation compared to the free aptamer due to unique spatial distribution and produce minimal immune response . [SEP]
[CLS] the synthesis of aptamer grafted sers nps B-nanoparticle was carried out by stepwise surface functionalization of the previously reported sers nps B-nanoparticle ( figure 2a , step 1 , 2 ) . [SEP]
[CLS] the synthesis of sers nps B-nanoparticle started with the formation of 70 nm aunp B-nanoparticle cores B-material using a modified protocol using a seed - mediated growth method described by perrault et . al . ( see experimental section for details ) . [SEP]
[CLS] we used hydroxylamine ( ha ) instead of hydroquinone ( hq ) as a reducing B-property agent I-property because aunps B-nanoparticle derived from the hq synthesis did not produce bright sers particles , likely due to high surface binding and subsequent passivation by hq molecules . [SEP]
[CLS] in a typical synthesis procedure , first 15 nm aunp B-nanoparticle seeds were synthesized and subsequently grown into 70 nm aunps B-nanoparticle using haucl 4 as a gold B-material precursor and ha as a reducing B-property agent I-property in the presence of 15 nm aunp B-nanoparticle seeds . [SEP]
[CLS] within a few seconds of the reaction , the color of the solution turned deep red confirming the synthesis of 70 nm aunp B-nanoparticle cores B-material . [SEP]
[CLS] further , synthesized core B-material aunps B-nanoparticle were silica coated in the presence of ir dyes ( ir780 perchlorate or ir792 perchlorate ) by a modified stober process to produce the sers nps B-nanoparticle . [SEP]
[CLS] the silica surface of the synthesized sers nps B-nanoparticle was stepwise modified in order to attach dna as explained in figure 2b . [SEP]
[CLS] in the first step , surface B-nanoparticle hydroxyl I-nanoparticle groups I-nanoparticle were thiolated using 10 % ( 3 - marcaptopropyl ) trimethoxysilane ( mptms ) in 85 % ethanol and 5 % water B-material . [SEP]
[CLS] next , the thiol B-material groups were reacted with maleimide - peg 2 - biotin to attach biotin moieties onto the silica surface . [SEP]
[CLS] in the last step , biotinylated sers nps B-nanoparticle were incubated B-technique with 1000 molar excess of premixed neutravidin and biotinylated dna ( 1 : 1 ratio ) at room temperature in 10 mm mes buffer . [SEP]
[CLS] the dna functionalized nps B-nanoparticle were purified by centrifugation and dispersion in 10 mm mes buffer . [SEP]
[CLS] we did not observe any change in raman signal intensities of sers nps B-nanoparticle during the functionalization procedure ( data not shown ) . [SEP]
[CLS] this strategy allowed us to functionalize the sers nps B-nanoparticle essentially with any dna sequence with a biotin modification , typically purchased from a commercial source ( idt dna inc . ) . [SEP]
[CLS] we characterized the sers nps B-nanoparticle using a transmission electron microscope ( tem ) and dynamic B-technique light I-technique scattering I-technique ( dls ) . [SEP]
[CLS] a typical tem image of the sers nps B-nanoparticle is presented in figure 2c ( see figure s1 for additional tem images ) , where the electron dense aunp B-nanoparticle cores B-material and the silica B-material shell I-material are clearly visible . [SEP]
[CLS] from the tem images , average aunp B-nanoparticle core B-material and the sers np B-nanoparticle diameters was found to be 69±7 nm and 132±9 nm respectively , demonstrating a high degree of monodispersity of sizes and shapes . [SEP]
[CLS] the dynamic B-technique light I-technique scattering I-technique ( dls ) measurements yielded hydrodynamic diameters of ~ 80 nm , ~ 140 nm and ~ 168 nm of aunps B-nanoparticle , sers nps B-nanoparticle and dna functionalized sers nps B-nanoparticle , respectively as shown in figure 2d . [SEP]
[CLS] the dls diameter was found to be ~ 10 nm larger than the average diameter in tem measurements due to the added hydration layer on the np B-nanoparticle surface . [SEP]
[CLS] the presence of dna molecules on the sers np B-nanoparticle surface was verified by two independent methods . [SEP]
[CLS] in the first method , we hybridized a complementary strand modified with ir 700 dye at the 5 ′ end of the dna - functionalized sers nps B-nanoparticle . [SEP]
[CLS] after removal of excess dye modified strands by centrifugation and redispersion of the nps B-nanoparticle , we measured the fluorescence B-property in two emission channels , 700 nm , and 800 nm . [SEP]
[CLS] we found a higher fluorescence B-property signal in the 700 nm channel compared to before hybridization , due to the specific dna hybridization of ir700 dna strands to the particles ( figure s2 ) . [SEP]
[CLS] the signal in the 800 nm channel was emitted by the ir780 dye present in the sers nps B-nanoparticle itself . [SEP]
[CLS] in the second method , we hybridized aunps B-nanoparticle with a diameter of 10 nm , functionalized with the complementary dna to the sers nps B-nanoparticle . [SEP]
[CLS] we acquired tem images of the sers nps B-nanoparticle after hybridization , showing sers - np B-nanoparticle - core B-material + 10 nm - aunp B-nanoparticle - satellite structures ( figure 2e and figure s3 ) . [SEP]
[CLS] these results clearly suggested that sers nps B-nanoparticle are successfully functionalized with dna . [SEP]
[CLS] we estimated the number of dna strands to be ~ 265 ± 30 per sers np B-nanoparticle ( see methods section for details ) . [SEP]
[CLS] in order to serve as an effective targeted imaging agent in vivo , the sers nps B-nanoparticle , as well as the dna corona , should exhibit considerable serum stability in vitro within the measurement time window . [SEP]
[CLS] therefore , we first investigated the in vitro serum stability of sers nps B-nanoparticle by measuring the raman spectrum of the sers nps B-nanoparticle after 0 , 2 , 4 and 16 hours of incubation B-technique in 50 % mouse serum at 37 °c , as shown in figure 3a . [SEP]
[CLS] we observed raman spectra to be very consistent in peak positions and intensities before and after incubation B-technique . [SEP]
[CLS] the measured peak intensities did not alter with time ; the 960 cm −1 peak intensity measured around 10 , 000 counts using the same acquisition conditions ( figure 3b ) . [SEP]
[CLS] we also examined the tem images of the same sers nps B-nanoparticle before and after 16 hours of serum incubation B-technique shown in figure 3c and 3d , respectively . [SEP]
[CLS] from the visual inspection of the tem images , we observed minimal degradation of silica B-material shells I-material after serum exposure . [SEP]
[CLS] these results suggested that the sers nps B-nanoparticle were optically and structurally stable in an in vivo milieu . [SEP]
[CLS] we further investigated the stability of the dna corona upon exposure to serum . [SEP]
[CLS] we first hybridized an ir700 dye labeled complementary dna with sers nps B-nanoparticle and incubated B-technique for 16 hours in 50 % mouse serum at 37 °c . [SEP]
[CLS] subsequently , we washed , re - dispersed in 10 nm mes buffer and measured the fluorescence B-property of the sers nps B-nanoparticle as shown in figure s2 . [SEP]
[CLS] we observed a decrease in the intensity of the 700 nm emission channel by only 10 % , suggesting that the dna corona was fairly stable in biological conditions . [SEP]
[CLS] this stability can be attributed to the spherical configuration of the dna chains which are resistant to nuclease degradation , consistent with previous reports . [SEP]
[CLS] these results suggested that both raman signal and dna corona of the sers nps B-nanoparticle should be stable in an in vivo environment . [SEP]
[CLS] since tumor B-material xenograft mouse models can exhibit passive uptake of sers nps B-nanoparticle due to the epr effect , we decided to include a nontargeted sers np B-nanoparticle control to decouple the active and passive targeting . [SEP]
[CLS] the multiplexing capabilities of sers nps B-nanoparticle allowed us to use two different flavors of sers nps B-nanoparticle with distinct raman signatures for targeted nps B-nanoparticle and nontargeted nps B-nanoparticle without changing the overall np B-nanoparticle architecture as shown in figure 4a . [SEP]
[CLS] both the muc1 targeted nps B-nanoparticle ( muc1 - nps B-nanoparticle ) and nontargeted nps B-nanoparticle ( nt - nps B-nanoparticle ) consisted of a similar internal structure of a 70 nm aunp B-nanoparticle core B-material , ir dye ( ir 780 for muc1 - nps B-nanoparticle and ir 792 for nt - nps B-nanoparticle ) and a silica B-material shell I-material . [SEP]
[CLS] however , the structure of the dna corona of these particles were significantly different . [SEP]
[CLS] the muc1 - np B-nanoparticle corona consisted of a 25 base double - stranded stem and a 25 base muc1 dna aptamer sequence separated by a t5 sequence . [SEP]
[CLS] the double stranded region acted as a rigid handle to spatially separate the muc1 aptamer sequence from the np B-nanoparticle surface . [SEP]
[CLS] the nt - nps B-nanoparticle were also functionalized with the double - stranded stem but lacked the muc1 aptamer region ( see si figure s4 for design details ) . [SEP]
[CLS] we observed a slightly larger ( 10 nm ) hydrodynamic diameter of muc1 - nps B-nanoparticle than nt - nps B-nanoparticle in dls measurements , due to the presence of the aptamer region of the size approximately 5 nm ( figure s5 ) . [SEP]
[CLS] these nps B-nanoparticle have distinct raman spectra due to the presence of different raman reporter molecules absorbed onto the aunp B-nanoparticle core B-material ( figure 4b ) . [SEP]
[CLS] we further determined the limit of detection of these nps B-nanoparticle in tissue phantoms using the same imaging conditions as used for in vivo imaging . [SEP]
[CLS] the direct classical least square ( dcls ) algorithm in the wire 3 . 4 software ( renishaw ) was used to generate the 2d raman maps of both sers nps B-nanoparticle . [SEP]
[CLS] from the raman map , the limit of detection for both nps B-nanoparticle was found to be 5 - 10 fm , consistent with our previous reports ( figure s4 ) . [SEP]
[CLS] we further tested whether the dcls algorithm could differentiate the signals from sers nps B-nanoparticle mixed at different ratios . [SEP]
[CLS] we mixed muc1 - nps B-nanoparticle ( ir 780 ) and nt - nps B-nanoparticle ( ir 792 ) in different ratios and used the dcls algorithm to deconvolute the composite spectra . [SEP]
[CLS] the algorithm was found to be sensitive up to a ratio of 20 : 1 showing the high sensitivity of the multiplexed measurements using the dcls algorithm ( figure 4c ) . [SEP]
[CLS] inspired by the excellent stability profile in serum and highly sensitive multiplexed imaging capability in tissue phantoms , we explored the targeting capabilities of the sers nps B-nanoparticle in muc1 overexpressing bc cells B-material in vitro . [SEP]
[CLS] first , we tested the binding of the muc1 aptamer to the muc1 overexpressing mda - mb - 468 and muc1 negative mda - mb - 453 cell B-material lines as verified by western blot ( si figure s6c ) . [SEP]
[CLS] we performed a flow cytometry - based assay for the binding of a fluorescently B-property labeled aptamer and a random dna sequence as a control with the cell B-material lines ( figure s6 a , b ) . [SEP]
[CLS] upon quantification of cellular fluorescence B-property intensity , we found that the random sequence exhibited a weak binding to both mda - mb - 468 and mda - mb - 453 cells B-material , presumably due to nonspecific binding . [SEP]
[CLS] however , the muc1 aptamer demonstrated a higher and specific binding with mda - mb - 468 cells B-material while showing the same fluorescence B-property intensity in mda - mb - 453 cells B-material as the non - aptamer control . [SEP]
[CLS] this result indicated specific targeting by the muc1 aptamers in a muc1 overexpressing cell B-material line . [SEP]
[CLS] in order to verify that the selective binding of the muc1 aptamer to muc1 overexpressing bc cells B-material was unaltered when grafted onto the sers np B-nanoparticle surface , we incubated B-technique mda - mb - 468 cells B-material adherent on a plate with an equal concentration of muc1 - nps B-nanoparticle and nt - nps B-nanoparticle ( both with ir780 dye in order to compare the peak intensity ) . [SEP]
[CLS] after subsequent washing steps , we mapped the raman intensity of the plates at 960 cm −1 . [SEP]
[CLS] we found that the muc1 - nps B-nanoparticle adhered to the mda - mb - 468 cells B-material more than nt - nps B-nanoparticle ( figure s6 d , e ) , demonstrating in vitro targeting of muc1 - nps B-nanoparticle . [SEP]
[CLS] these findings motivated us to further test the targeting capabilities of muc1 - nps B-nanoparticle in vivo . [SEP]
[CLS] in order to validate the targeting capabilities of muc1 - nps B-nanoparticle in vivo , an equimolar cocktail of muc1 - nps B-nanoparticle and nt - nps B-nanoparticle was injected via tail vein into athymic nude mice ( n = 4 ) that were inoculated with muc1 overexpressing mda - mb - 468 cells B-material . [SEP]
[CLS] this strategy allowed us to evaluate the targeting capabilities of sers nps B-nanoparticle in the same animal , considering the epr effect can be variable between different animals even if the tumor B-material type and size are identical . [SEP]
[CLS] the raman spectrum of the injected sers np B-nanoparticle cocktail contained the major peaks from muc1 - nps B-nanoparticle ( 740 and 960 cm −1 ) and nt - nps B-nanoparticle ( 1210 cm −1 ) ( figure 5a ) . [SEP]
[CLS] after 16 - 18 hours post - injection , tumors B-material and reticuloendothelial system ( res ) rich organs such as liver were excised and imaged with a confocal raman microscope . [SEP]
[CLS] we observed that the injected sers nps B-nanoparticle primarily accumulated in res organs like liver and spleen as shown in the figure s7 , consistent with previous reports . [SEP]
[CLS] we also found a commensurate accumulation of both the nps B-nanoparticle in the res organs and observed the major peak heights to be equal to the injected dose shown in figure s8 . [SEP]
[CLS] interestingly , when we imaged the tumor B-material tissue in a raman microscope , we observed preferential accumulation of muc1 - nps B-nanoparticle ( figure 5c , d ) . [SEP]
[CLS] we observed foci of muc1 - nps B-nanoparticle to be scattered all over the tumor B-material volume , shown as red dots . [SEP]
[CLS] interestingly , the nt - nps B-nanoparticle did not have significant accumulation , with very few observed foci ( green color ) in the tumor B-material . [SEP]
[CLS] these results suggested the targeting capabilities of nps B-nanoparticle were operational even after systemic delivery in the mouse model and the epr mediated targeting was minimal . [SEP]
[CLS] histological evaluation of the resected tumors B-material using hematoxylin and eosin ( h & e ) staining confirmed the presence of tumor B-material tissue in the imaged specimen ( figure 5e ) . [SEP]
[CLS] furthermore , we performed immunohistochemical ( ihc ) staining of muc1 expression levels of the tumor B-material tissue ( figure 5f ) validating the muc1 overexpression was scattered throughout the whole tumor B-material volume . [SEP]
[CLS] these findings further led us to further investigate whether the muc1 negative tumors B-material would exhibit any uptake of the targeted and the non - targeted sers nps B-nanoparticle in vivo . [SEP]
[CLS] in order to verify the specific uptake of muc1 - nps B-nanoparticle in muc1 positive tumors B-material , we developed bilateral human bc xenograft model for such a study by staggered inoculation of mda - mb - 453 ( muc1 negative ) and mda - mb - 468 ( muc1 positive ) cell B-material lines into both flanks of athymic nude mice ( n = 3 ) . [SEP]
[CLS] after the tumors B-material had grown to similar sizes , we injected the cocktail of muc1 - nps B-nanoparticle and nt - nps B-nanoparticle and excised the tumors B-material 16 - 18 hours post injection . [SEP]
[CLS] a representative ex vivo raman image of both tumors B-material acquired in the same scan is shown in figure 6b , c . [SEP]
[CLS] we detected abundant signal from the muc1 - nps B-nanoparticle shown in red throughout the muc1 positive tumor B-material derived from the mda - mb - 468 cells B-material , and in contrast very few , sparsely distributed foci from nt - np B-nanoparticle ( green dots ) . [SEP]
[CLS] more interestingly , we observed a 10 - fold lower uptake of the targeted muc1 - np B-nanoparticle in the tumor B-material derived from the mda - mb - 453 cell B-material line . [SEP]
[CLS] the signal from nt - nps B-nanoparticle was scarce , similar to the contralateral muc1 positive tumors B-material and consistent with the results of the one - tumor mouse model . [SEP]
[CLS] the raman mapping of these tumors B-material clearly suggested selective targeting of muc1 - nps B-nanoparticle to mda - mb - 468 tumors B-material , while non - specific epr - based uptake remained negligible . [SEP]
[CLS] we further corroborated the raman imaging results by histological correlation of the fixed tissue sections . [SEP]
[CLS] ihc staining confirmed high muc1 overexpression in tumors B-material derived from the mda - mb - 468 cell B-material line overexpressed muc1 , in contrast to the tumors B-material derived from mda - mb - 453 cells B-material line , in contrast to the tumors B-material derived from mda - mb - 453 cells B-material ( figure 6d , e ) . [SEP]
[CLS] these findings strongly supported the selective targeting of muc1 overexpressing bc tumors B-material by muc1 - nps B-nanoparticle . [SEP]
[CLS] we further verified the muc1 aptamer - mediated targeting , by attaching a blocking module in the same manner as we used to verify successful dna functionalization . [SEP]
[CLS] the 10 nm aunps B-nanoparticle functionalized with a complementary dna sequence were attached to the muc1 - np B-nanoparticle to shield the aptamer from being exposed and able to bind its target . [SEP]
[CLS] the schematic representation and the tem images of the construct are shown in figure 7a and figure 7b respectively . [SEP]
[CLS] we incubated B-technique the blocked muc1 - nps B-nanoparticle in serum at 37 °c to assess the stability in vitro . [SEP]
[CLS] we did not observe significant dissociation of the 10 nm aunp B-nanoparticle shields from the blocked muc1 - nps B-nanoparticle after the serum exposure , suggesting sufficient serum stability ( figure s3 ) . [SEP]
[CLS] these blocked nps B-nanoparticle when injected in the two tumor xenograft mouse model , did not exhibit considerable uptake in either the muc1 negative or muc1 positive tumors B-material as shown in figure 7c , d . [SEP]
[CLS] this result strongly supported the fact that targeting was dependent on the dna aptamers on the sers np B-nanoparticle surface . [SEP]
[CLS] in conclusion , we have developed a strategy to functionalize sers nps B-nanoparticle with a muc1 aptamer sequence . [SEP]
[CLS] these targeted sers nps B-nanoparticle were found to be stable in biological conditions and could be detected even at low fm concentrations . [SEP]
[CLS] further , we showed that muc1 aptamers and muc1 - nps B-nanoparticle exhibited selective binding to muc - 1 overexpressing breast cancer cells B-material in vitro . [SEP]
[CLS] the multiplexing capability inherent to the methodology of raman imaging allowed us to inject differently functionalized nps B-nanoparticle into the same animal , thus comparing directly passive and targeted homing of tumor B-material lesions in vivo . [SEP]
[CLS] we were able to demonstrate that the major uptake of the nps B-nanoparticle was indebted to active rather than passive / epr driven targeting . [SEP]
[CLS] the lack of passive uptake could be attributed to the fact that the np B-nanoparticle surface is different from the traditional peg - based polymers B-material , as supported by previous findings . [SEP]
[CLS] these results advocate the use of dna aptamer sequences as targeting B-property agents I-property for sers or other imaging modalities considering the existing library of aptamer sequences for cancer biomarkers B-property and the ease of discovering one for suitable targets by sytematic evolution of ligands by exponential enrichment ( selex ) . [SEP]
[CLS] due to the high multiplexing potential of different sers probes , we envision multiplexed detection of cancer in vivo using different dna aptamers as targeting moieties . [SEP]
[CLS] we also believe stimuli - responsive sers imaging agents that will selectively light up tumors B-material overexpressing a specific antigen is not far from reality . [SEP]
[CLS] all reagents were purchased of the highest purity from sigma - aldrich ( st . louis , mo ) and were used as received . [SEP]
[CLS] the maleimide - peg 2 - biotin linker and neutravidin were obtained from thermo fisher scientific . [SEP]
[CLS] dialysis cassettes ( mwco 3 . 5 kda ; slide - a - lyzer g2 ) were purchased from thermo - fisher scientific ( waltham , ma ) . [SEP]
[CLS] all dna sequences were purchased from idt dna inc . as hplc purified grade and used without further purifications . [SEP]
[CLS] synthesis of 70 nm aunp B-nanoparticle core B-material - the aunp B-nanoparticle core B-material was synthesized using a modified protocol using a seed mediated growth method described by perrault et al . we used hydroxylamine instead of hydroquinone as a reducing B-property agent I-property . [SEP]
[CLS] in a typical synthesis procedure , we first synthesized ~ 15 nm aunp B-nanoparticle cores B-material . [SEP]
[CLS] to 99 ml deionized B-material water I-material , 1 ml 25 mm haucl 4 was added and the solution was heated on a heating plate to boil . [SEP]
[CLS] to the boiling solution , 1 ml of 3 . 3 % sodium B-material citrate solution was added . [SEP]
[CLS] after 15 minutes the color of the solution had changed to red confirming the formation of 15 nm aunp B-nanoparticle cores B-material . [SEP]
[CLS] in the next step , to 100 ml deionized B-material water I-material 125 μl of 200 mm haucl 4 , 30 μl 500 mm trisodium citrate and 700 μl 15 nm aunp B-nanoparticle cores B-material were added under stirring . [SEP]
[CLS] further , 250 μl of 1m hydroxylamine hydrochloride solution was mixed . [SEP]
[CLS] within few seconds the color of the solution turned deep red confirming the synthesis of 70 nm aunp B-nanoparticle . [SEP]
[CLS] synthesis of sers nps B-nanoparticle - synthesis of sers nps B-nanoparticle was carried out using a modified version of the previously described protocol . [SEP]
[CLS] in brief , in a 50 ml falcon tube , 10 ml 2 - propanol , 500 μl tetraethylorthosilicate ( teos ) , and 20 μl 25 mm ir dye ( ir780 or ir792 perchlorate ) dissolved in anhydrous n , n - dimethylformamide ( dmf ) were mixed . [SEP]
[CLS] in another tube , 3 ml ethanol , 1 . 2 ml of 4 nm 70 nm aunp B-nanoparticle solution and 200 μl ammonia solution were combined . [SEP]
[CLS] then the contents of two tubes were mixed under vigorous stirring and kept gently shaking for 15 minutes at room temperature . [SEP]
[CLS] after the reaction was completed , the particles were washed three times with pure ethanol . [SEP]
[CLS] dna functionalization of sers nps B-nanoparticle - as synthesized sers nps B-nanoparticle were functionalized with dna in several steps . [SEP]
[CLS] in step one , 1 nm sers nps B-nanoparticle were thiolated in 1 ml 85 % ethanol , 5 % di water B-material and 10 % ( 3 - marcaptopropyl ) trimethoxy silane ( mptms ) at 70 °c temperature . [SEP]
[CLS] after 2 hours of reaction , the particles were washed 3 times with ethanol and 1 time with di water B-material . [SEP]
[CLS] in the next step , the thiolated particles were biotinylated using a maleimide - peg 2 - biotin linker . [SEP]
[CLS] to 1 nm thiolated sers np B-nanoparticle solution in 10 mm mes buffer ( ph 7 . 5 ) , 100 μl of 10 mm maleimide - peg 2 - biotin solution was added and kept for 2 hours at room temperature . [SEP]
[CLS] next , the particles were washed with water B-material 3 times and redispersed in 10 mm mes buffer ( ph 7 . 4 ) . [SEP]
[CLS] biotinylated sers nps B-nanoparticle were then added to 1000 times excess neutravidin and biotinylated dna ( premixed 1 : 1 ratio in 10 mm mes buffer ) . [SEP]
[CLS] the sers nps B-nanoparticle were incubated B-technique for 3 - 4 hours and centrifuged 2 times to remove excess neutravidin and dna , and redispersed in 10 mm mes buffer for injection . [SEP]
[CLS] characterizations of sers nps B-nanoparticle - the sers nps B-nanoparticle were characterized by transmission B-technique electron I-technique microscopy I-technique ( jeol 1200 , 80 kv , 80 , 000× - 120 , 000× magnification ) to study the sers np B-nanoparticle structures . [SEP]
[CLS] the size and concentration of the sers nps B-nanoparticle were measured on a nanoparticle B-nanoparticle tracking analyzer ( malvern instruments , malvern , uk ) . [SEP]
[CLS] dls data was measured using zetasizer nano zs ( malvern instruments ) . [SEP]
[CLS] a raman spectra were obtained in an invia system equipped with a 785 - nm laser ( renishaw inc . , hoffman estates , il ) . [SEP]
[CLS] estimation of the grafting density of dna - the number of dna per sers np B-nanoparticle was measured using a fluorophore - modified biotinylated dna strand . [SEP]
[CLS] the fluorescence B-property intensity was measured before and after the incubation B-technique with the sers nps B-nanoparticle ( biotinylated ) . [SEP]
[CLS] the difference between the fluorescence B-property intensity is proportional to the number of the biotinylated dna attached to the particle . [SEP]
[CLS] we determined the numbers of dna by comparing the difference with the fluorescence B-property intensity of known concentration of the same dna . [SEP]
[CLS] sers nanoprobe B-nanoparticle limit of detection - to determine the limit of detection of the muc1 - nps B-nanoparticle and nt - nps B-nanoparticle , the nps B-nanoparticle were mixed in desired concentration ratios in 1 % agarose and casted in a 96 well plate . [SEP]
[CLS] the phantom was scanned using the exact setup used for the actual ex vivo tumor B-technique imaging I-technique ( 100 % laser power , 1 . 5 second integration time , 5× objective ) . [SEP]
[CLS] the direct classical least square ( dcls ) algorithm in the wire 3 . 4 software ( renishaw ) was used to generate the 2d raman maps of two sers nps B-nanoparticle . [SEP]
[CLS] the raman maps were analyzed in imagej ( https : / / imagej . nih . gov / ij / ) software to determine the limit of detection . [SEP]
[CLS] cells B-material were lysed 15 minutes on ice in 10 % ripa buffer ( 9806 , cell B-material signaling technology ) , 0 . 5 % pmsf ( 8553s , cell B-material signaling technology ) , 1 % protease / phosphatase inhibitor cocktail ( 5872 , cell B-material signaling technology ) . [SEP]
[CLS] after protein B-material quantification by using bca assay kit ( 23227 , thermofisher scientific ) , 20 μg protein B-material was mixed with reducing B-property agent I-property and lds sample buffer ( np0004 + np0007 B-nanoparticle , thermofisher scientific ) . [SEP]
[CLS] samples were resolved by 10 % sds - polyacrylamide B-technique gel I-technique electrophoresis I-technique , and electrotransfer to a pvdf membrane . [SEP]
[CLS] immunoblotting was done with the muc - 1 antibody B-material ( 4538 , cell B-material signaling technology ) or β - actin antibody B-material in 4 °c for overnight staining . [SEP]
[CLS] the staining was completed with horseradish peroxidase - conjugated secondary antibody B-material ( diluted at 1 : 10000 ) , and the protein B-material band was detected by chemiluminescence B-property ( ecl ) system ( 32109 , thermofisher scientific ) on autoradiography film . [SEP]
[CLS] the cellular binding of aptamers was determined by flow B-technique cytometry I-technique analysis of cells B-material after incubation B-technique with an af488 labeled aptamer , or a control dna ( figure s5 a and b ) . [SEP]
[CLS] the cells B-material mda - mb - 468 or mda - mb - 453 cells B-material were washed two times in facs buffer ( 1×pbs , 0 . 5 % bsa ) and suspended in the binding buffer ( 1×pbs , 5 mm mgcl 2 , ph 7 . 2 ) and incubated B-technique with muc1 aptamer or control dna , at the concentration of 100 nm for 30 min . [SEP]
[CLS] then the cells B-material were washed with facs buffer three times and run in a flow cytometer . [SEP]
[CLS] all animal experiments were approved by the institutional animal care and use committees of memorial sloan kettering cancer center . [SEP]
[CLS] four to six week old female outbred homozygous nude mice ( foxn - 1 nu / foxn - 1 nu , jackson laboratory ) were subcutaneously injected with 1 - 2 × 10 6 mda - mb - 468 and / or mda - mb - 453 cells B-material ( htb - 132 & htb - 131d , atcc ) mixed with 0 . 04 ml of matrigel ( 354248 , corning ) into each lower ventral side of the mammary fat pad . [SEP]
[CLS] since mda - mb - 453 grows less rapidly , we injected mda - mb - 453 2 weeks before the injection of mda - mb - 468 to create the 2 - tumor mouse model . [SEP]
[CLS] we waited for four weeks post inoculation or until tumor B-material sizes reached 0 . 5 × 0 . 5 cm . [SEP]
[CLS] mice were administered 150 μl of 2 nm sers np B-nanoparticle ( corresponding to a dose of 35 mg / kg ) in 10 mm mes buffer ( ph 7 . 1 - 7 . 3 ) via tail vein injection 18 - 20 hours prior to imaging . [SEP]
[CLS] mice were euthanized by carbon B-material dioxide asphyxiation and organs were imaged ex vivo . [SEP]
[CLS] all raman scans were performed on an invia raman microscope ( renishaw ) equipped with a piezo - controlled stage for spatial mapping , a 300 - mw , 785 - nm diode laser and a 1 - inch ccd detector with a spectral resolution of 1 . 07 cm −1 . [SEP]
[CLS] the sers spectra were acquired through a 5× objective lens ( leica ) . [SEP]
[CLS] typically , raman scans were performed at 100 mw laser power , with 1 . 5 second acquisition time , using the streamline high - speed acquisition mode . [SEP]
[CLS] all raman images were acquired and analyzed under the same conditions , with the same laser power , raman integration times , focal plane ( same objective lens ) , and a threshold setting of 0 . 1 . [SEP]
[CLS] the focal plane was found by focusing on the region of interest with a white light camera . [SEP]
[CLS] the raman scans have a resolution of 14 μm in the x - direction , and 80 - 200 μm in the y - direction . [SEP]
[CLS] a typical scan of tumors B-material took around 50 - 70 minutes to complete depending on the scan area . [SEP]
[CLS] schematic representation of the proposed concept : ( a ) sers nps B-nanoparticle , consisting of aunp B-nanoparticle core B-material , ir dye coating B-material , and silica B-material shell I-material , were functionalized with muc1 dna aptamers . [SEP]
[CLS] ( b ) the muc1 - nps B-nanoparticle were administered in a human bc xenograft mouse model via intravenous injection . [SEP]
[CLS] the muc1 - nps B-nanoparticle selectively homed to muc1 overexpressing bc tissue and were detected by identifying the fingerprint - like spectral signature of the sers nps B-nanoparticle with a confocal raman microscope . [SEP]
[CLS] 2 . ( a ) synthesis and dna functionalization of sers nps B-nanoparticle . [SEP]
[CLS] first , 70 nm spherical aunp B-nanoparticle cores B-material were silica coated in the presence of ir dye ( ir 780 perchlorate or ir 792 perchlorate ) to produce sers nps B-nanoparticle . [SEP]
[CLS] the sers nps B-nanoparticle were functionalized with dna molecules by sequential modification of the silica B-material shell I-material surface . [SEP]
[CLS] to verify the presence of the dna sequence , a 10 nm aunp B-nanoparticle functionalized with the complementary dna strand was hybridized to produce sers np B-nanoparticle core - 10 nm aunp B-nanoparticle satellite hybrid nanostructures . [SEP]
[CLS] ( b ) illustration of dna functionalization : first , the surface B-nanoparticle hydroxyl I-nanoparticle groups I-nanoparticle were converted to sulfhydryl groups using ( 3 - mercaptopropyl ) trimethoxysilane ( mptms ) in a water - ethanol mixture . [SEP]
[CLS] next , sulfhydryl groups were reacted with a maleimide - peg 2 - biotin linker to attach biotin molecules to the surface . [SEP]
[CLS] subsequently , biotin labeled dna molecules were attached to the biotinylated surface of the sers nps B-nanoparticle using neutravidin , a biotin - binding protein B-material as a crosslinker . [SEP]
[CLS] ( c ) representative tem images of the dna functionalized sers nps B-nanoparticle . [SEP]
[CLS] ( d ) the dls hydrodynamic diameter measurements show an increase in size from ~ 80 nm to ~ 137 nm after silica coating B-material and ~ 170 nm after dna functionalization . [SEP]
[CLS] error bars represent standard deviations of three measurements . [SEP]
[CLS] ( e ) representative tem images of the sers np B-nanoparticle core - 10nm aunp B-nanoparticle satellite hybrid nanostructures . [SEP]
[CLS] the inset shows 10 nm aunp B-nanoparticle satellites being attached to the sers np B-nanoparticle surface . [SEP]
[CLS] all the scale bars are 100 nm . [SEP]
[CLS] ( a ) raman spectra of the muc1 - nps B-nanoparticle after 0 ( black ) , 2 ( red ) , 4 ( blue ) and 16 ( purple ) hours post incubation B-technique in 50 % mouse serum at 37 °c temperature . [SEP]
[CLS] the raman spectra were acquired at 0 . 05 % laser power , 1 - second exposure time and using a 5× objective . [SEP]
[CLS] ( b ) the intensity of 960 cm −1 peak was plotted against incubation B-technique time showing an excellent signal stability of the sers nps B-nanoparticle . [SEP]
[CLS] the error bars are the standard deviation of three independent measurements . [SEP]
[CLS] ( c ) , ( d ) the tem images of the sers nps B-nanoparticle before and after 16 hours of serum exposure showing integrity of silica B-material shell I-material . [SEP]
[CLS] scale bars for inset images are 100 nm . [SEP]
[CLS] 4 . ( a ) a schematic representation of the nt - np B-nanoparticle and muc1 - np B-nanoparticle . [SEP]
[CLS] ir 792 and ir 780 dyes are used as raman reporters for nt - nps B-nanoparticle and muc1 - nps B-nanoparticle , respectively . [SEP]
[CLS] muc1 - nps B-nanoparticle are functionalized with muc1 aptamers on a ds - dna corona , while the nt - nps B-nanoparticle lack the muc1 aptamer region . [SEP]
[CLS] ( b ) the raman spectra of nt - np B-nanoparticle ( green ) and muc1 - np B-nanoparticle ( red ) showing distinct spectral signatures . [SEP]
[CLS] ( c ) determination of limit of detection of nt - nps B-nanoparticle and muc1 - nps B-nanoparticle in tissue phantoms ( 100 % laser power , 1 . 5 s integration time , 5× objective ) . different wells have different concentrations and ratio of muc1 - nps B-nanoparticle and nt - nps B-nanoparticle as described in the table above . [SEP]
[CLS] the limit of detection of individual nps B-nanoparticle was found to be ~ 10 fm and ratios up to 1 : 20 ratio of either particle can be distinguished using the dcls algorithm inbuilt in the wire 3 . 4 software . [SEP]
[CLS] 5 . ( a ) the raman spectra of nt - np B-nanoparticle ( green ) and muc1 - np B-nanoparticle ( red ) showing distinct spectral signatures [SEP]
[CLS] the spectra of the injected np B-nanoparticle cocktail ( black ) contain the major peaks from nt - np B-nanoparticle ( 1210 cm −1 ) and muc1 - nps B-nanoparticle ( 740 and 960 cm −1 ) ( b ) photograph of a nude mouse with an mda - mb - 468 tumor B-material xenograft in the left flank . [SEP]
[CLS] ( c ) bright field image of the excised tumor B-material . [SEP]
[CLS] ( d ) ex vivo raman image of the tumor B-material ( 100 % laser power , 1 . 5 s integration time , 5× objective ) . [SEP]
[CLS] the predominantly red signal corresponds to the prevalence of muc1 - nps B-nanoparticle throughout the tumor B-material volume . [SEP]
[CLS] very few foci of nt - nps B-nanoparticle accumulation represented by the green signal . [SEP]
[CLS] ( e ) hematoxylin and eosin ( h & e ) staining and ( f ) immunohistochemical staining of muc1 in the fixed tumor B-material tissue demonstrated overexpression of muc1 in the sectioned and imaged specimen . [SEP]
[CLS] ( a ) photograph of a nude athymic mouse with mda - mb - 468 tumor B-material ( r ) and mda - mb - 453 ( l ) xenograft . [SEP]
[CLS] ( b ) bright field image of the excised tumors B-material . [SEP]
[CLS] ( c ) ex vivo raman image of the tumors B-material ( 100 % laser power , 1 . 5 s integration time , 5× objective ) . [SEP]
[CLS] the predominantly red signal corresponds to the prevalence of muc1 - nps B-nanoparticle throughout the tumor B-material volume . [SEP]
[CLS] on the other hand , the mda - mb - 453 ( l ) tumor B-material showed minimal uptake of both , muc1 - nps B-nanoparticle and nt - nps B-nanoparticle . [SEP]
[CLS] ( d ) hematoxylin and eosin ( h & e ) staining of the cancer tissue . [SEP]
[CLS] ( e ) immunohistochemical ( ihc ) staining of muc1 in the fixed tumor B-material tissue demonstrating overexpression of muc1 in the mda - mb - 468 tumor B-material in contrast to the mda - mb - 453 tumor B-material . [SEP]
[CLS] 7 . ( a ) schematic representation of the blocked muc1 - nps B-nanoparticle . [SEP]
[CLS] the aptamer moiety is sterically shielded by 10 nm aunps B-nanoparticle . [SEP]
[CLS] ( b ) tem images of the blocked muc1 - nps B-nanoparticle after 16 hours of serum incubation B-technique at 37 °c showing excellent structural integrity of the hybrid structures . [SEP]
[CLS] ( c ) bright field image of the excised tumors B-material . [SEP]
[CLS] ( d ) ex vivo raman image of the tumor B-material ( 100 % laser power , 1 . 5 s integration time , 5× objective ) . [SEP]
[CLS] both tumors B-material exhibited very low uptake of the blocked nps B-nanoparticle , suggesting that np B-nanoparticle homing is due to the active targeting of the aptamers grafted on the muc1 - nps B-nanoparticle . [SEP]
[CLS] gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) show potential for transfecting target cells B-material with small interfering rna ( sirna ) , but the influence of key design parameters such as the size and shape of the particle core B-material is incomplete . [SEP]
[CLS] this paper describes a side - by - side comparison of the in vitro response of u87 glioblastoma cells B-material to different formulations of sirna - conjugated gold B-material nanoconstructs targeting the expression of isocitrate dehydrogenase 1 ( idh1 ) based on 13 - nm spheres , 50 - nm spheres , and 40 - nm stars . [SEP]
[CLS] 50 - nm spheres and 40 - nm stars showed much higher uptake efficiency compared to 13 - nm spheres . [SEP]
[CLS] confocal fluorescence B-technique microscopy I-technique showed that all three formulations were localized in the endosomes at early incubation B-technique times ( 2 h ) but that after 24 h , 50 - nm spheres and 40 - nm stars were neither in endosomes nor lysosomes while 13 - nm spheres remained in endosomes . [SEP]
[CLS] transmission B-technique electron I-technique microscopy I-technique images revealed that the 13 - nm spheres were enclosed and dispersed within endocytic vesicles while 50 - nm spheres and 40 - nm stars were aggregated , and some of these nps B-nanoparticle were outside of endocytic vesicles . [SEP]
[CLS] in our comparison of nanoconstructs with different sizes and shapes , while holding sirna surface density and nanoparticle B-nanoparticle concentration constant , we found that larger particles ( 50 - nm spheres and 40 - nm stars ) showed higher potential as carriers for the delivery of sirna . [SEP]
[CLS] glioblastoma multiforme ( gbm ) is the most common and aggressive form of malignant primary brain tumors B-material and has one of the poorest survival rates ( 14 - 16 months ) after diagnosis . [SEP]
[CLS] isocitrate dehydrogenase 1 ( idh1 ) has been recognized as a metabolic target for the development of small interfering rna ( sirna ) - based therapies against gbm . [SEP]
[CLS] however , the use of sirna as a therapeutic agent requires carriers for delivery and stabilization against nuclease degradation . [SEP]
[CLS] gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) can serve as potential sirna carriers since " free " sirna can be chemically conjugated and presented by the particle cores B-material at high surface densities . [SEP]
[CLS] aunp B-nanoparticle - sirna conjugates have been shown to enter cells B-material through scavenger receptor a I-event - I-event mediated I-event endocytosis I-event and to knock down gene expression in vitro and in vivo . [SEP]
[CLS] when densely packed on the surface of spherical aunps B-nanoparticle , sirna is more resistant to nuclease degradation compared to their free forms . [SEP]
[CLS] further development of aunps B-nanoparticle as sirna delivery agents for therapeutic applications requires an understanding of a critical biological property : cellular uptake and subsequent trafficking within cells B-material , particularly to the cytosol ( the location of the rna - induced silencing complex ) . [SEP]
[CLS] structural features of nanoconstructs that influence cellular uptake include size , shape , and surface chemistry . [SEP]
[CLS] size - dependent cellular uptake has been observed in a variety of spherical aunps B-nanoparticle , but consensus for an optimal size to maximize levels of cellular uptake has not been reached . [SEP]
[CLS] while studies of citrate - capped or protein - functionalized aunps B-nanoparticle have shown that 50 - nm aunps B-nanoparticle led to the greatest uptake by cervical cancer cells B-material ( hela ) , aunps B-nanoparticle functionalized with the drug tiopronin showed that ultra - small nps B-nanoparticle ( 2 nm ) showed the greatest cellular uptake by breast cancer cells B-material ( mcf - 7 ) . [SEP]
[CLS] such contrasting observations indicate that uptake also depends on the type of biomolecules presented by the aunp B-nanoparticle surface and interactions with cell B-material - I-material surface I-material receptors I-material . [SEP]
[CLS] other properties of nps B-nanoparticle , such as pegylation , surface charge , and the formation of protein B-material coronas I-material in vitro and in vivo also influence cellular uptake . [SEP]
[CLS] np B-nanoparticle shape is an additional structural feature that can influence uptake . [SEP]
[CLS] for example , gold B-material nanorods B-nanoparticle showed lower uptake efficiency compared to spherical particles , with the rate of cellular uptake decreasing with increasing aspect ratio . [SEP]
[CLS] gold B-material nanostars ( auns ) , anisotropic nps B-nanoparticle having multiple branches , have shown efficient delivery of magnetic B-property resonance imaging t 1 contrast B-technique agents I-technique in vitro ; these high relaxivity gd ( iii ) - dna auns showed significant contrast enhancement over their spherical counterparts . [SEP]
[CLS] furthermore , auns have shown great potential as carriers for the delivery of therapeutic aptamers in a variety of cancer cell B-material lines . [SEP]
[CLS] however , an evaluation of the biological properties of auns and spherical aunps B-nanoparticle functionalized with oligonucleotides to determine the effects of particle shape has not yet been performed . [SEP]
[CLS] moreover , although a range of np B-nanoparticle - sirna conjugates has been tested as sirna delivery vehicles B-material , a side - by - side comparison of cellular uptake and intracellular distribution of aunp B-nanoparticle - sirna conjugates that vary in size and shape is needed to accelerate the design of these nanoconstructs . [SEP]
[CLS] here we compare the in vitro biological properties of sirna nanoconstructs ( np B-nanoparticle - sirna ) with different sirna surface densities and different aunps B-nanoparticle cores B-material : 13 - nm and 50 - nm au spheres and 40 - nm au stars . [SEP]
[CLS] we tested nine combinations of cores B-material and ligand surface densities and found that np B-nanoparticle size and shape not only influenced the kinetics of cellular uptake but also affected intracellular distribution . [SEP]
[CLS] at short times ( up to 8 h ) , each nanoconstruct formulation was taken up by human primary glioblastoma u87 cells B-material in similar amounts based on the number of internalized aunps B-nanoparticle . [SEP]
[CLS] at longer periods of time ( 16 - 24 h ) , however , cellular uptake was greater for 50 - nm spheres and 40 - nm stars ( with greatest uptake by 50nm spheres ) compared to 13 - nm spheres . [SEP]
[CLS] these higher uptake levels for 50 - nm spheres and 40 - nm stars were correlated with changes in cell B-material morphology and differences in subcellular localization . [SEP]
[CLS] confocal fluorescence B-technique microscopy I-technique and immunostaining of the subcellular compartments showed that after 24 h , 13 - nm sphere sirna conjugates remained in endosomes , while most of the 50 - nm spheres and 40 - nm star carriers were neither localized to endosomes nor lysosomes . [SEP]
[CLS] tem images provided improved insight to the confocal images , and we found that 50 - nm - sphere and 40 - nm - star conjugates within cells B-material were often aggregated and in many instances , outside of endocytic vesicles . [SEP]
[CLS] to compare effects of size and shape , we prepared np B-nanoparticle - sirna nanoconstructs with different particle cores B-material ( 13 - nm spheres , 50 - nm spheres and 40 - nm stars ) ( figure 1a ) but with comparable sirna surface densities . [SEP]
[CLS] aunps B-nanoparticle were functionalized with thiol - terminated sirna and mercaptoundecyl hexa ( ethylene glycol ) ( eg 6 ) ( figure 1b ) according to literature . [SEP]
[CLS] in brief , we synthesized np B-nanoparticle - sirna constructs by adding sirna to citratecapped au spheres ( 13 - nm and 50 - nm ) and hepes - capped au stars in a nacl solution ( experimental procedures ) . [SEP]
[CLS] we used a model sirna sequence specific for idh1 , which is a potential therapeutic gene target for gbm consisting of double - stranded rna with a 3 ' thiol B-material group on the " sense " oligonucleotide strands and several 2 ' - och 3 groups in both the " sense " and " antisense " oligonucleotides , to increase the resistance of sirna to nuclease activity . [SEP]
[CLS] the addition of eg 6 following the conjugation of sirna passivated the au surface , which not only prevented aggregation of np B-nanoparticle - sirna during synthesis and purification , but also displaced remaining citrate ( 13 - nm and 50 - nm spheres ) or hepes ( 40 - nm nanostars ) from the aunp B-nanoparticle surfaces . [SEP]
[CLS] previous work has shown that oligonucleotide - functionalized spherical aunps B-nanoparticle can enter cells B-material through receptor - I-event mediated endocytosis B-event , which involves the multivalent binding of oligonucleotide to scavenger receptors . [SEP]
[CLS] because the surface density of oligonucleotides is a key parameter for cellular uptake , we compared aunp B-nanoparticle - sirna constructs with similar sirna surface densities . [SEP]
[CLS] we quantified the number of antisense ( as ) and sense ( s ) strands by selectively dissociating the oligonucleotides from the aunp B-nanoparticle surfaces and then analyzing oligreen fluorescence B-property , a nonspecific intercalator of single - stranded oligonucleotides ( experimental procedures ) . [SEP]
[CLS] next , we tuned the loading of rna strands by adjusting the molar ratio of sirna added to aunps B-nanoparticle ( r ) . [SEP]
[CLS] to prevent np B-nanoparticle aggregation during the salt - aging process , a minimum r was required for 13 - nm spheres ( 200 : 1 ) , 50 - nm spheres ( 1200 : 1 ) and 40 - nm stars ( 1200 : 1 ) , respectively . [SEP]
[CLS] 2 shows that loading of rna strands increased with increased r , and a maximum loading of rna strands was achieved when r was higher than 800 for 13 - nm spheres or 3200 for 50 - nm spheres and 40 - nm stars . [SEP]
[CLS] 1 summarizes the chemical and physical properties of sirna nanoconstructs . [SEP]
[CLS] notably , the stoichiometry of as oligonucleotides per np B-nanoparticle ( those hybridized to the sense strand ) was lower than that of the sense oligonucleotides ( bound to the au surface at the 3 ' - thiol B-material group ) for all three formulations . [SEP]
[CLS] this observation is consistent with previous work on 13 - nm aunp B-nanoparticle - sirna and indicates partial dehybridization during adsorption and purification steps . [SEP]
[CLS] we chose to compare np B-nanoparticle - sirna that were similar in surface density of total oligonucleotide ( s and as strands per np B-nanoparticle or per nm 2 ) since this property is hypothesized to contribute to the physico - chemical properties and cellular uptake of np B-nanoparticle - sirna constructs . [SEP]
[CLS] after functionalization , hydrodynamic diameters of np B-nanoparticle - sirna constructs increased by a comparable extent ( 12 - 22 nm ) . [SEP]
[CLS] all three formulations had negative surface charge and similar zeta B-property potential I-property values , which ensured a similar surface chemical environment for comparison . [SEP]
[CLS] the cellular uptake of np B-nanoparticle - sirna constructs was evaluated in a model cancer cell B-material line u87 , a grade iv gbm cell B-material line with high levels of expression of idh1 . [SEP]
[CLS] before incubation B-technique with np B-nanoparticle - sirna constructs , u87 cells B-material were grown for a time longer than that of the doubling time ( 34 h ) until > 80 % confluence was achieved to minimize the influence of cell B-material - cycle on the uptake . [SEP]
[CLS] to quantify np B-nanoparticle cellular uptake , we used inductively coupled mass spectrometry ( icp - ms ) to determine the gold B-material content per cell B-material . [SEP]
[CLS] using a two - dimensional projection of aunps B-nanoparticle from tem images to estimate particle volume , we converted au content ( number of atoms B-material ) to number of aunps B-nanoparticle per cell B-material . [SEP]
[CLS] to examine the effect of np B-nanoparticle size , we compared 13nm spheres ( 23 ± 8 as per np B-nanoparticle ) with 50 - nm spheres ( 329 ± 42 as per np B-nanoparticle ) ; these structures had similar surface density of total rna ( ~ 0 . 16 molecules nm −2 ) . [SEP]
[CLS] we treated u87 cells B-material ( 2 × 10 4 cells B-material per well in a 24 - well plate ) with np B-nanoparticle - sirna constructs under the same concentration of np B-nanoparticle ( 0 . 5 nm ) in dulbecco ' s modified eagle medium ( dmem ) supplemented with 10 % fbs . [SEP]
[CLS] the sirna concentration under these conditions was greater for 50 - nm spheres than for 13 - nm spheres by a factor of ~ 14 . [SEP]
[CLS] we treated cells B-material with these nanoconstructs at equivalent concentrations of nps B-nanoparticle instead of sirna because we aimed to understand the cellular recognition and entry of the nanostructures based on size and shape . [SEP]
[CLS] 3a shows that uptake of 13 - nm spheres and 50 - nm spheres was comparable over the first 8 h of incubation B-technique with ca . 10 4 particles per cell B-material for each np B-nanoparticle - sirna . [SEP]
[CLS] note that total sirna uptake was 10 - fold greater for 50 - nm spheres than for 13 - nm spheres because of the greater surface area and sirna stoichiometry per particle . [SEP]
[CLS] furthermore , cellular uptake of np B-nanoparticle - sirna constructs accelerated for 50 - nm spheres after this period ( 2 h and 8 h ) , which ultimately resulted in cellular uptake levels greater than those of 13 - nm spheres by a factor of 3 . 6 , and a corresponding level of sirna delivery that was greater by 50 - fold after 24 h . [SEP]
[CLS] to investigate the effect of np B-nanoparticle shape , we compared 50 - nm spheres ( 221 ± 34 as per np B-nanoparticle ) to 40 - nm stars ( 251 ± 41 as per np B-nanoparticle ) with similar surface densities of overall rna ( 0 . 13 molecules nm −2 ) . [SEP]
[CLS] 3b indicates that cellular uptake of spherical nps B-nanoparticle increased at a higher rate compared to the nanostars , and uptake efficiency of the 50 - nm spheres at 24 h was 1 . 6 times higher than that of 40 - nm stars . [SEP]
[CLS] although the uptake levels of np B-nanoparticle - sirna with different shapes were similar in the first 8 h of incubation B-technique ( ~ 8 × 10 3 particles per cell B-material ) , major differences in cellular uptake emerged at later time points . [SEP]
[CLS] since the sirna density and hydrodynamic diameter of these constructs were comparable , we hypothesized that the greater uptake of spheres over that of stars may be attributed to differences in interactions between oligonucleotides presented by spherical surfaces and those from branches directed by nanostars and cell B-material membrane - class a scavenger receptors implicated in the uptake of oligonucleotides presented by spherical nps B-nanoparticle . [SEP]
[CLS] to determine whether np B-nanoparticle - sirna constructs entered u87 cells B-material by scavenger - receptor - mediated endocytosis B-event , we investigated uptake of np B-nanoparticle - sirna by u87 cells B-material pre - treated with polyinosinic acid ( poly ( i ) , an inhibitor that can bind with scavenger receptors and thus block scavenger - receptor - mediated endocytosis B-event ) . [SEP]
[CLS] pre - treatment of u87 cells B-material with poly ( i ) resulted in a reduction of cellular uptake : 53 % ( for 13 - nm spheres ) , 54 % ( for 50 - nm spheres ) and 63 % ( for 40 - nm stars ) ( supporting information , figure s2 ) , which support that scavenger receptors play an important role in cellular uptake of np B-nanoparticle - sirna . [SEP]
[CLS] taken together , the results indicate ( 1 ) distinct increases in cellular uptake of nps B-nanoparticle driven by an increase in particle size ; ( 2 ) a dependence on np B-nanoparticle shape , where uptake of spherical particles was greater than that of nanostars of comparable surface area ; and ( 3 ) the rates of cellular uptake increased after an initial 8 - h period for larger particles , which is different from uptake kinetics previously reported for 13 - nm aunpoligonucleotide conjugates , where the most rapid uptake occurred within the first 60 min . [SEP]
[CLS] to determine how the loading density of rna strands on nps B-nanoparticle affected uptake , we quantified the cellular uptake of np B-nanoparticle - sirna constructs under three different surface rna densities . [SEP]
[CLS] the range of surface density of sirna for 13 - nm spheres ( 0 . 16 - 0 . 6 molecules nm −2 ) is higher than 50 - nm spheres ( 0 . 07 - 0 . 16 molecules nm −2 ) and 40 - nm stars ( 0 . 06 - 0 . 19 molecules nm −2 ) ( table 1 ) because of large differences of surface curvature . [SEP]
[CLS] therefore , we independently tested density effects on cellular uptake for each formulation . [SEP]
[CLS] 4 shows cellular uptake with varying surface densities of rna strands . [SEP]
[CLS] for 13 - nm spheres , when the density of rna strands increased from 0 . 16 to 0 . 44 molecules nm −2 , cellular uptake increased by 28 % , while decreased uptake was observed when the density was further increased to 0 . 60 molecules nm −2 . [SEP]
[CLS] previous work has shown that polyvalent interactions between single - stranded dna 13 - nm spherical aunps B-nanoparticle ( ssdna - aunp B-nanoparticle ) and scavenger receptors on cell B-material membranes determined the cell B-material uptake efficiency of ssdna - aunps B-nanoparticle , with higher ssdna densities leading to increased cellular uptake . [SEP]
[CLS] this discrepancy may be due to the different surface compositions ( ssdna vs . sirna ) of the aunps B-nanoparticle . [SEP]
[CLS] for 50 - nm spheres and 40 - nm stars , we found that the trend of cellular uptake vs . rna density followed that of 13 - nm spheres , where the median density of total rna showed the highest cellular uptake efficiency . [SEP]
[CLS] one explanation for the decrease in levels of cellular uptake at the highest surface densities may be from steric effects and reduced accessibility of sirna to interact with scavenger receptors . [SEP]
[CLS] surface densities at which cellular uptake decreased were considerably higher for np B-nanoparticle - sirna with 13 - nm spheres ( 0 . 6 molecules nm −2 ) compared with 50 - nm spheres and 40 - nm stars and may be due to differences of surface curvature . [SEP]
[CLS] literature on targeting systems , where np B-nanoparticle constructs enter cells B-material by receptor - I-event mediated I-event endocytosis I-event , have shown that overcrowding of ligands on the np B-nanoparticle surface prevent ligands from obtaining the correct orientation necessary for binding B-event to I-event receptors I-event . [SEP]
[CLS] these results also indicate that size and shape of np B-nanoparticle - sirna have a stronger influence on cellular uptake at longer incubation B-technique times ( 24 h ) compared to surface loading density . [SEP]
[CLS] at the 24 - h point , larger 50 - nm spheres demonstrated the highest average uptake of ca . 5 . 4 × 10 4 nps B-nanoparticle / cell B-material , while 40 - nm nanostars had slightly less uptake ( ~ 3 . 4 × 10 4 nps B-nanoparticle / cell B-material ) , and 13 - nm spheres showed the lowest uptake of ca . 1 . 9× 10 4 nps B-nanoparticle / cell B-material . [SEP]
[CLS] to visualize np B-nanoparticle - sirna within u87 cells B-material , we prepared np B-nanoparticle - sirna functionalized with single - stranded dna ( dt 20 with a 3 ' - thiol B-material ) labelled with 5 ' - cy5 ( dt 20 - cy5 ) for analysis by confocal B-technique laser I-technique scanning I-technique microscopy I-technique . [SEP]
[CLS] during the functionalization process , we pre - mixed dt 20 - cy5 with sirna ( molar ratio 1 : 8 ) before adding to the aunp B-nanoparticle solution and assumed that the relatively small amount of dt 20 - cy5 would not adversely influence the final surface composition of np B-nanoparticle - sirna constructs . [SEP]
[CLS] we analyzed the surface composition of nanoconstructs functionalized with sirna and dt 20 - cy5 , and indeed found that their loading of total rna ( as and s strands ) were not significantly different from those of without dt 20 - cy5 . [SEP]
[CLS] the proportion of dt 20 - cy5 strands to the sirna oligonucleotides was small and similar among three formulations ( 5 . 5 % , 4 . 7 % and 4 . 4 % for 13 - nm spheres , 50nm spheres and 40 - nm stars , respectively ) . [SEP]
[CLS] to test whether the addition of dt 20 - cy5 perturbed the cellular uptake of np B-nanoparticle - sirna , we compared the cellular uptake of nanoconstructs with dt 20 - cy5 to those without dt 20 - cy5 by icp - ms . [SEP]
[CLS] we found cellular uptake of np B-nanoparticle - sirna / cy5 compared to unlabeled constructs was enhanced by 40 % , 54 % and 33 % for 13 - nm spheres , 50 - nm spheres and 40 - nm stars respectively ( supporting information , figure s3 ) , which may be from the small fraction of oligonucleotides presenting positively charged cy5 at the aunp B-nanoparticle surfaces . [SEP]
[CLS] however , the data showed similar trends on cellular uptake ( greatest uptake efficiency for 50 - nm spheres , followed by 40 - nm stars , followed by 13 - nm spheres ) , which suggests that the cy5 labels did not change the nature of the interaction of the np B-nanoparticle - sirna with u87 cells B-material . [SEP]
[CLS] next , we monitored the cellular uptake of three formulations of cy5 - labeled nanoconstructs with similar density of rna strands ( 0 . 21 , 0 . 19 and 0 . 18 molecules nm −2 for 13 - nm spheres , 50 - nm spheres and 40 - nm stars , respectively ) by confocal microscopy B-technique at two representative time points ( 2 h and 24 h ) , corresponding to the two major regimes found in the kinetic analysis of figure 3 . [SEP]
[CLS] 5a shows u87 cells B-material following the incubation B-technique with np B-nanoparticle - sirna / cy5 at an equivalent np B-nanoparticle concentration for the three formulations or the equivalent volume of pbs as control . [SEP]
[CLS] at equal concentrations of nps B-nanoparticle , the levels of sirna and cy5 for the 50 - nm spheres and 40 - nm stars were ca . 12 and 9 - fold greater than that of 13 - nm spheres . [SEP]
[CLS] to distinguish whether nanoconstructs were internalized by cells B-material or just associated within the extracellular matrix ( ecm ) , we stained the actin cytoskeleton with phalloidin , which can differentiate between intracellular compartments and the ecm . [SEP]
[CLS] at short time periods ( 2 h ) , the cy5 fluorescence B-property was surrounded by actin signals , which supported that np B-nanoparticle - sirna were distributed inside cells B-material . [SEP]
[CLS] at longer incubation B-technique times ( 24 h ) , we found that u87 cells B-material showed significant accumulation of 50 - nm spheres and 40 - nm stars and lower levels for 13 - nm spheres . [SEP]
[CLS] in a separate experiment , we treated u87 cells B-material with np B-nanoparticle - sirna with equivalent concentrations of sirna and cy5 and also observed higher levels of cy5 within cells B-material treated with 50 - nm spheres or 40 - nm stars compared to 13 - nm spheres ( supporting information , figure s4 ) . [SEP]
[CLS] both quantitative analysis of aunp B-nanoparticle uptake ( figure 3 ) and qualitative analysis of cy5 fluorescence B-property confirmed that larger np B-nanoparticle - sirna constructs entered u87 cells B-material in greater amounts ( on a particle - to - particle basis ) and are more efficient carriers for delivering sirna . [SEP]
[CLS] interestingly , we discovered differences in cell B-material morphology among the different formulations at both time points of observation ( 2 and 24 h ) ( figure 5a ) . [SEP]
[CLS] cells B-material treated with 13 - nm spheres had long , narrow , branched extensions like control cells B-material treated with pbs , while cells B-material treated with 50 - nm spheres and 40 - nm stars showed fewer structures of this type . [SEP]
[CLS] to investigate whether changes in cell B-material morphology were induced by possible toxicity B-property of nanoconstructs , we tested the viability B-property of I-property u87 I-property cells I-property by 2 - ( 4 - sulfophenyl ) - 2h - tetrazolium ( mts ) assay after treatments with the same concentrations of np B-nanoparticle - sirna / cy5 used in the confocal studies ( experimental procedures ) . [SEP]
[CLS] 5b shows that the cell B-material viabilities following treatment with all three np B-nanoparticle - sirna were greater than 80 % , which suggested minimal cytotoxicity B-property . [SEP]
[CLS] endocytotic processes may involve the rearrangement ( assembly and disassembly ) of actin filaments , leading to reshaping of the plasma membrane and facilitation of internalization . [SEP]
[CLS] to test whether the morphological changes of u87 cells B-material were correlated with cellular uptake of np B-nanoparticle - sirna , we pre - treated u87 cells B-material with an actin polymerization inhibitor ( cytochalasin d , cytod ) and then investigated the cellular uptake of the three np B-nanoparticle - sirna in the presence of cytod . figure 5c shows that inhibition of actin polymerization resulted in the inhibition of cellular uptake of np B-nanoparticle - sirna , and that the magnitude depended on np B-nanoparticle size and shape . [SEP]
[CLS] cellular uptake of 13 - nm spheres showed only a minor decrease ( ~ 10 % ) in uptake in cells B-material with arrested polymerization , while 50 - nm spheres and 40 - nm stars showed greater levels of inhibition ( 20 - 30 % ) . [SEP]
[CLS] these observations suggest ( 1 ) that endocytosis B-event of 50 - nm spheres and 40 - nm stars involve greater levels of actin polymerization compared to 13 - nm stars ; and ( 2 ) that the morphological changes observed in u87 cells B-material from 50 - nm spheres and 40 - nm stars may be related to cytoskeletal rearrangements and actin polymerization involved in the endocytosis B-event of these constructs . [SEP]
[CLS] besides cellular uptake , the subcellular location of nanoconstructs is another critical parameter that can influence therapeutic efficacy and especially for sirna , delivery of the constructs to the cytoplasm is needed . [SEP]
[CLS] one challenge for np B-nanoparticle - based constructs is that following endocytosis B-event , the majority of nanoconstructs reside in early endosomes , and from there are sorted to late endosomes and lysosomes . [SEP]
[CLS] to investigate the subcellular trafficking of np B-nanoparticle - sirna and the dependence of trafficking on the size and shape of np B-nanoparticle - sirna , we treated u87 cells B-material with three different formulations of np B-nanoparticle - sirna / cy5 and monitored their localization . [SEP]
[CLS] using antibodies B-material targeting the early endosome antigen 1 ( eea1 ) and lysosomeassociated membrane glycoprotein 1 ( lamp1 ) , we labeled the endosomes and lysosomes with different fluorophores to establish independently the co - localization of np B-nanoparticle - sirna with endosomes and lysosomes . [SEP]
[CLS] 6 shows confocal microscopy B-technique images of np B-nanoparticle - sirna / cy5 constructs in u87 cells B-material at two time points ( 2 h and 24 h ) . [SEP]
[CLS] at 2 h , nearly all the construct signals , regardless of np B-nanoparticle size and shape , overlapped well with endosomal signals ( representative areas indicated by solid yellow arrows ) , which suggests that np B-nanoparticle - sirna constructs first localize in endosomes . [SEP]
[CLS] there is no significant overlap of cy5 fluorescence B-property with lysosomes at this early incubation B-technique time . [SEP]
[CLS] as the incubation B-technique time increased to 24 h , the location of np B-nanoparticle - sirna constructs within u87 cells B-material varied among the three formulations . [SEP]
[CLS] for 13 - nm spheres , only a small amount of signal overlapped with lysosomal signals ( solid white arrows , figure 6 ) , and most of the np B-nanoparticle - sirna constructs remained trapped within endosomes . [SEP]
[CLS] for 50 - nm spheres , most of the nanoconstructs showed little co - localization with either endosomes or lysosomes ( hollow white arrows , figure 6 ) . [SEP]
[CLS] 40 - nm stars showed distributions like the 50 - nm spheres with only slight differences in intracellular distribution . [SEP]
[CLS] taken together , the results indicate that np B-nanoparticle - sirna constructs follow the endosomal pathway at the beginning of endocytosis B-event ( within 2 h ) but that over long incubation B-technique times ( 24 h ) , the 13 - nm spheres remained in endosomes while 50 - nm spheres and 40 - nm stars were in neither early endosomes nor lysosomes . [SEP]
[CLS] to test whether 50 - nm spheres and 40 - nm star constructs were distributed in the cytoplasm , we visualized their intracellular location with transmission B-technique electron I-technique microscopy I-technique ( tem ) after 24 h ( figure 7 ) . [SEP]
[CLS] to increase the contrast of the cellular organelles , we double - stained cell B-material sections by uranyl acetate and lead citrate ( experimental procedures ) . [SEP]
[CLS] for cells B-material treated with 13 - nm spheres , nearly all the nps B-nanoparticle were enclosed by a clear , intact membrane ( figure 7a ) , indicating that 13 - nm spheres were trapped in vesicles . [SEP]
[CLS] within the vesicles , 13 - nm spheres were well separated . [SEP]
[CLS] for cells B-material treated with 50 - nm spheres however , many nps B-nanoparticle were found outside the vesicles ( figure 7b , yellow arrows ) . [SEP]
[CLS] we also observed 50 - nm spheres trapped in vesicles , which appeared as large aggregates of particles . [SEP]
[CLS] correlating this data with confocal images in figure 6 , we suggest that the vesicles are endocytic compartments other than early endosomes or lysosomes . [SEP]
[CLS] interestingly , some vesicle membranes enclosing the 50 - nm spheres were not well resolved compared to those around 13 - nm spheres , and some membranes seemed to be locally disrupted and discontinuous ( figure 7b , orange arrows ) . [SEP]
[CLS] in u87 cells B-material treated with 40 - nm stars , we also observed stars outside of vesicles ( figure 7c , yellow arrows ) , while those that remained trapped in vesicles appeared as np B-nanoparticle aggregates with very few isolated nps B-nanoparticle . [SEP]
[CLS] s5 summarizes a more extensive set of tem images of u87 cells B-material treated with np B-nanoparticle - sirna that further support these observations . [SEP]
[CLS] previous tem work has indicated a wide range of intracellular distributions of aunps B-nanoparticle , most likely because of differences in the nanoconstructs ( for example , size / shape of aunps B-nanoparticle and surface functionalization ) , cell B-material type , and culture conditions . [SEP]
[CLS] we have summarized the literature in table s3 ( supporting information ) . [SEP]
[CLS] for example , citrate or polyethylene glycol coated 15 - nm spherical aunps B-nanoparticle were mainly distributed in vesicles in human alveolar epithelial cells B-material ( a549 ) ; vesicle sizes containing nps B-nanoparticle increased from less than 150 nm to greater than 1000 nm as incubation B-technique time increased from 1 h to 24 h . [SEP]
[CLS] in contrast , similarly sized spherical aunps B-nanoparticle functionalized with cell - penetrating peptides B-material showed unusual distribution in hela cells B-material , where aunps B-nanoparticle were initially ( 2 h ) found in the cytosol , nucleus , and mitochondria but were later ( 24 h ) found densely packed within vesicles and then finally distributed into the cytosol after even longer periods of time ( 48 h ) . [SEP]
[CLS] for np B-nanoparticle - sirna , we found that subcellular distribution most strongly depended on size , where 13 - nm spheres were only found in vesicles , and 50 - nm spheres and 40 - nm stars were found in vesicles and in the cytoplasm . [SEP]
[CLS] in summary , we examined the influence of the structural features of np B-nanoparticle diameter and np B-nanoparticle shape on the ability of np B-nanoparticle - sirna to enter u87 cells B-material . [SEP]
[CLS] we fabricated nine formulations of sirna conjugated au nanoconstructs based on cores B-material of 13 - nm spheres , 50 - nm spheres and 40 - nm stars . [SEP]
[CLS] using a set of techniques ( quantitative analysis of aunps B-nanoparticle and fluorescence B-property and electron B-technique microscopy I-technique imaging of dye - labelled oligonucleotides and aunps B-nanoparticle ) , we differentiated the cellular response and the intracellular distribution of the np B-nanoparticle - sirna . [SEP]
[CLS] sideby - side comparison of cellular uptake of the formulations indicated much higher uptake efficiency for 50 - nm spheres and 40 - nm stars compared to 13 - nm nps B-nanoparticle . [SEP]
[CLS] by means of confocal fluorescence B-technique microscopy I-technique , we found all the formulations were localized in the endosomes at an early incubation B-technique time ( 2 h ) ; however , after 24 h , a majority of 50 nm spheres and 40 - nm stars were neither in endosomes nor in lysosomes , while 13 - nm spheres remained in the endosomes . [SEP]
[CLS] interestingly , we found that 50 - nm spheres and 40 - nm stars formed clusters within cells B-material , and to some extent were distributed to the cytoplasm , while 13nm spheres were mainly distributed inside the vesicles in a form of single np B-nanoparticle . [SEP]
[CLS] these observations indicate that the size of aunps B-nanoparticle is a major design feature that can influence uptake of sirna ( from greater numbers of nps B-nanoparticle as well greater amounts of sirna delivered per particle ) and that the intracellular distribution of the larger np B-nanoparticle - sirna can be differentiated from that of smaller npsirna . [SEP]
[CLS] the underlying cause for these differences , as well as the consequences on the function of sirna delivered by nps B-nanoparticle , requires further study . [SEP]
[CLS] larger np B-nanoparticle diameters in the design of np B-nanoparticle - sirna should be considered as a key feature in the development of np B-nanoparticle agents for in vitro and potentially in vivo delivery of sirna . [SEP]
[CLS] spherical aunps B-nanoparticle with 13 - nm diameter were synthesized by reducing chloroauric acid ( haucl 4 ) in sodium B-material citrate solution . [SEP]
[CLS] spherical aunps B-nanoparticle with 50 - nm diameter were obtained from ted pella . [SEP]
[CLS] gold B-material nanostars ( auns ) were synthesized by reducing 0 . 2 mm of haucl 4 at room temperature in 110 mm of hepes buffer ( atlanta biologicals ) . [SEP]
[CLS] to prepare duplex sirna , the sense strand ( 3 ' - dtdtu ome u ome u ome gc ome c ome c ome u ome au ome agau ome acu ome g ome u ome - 5 ' , 200 µm ) and anti - sense strand ( 5 ' - aaacgggau ome au ome c ome u ome au ome gac ome adtdt - 3 ' , 200 µm ) were hybridized in nuclease - free duplex buffer ( 30 mm hepes and 100 mm potassium B-material acetate , ph 7 . 5 , idt ) . [SEP]
[CLS] the hybridization was carried out by first heating the solution to 45 °c for 15 min , then cooling to 37 °c over 30 min and 4 °c for another 30 min . [SEP]
[CLS] duplex sirna was added to the aunp B-nanoparticle solution at a molar ratio of ≥ 200 for 13 - nm spheres and ≥ 1200 for 50nm spheres and auns . [SEP]
[CLS] sds was then added to the reaction mixture to a final concentration of 5 mm , and the mixture was shaken at room temperature for 2 h to allow the adsorption of sirna on np B-nanoparticle surface . [SEP]
[CLS] nacl was added to the mixture , while shaking , to result in a sequence of increasing concentrations of nacl in the reaction mixtures for the following time periods : ( 1 ) 50 mm for 1h ; ( 2 ) 100 mm for 1 h ; ( 3 ) 200 mm for 1 h ; ( 4 ) 400 mm for 16 h . [SEP]
[CLS] subsequently , 11 - mercaptoundecyl hexa ( ethylene glycol ) ( eg 6 , aldrich ) was added to the mixture at a molar ratio ( to aunp B-nanoparticle ) of 2000 for 13 - nm spheres and 10 , 000 for 50 - nm spheres and auns . [SEP]
[CLS] the mixture was shaken at room temperature for 5 h , followed by centrifugation at 4 °c using different speeds for different nps B-nanoparticle ( 13 - nm spheres : 21 , 000 ×g for 30 min ; 50nm spheres : 8000 ×g for 10 min auns : 9000 ×g for 10 min ) . [SEP]
[CLS] the supernatant was discarded and the pellet was resuspended in nuclease - free pbs buffer ( thermo ) . [SEP]
[CLS] two more centrifugation cycles were carried out to completely remove the unconjugated sirna and eg 6 . [SEP]
[CLS] to determine the stoichiometry of the rna oligonucleotide to aunp B-nanoparticle , the noncovalently associated anti - sense strands and covalently conjugated sense strands were sequentially separated from the aunps B-nanoparticle . [SEP]
[CLS] gold B-material nanoconstructs ( 2 × 10 −12 mol ) were suspended in 400 µl of 8 m urea and heated to 45 °c with shaking for 20 min . [SEP]
[CLS] the solution was diluted with 0 . 1 % tween - 20 to a final concentration of 4 m urea and centrifuged , followed by the separation of the supernatant solution , containing as oligonucleotides , and aunp B-nanoparticle pellet . [SEP]
[CLS] the aunp B-nanoparticle pellet was re - suspended in 10 µl of iodine B-material solution ( 0 . 16 m i 2 and 1 . 0 m ki ) and the mixture was shaken at room temperature for 5 min , followed by addition of 10 µl of 1 . 0 m nah 2 po 4 . [SEP]
[CLS] reaction with nabh 4 and dithiothreitol ( dtt ) ( 1 : 5 mixture of 2 m nabh 4 : 0 . 3 m dtt ) for 5 min was followed by centrifugation at 21 , 000 ×g for 10 min to obtain a supernatant containing sense strands . [SEP]
[CLS] the concentrations of antisense strands ( c as ) and sense strands ( c s ) in each supernatant were analyzed by the quant - it oligreen ( invitrogen ) assay , where oligreen reagent was added to the supernatant for measurements of the fluorescence B-property at λ ex = 485 nm . [SEP]
[CLS] a calibration curve was generated using reaction mixtures containing aunps B-nanoparticle and standards of c as or c s , prepared in parallel and with the same chemical reactions used to dissociate as and s from the nanoconstructs . [SEP]
[CLS] finally , the number of anti - sense strands ( or sense strands ) per aunp B-nanoparticle was calculated by dividing c as or c s by the concentration of aunps B-nanoparticle which was measured by icp - ms . [SEP]
[CLS] the human primary glioblastoma cell B-material line u87 ( atcc ) maintained in dulbecco ' s modified eagle ' s medium ( dmem ) ( gibco ) supplemented with 10 % fetal bovine serum ( fbs ) ( gibco ) was used as the target cell B-material line for in vitro efficiency evaluations . [SEP]
[CLS] cells B-material were plated on 12 - well plates with the density of 2 × 10 4 cells B-material / well and cultured in complete growth media at 37 °c with 5 % co 2 until cells B-material reached ~ 80 % confluence . [SEP]
[CLS] cells B-material were treated with 0 . 5 nm of gold B-material nanoconstructs for different time periods ( 2 h , 8 h , 16 h , and 24 h ) , after which the supernatant was removed and cells B-material were washed 3 times with pbs . [SEP]
[CLS] cells B-material were then treated with trypsin to release the cells B-material from the plates . [SEP]
[CLS] trypsinized cells B-material were transferred to a 1 . 5 ml of eppendorf tube and washed 3 times with pbs by centrifugation at 300 g × 5 min . [SEP]
[CLS] after washing , cell B-material pellets were resuspended in pbs and counted with a hemocytometer . [SEP]
[CLS] finally , cells B-material were treated with an acid solution ( 2 % hcl + 2 % hno 3 ) at 70 °c overnight to prepare lysates for the quantification of gold B-material by inductively coupled plasma mass spectrometry ( icp - ms ) . [SEP]
[CLS] to visualize the np B-nanoparticle distributions within cells B-material , all the three formulations were backfilled with 5 ' - cy5 - conjugated ( dt ) 20 oligonucleotides with 3 ' - disulfide . [SEP]
[CLS] u87 cells B-material were plated on a round coverslip ( d = 12mm , bd biosciences ) with the density of 10 4 cells B-material / coverslip and cultured in complete growth media overnight . [SEP]
[CLS] cells B-material were treated with 0 . 5 nm of cy5 - labeled nanoconstructs for 2 h or 24 h , followed by 3 - time washing with pbs . [SEP]
[CLS] cells B-material were fixed with 4 % paraformaldehyde at room temperature for 10 min , washed 3 times with pbs , permeabilized with triton x100 ( 0 . 1 % , at room temperature for 5 min ) , and then washed 3 times again with pbs . [SEP]
[CLS] the actin cytoskeleton of permeabilized cells B-material was stained with alexa fluor® 594 phalloidin at room temperature for 30 min . [SEP]
[CLS] finally , the coverslips were mounted on glass slides using dapi - containing [UNK] antifade mounting medium ( invitrogen ) . [SEP]
[CLS] after plating on the coverslips , cells B-material were treated with cy5 - labeled 13 - nm , 50 - nm or auns nanoconstructs which had the similar density of rna strands ( 0 . 21 , 0 . 19 and 0 . 18 molecules nm −2 for 13 - nm spheres , 50 - nm spheres and 40 - nm stars , respectively ) . [SEP]
[CLS] after 2 h or 24 h incubation B-technique , the coverslips were washed 3 times with pbs , fixed with 4 % paraformaldehyde and permeabilized with 0 . 1 % triton x100 . [SEP]
[CLS] cells B-material were incubated B-technique with the blocking solution ( 1 % bsa and 10 % rabbit serum in pbs ) at room temperature for 1 h . [SEP]
[CLS] cells B-material were then incubated B-technique at 4 °c overnight with a solution of primary antibodies B-material ( mouse anti - human lamp - 1 antibody B-material and goat anti - human eea - 1 antibody B-material ; 300 - fold dilutions of stocks from santa cruz biotechnology ) in blocking solution . [SEP]
[CLS] after overnight incubation B-technique , coverslips were washed 6 times with pbst ( 0 . 1 % tween 20 in pbs ) over 1 h . [SEP]
[CLS] secondary antibodies B-material ( rabbit anti - mouse igg ( h + l ) alexa fluor 488 and rabbit antigoat igg ( h + l ) alexa 594 , thermo ) in blocking solution were incubated B-technique with the cells B-material for 1 h at room temperature . [SEP]
[CLS] following an additional 6 washes with pbst buffer over 1 h , coverslips were mounted on glass slides using dapi - containing antifade mounting medium ( invitrogen ) . [SEP]
[CLS] u87 cells B-material were seeded in a 12 - well plate with the density of 4 × 10 5 cells B-material / well . [SEP]
[CLS] after 48 h , cells B-material were incubated B-technique with 0 . 5 nm of np B-nanoparticle - sirna nanoconstructs in dmem supplemented with 10 % fbs for 24 h at 37 °c in 5 % co 2 . [SEP]
[CLS] cells B-material were then washed with pbs for 3 times and harvested by treating with 0 . 25 % trypsin - edta for 5 min at the room temperature . [SEP]
[CLS] the cells B-material were then centrifuged at 300 ×g for 5 min . [SEP]
[CLS] the supernatant was removed , and the pellets were transferred to the karnovsky ' s fixative and fixed using a pelco biowave microwave for 4 min . [SEP]
[CLS] primary fixative was exchanged for fresh fixative , and microwave fixation was carried out for another 4 min , followed by two washes with phosphate buffer ( pb ) . [SEP]
[CLS] a subsequent fixation step was carried out with the secondary fixative of 1 % osmium tetroxide in di water B-material , followed by 3 washes with di water B-material . [SEP]
[CLS] a series of acetone solutions ( 30 % , 50 % , 70 % , 90 % in di water B-material ) , followed by 2 treatments with 100 % acetone was used to dehydrate the specimens . [SEP]
[CLS] cells B-material were then infiltrated with 25 % and 50 % of embed 812 resin with microwaving , followed by bench infiltration with 75 % of resin in acetone overnight . [SEP]
[CLS] two further infiltrations with 100 % resin were carried out over 5 h . [SEP]
[CLS] polymerization took place in a 60 °c oven for 24 h . [SEP]
[CLS] a leica ultracut s or rmc mt - 6000 xl microtome was used to collect 90 nm thick sections . [SEP]
[CLS] cell B-material sections were double - stained with uranyl acetate and lead citrate . [SEP]
[CLS] finally , imaged were captured by a jeol1230 tem and gatan 831 bottom - mounted ccd camera . [SEP]
[CLS] tunable loading of rna strands on aunps B-nanoparticle by adjusting the molar ratio of sirna to aunp B-nanoparticle used for conjugation . s and as represent sense and anti - sense strands , respectively . [SEP]
[CLS] composition of core - shell structures of np B-nanoparticle - sirna constructs ( a ) transmission B-technique electron I-technique microscopy I-technique ( tem ) images of au np B-nanoparticle cores B-material . [SEP]
[CLS] ( b ) chemical composition of the ligand shell B-material . [SEP]
[CLS] nucleotides highlighted in red have 2 ' - och 3 substitutions to increase their resistance to nuclease - catalyzed degradation . [SEP]
[CLS] ( a ) size - and ( b ) shape - dependent cellular uptake kinetics of au np B-nanoparticle - sirna constructs . [SEP]
[CLS] in ( a ) , 13 - nm spheres ( 23 ± 8 as per np B-nanoparticle ) and 50 - nm spheres ( 329 ± 42 as per np B-nanoparticle ) had similar oligonucleotide densities of ~ 0 . 16 molecules nm −2 . [SEP]
[CLS] in ( b ) , 50 - nm spheres ( 221 ± 34 as per np B-nanoparticle ) and 40 - nm stars ( 251 ± 41 as per np B-nanoparticle ) had oligonucleotide densities of ~ 0 . 13 molecules nm −2 . [SEP]
[CLS] 4 . np B-nanoparticle - sirna constructs with median density of total rna exhibited the highest cellular uptake efficiency ( * p < 0 . 05 ; * * p > 0 . 05 ) . [SEP]
[CLS] 5 . intracellular distribution of np B-nanoparticle - sirna constructs and cell B-material morphology changes induced by larger constructs . [SEP]
[CLS] ( a ) confocal fluorescence B-property images of cells B-material treated with pbs ( control ) or three different formulations of constructs with the same concentration of nps B-nanoparticle ( 0 . 2 nm ) ; the actin ( a ) cytoskeleton and nucleus ( n ) were stained with alexa fluor® 594 phalloidin ( green ) and dapi ( blue ) , respectively ; nanoconstructs were labeled with cy5 ( red ) . [SEP]
[CLS] the scale bar is 20 µm for all images . [SEP]
[CLS] ( b ) mts assay of the cell B-material viabilities of u87 cells B-material treated with three different formulations of np B-nanoparticle - sirna / cy5 ( 0 . 2 nm ) or pbs as the control ; ( c ) cell B-material uptake inhibition by cytod for three formulations . [SEP]
[CLS] following 30 - min pre - treatment with cytod , u87 cells B-material were incubated B-technique with np B-nanoparticle - sirna in presence of cytod for another 30 min and cell B-material uptake was measured by icp - ms . [SEP]
[CLS] as a control , u87 cells B-material were also treated with constructs under the same conditions , but without cytod . [SEP]
[CLS] confocal fluorescent B-property immunostaining studies indicate size - and shape - dependent subcellular localizations of np B-nanoparticle - sirna constructs . [SEP]
[CLS] cy5 - labeled nanoconstructs ( 0 . 2 nm in np B-nanoparticle , red color ) were treated with u87 cells B-material for 2 h or 24 h , followed by immunostaining of endosomes and lysosomes by eea - 1 antibody B-material ( blue ) and lamp - 1 antibody B-material ( green ) , respectively . [SEP]
[CLS] the solid yellow arrows indicate co - localizations of constructs with endosomes ; solid white arrows indicate the co - localization of constructs with lysosomes ; hollow white arrows indicate constructs that are neither localized in endosomes nor in lysosomes . [SEP]
[CLS] the scale bar is 10 µm for all images . [SEP]
[CLS] representative tem images of u87 cells B-material after treatment with np B-nanoparticle - sirna constructs indicate larger constructs can distribute in the cytoplasm . [SEP]
[CLS] u87 cells B-material were treated with 0 . 5 nm of ( a ) 13 - nm spheres , ( b ) 50 - nm spheres , and ( c ) 40 - nm stars for 24 h . [SEP]
[CLS] images in boxes ( lower panel ) indicate zoomed - in views . [SEP]
[CLS] yellow arrows indicate nps B-nanoparticle distributed outside vesicles ; orange arrows indicate locally disrupted vesicle membranes . [SEP]
[CLS] the nanoassembly behavior of trivalent small molecule - dna hybrids ( smdh 3 s ) were investigated as a function of core B-material geometry and supramolecular flexibility through a synergistic experimental - modeling study . [SEP]
[CLS] while complementary smdh 3 s possessing a highly flexible tetrahedral trivalent core B-material primarily assemble into nanoscale caged dimers , the nanoassemblies of smdh 3 comonomers with rigid pyramidal and trigonal cores B-material yields fewer caged dimers and more large - oligomer networks . [SEP]
[CLS] specifically , the rigid pyramidal smdh 3 comonomers tend to form smaller nanosized aggregates ( dimer , tetramer , and hexamer ) upon assembly , attributable to the small ( < 109 ° ) branch - core - branch angle of the pyramidal core B-material . [SEP]
[CLS] in contrast , the more - rigid trigonal planar smdh 3 comonomers have a larger ( ~ 120 ° ) branch - core - branch angle , which spaces their dna arms farther apart , facilitating the formation of larger nanoassemblies ( ≥ nonamer ) . [SEP]
[CLS] the population distributions of these nanoassemblies were successfully captured by coarse - grained molecular dynamics ( cgmd ) simulations over a broad range of dna concentrations . [SEP]
[CLS] cgmd simulations can also forecast the effect of incorporating t n spacer B-material units between the hydridizing dna arms and the rigid organic B-material cores I-material to increase the overall flexibility of the smdh 3 comonomers . [SEP]
[CLS] such " decoupling " of the dna arms from the organic B-material core I-material was found to result in preferential formation of nanoscale dimers up to an optimal spacer B-material length , beyond which network formation takes over due to entropic factors . [SEP]
[CLS] the excellent agreement between simulation and experimental results confirm the versatility of the cgmd model as a useful and reliable tool for elucidating the nanoassembly of smdh - based building blocks . [SEP]
[CLS] as a versatile , scalable synthon , synthetic dna has been tethered to many building blocks such as small organic molecules , dendrimers B-nanoparticle , polymers B-material , and metallic B-nanoparticle nanoparticles I-nanoparticle , which can then form a variety of hybrid nanomaterials B-material with many applications in diagnostics and therapeutics . [SEP]
[CLS] given the synthetic ease with which the sugar - phosphate backbone can be manipulated and the high sequence - specificity of nucleotide base pairing , dnafunctionalized supramolecular assemblies and nanostructures have also provided a in a previous study , we have shown that the dynamic nanoassembly of flexible smdhs ( fsmdh 3 s ) comonomers with three dna arms surrounding a highly flexible tris ( oxypropyloxymethyl ) methyl core B-material 1 ( fig . 1 ) can result in either nanoscale caged dimers or ill - defined networks depending on the length of the dna arms . [SEP]
[CLS] in addition , a coarsegrained molecular dynamics ( cgmd ) simulation strategy was successfully used to provide mechanistic insights into this assembly process as well as the most suitable range of dna arm lengths that can be used to favor the formation of caged dimers . [SEP]
[CLS] however , as core B-material 1 is not representative of the broad ranges of currently known smdh nanoassemblies , many of which have employed highly rigid aromatic organic B-material cores I-material ( e . g . , 2 , 3 , and 4 in fig . 1 ) , a more general model must be developed that can account for the roles that core - centric factors - such as the rigidity and geometry of the organic B-material cores I-material and the connection between the core and the dna arms - may play in the nanoassemblies of smdhbased building blocks . [SEP]
[CLS] herein , we demonstrate that the nanoassembly of smdh 3 comonomers can be significantly affected by core B-material geometry and flexibility that ranges from a highly flexible tetrahedral trivalent core B-material 1 to more - rigid pyramidal and trigonal cores B-material 2 and 3 ( fig . 1 ) . [SEP]
[CLS] in addition , the cgmd model has been significantly expanded to accurately forecast the experimentally observed nanoassembly population distributions over a broad range of dna concentrations as a function of the overall flexibility of the comonomers . [SEP]
[CLS] it can now capture the subtle interplay between the intrinsic rigidity of the organic B-material core I-material and the flexibility afforded by incorporating t n spacer B-material units between the hydridizing dna arms and the organic B-material core I-material . [SEP]
[CLS] the experiments are successfully validated by cgmd simulations at every stage , demonstrating the versatility of our cgmd model as a useful and reliable tool for systematically understanding the nanoassembly behavior of smdh 3 comonomers with different core B-material types and spacer B-material lengths . [SEP]
[CLS] such successes bode well for the eventual evolution of this gcmd model into a comprehensive framework for designing dna - hybrid nanomaterials B-material for various applications . [SEP]
[CLS] to demonstrate the effects of core B-material flexibility and geometry , we carried out the nanoassembly of smdh 3 comonomers possessing two different rigid organic B-material cores I-material : the pyramidal core B-material 2 and the trigonal planar core B-material 3 ( fig . 1 ) . [SEP]
[CLS] these two cores B-material were chosen to represent the two fundamental manifestations of trivalent geometries that a rigid organic molecule can exhibit . [SEP]
[CLS] both organic B-material cores I-material have three branches to be conjugated with dna arms , but their branchcenter - branch ( bcb ) angles ( 107° for the pyramidal core B-material , and 120° for the trigonal planar core B-material ) are different . [SEP]
[CLS] for consistency , the dna arm ( 8 mer ; 3 ′ - tcc gcc ga - core B-material and 3 ′ - tcg gcg ga - core B-material ; also see esi † , table s1 ) was chosen to be the same as those in the previously reported fsmdh 3 nanoassemblies . [SEP]
[CLS] the short 8 base length was also chosen to allow for many structures to be sampled and enable the final population of the nanoassemblies to be determined primarily by the relative thermodynamic stabilities of the various species . [SEP]
[CLS] we also design the dna arms to counter the potential formation of assemblies via hydrophobic B-property stacking of the cores B-material , as has been previously reported . [SEP]
[CLS] the specific matching of the sequences between complementary comonomers imposed an orientation control that favors the formation of caged dimers over face - to - face analogs . [SEP]
[CLS] the high gc content of the arms stabilizes duplex formation , which also promotes the formation of caged dimers via intramolecular hybridization between complementary comonomers . [SEP]
[CLS] finally , the " relatively larger " size of the arms compared to that of the cores B-material would lead to strong electrostatic repulsions between non - hybridizing arms . [SEP]
[CLS] together , these design features can help to counter interactions between the hydrophobic B-property cores B-material and prevent the formation of largeoligomer networks via hydrophobic B-property nucleation mechanism . [SEP]
[CLS] following a procedure previously established for the nanoassembly of fsmdh 3 comonomers , equimolar amounts of complementary pyrsmdh 3 ( pyr = core B-material 2 ) comonomers ( or tpsmdh 3 comonomers , tp = core 3 ) were combined at various concentrations ( [ smdh 3 ] = 4 , 8 , 16 , and 32 μm ; [ dna ] = 12 , 24 , 48 , and 96 μm ) and assembled ( see esi † , section s4 for details ) . [SEP]
[CLS] analysis of the product mixtures from both pyrsmdh 3 and tpsmdh 3 nanoassemblies by native polyacrylamide B-technique gel I-technique electrophoresis I-technique ( page ) analysis ( fig . 2a and 2b ) shows broad ranges of species , from dimers to large oligomers , over the whole range of explored concentrations . [SEP]
[CLS] these behaviors are in stark contrast to those observed in our previous study for the nanoassemblies of the fsmdh 3 comonomers possessing the highly flexible core B-material 1 , where almost no oligomers formed and caged dimer is a predominant species up to 100 μm [ dna ] . [SEP]
[CLS] indeed , the amount of nanoscale caged dimers assembled from pyrsmdh 3 and tpsmdh 3 comonomer pairs are dramatically decreased ( 18 - 30 % ; see esi † , table s2 ) compared to the corresponding nanoassemblies of the fsmdh 3 comonomers ( ≥ 95 % ; see esi † , table s2 ) . [SEP]
[CLS] † electronic supplementary information ( esi ) available : descriptions of experimental procedures ; maldi and hplc data for the smdh 3 s ; population distribution data from the analyses of the page gels ; detailed descriptions of the force - field parameters for the cg model ; and discussions of the various parameters that impact the cg model and cgmd calculations . [SEP]
[CLS] see doi : 10 . 1039 / x0xx00000x [SEP]
[CLS] interestingly , cores B-material 2 and 3 also exhibited significant differences in nanoproduct formation . [SEP]
[CLS] at the same smdh 3 concentration ( [ smdh 3 ] = 32 μm ) , the nanoassembly of pyrsmdh 3 comonomers gave a higher number of smaller networks ( up to octamer ; 63 % of the total population ) ( fig . 2a ; see also esi † , table s2 ) ; whereas that between tpsmdh 3 comonomers resulted in a higher population of large - oligomer networks ( ≥ nonamers ; 60 % of the total population ) ( fig . 2b ; see also esi † , table s2 ) . [SEP]
[CLS] as discussed further below in the simulation section , this behavior appears to be caused by the difference in core B-material geometries and bcb angles . [SEP]
[CLS] the pyramidal core B-material 2 has a bent geometry and smaller bcb angle , which brings the dna arms closer to each other , facilitating the formation of smaller networks . [SEP]
[CLS] on the other hand , the dna arms of the trigonal planar core B-material 3 are farther from each other , making it easier for them to form larger nanoassemblies . [SEP]
[CLS] this effect is most clear at high assembly concentrations : while the percentage of the caged dimers remains stable throughout the range of tested concentrations ( 4 - 32 μm ) for both pyrsmdh 3 and tpsmdh 3 systems , the population of large - oligomer networks increases at a much faster rate for the tpsmdh 3 nanoassemblies ( fig . 2c and 2d ) . [SEP]
[CLS] to further elucidate the roles that core B-material flexibility and geometry play in the nanoassembly of smdh 3 , especially as functions of concentration changes , we carried out a systematic series of md simulations for the nanoassemblies of the tpsmdh 3 and pyrsmdh 3 comonomers under a broad range of concentrations ( [ smdh 3 ] = 16 , 24 , 32 , and 40 μm ) . [SEP]
[CLS] because such high concentrations and the long timescale for assembly render full - atomistic simulations infeasible , we adopted a coarse - grained ( cg ) approach , which reduces the complexity of the system by grouping atoms B-material into beads . [SEP]
[CLS] previously , we have successfully employed such a cgmd model in md simulations to unravel the effect of dna arm length and smdh concentration on the outcomes of the nanoassemblies of fsmdh 3 s under various conditions . [SEP]
[CLS] that early model , however , cannot readily be extended to account for the roles that corecentric factors - such as the rigidity and geometry of the organic B-material cores I-material and the connection between the core B-material and the dna arms - may play in smdh - based nanoassembly . [SEP]
[CLS] this has now been greatly improved in the current study through incorporation of new parameters to account for those core - centric factors , thus significantly widening the applicability and scope for predicting the nanoassembly of smdh 3 comonomers . [SEP]
[CLS] briefly , our improved cg model ( fig . 3 ) also grouped three dna nucleotides into one large cg bead that can form hydrogen B-material bonds with a complimentary cg bead , just as in our previously used model . [SEP]
[CLS] while this grouping causes the dna arms in our cgmd model to have one more dna base in comparison to those in our experimental systems , it does not alter the cgmd nanoassembly behaviors . [SEP]
[CLS] as previously reported , our cgmd model fully captures the nanoassembly behaviors of the 9 - fsmdh 3 ( i . e . , a three - arm smdh based on the flexible core B-material 1 and 9 bases on each of the dna arms ) experimental system , which is virtually indistinguishable from those for the 8 - fsmdh 3 . [SEP]
[CLS] as such , for the remainder of this manuscript , we will consider the 9 - hybridizing - dna - base arms in the cgmd constructs to be adequate representation of the experimental 8 - hybridizing - dna - base arms . [SEP]
[CLS] the orientation of the hydrogen B-material bonds between each dna " base pair " is restricted by two or three smaller beads that are attached to the hybridizable cg bead . [SEP]
[CLS] the small - molecule organic B-material core I-material is also treated as a sum of coarse - grained ( cg ) beads , with one " center " cg bead ( green ) representing the core B-material , and other " branch " cg beads ( yellow ) representing the junctions to which dna arms are attached . [SEP]
[CLS] however , to account for the roles that core - centric factors - such as the rigidity and geometry of the organic B-material cores I-material and the connection between the core B-material and the dna arms - may play in smdh - based nanoassembly , we incorporated three additional enhancements . [SEP]
[CLS] first , the effects of core B-material flexibility and geometry are integrated into the cg model by modulating two variables : ( i ) the branchcenter - branch angle , which registers the core B-material geometry ( pyramidal , trigonal planar , etc . ) , and ( ii ) the stiffness of this angle , which corresponds to the value of k 0 ( i . e . , the force constant ) in the angle harmonic potential of this model ( see esi † , section s5 for details ) . [SEP]
[CLS] second , directionality is introduced to the cg beads between two smdh comonomers by assigning different bead types to each cg bead within a single dna arm , a " sense - antisense " feature that was not included in our early model . [SEP]
[CLS] such constraint prohibits the formation of faceto - face dimers , allowing us to closely mimic the experimental design of the dna strands ( see esi † , table s1 ) . [SEP]
[CLS] however , the internal twist between the cg beads and the core B-material does not have a torsional barrier B-property in our model and can freely twist to sample more configurations . [SEP]
[CLS] third , a varying number of spacer cg beads ( n = 1 - 5 , where each bead represents three non - base - pairing thymine nucleotides ( t 3 ) ) between the organic B-material core I-material and the dna arms were introduced , thereby adding more length and flexibility to the overall smdh cg constructs . [SEP]
[CLS] together , these changes allow us to systematically explore the effect of the structural variation of the smdh comonomers on their nanoassembly behavior . [SEP]
[CLS] we note that a key advantage of a cgmd model , such as that described above , is the significant saving in computational cost . [SEP]
[CLS] given the large number of atoms B-material in each smdh 3 monomer B-material and the long timescale needed to observe nanoassembly in systems that are large enough to generate statistically significant population distributions , fully atomistic md simulations would have been infeasible . [SEP]
[CLS] for example , yildirim et al . studied a rigid smdh 3 cage - dimer system with full atomistic simulations and were able to deduce the relationship between the hydrophobic B-property surfaces of the organic B-material core I-material and nanoassembly formation . [SEP]
[CLS] however , they could only indirectly infer the formation of ill - defined networks from the nanoassembly of two complementary monomers B-material . [SEP]
[CLS] in contrast , our model can analyze up to 100 comonomer pairs in a single simulation box ( see esi † , section s6 for details ) and directly observe the formation of networks of various sizes , far beyond nanoscale caged dimers , as well as the equilibration of monomers B-material between clusters . [SEP]
[CLS] additionally , since the number of cg beads in our cgmd strategy is much smaller than the number of individual atoms B-material in the corresponding all - atom simulations , and the number of bonding interactions are greatly reduced by coarse - graining , it is much easier for us to change parameters and systematically elucidate how such changes affect the nanoassembly than in the corresponding fully atomistic md simulations . [SEP]
[CLS] in this sense , our cg model can efficiently provide us with key insights into the formation of smdh - based nanoassemblies and allow us to formulate design parameters in a more fluid manner , in spite of its simplicity . [SEP]
[CLS] these advantages will be illustrated in the remainder of this manuscript . [SEP]
[CLS] as shown in fig . 4a and 4b , the cgmd simulations showed smdh 3 comonomers with rigid cores B-material 2 and 3 to have assembled into populations that comprise a significant amount of large - oligomer networks ( ≥ nonamers ) . [SEP]
[CLS] these behaviors are in stark contrast to those observed in our previous study for the assemblies of the fsmdh 3 comonomers possessing the highly flexible core B-material 1 ( cf . fig . 4a and 4b vs 4c ) . [SEP]
[CLS] for example , the number of caged dimers assembled from smdh 3 comonomers comprising either core B-material 2 or 3 are significantly decreased ( 15 - 40 % vs 52 - 85 % for core B-material 1 ) . [SEP]
[CLS] in close reflection of the experimental results ( fig . 2a and 2b ; see also esi † , table s2 ) , the simulated nanoassemblies of the pyrsmdh 3 comonomers gave more dimers and tetramers than large - oligomer networks ( fig . 4a ) , whereas the nanoassemblies of tpsmdh 3 comonomers resulted in a much - higher amount of large - oligomer networks in comparison to dimers and tetramers ( fig . 4b ) . [SEP]
[CLS] this behavior appears to be caused by the difference in the angle between the center and branch cg beads within these cores B-material . [SEP]
[CLS] the pyramidal core B-material 2 has a smaller bcb angle between cg beads , which brings the dna arms closer to each other , facilitating the formation of smaller networks . [SEP]
[CLS] on the other hand , the dna arms of the trigonal planar core B-material 3 are farther from each other , making it easier for them to form larger assemblies . [SEP]
[CLS] interestingly , the nanoassemblies of pyrsmdh 3 and tpsmdh 3 comonomer pairs comprise a broad range of species ( monomer B-material to large - oligomer networks ) over all simulated concentrations . [SEP]
[CLS] this is very different from the simulations of the nanoassemblies of fsmdh 3 comonomers , where pentamer , the largest species found , only shows up at the high [ smdh 3 ] of 32 μm ( d = 80 ) ( fig . 4c ) . [SEP]
[CLS] over the range of concentrations ( 8 μm ≤ [ smdh 3 ] ≤ 32 μm ) that we examined in the simulation , the nanoassemblies of fsmdh 3 comonomers unfailingly afforded caged dimers as the predominant species ( fig . 4c ) , consistent with the experimentally observed results ( esi † , table s2 ) . [SEP]
[CLS] in contrast , large - oligomer networks begin to dominate over caged dimers in the nanoassemblies of pyrsmdh 3 comonomer pairs at [ smdh 3 ] = 32 μm ( d = 80 ) ( fig . 4a ) . [SEP]
[CLS] the population of large - oligomer networks overtook that for the dimers at an even - lower [ smdh 3 ] of 24 μm ( d = 60 ) ) in the nanoassemblies of tpsmdh 3 comonomer pairs ( fig . 4b ) . [SEP]
[CLS] these observations suggest that both core B-material rigidity and smdh 3 concentration contributed significantly to the simulated population distributions for smdh 3 systems . [SEP]
[CLS] the more rigid nature of cores B-material 2 and 3 make the nanoassembly more sensitive to the concentration profiles , favoring a much broader and diverse simulated population distribution than the fsmdh 3 counterpart ( cf . fig . 4a and 4b vs 4c ) . [SEP]
[CLS] remarkably , the cgmd - simulated population distributions in the nanoassemblies of pyrsmdh 3 and tpsmdh 3 comonomers are very similar to those obtained from the experiments ( fig . 5 ) . [SEP]
[CLS] together with the aforementioned concentration - dependent data , this excellent agreement confirms that our new cgmd model can fully capture the physicality of the experimental systems in two key aspects : 1 ) the more - rigid cores B-material 2 and 3 led to a significant amount of oligomers , and 2 ) the overall population distributions vary significantly as a function of both core B-material rigidity and smdh 3 concentration . [SEP]
[CLS] these two observations can be qualitatively rationalized if one considers the potential constraint imposed on the dna arms by these rigid cores B-material and their effects on the stability of caged dimers . [SEP]
[CLS] indeed , our previous all - atom simulation of the nanoassembly of rsmdh 3 comonomers based on the highly rigid core B-material 4 has found that stable caged dimers can form better when a flexible spacer B-material ( t 3 or t 6 ) is added between 4 and the dna arms , consistent with experimental results . [SEP]
[CLS] in our recent cgmd study , because the flexible and relatively hydrophilic B-property core B-material 1 imposed little constraint on the dna arms , the 9 - fsmdh 3 caged dimers can be formed and are most stable energetically when all three dna arms of a smdh 3 comonomer are hybridizing with those of another . however , the formation of such nanoscale caged dimers would be energetically less favorable for pyrsmdh 3 and tpsmdh 3 comonomers , which have rigid and hydrophobic B-property cores B-material . [SEP]
[CLS] not only that their dna arms will potentially have to undergo distortion to become available for hybridization , they may also interact substantially with the cores B-material . [SEP]
[CLS] we find that with the more - rigid cores B-material 2 and 3 , possibly as a result of the constraint induced by the core B-material , most of the dimers formed by the nanoassembly of pyrsmdh 3 and tpsmdh 3 comonomers will have partial hybridization , where only one or two out of their three dna arms will be participating in hybridization , with the remaining non - hybridizing dna arm ( s ) dangling freely . this / these dangling dna arm ( s ) can then hybridize with either the dangling arm ( s ) of another smdh 3 comonomer or the dna arms of free smdh 3 to form networks higher than dimer , sometimes even ≥ nonamer . in addition , it is worth noting that oddnumbered nanoassemblies ( trimers , pentamers , etc . ) , with unhybridized dna arms , are less prevalent than fully hybridizing , even - numbered nanoassemblies ( tetramer , hexamer , etc . ; see esi † , section s4e and fig . s18 ) . [SEP]
[CLS] this statistical result was indeed confirmed by our cgmd simulations ( fig . 4a and 4b ) : even - numbered nanoassemblies are more dominant than odd - numbered ones . [SEP]
[CLS] interestingly , odd - and even - numbered nanoassemblies larger than hexamers appear to have no significant difference in population , as : ( i ) their populations are already quite small , and ( ii ) the energetic cost of having a unhybridized dna arm diminishes in assemblies that have more dna duplexes than pentamers . [SEP]
[CLS] together , these results suggest that in the nanoassemblies of pyrsmdh 3 and tpsmdh 3 comonomers , enthalpy - driven effects dominate over entropic ones for oligomers formation . [SEP]
[CLS] the partial hybridization of dimers formed by pyrsmdh 3 and tpsmdh 3 comonomers can also explain the strong observed effect of smdh 3 concentration on the nanoassembly . [SEP]
[CLS] for an " imperfect " ( i . e . , partially hybridizing ) dimer to grow into a larger nanoassembly , its dangling dna arms have to hybridize with either the dna arms of free smdh 3 monomers B-material or the dangling dna arms of another assembly . [SEP]
[CLS] as the smdh 3 concentration is increased , the local density of either free smdh 3 monomers B-material or their nanoassemblies around the dangling dna arm of the " imperfect " dimer will become higher , giving the system more opportunity to form large networks . [SEP]
[CLS] this is in stark contrast to the nanoassembly of the 9 - fsmdh 3 comonomers ( cf . fig . 4a and 4b vs 4c ) , wherein their flexible core B-material allowed for more facile formation of " perfect " nanoscale caged dimers ( i . e . , fully hybridized , " caged " dimers with no dangling dna arms ) as the majority component of the population . [SEP]
[CLS] doubling the 9 - fsmdh 3 concentration from 16 to 32 μm only has a small effect on the dimer population given the lack of the dangling dna arms . [SEP]
[CLS] as discussed thus far , introducing rigidity constraints into the trivalent organic B-material cores I-material , such as is present in 2 and 3 , can lead to preferential formation of large - oligomer networks in the nanoassemblies of the corresponding smdh 3 comonomers . [SEP]
[CLS] however , such constraints may potentially be alleviated by introducing spacer B-material sequences between the rigid organic B-material core I-material and its hybridizing dna arms , as successfully demonstrated for the nanoassembly of smdh 3 comonomers possessing a highly rigid 1 , 3 , 5 - tris ( p - ethynylphenyl ) benzene core B-material 4 . [SEP]
[CLS] hence , we set out to further utilize our new cgmd model to understand the interplay between core B-material rigidity and flexibility of dna arms by incorporating thymine oligonucleotide ( t n ) spacers B-material in between cores B-material 2 and 3 and their hybridizing arms and explore the subsequent nanoassemblies of the corresponding pyr - t n - smdh 3 and tp - t n - smdh 3 comonomers by both experiments and computation . [SEP]
[CLS] as the experiments are much more time - consuming than the modeling , our initial plan was to limit the experimental work to the nanoassemblies of systems with t 3 and t 6 spacers B-material , to be consistent with the aforementioned , previously reported assemblies of smdh 3 comonomers possessing the highly rigid core B-material 4 . [SEP]
[CLS] simultaneously , the modeling work was expanded to include a much larger range of spacer B-material lengths ( t 3 , t 6 , t 9 , t 12 , and t 15 ) , conveniently deployed by modularly including one " spacer B-material " cg bead for each additional t 3 repeating B-material unit I-material ( see further discussion below ) . [SEP]
[CLS] given precedents from our previous work with smdh 3 comonomers possessing the highly rigid core B-material 4 , the formation of large oligomers from pyrsmdh 3 and tpsmdh 3 comonomers can indeed be dramatically reduced in experiments by incorporating flexible t 3 and t 6 spacers B-material between organic B-material cores I-material 2 and 3 and their hybridizing dna arms . [SEP]
[CLS] as shown by page analysis ( fig . 6a and 6b ) , the nanoassemblies of pyr - t 3 - smdh 3 and tp - t 3 - smdh 3 comonomer pairs , where a simple t 3 spacer B-material is incorporated between the core B-material and the dna arms in each comonomer , resulted primarily in caged dimers , even at the highest attempted experimental concentration ( [ smdh 3 ] = 32 μm ) . [SEP]
[CLS] these results are dramatically different from the results shown in fig . 2 where the analogous nanoassemblies of spacer - free pyrsmdh 3 and tpsmdh 3 comonomer pairs led to large amounts of oligomers , even at the lowest attempted experimental concentration ( [ smdh 3 ] = 4 μm ) . [SEP]
[CLS] this spacer B-material effect became even more significant in the nanoassemblies of the longer - spacer B-material pyr - t 6 - smdh 3 and tp - t 6 - smdh 3 comonomer pairs , where only caged dimers can be visually observed on the pagegel images ( fig . 6c and 6d ) ; the tiny amount of tetramers that formed were barely detectable at high concentrations ( [ smdh 3 ] = 16 and 32 μm ) . [SEP]
[CLS] the distinctive structural difference between the nanoscale caged dimers and large - oligomer networks can be visulaized through cryogenic scanning B-technique transmission I-technique electron I-technique microscopy I-technique . [SEP]
[CLS] as shown in fig . 7a and 7b , the nanoassembly of tp - t 6 - smdh 3 comonomers resulted in " discrete particles " with sizes of approximately 5 - 10 nm , corresponding to caged dimers and small clusters of caged dimers . [SEP]
[CLS] this is in stark contrast to that for the nanoassembly of spacer - free tpsmdh 3 comonomers ( fig . 7c and 7d ) , which showed network structures with much larger sizes . [SEP]
[CLS] as mentioned at the beginning of this section , to model the aforementioned spacer B-material effect in our simulations , we introduced " spacer B-material " cg beads equivalent to t 3 segments into our models of the pyrsmdh 3 and tpsmdh 3 comonomers . [SEP]
[CLS] as in the experimental systems , these beads were modularly introduced between the organic B-material core I-material and the dna arms in each monomer B-material structure to increase the overall flexibility without changing the originally implemented core B-material flexibility and dna arm parameters . [SEP]
[CLS] as each spacer B-material cg bead corresponds to three thymine nucleotides in a t 3 repeating B-material unit I-material , we chose to sequentially add up to five spacer B-material cg beads ( e . g . , t 15 spacer B-material ) to represent smdh 3 systems with dna arms as long as 24 bases but with only 9 of those bases capable of base pairing . [SEP]
[CLS] as previously reported , this so - called equal - enthalpy ( ee ) design of hybridization allows for the facile sampling of many structures during simulation , " with the final population distributions determined primarily by the relative thermodynamic stabilities of the various species " . [SEP]
[CLS] this in turn , allows us to systematically compare the relative population distributions in the nanoassemblies of the two sets ( i . e . , those based on cores B-material 2 and 3 ) of smdh 3 comonomers as the sole function of spacer B-material flexibility added next to the organic B-material cores I-material . [SEP]
[CLS] our cgmd simulations were carried out at a total smdh 3 concentration of 32 μm for the nanoassemblies of pyr - t n - smdh 3 and tp - t n - smdh 3 comonomer pairs ( n = 3 , 6 , 9 , 12 , and 15 ) possessing different numbers ( from 1 to 5 ) of spacer B-material cg beads . [SEP]
[CLS] as shown in fig . 8 , the inclusion of only one cg spacer B-material bead in between the dna arms and cores B-material 2 and 3 significantly increases the simulated populations of nanoscale caged dimers upon assembly , particularly in comparison to the corresponding no - spacer B-material cases ( from 25 % for pyrsmdh 3 to 33 % for pyr - t 3 - smdh 3 ; from 16 % for tpsmdh 3 to 42 % for tp - t 3 - smdh 3 ) . [SEP]
[CLS] interestingly , these increases occurred at the expense of the large - oligomernetwork ( i . e . , ≥ nonamers ) populations ( from 24 % for pyrsmdh 3 to 5 % for pyr - t 3 - smdh 3 ; from 37 % for tpsmdh 3 to 8 % for tp - t 3 - smdh 3 ) without significant changes in the intermediate B-property - sized nanoassemblies ( esi † , fig . s22 ) . [SEP]
[CLS] quantification of the corresponding dimer and oligomer spots on the page - gel profiles ( cf . fig . 6a vs 2a and fig . 6b vs . 2b ) further confirm this trend : the dimer populations increase ( from 30 % for pyrsmdh 3 to 68 % for pyr - t 3 - smdh 3 ; from 18 % for tpsmdh 3 to 68 % for tp - t 3 - smdh 3 ) while the largeoligomer - network populations decrease ( from 37 % for pyrsmdh 3 to 8 % for pyr - t 3 - smdh 3 ; from 60 % for tpsmdh 3 to 7 % for tp - t 3 - smdh 3 ) . [SEP]
[CLS] together , these data confirm the remarkable ability of our cgmd model in describing trends in the nanoassembly behavior of pyr - t 3 - smdh 3 and tp - t 3 - smdh 3 comonomer pairs . [SEP]
[CLS] that the " addedflexibility " effect created by inserting oligo t 3 spacers B-material in between cores B-material 2 and 3 and their dna arms can be captured by the model supports our hypothesis that such a strategy can be used to offset the rigidity of the cores B-material to favor the assembly of nanoscale caged dimers . [SEP]
[CLS] interestingly , adding more spacer B-material beads ( beyond the first t 3 spacer B-material ) in between cores B-material 2 and 3 and their dna arms led to significant increases in large - oligomer - network formation in the corresponding simulated nanoassemblies . [SEP]
[CLS] to our surprise , however , these increases do not seem to strongly correlate with changes in the simulated dimer populations . [SEP]
[CLS] as shown in fig . 8 , when the spacer B-material length increases from t 3 to t 15 , the populations of large - oligomernetworks ( ≥ nonamers ) steadily increase by 4 - 6 fold ( from 5 to 23 % for the nanoassemblies of pyr - t n - smdh 3 comonomers ; and from 7 to 42 % for the assemblies of tp - t n - smdh 3 comonomers ) . [SEP]
[CLS] in contrast , the dimer populations decrease by much smaller proportions , from 33 to 30 % for pyr - t n - smdh 3 comonomers ; and from 42 % to 26 % for tp - t n - smdh 3 comonomers ) . [SEP]
[CLS] these simulation results effectively forecast that adding spacers B-material longer than t 3 can induce pyr - t n - smdh 3 and tp - t n - smdh 3 comonomer pairs ( n > 3 ) to form more large - oligomer networks . [SEP]
[CLS] as such , there exists an optimal spacer B-material length for pyr - t n - smdh 3 and tp - t n - smdh 3 comonomer pairs to form the largest amount of dimers while minimizing the formation of large - oligomer networks . [SEP]
[CLS] that our cgmd simulations unexpectedly show an increase in the formation of largeoligomer networks that is proportional to the spacer B-material - length increase beyond t 3 prompted us to carry out an additional experimental verification . [SEP]
[CLS] similar to the experiments described above for the t 3 and t 6 spacer B-material systems , the nanoassemblies of pyr - t 15 - smdh 3 and tp - t 15 - smdh 3 comonomer pairs were carried out at total smdh 3 concentrations of 4 , 8 , 16 , and 32 μm , and subjected to page - gel analysis ( fig . 9 ) . [SEP]
[CLS] while caged dimers still dominate even at the highest experimental concentration ( [ smdh 3 ] = 32 μm ) attempted for both systems , more large - oligomer networks ( ≥ nonamers ) can be observed in comparison to the corresponding nanoassemblies of pyr - t 3 - smdh 3 and tp - t 3 - smdh 3 comonomer pairs ( cf . fig . 6 vs 9 ) , and this trend becomes more significant at higher smdh 3 concentration . [SEP]
[CLS] our results thus far with the spacer B-material additions can be understood if one considers the two competing effects that come into play when the spacer B-material cg beads are introduced . [SEP]
[CLS] adding the spacer B-material cg beads will extend the hybridizable sections of the dna arms away from the core B-material and give them more flexibility by reducing steric hindrance . [SEP]
[CLS] the farther these hybridizable sections are from the core B-material , the more space they can explore without being constrained by the core B-material geometry . [SEP]
[CLS] this increased flexibility negates the effect of the rigid core B-material and allows two complementary smdh 3 comonomers to form caged dimers more readily , as their dna arms have more conformational freedom to hybridize with each other . [SEP]
[CLS] even with the relatively short t 3 and t 6 spacers B-material , the increased flexibility significantly enhances dimer formation by reducing any enthalpic penalties associated with conformational rigidity . [SEP]
[CLS] however , as the spacers B-material becomes much longer than t 6 , the dna arms of an smdh 3 comonomer will have significant freedom to move around in space , making it harder for them to hybridize with the complementary arms from another comonomer to form perfect nanoscale caged dimers . [SEP]
[CLS] as was in the case with the no - spacer smdh 3 comonomers , this will increase the formation of imperfect dimers with dangling dna arms , which can hybridize with either dangling arms from other assemblies or the dna arms of unhybridized smdh 3 in their proximity . [SEP]
[CLS] this entropic effect becomes dominant as the spacer B-material lengthens and can explain the increase in the populations of large - oligomer networks at the expense of dimer formation as the spacers B-material becomes longer than t 6 . [SEP]
[CLS] quantitative evaluation of the populations of dimers and large - oligomer networks for the nanoassemblies of pyr - t n - smdh 3 and tp - t n - smdh 3 comonomers pairs ( n = 0 , 3 , 6 , and 15 ) were calculated from the page - gel images in fig . 2 , 6 , and 9 using the imagej analysis tool and the results were plotted together with those obtained by the corresponding cgmd simulations . [SEP]
[CLS] as shown in fig . 10 , the experimentally observed trends for dimer and largeoligomer - network distributions again mirrored the corresponding gcmd - simulated trends . [SEP]
[CLS] the agreement between experiment and simulation is quite excellent for the large - oligomernetwork populations ( fig . 10b and 10d ) even though the experimental dimer populations were not - as - perfectly captured by the simulations ( fig . 10a and 10c ) . [SEP]
[CLS] part of this underestimation may be due to the previously reported strong hydrophobic B-property interaction I-property between the core B-material and the t n spacer B-material in aqueous media , which can result in the insertion of the core B-material into the spacer B-material unit , as observed in full atomistic md simulations . [SEP]
[CLS] this would in turn shorten the effective length of the spacer B-material and increase the overall stability of the caged dimer beyond what can be captured by the simple cgmd methodology reported herein ( see esi † , section s5 for further discussion ) . [SEP]
[CLS] in spite of this , the cgmd model captures the population turnover for the caged dimers while still allowing us to easily extend our simulation to t 15 spacer B-material , whose length would have rendered full - molecular md simulations impractical . [SEP]
[CLS] another reason for the mismatch in dimer populations between the experimental data and the cgmd simulation may be attributed to neglects by the latter of low - energy contributions , such as chain contraction due to hydrophobic B-property shielding and structural degrees of freedom of the dna arms . [SEP]
[CLS] in an experimental situation , the entropic differences between any two aggregate species in the nanoassembly ( i . e . , tδs ≈ - k b t log ( p 1 / p 2 ) , p 1 and p 2 are the properly normalized populations of the two aggregates , assuming the equal - enthalpy scenario ) would be quite small ( ~ 1 - 2 kcal / mol ; see esi † , section s10 ) . [SEP]
[CLS] as this value is quite similar to the thermal energy , we expect the aforementioned low - energy contributions to play important roles in the population distributions . [SEP]
[CLS] however , they are ignored by restrictions placed by the cg model on the degrees of freedom of the dna arms in smdh 3 ( i . e . , all the hybridizable beads are rigidly linked together and cannot move as independently as the beads in the spacer B-material portions ) , which essentially " freezes " them to increase the rate of hybridization and make the simulations tractable . [SEP]
[CLS] this results in a quantitative mismatch of the predicted dimer populations . [SEP]
[CLS] we note in passing that while ionic contributions , such as explicit treatment of the counterions and polyanionic effects of the dna backbone , have been incorporated into cgmd model , their deployment herein would only impact the predicted melting behavior via the size and range of thermal fluctuations of the dna arms . [SEP]
[CLS] the apparent underestimation of the cgmd - predicted population of dimers may contribute to the partial preference that our cgmd model gives to the formation of intermediate B-property nanosized oligomers ( trimers to octamers ) ( esi † , fig . s21 ) at the expense of nanoscale caged dimers . [SEP]
[CLS] this hypothesis is supported by the larger combined amount of intermediate B-property nanosized oligomers observed in simulation than in experiment ( esi † , fig . s22 ) , as well as the excellent agreement in the large - oligomer - network population profiles ( fig . 10b and 10d ) . [SEP]
[CLS] as discussed above , part of this overestimation can be attributed to the treatment of the hybridizable section of the dna arms as rigid bodies by our cgmd model . [SEP]
[CLS] this will kinetically favor the formation of slightly larger networks : during the nanoassembly , as the cgmd - rigidified dna arms of an smdh 3 comonomer sample the hybridization space , they do not have many degrees of freedom and are more likely to hybridize in a kinetically irreversible fashion with the complementary dna arms of neighboring smdh 3 to form oligomers , especially as smdh 3 concentration increases . [SEP]
[CLS] however , this overestimation diminishes as the size of the oligomer increases and thus does not significantly affect the amount of large - oligomer networks . [SEP]
[CLS] interestingly , in both experiment and cgmd simulation results for the nanoassemblies of the smdh 3 comonomers of both cores B-material 2 and 3 , the population of large - oligomer networks significantly decreases from the no - spacer B-material case to when t 3 and t 6 spacers B-material were added to each of the dna arms ; and increases again when the longest t 15 spacer B-material was added ( fig . 10b and 10d ) . [SEP]
[CLS] in contrast , the dimer populations for both systems increase with t 3 and t 6 spacers B-material and then decrease with t 15 spacer B-material ( fig . 10a and 10c ) . [SEP]
[CLS] that the cgmd model can successfully capture these qualitative trends in nanoassemblies formation over a large range of spacer B-material lengths suggests that our cgmd model can be used more generally in predicting smdh 3 nanoassembly structures . [SEP]
[CLS] importantly , the agreement between modeling and experiments demonstrates the potential for biasing the assembly to promote the formation of smaller nanosized aggregates ( rather than large - oligomer networks ) through the incorporation of an optimal spacer B-material length ( e . g . , t 3 - t 6 ) . [SEP]
[CLS] the clear difference in population trends between dimers / tetramers and large - oligomer networks as a function of core B-material flexibility can serve as an enticing first system to study the rational design of smdh 3 nanoassemblies . [SEP]
[CLS] in summary , we have demonstrated that the nanoassemblies of small molecule - dna hybrids can be controlled by tuning the interplay between the geometry and rigidity of the organic B-material cores I-material and the flexible t n spacers B-material between these cores B-material and the dna arms our synergistic experimental - modeling study showed that the smdh 3 comonomers possessing tetrahedral cores B-material 1 and 2 assemble into primarily nanoscale caged dimers , in stark contrast to those based on the trigonal core 3 , whose rigid , sterically hindered planar geometry force the assembly into more large - oligomer networks ( ≥ nonamers ) , reflecting a higher enthalpic barrier B-property to the formation of dimers . [SEP]
[CLS] this effect can be accentuated by high smdh 3 concentrations , which also distinguishes the two tetrahedral systems based on cores B-material 1 and 2 : the nanoassembly of the smdh 3 comonomers possessing the highly flexible core B-material 1 remains strongly favored for the formation of nanoscale caged dimers up to 32 μm , while that for the less - flexible core B-material 2 forms a significant amount of large - oligomer networks ( fig . 4d ) . [SEP]
[CLS] notably , the overall flexibility of the sterically hindered pyrsmdh 3 and tpsmdh 3 structures can be enhanced by incorporating non - hybridizing t n spacer B-material units between the hydridizing dna arms and the organic B-material core I-material . [SEP]
[CLS] our cgmd model successfully captures the experimentally observed trends for the nanoassemblies of pyr - t n - smdh 3 and tp - t n - smdh 3 comonomer pairs ( n = 0 , 3 , 6 , and 15 ) , with data for the n = 15 point being predicted before the experiments were carried out . [SEP]
[CLS] as such , these experimental and simulation results can be utilized to guide the design of smdh 3 comonomers with different types of cores B-material to favor a particular nanoassembly outcome based on flexibility . [SEP]
[CLS] for example , to favor nanoscale caged dimers , systems with more rigid cores B-material can be made flexible with the insertion of non - hybridizing t n spacers B-material between the core B-material and the hybridizable dna arms . [SEP]
[CLS] however , the spacer B-material cannot be too long that entropic contributions begin to take over . [SEP]
[CLS] from a broader design perspective , our combined experimental - modeling study demonstrates that the geometry and flexibility of organic B-material core I-material as well as the presence of t n spacers B-material are both crucial parameters in the design of nanoscale assemblies from smdh comonomers , akin to the non - hybridizing spacers B-material used in dna - directed aunp B-nanoparticle assembly or in dna origami . [SEP]
[CLS] while the complicated relationship between core B-material geometry and flexibility can certainly be rationalized as functions of the interplay between enthalpy and entropy , such analysis is often made in hindsight after the experiment . [SEP]
[CLS] to this end , our cgmd model can allow chemists and material B-material scientists to begin to predict the outcomes of dna - directed nanoassembly of smdh 3 comonomers as a function of the supramolecular design parameters . [SEP]
[CLS] thanks to the simplicity of cgmd modeling and its low computational cost , this innovative model can be further improved , with inputs from spectroscopic studies , to capture factors such as core - dna hydrophobic B-property collapse , polyanionic repulsions between cg beads , and degrees of freedom of the dna arms . [SEP]
[CLS] with these improvements , it can continually evolve into a comprehensive framework for the design and improvement of dna - hybrid nanomaterials B-material for various applications . [SEP]
[CLS] refer to web version on pubmed central for supplementary material B-material . [SEP]
[CLS] supplementary materialmodel , their degrees - of - freedom are restricted and their molecular motions are not fully coupled to the thermal bath . [SEP]
[CLS] removing this constraint , however , can lead to non - physical scenarios such as rotated bonding conformations that are responsible for partially bonded states . [SEP]
[CLS] 40 . [SEP]
[CLS] edwardson [SEP]
[CLS] small - molecule cores B-material of smdh 3 comonomers with three dna arms . [SEP]
[CLS] cores B-material 1 and 2 are both pyramidal but 1 is flexible and 2 is more rigid . [SEP]
[CLS] cores B-material 3 and 4 are both trigonal planar and rigid . [SEP]
[CLS] for the remainder of this paper , we will use the notation fsmdh 3 , pyrsmdh 3 , tpsmdh 3 , and rsmdh 3 to denote smdhs with 3 arms based on cores B-material 1 , 2 , 3 , and 4 , respectively . [SEP]
[CLS] when there is a t n spacer B-material between the core B-material and the dna arm , the notation t n will be inserted in between the core B-material notations and the smdh 3 notation . [SEP]
[CLS] for example , pyr - t 3 - smdh 3 indicates a three - arm smdh based on the pyramidal core B-material 2 , with a t 3 spacer B-material between the core B-material and each of the dna arms . [SEP]
[CLS] in addition , we will use the notation 9 - fsmdh 3 to indicate a three - arm smdh based on the flexible core B-material 1 and 9 bases on each of the dna arms . [SEP]
[CLS] a schematic description of the coarse - grained ( cg ) model used in the simulations of the nanoassemblies of smdh 3 comonomers with 9 - base dna arms and three different core B-material types ( 1 , 2 , and 3 ) . [SEP]
[CLS] as shown , the cores B-material are represented by one " center " cg bead ( green ) and three surrounding " branch " cg beads ( yellow ) at different angles , as viewed down the z axis of each smdh 3 building block . [SEP]
[CLS] following the convention that was originally developed by knorowski et al . , three dna nucleotides are grouped into one large hybridizable bead ( blue ) that can form hydrogen B-material bonds with a complimentary cg bead . [SEP]
[CLS] to each of the hybridizable beads are attached two or three smaller beads ( flank beads , white ) that restrict the orientation of the hydrogen B-material bond . [SEP]
[CLS] the large grey shape that outlines the hybridizable and flank beads comprises of " overlapping " scaffold beads that are used to prevent unphysical hybridization and promote directional binding in the hybridizable dna arms . [SEP]
[CLS] a t n spacer B-material section ( unhybridizable beads , red ) can be inserted between the core B-material and the hybridizable arm to increase the overall flexibility of the smdh 3 . [SEP]
[CLS] for simplicity of representation , the hydroxymethyl group attached to the central carbon B-material atom I-material of core B-material 1 is omitted in the fourbead model figure on the bottom left cartoon . [SEP]
[CLS] however , that group was formally included into the model as a fifth , non - interacting branch bead in all simulations ( see the esi † that accompanies reference 11 ) . [SEP]
[CLS] native page - gel ( 6 % ) images of the products obtained from the nanoassemblies of ( a ) pyr - t 15 - smdh 3 and ( b ) tp - t 15 - smdh 3 comonomer pairs . [SEP]
[CLS] for each page - gel image : lane 1 = dsdna ladder , lane 2 = smdh 3 monomer B-material , lane 3 = complementary smdh 3 monomer B-material , and lanes 4 - 7 = products from the nanoassemblies of the two smdh 3 comonomers at total smdh 3 concentrations of ( left to right ) 4 , 8 , 16 , and 32 μm , respectively [SEP]
[CLS] these gel bands are broader than those obtained for the nanoassemblies of system with either no spacer B-material ( fig . 2 ) or short t 3 and t 6 spacers B-material ( fig . 6 ) , and are widely spread along the gel - loading lanes . [SEP]
[CLS] these characteristics are very similar to those observed for the nanoassembly of fsmdh 3 comonomers comprising core B-material 1 and long ( 24 - base ) hybridizable dna arms , and may be attributable to small dynamic changes in structure . [SEP]
[CLS] presumably , the presence of the long t 15 spacers B-material between the cores B-material and their hybridizable dna arms confers a small variance in flexibility on the assembled dimers and oligomers , allowing structures with the same mass to diffuse somewhat differently down the gel . [SEP]
[CLS] 2 . ( a - b ) native page - gel ( 6 % ) images of products obtained from the nanoassemblies of pyrsmdh 3 ( a ) and tpsmdh 3 ( b ) . [SEP]
[CLS] for each page - gel image : lane 1 = dsdna ladder , lane 2 = smdh 3 monomer B-material , and lanes 3 - 6 = products from the nanoassemblies of the two smdh 3 comonomers at total smdh 3 concentrations of ( left to right ) 4 , 8 , 16 , and 32 μm , [SEP]
[CLS] fig . 2 . respectively . [SEP]
[CLS] assignments of the oligomer identities obtained from the nanoassemblies were made based on corresponding positions to the dna ladder . [SEP]
[CLS] the lack of intermediate B-property - size odd oligomers ( trimers , pentamers , etc . ) makes sense given the presence of dangling arms in those species ( for additional discussion , see the text near the end of the section entitled " coarse - grained molecular dynamics ( cgmd ) model and simulations " and esi † , section 4e ) . [SEP]
[CLS] however , as the size of the oligomers grow larger than 9 , this effect is diminished and large odd oligomers may be present . [SEP]
[CLS] ( c - d ) plots of relative populations of nanoscale caged dimers ( c ) and large - oligomer networks ( ≥ nonamers ) ( d ) that were formed from the nanoassemblies of pyrsmdh 3 ( blue solid circle ) and tpsmdh 3 ( red solid triangle ) at total smdh 3 concentrations of 4 , 8 , 16 , and 32 μm . [SEP]
[CLS] ( a - b ) population distributions from the nanoassemblies of smdh 3 comonomers possessing pyramidal ( a ) and trigonal planar ( b ) using our improved cgmd model . [SEP]
[CLS] ( c ) population distributions from the nanoassemblies of fsmdh 3 comonomers possessing a highly flexible core B-material 1 as found in our initial cgmd simulation 11 ( data was not reported previously ) . [SEP]
[CLS] the product distribution of these fsmdh 3 nanoassemblies does not change when directionality constraint was imposed , as in the current model . [SEP]
[CLS] the parameter d denotes the number of comonomer pairs in the simulation box , corresponding to [ smdh 3 ] = 16 , 24 , 32 , and 40 μm . each population was averaged over three repeated simulations . [SEP]
[CLS] ( d ) comparison of the nanoassemblies found with these two core B-material types at d = 80 ( [ smdh 3 ] = 32 μm ) . [SEP]
[CLS] for comparison , unpublished data previously obtained for the nanoassemblies of the fsmdh 3 comonomers possessing the highly flexible core B-material 1 11 are also included . [SEP]
[CLS] 5 . the remarkable similarities in population distribution profiles from comparative experiment and cgmd simulation studies for the nanoassemblies of pyrsmdh 3 ( a ) and tpsmdh 3 ( b ) comonomers pairs . [SEP]
[CLS] the blue circles represent the experimentally observed populations , whereas the red triangles represent the corresponding results from cgmd simulations . [SEP]
[CLS] the correspondingly colored lines were only included as visual guides . [SEP]
[CLS] both experiment and cgmd simulation studies were carried out at [ smdh 3 ] = 32 μm . [SEP]
[CLS] the populations of nanoassemblies in the page gels were calculated using imagej analysis tool . [SEP]
[CLS] ( a - d ) native page - gel ( 6 % ) images of the products obtained from the nanoassemblies of pyr - t 3 - smdh 3 comonomers ( a ) , tp - t 3 - smdh 3 comonomers ( b ) , pyr - t 6 - smdh 3 comonomers ( c ) , and tp - t 6 - smdh 3 comonomers ( d ) . [SEP]
[CLS] for each page - gel image : lane 1 = dsdna ladder , lane 2 = smdh 3 monomer B-material , lane 3 = complementary smdh 3 monomer B-material , and lanes 4 - 7 = products from the nanoassemblies of the two smdh 3 comonomers at total smdh 3 concentrations of ( left to right ) 4 , 8 , 16 , and 32 μm , respectively [SEP]
[CLS] cryo - scanning transmission B-technique electron I-technique microscopy I-technique images for : ( a and b ) nanoscale caged dimers from the nanoassembly of tp - t 6 - smdh 3 comonomer pairs and ( c and d ) largeoligomer networks from the nanoassembly of spacer - free tpsmdh 3 comonomer pairs . [SEP]
[CLS] the bright dots shown in the top panels ( a and b ) represent dimers and small clusters of dimers ; the latter are presumably formed through the preparation of the tem sample , since dimers are the primary species observed in the page - gel image ( fig 6d ) . [SEP]
[CLS] in stark contrast , larger network - like features predominates in the bottom panels ( c and d ) . [SEP]
[CLS] total smdh 3 concentration was 32 μm . [SEP]
[CLS] 8 . ( a and b ) populations of dimers and large - oligomer networks ( ≥ nonamers ) found in the cgmd simulation for the nanoasemblies of pyr - t n - smdh 3 ( a ) and tp - t n - smdh 3 ( b ) comonomers pairs ( n = 3 , 6 , 9 , 12 , and 15 ) . [SEP]
[CLS] the simulations were carried out at [ smdh 3 ] = 32 μm . [SEP]
[CLS] each population result was the average of three and more repeated simulations . [SEP]
[CLS] standard deviations = ± 1 % [SEP]
[CLS] 10 . changes in the population distributions for dimers ( a and c ) and large - oligomer networks ( i . e . , ≥ nonamers ; b and d ) ) found in comparative experiment and cgmd simulation studies for the nanoassemblies of pyr - t n - smdh 3 ( a and b ) and tp - t n - smdh 3 ( c and d ) comonomers pairs ( n = 0 , 3 , 6 , and 15 ) . [SEP]
[CLS] the blue circles represent the experimentally observed populations of dimers and oligomers , whereas the red triangles represent the corresponding results from cgmd simulations . [SEP]
[CLS] the correspondingly colored lines were only included as visual guides . [SEP]
[CLS] both experiment and cgmd simulation studies were carried out at [ smdh 3 ] = 32 μm . [SEP]
[CLS] the populations of dimers and oligomers in the page gels were calculated using the imagej analysis tool . [SEP]
[CLS] tgw , lau kl , bousmail d , serpell cj , sleiman hf . nat chem . 2016 ; [SEP]
[CLS] 8 : 162 - 170 [SEP]
[CLS] [ pubmed : 26791900 ] 41 . goodman rp , schaap iat , tardin cf , erben cm , berry rm , schmidt cf , turberfield aj . science . [SEP]
[CLS] liposomal B-nanoparticle spherical nucleic B-material acids I-material ( lsnas ) are an attractive therapeutic platform for gene regulation and immunomodulation B-property due to their biocompatibility B-property , chemically tunable structures , and ability to enter cells B-material rapidly without the need for ancillary transfection agents . [SEP]
[CLS] such structures consist of small ( < 100 nm ) liposomal B-nanoparticle cores B-material functionalized with a dense , highly oriented nucleic B-material acid I-material shell B-material , both of which are key components in facilitating their biological activity . [SEP]
[CLS] here , the properties of lsnas synthesized using conventional methods , anchoring cholesterol terminated oligonucleotides into a liposomal B-nanoparticle core B-material , are compared to lsnas made by directly modifying the surface of a liposomal B-nanoparticle core B-material containing azide - functionalized lipids B-material with dibenzocyclooctylterminated oligonucleotides . [SEP]
[CLS] the surface densities of the oligonucleotides are measured for both types of lsnas , with the lipid - modified structures having approximately twice the oligonucleotide surface coverage . [SEP]
[CLS] the stabilities and cellular uptake properties of these structures are also evaluated . [SEP]
[CLS] the higher density , lipid - functionalized structures are markedly more stable than conventional cholesterol - based structures in the presence of other unmodified liposomes B-nanoparticle and serum proteins B-material as evidenced by fluorescence B-property assays . [SEP]
[CLS] significantly , this new form of lsna exhibits more rapid cellular uptake and increased sequence - specific toll - like receptor B-material activation in immune reporter cell B-material lines , making it a promising candidate for immunotherapy . [SEP]
[CLS] lipid - functionalized oligonucleotides ( lons ) are an attractive platform for many uses that include materials assembly , detection , and therapeutic design , due to their attractive chemical and biological properties that include increased stability in serum and straightforward assembly into nanocarriers through hydrophobic B-property interactions I-property . [SEP]
[CLS] lons can also be used to synthesize spherical nucleic B-material acids I-material ( snas ) , a class of nanomaterial B-material that consists of a small spherical core B-material ( < 100 nm ) functionalized with a dense and highly oriented oligonucleotide shell B-material . [SEP]
[CLS] the nucleic B-material acid I-material shell B-material allows snas to readily enter cells B-material without the need for transfection agents , enhances their binding affinity for protein B-material receptors and complementary oligonucleotides , and reduces their susceptibility to degradation by endonucleases . [SEP]
[CLS] because of these enhanced properties , snas have emerged as attractive agents for biodetection , drug delivery , gene regulation , and immunomodulation B-property . [SEP]
[CLS] since the biological properties of snas are independent of the core B-material type , they have been synthesized with a variety of templates including gold B-material particles , micelles B-material , infinite coordination polymer B-material particles , liposomes B-nanoparticle , and proteins B-material . [SEP]
[CLS] for in vivo biological applications , the liposomal B-nanoparticle variants are more appealing since the vesicle cores B-material are highly biocompatible B-property , able to encapsulate a diverse range of molecules , and readily functionalized in a modular fashion . [SEP]
[CLS] lsnas are typically synthesized by anchoring nucleic B-material acids I-material modified with hydrophobic B-property moieties , such as cholesterol or tocopherol , into the lipid B-material bilayer I-material of a liposomal B-nanoparticle template . [SEP]
[CLS] however , the fluidity of the liposomal B-nanoparticle core B-material and the hydrophilicity B-property of the nucleic B-material acid I-material shell B-material make this structure intrinsically dynamic . [SEP]
[CLS] for lsnas , interparticle component exchange could reduce the stability of the nucleic B-material shell I-material and ultimately the entire sna structure . [SEP]
[CLS] the nature of the hydrophobic B-property groups covalently attached to the nucleic B-material acids I-material can modulate the dynamics of interparticle exchange ; cholesterol - modified nucleic B-material acids I-material have weaker and more dynamic interactions with lipid B-material bilayers I-material , while nucleic B-material acids I-material modified with diacyl chains are significantly more stable . [SEP]
[CLS] furthermore , in biological environments , the presence of serum proteins B-material and natural lipid - based structures , both of which are known to readily interact with liposome - based conjugates , could further destabilize lsnas by interacting with dissociated nucleic B-material acid I-material strands . [SEP]
[CLS] any loss of the nucleic B-material acid I-material shell B-material from the lsnas is likely to alter their interactions with cells B-material . [SEP]
[CLS] indeed , prior sna research has shown that greater oligonucleotide density leads to increased cellular uptake . [SEP]
[CLS] along with the dynamic behavior of lsnas , conventional approaches to synthesizing lsnas often result in lower oligonucleotide densities in comparison to snas with inorganic B-material cores I-material . [SEP]
[CLS] these observations point toward the importance of increasing oligonucleotide density and maintaining stability of lsna architectures in physiological environments . [SEP]
[CLS] herein , we synthesized lsna structures with different nucleic B-material acid I-material loadings and dynamic behaviors in an effort to establish structure - function relationships between these structures and cellular environments . [SEP]
[CLS] to accomplish this goal , we synthesized lsnas using different hydrophobic B-property anchors , lipid B-material or cholesterol , that are covalently linked to dna ( scheme 1 ) and measured their stability in buffer and serum , dissociation kinetics , cellular uptake properties , and ability to engage with toll - like receptors ( tlrs ) critical for immune - modulation . [SEP]
[CLS] from these studies , we determined that the lsnas synthesized with lipid - modified dna lead to twice the oligonucleotide loading , resulting in substantively enhanced stability , more rapid cellular internalization , and greater sequence - specific tlr - mediated immune activation . [SEP]
[CLS] lsnas were synthesized by modifying the surface of small B-material unilamellar I-material vesicle I-material ( suv ) templates ( 50 nm size ; figure s1 , supporting information ) using two different functionalization strategies ( scheme 1 ) . [SEP]
[CLS] in the first strategy , cholesterol - tail lsnas were synthesized via literature methods by adding cholesterol - modified dna to 1 , 2 - dioleoyl - snglycero - 3 - phosphocholine ( dopc ) - based suvs in 4 - ( 2 - hydroxyethyl ) - 1piperazineethanesulfonic acid ( hepes ) - buffered saline ( hbs ; scheme 1a ) . [SEP]
[CLS] in the second strategy , an azide - functionalized 1 , 2 - dipalmitoyl - sn - glycero - 3 - phosphoethanolamine ( dppe ) derivative , 1 , 2 - dipalmitoyl - sn - glycero - 3 - phosphoethanolamine - n - ( 6 - azidohexanoyl ) ( dppe - az ) , was used as the minor lipid B-material component ( 0 . 5 - 10 mol % ) in the formation of dopc - based suvs with azide groups presented on the surface of the vesicle ( scheme 1b ) . [SEP]
[CLS] dibenzocyclooctyl ( dbco ) - modified dna strands were then covalently conjugated to the azide groups through cu - free click chemistry to synthesize lipid - tail lsnas . [SEP]
[CLS] this second strategy has two potential advantages : ( 1 ) the number of potential anchoring sites ( i . e . , azides ) can be controlled prior to surface modification through the lipid B-material stoichiometry used to create the initial vesicle , and ( 2 ) the saturated lipid B-material tail is more hydrophobic B-property than cholesterol , minimizing the dissociation of oligonucleotides already anchored to the liposomal B-nanoparticle vesicle . [SEP]
[CLS] since the hydrophobic B-property anchors for the two different strategies have different affinities for the liposomal B-nanoparticle template , the dna loading for each was determined . [SEP]
[CLS] to determine the stoichiometry that led to maximum dna loading , increasing amounts of either dbco - dna or cholesterol - dna were incubated B-technique with their respective liposomal B-nanoparticle templates overnight at 25 °c ( table 1 ) . [SEP]
[CLS] following purification by size - exclusion chromatography B-technique , the number of strands per liposome B-nanoparticle was assessed . [SEP]
[CLS] importantly , lipid B-material - tail lsnas had significantly greater dna loading compared to cholesterol - tail analogs ( table 1 ) . [SEP]
[CLS] this constitutes a substantive increase in dna shell B-material density , which should affect particle interactions with proteins B-material and cells B-material . [SEP]
[CLS] although the surface dna density is important , lsna constructs also must remain assembled in physiological environments for it to exhibit its architecture - dependent properties . [SEP]
[CLS] to determine the stability of the lsnas , the kinetics of interparticle dna exchange for both cholesterol - tail and lipid - tail lsnas were measured in liposome - containing buffers and serum protein B-material environments . [SEP]
[CLS] as a reporter of the assembly state of the structures , forster resonance energy transfer ( fret ) lsnas were synthesized using a rhodamine - labeled lipid B-material and cy5 - labeled dna ( detailed sequences are listed in table s1 of the supporting information ) , so that fret can occur between the fluorophore - labeled lipids B-material and dna when the lsna is fully assembled ( figure s2 , supporting information ) . to assess the rate at which the dna shell B-material dissociated from its original liposomal B-nanoparticle template and inserted into a different lipid B-material bilayer I-material , fret reporter particles , synthesized with ≈150 strands / particle , were mixed with an excess ( ≈100 fold by liposome B-nanoparticle ) of 50 nm dopc liposomes ( without dna ) . [SEP]
[CLS] the addition of the cholesterol - tail fret reporter lsna to the dopc - derived liposomes B-nanoparticle at room temperature ( 21 °c ) and physiologic temperature ( 37 °c ) resulted in an exponential decay of fret signal ( k obs = ( 2 . 1 ± 0 . 1 ) × 10 −3 and ( 1 . 0 ± 0 . 08 ) × 10 −2 s −1 respectively figure 1b ) indicating rapid dissociation of the lsna . [SEP]
[CLS] in contrast , lipid - tail lsnas showed minimal decay at room temperature and dissociation rates of 7 . 1 ± 0 . 2 × 10 −5 s −1 at 37 °c . [SEP]
[CLS] consistent with our hypothesis and previous findings , the rate of exchange between particles is significantly slower for lipid B-material - tail lsnas in comparison to the cholesterol - tail analogs . [SEP]
[CLS] as a control , the lsnas were incubated B-technique in buffer over the same time and displayed no decay ( figure 1c ) , indicating that disassembly of the lsnas only occurs in the presence of other liposomes B-nanoparticle . [SEP]
[CLS] previous studies have reported that the rate of spontaneous dissociation of fluorophoremodified dope from liposomal B-nanoparticle bilayers is significantly slower ( 1 . 16 × 10 −5 s −1 at 37 °c ) than the dissociation rates reported here for the dna strands modified with either cholesterol or lipid B-material . [SEP]
[CLS] since the rate of dissociation for lipids B-material is significantly slower than those observed in our particles , we hypothesized that the dna shell B-material was dissociating much quicker than the lipid B-material components . [SEP]
[CLS] to confirm this hypothesis , control particles featuring rhodamine - labeled lipids B-material and unlabeled dna were mixed with particles containing carboxyfluorescein - labeled lipids B-material and unlabeled dna ( see table s2 of the supporting information for composition ) . [SEP]
[CLS] the fluorescence B-property spectrum was measured after 0 , 0 . 25 , and 24 h incubation B-technique at 25 °c , revealing only minimal exchange of the dye - labeled lipids B-material ( figure s3 , supporting information ) . [SEP]
[CLS] suspecting that the protein - rich environment in serum can destabilize lsnas by interacting with the lipid B-material components or the dna , either on the particle or when dissociated , we incubated B-technique our fret reporter lsnas in a 10 vol % serum - containing medium . [SEP]
[CLS] disruption of the lsnas due to interactions with serum proteins B-material would result in decreased fret ( figure 2a ) . [SEP]
[CLS] indeed , the fret signals decreased and the fluorescence B-property of rhodamine - labeled lipids B-material increased for both lsna structures over time ( figure 2b , c ) , although at different rates . [SEP]
[CLS] the lipid B-material - tail lsnas show a > 20 - fold extended half - life in comparison to the cholesterol - tail analogs with observed dissociation rates of ( 2 . 8 ± 0 . 4 ) × 10 −4 and ( 7 . 9 ± 1 . 1 ) × 10 −3 s −1 , respectively . [SEP]
[CLS] the increased stability of lipid - tail lsnas should allow such structures to remain intact and enter cells B-material via known endosomal pathways , which , in the context of immunotherapy , should equate to a larger therapeutic payload . [SEP]
[CLS] in addition , this result suggests that serum proteins B-material actively disrupt lsnas as evidenced by the four - fold increase in exchange rate for the lipid B-material - tail lsnas in the presence of serum proteins B-material when compared to the system containing only lsnas mixed with unmodified liposomes B-nanoparticle , but in the absence of proteins B-material where only passive dissociative exchange is possible . [SEP]
[CLS] note that although there is a significant decrease in the fret signal for both classes of lsnas over the course of the experiments , a baseline level of fret remains , which indicates that some dna strands may remain attached to the lipid B-material bilayer I-material . [SEP]
[CLS] to confirm that the decrease in fret is due to the loss of the dna shell B-material and not the fluorophore - labeled lipids B-material in the core B-material , a lsna composed of carboxyfluorescein - and rhodamine - labeled lipids B-material ( 1 mol % each ) with unlabeled cholesterol - tail dna was incubated B-technique in a 10 vol % serum solution . [SEP]
[CLS] the dissociation rate of the lipids B-material from the core B-material was significantly slower ( k obs = ( 5 . 7 ± 0 . 2 ) × 10 −5 s −1 ) than that measured between the dna shell B-material and the template ( figure s4 , supporting information ) . [SEP]
[CLS] this supports the conclusion that the dissociation rate of the dna shell B-material is faster than the disassembly of the lipid B-material core B-material in protein - rich environments . [SEP]
[CLS] since the density of the dna shell B-material can potentially alter interactions between lsnas and serum proteins B-material due to electrostatic considerations and oligonucleotide sequence - and density - specific interactions , the stabilities of the lipid B-material - tail lsnas were evaluated as a function of shell B-material density . [SEP]
[CLS] the particles with higher dna shell B-material densities reached equilibrium , as determined by the fret ratio , at later time points compared to those with lower shell B-material densities ( figure s5 , supporting information ) , showing that increased dna loading leads to longer structural retention . [SEP]
[CLS] since the concentration of the particles and serum proteins B-material could potentially impact the dissociation rates of the system , we measured the dissociation rate as a function of different serum and particle concentrations . [SEP]
[CLS] to examine the serum - protein B-material - concentration - dependent dissociation , the lsnas were incubated B-technique with media containing increasing serum protein B-material concentrations ( 10 , 20 , and 30 vol % fetal bovine serum ( fbs ) ) . [SEP]
[CLS] there was no observable increase in dissociation rate at higher serum concentrations ( figure s6a , supporting information ) . [SEP]
[CLS] in addition , the effect of particle concentration was evaluated by incubating B-technique two different particle concentrations , 100 × 10 −9 and 500 × 10 −9 m by dna , with 10 vol % fbs ( figure s6b , supporting information ) . [SEP]
[CLS] again , no significant concentration dependence was observed . [SEP]
[CLS] taken together , these results suggest that lsna dissociation is independent of serum protein B-material and particle concentration for ranges typically used in cell B-material experiments and is primarily dependent on the hydrophobic B-property anchor attached to the dna . [SEP]
[CLS] a characteristic property of snas is that the nucleic B-material acid I-material shell B-material facilitates their rapid cellular internalization by engaging scavenger class a receptors , among others , on the cell B-material membrane . [SEP]
[CLS] as such , increased surface loading of dna on the lsnas should lead to higher rates of cellular uptake . [SEP]
[CLS] structures that have slower dissociation rates should result in higher dna densities facilitating cellular uptake . [SEP]
[CLS] to study the effect of lsna stability and dna shell B-material density on cellular uptake , we employed two different cell B-material lines , u87 - mg glioblastoma cells B-material and raw - blue macrophages . [SEP]
[CLS] the cells B-material were incubated B-technique with both types of lsnas , cholesterol - modified dna , and dbco - modified dna , which were all synthesized with cy5 - labeled phosphorothioate ( ps ) dna , and then evaluated using flow B-technique cytometry I-technique . [SEP]
[CLS] notably the u87 - mg cells B-material showed increased uptake of the lipid B-material - tail lsnas after 1 h compared to the cholesterol - tail lsnas ( figure 3a ) . [SEP]
[CLS] after 2 h incubation B-technique , however , the lipid B-material - tail lsnas no longer displayed an advantage ( figures 3a ) . [SEP]
[CLS] confocal imaging of the cells B-material corroborated this result , with greater cy5 fluorescence B-property intensity after 1 h of incubation B-technique with the lipid B-material - tail lsnas ( figure s8 , supporting information ) . [SEP]
[CLS] significantly , the uptake of both types of lsnas as well as cholesterol - tail dna is greater than that observed for dbco - modified ps dna , which is not capable of assembling into a spherical architecture like the other dna structures used . [SEP]
[CLS] the raw - blue macrophages also displayed enhanced uptake of the lipid B-material - tail lsnas in comparison to cholesterol - tail lsnas ( figure 3b ) . [SEP]
[CLS] these cells B-material showed rapid uptake of the lipid B-material - tail lsnas and greater total uptake even after 4 h of incubation B-technique ( figure 3b ) . [SEP]
[CLS] surprisingly , the cholesterol - tail lsnas and cholesterol - modified dna did not have any enhancement in uptake over the dbco - modified dna . [SEP]
[CLS] this stands in contrast to what we observed for the glioblastoma derived u87 - mg cells B-material . [SEP]
[CLS] the differences between these two cell B-material lines are likely due to differences in the levels of expression of cell B-material membrane receptors . [SEP]
[CLS] we hypothesized that the increased uptake of the lipid B-material - tail lsnas stems from its morestable and higher - density dna shell B-material facilitating interactions with scavenger receptors on the cell B-material surface , thus , enhancing cellular internalization . [SEP]
[CLS] to test this hypothesis , we treated u87 - mg cells B-material with fucoidan , an inhibitor of scavenger receptors , prior to incubation B-technique with lsnas . [SEP]
[CLS] following this treatment , the overall uptake of the lsnas decreased , and no significant difference between the uptake of cholesterol - tail and lipid B-material - tail particles was observed ( figure s9 , supporting information ) . [SEP]
[CLS] this result is consistent with the conclusion that the enhanced uptake of the lipid B-material - tail structures at early time points stems largely from scavenger receptor - I-event mediated I-event endocytosis B-event . [SEP]
[CLS] since both cholesterol - tail and lipid - tail dnas are capable of cellular internalization , due to their ability to independently form self - assembled micellar structures that mimic the sna architecture or bind nonspecifically to cell B-material membranes , confocal microscopy B-technique and flow B-technique cytometry I-technique data alone are not sufficient to determine if the constructs are being internalized as fully or partially intact structures . to answer this question , we measured the fret efficiency of the particles inside cells B-material by imaging the rhodamine fluorescence B-property before / after photobleaching of the cy5 dye ( figure 4 ) . [SEP]
[CLS] intact particles have increased rhodamine fluorescence B-property after photobleaching of the cy5 - labeled dna ( figure 4a ) . [SEP]
[CLS] in contrast , disassembled particles , where the cy5 - labeled dna has detached from the initial liposomal B-nanoparticle structure , have minimal increases in rhodamine fluorescence B-property . [SEP]
[CLS] after 1 h of incubation B-technique , cells B-material treated with the lipid B-material - tail lsnas exhibit increased fret compared to those treated with the cholesterol - tail analogs ( figure 4c ) , consistent with the former being more stable . [SEP]
[CLS] significantly , lipid B-material - tail lsnas loaded with higher dna densities ( 300 strands / lsna ) continued to retain more dna strands per liposome B-nanoparticle after 1 and 2 h of incubation B-technique compared to those assembled with lower dna densities ( ≈150 strands / lsna ) . [SEP]
[CLS] the amount of observed fret continued to decrease for the lipid B-material - tail lsnas at the 2 h time point . [SEP]
[CLS] after 24 h of incubation B-technique , no significant differences in fret were detected and only minimal fret was observed for any of the structures , suggesting that the lsnas gradually disassemble inside cells B-material as a function of time . [SEP]
[CLS] to assess the ability of both types of lsnas to activate therapeutic targets inside cells B-material , we synthesized lsnas with immunostimulatory unmethylated cpg - rich dna ( 1826 odn ) [SEP]
[CLS] these dna sequences are known to activate toll - like receptor B-material 9 ( tlr9 ) , which modulates innate immunity by stimulating cytokine production . [SEP]
[CLS] incubating B-technique these lsnas with raw - blue macrophages , which are modified to secrete alkaline phosphatase upon stimulation of tlrs for a colorimetric readout of immune - stimulation , allows us to compare their immune stimulatory activity . [SEP]
[CLS] as shown in figure 5a , the lipid B-material - tail lsnas show modestly increased activity at lower concentrations compared to cholesterol - tail analogs . [SEP]
[CLS] more importantly , pulse - chase experiments conducted at 250 × 10 −9 m concentration reveal significantly faster activation of the macrophages ( figure 5b ) by the lipid B-material - tail lsna formulations , presumably a consequence of more rapid uptake by cells B-material ( figure 3b ) . [SEP]
[CLS] in summary , we have evaluated the stability and biological behavior of lsnas synthesized with either lipid - or cholesterol - modified oligonucleotides . [SEP]
[CLS] importantly , this work demonstrates that a synthetic route of directly modifying lipid B-material - head groups on liposomes B-nanoparticle with dna leads to higher nucleic B-material acid I-material shell B-material densities and increased stability in physiological environments . [SEP]
[CLS] these combined properties result in enhanced interactions with cells B-material and significant advantages in the context of sequence - specific immune modulation . [SEP]
[CLS] taken together , this work outlines important structure - function principles for lsnas that will directly impact the design of nanomaterials B-material for novel therapeutic platforms . [SEP]
[CLS] unless otherwise noted , all reagents were purchased from commercial sources and used as received . [SEP]
[CLS] for oligonucleotide synthesis , all phosphoramidites and reagents were purchased from glen research , co . ( sterling , va , usa ) . [SEP]
[CLS] all lipids B-material were purchased from avanti polar lipids B-material , inc . ( alabaster , al , usa ) either in dry powder form or as a chloroform solution and used without further purification . [SEP]
[CLS] all other reagents were purchased from sigma - aldrich , co . ( st . louis , mo , usa ) . [SEP]
[CLS] ultrapure deionized ( di ) h 2 o ( 18 . 2 mω cm resistivity ) was obtained from a milli - q biocel system ( millipore co . , billerica , ma , usa ) . [SEP]
[CLS] uv - vis absorbance spectra were collected on a varian cary 5000 uv - vis spectrometer ( varian inc . , palo alto , ca , usa ) using quartz cuvettes with a 1 cm path length . [SEP]
[CLS] matrix - assisted laser desorption / ionization B-property time - of - flight ( maldi - tof ) mass spectrometric data were obtained on a bruker autoflex iii maldi - tof mass spectrometer ( bruker daltonics inc . , billerica , ma , usa ) . [SEP]
[CLS] for maldi - tof analysis , the matrix was prepared by mixing an aqueous solution of ammonium hydrogen B-material citrate ( 0 . 6 μl of a 35 wt % solution ( 15 mg in 30 μl of h 2 o ) ) and 3 - hydroxypicolinic acid ( sigma - aldrich , 2 mg in h 2 o : mecn ( 30 μl of a 1 : 1 v / v mixture ) ) . [SEP]
[CLS] an aliquot of the dna ( ≈0 . 5 μl of a 150 × 10 −6 m solution ) was then mixed with the matrix ( 1 : 1 ) and the resulting solution was added to a steel maldi - tof plate and dried under ambient conditions for 1 h before analysis . [SEP]
[CLS] samples were detected as negative ions B-material using the linear mode . [SEP]
[CLS] the laser was typically operated at 10 - 20 % power with a sampling speed of 10 hz . [SEP]
[CLS] each measurement averaged 500 scans with the following parameters : ion B-material source voltage 1 = 20 kv , ion B-material source voltage 2 = 18 . 5 kv , lens voltage = 8 . 5 kv , linear detector voltage = 0 . 6 kv , deflection mass = 3000 da . [SEP]
[CLS] centrifugation was carried out in a temperature - controlled eppendorf centrifuge 5430r ( eppendorf ag , hauppauge , ny , usa ) . [SEP]
[CLS] dynamic B-technique light I-technique scattering I-technique ( dls ) and zeta B-property potential I-property measurements were collected on a zetasizer nano zs ( malvern instruments , uk ) equipped with a he - ne laser ( 633 nm ) . [SEP]
[CLS] phosphorothioate oligonucleotides were synthesized on cpg supports using an automated nucleotide system ( model : mm12 , bioautomation inc . , plano , tx , usa ) and were purified using high - performance liquid B-technique chromatography I-technique ( hplc ) . [SEP]
[CLS] all oligonucleotides used in this study have ps backbone modifications . [SEP]
[CLS] for details on sequences synthesized ( table s1 , supporting information ) and purification methods , see section s1 ( supporting information ) . [SEP]
[CLS] suvs were synthesized using literature methods . [SEP]
[CLS] a brief description of the synthesis , purification , and characterization of these structures along with the composition of the liposomes B-nanoparticle used for the studies ( table s2 , supporting information ) can be found in section s2 ( supporting information ) . [SEP]
[CLS] for cholesterol - tail lsnas , a 3 ′ - cholesterol - tail oligonucleotide was added to the suv colloids ( 1 . 3 × 10 −3 m phospholipid concentration , final volume 1 ml ) and was shaken overnight . [SEP]
[CLS] for lipid - tail lsnas , an aliquot of the desired dbco - tail oligonucleotides was added to a n 3 - dppe containing suv ( 0 . 5 × 10 −3 m total phospholipid concentration , final volume 1 ml ) with a dna - dbco : surface n 3 - dppe lipid molar ratio of 2 : 1 . [SEP]
[CLS] the mixture was shaken overnight . [SEP]
[CLS] both structures were purified via size - exclusion chromatography B-technique on a sepharose cl - 4b column ( sigma - aldrich ) and the particle size distribution was analyzed using dls . [SEP]
[CLS] for details on the dls measurements , see section s3 ( supporting information ) . [SEP]
[CLS] the density of the nucleic B-material acid I-material shell B-material was determined by first dissociating the particles in sodium B-material dodecyl sulfate and then measuring the absorbance at 260 nm via uv - vis spectroscopy B-technique to calculate the dna concentration . [SEP]
[CLS] the amount of lipid B-material was determined by measuring the total phosphorus B-material content via inductively coupled plasma mass spectroscopy B-technique ( thermo fisher x series ii , thermo fisher scientific inc . , waltham , ma , usa ) and subtracting the phosphorus B-material content contributed by the dna backbone . [SEP]
[CLS] an alternative method to determine the density was also used . [SEP]
[CLS] the absorbance spectrum of lsnas synthesized with cy5 labeled dna and 1 mol % rhodamine labeled lipid B-material were measured . [SEP]
[CLS] the peak absorbance of the cy5 and rhodamine ( corrected to remove any cy5 absorbance contribution ) were used to measure the relative amount of dna to lipid B-material . [SEP]
[CLS] a solution of fret reporter lsna ( table s2 , supporting information , 0 . 1 × 10 −6 m by final [ oligonucleotide ] , final volume 1 ml ) was aliquoted into a quartz cuvette at room temperature . [SEP]
[CLS] a ≈100 - fold excess of dopc liposomes B-nanoparticle was added and quickly pipetted up and down ( within 3 s ) to mix uniformly . [SEP]
[CLS] the fluorescence B-property of the fret reporter particles was monitored over 3 h ( rhodamine / cy5 , excitation at 560 nm , emission at 583 and 672 nm , 3 nm slit width ) using a fluorlog - 3 ( horiba jobin yvon inc . , edison , nj , usa ) . [SEP]
[CLS] for experiments measuring lipid B-material dissociation , cholesterol - control rhodamine particles ( table s2 , supporting information , 0 . 1 × 10 −6 m by final [ oligonucleotide ] , volume 1 ml ) were aliquoted into an eppendorf tube at room temperature . [SEP]
[CLS] an equimolar aliquot of cholesterolcontrol fluorescein particles ( table s2 , supporting information ) was added and quickly pipetted up and down ( within 3 s ) to mix uniformly . [SEP]
[CLS] the fluorescence B-property spectrum was then taken after incubation B-technique at 25 °c at 0 , 0 . 25 , and 24 h , with a plate reader ( synergy h4 , biotek instruments , inc . , winooski , vt , usa , 9 . 0 nm slit width , excitation 480 nm ) . [SEP]
[CLS] the fret ratio was calculated using the formula fret ratio = [SEP]
[CLS] where i a is the acceptor intensity and i d is the donor intensity measured at their respective peak wavelengths . [SEP]
[CLS] fret reporter particles ( 100 × 10 −9 m [ oligonucleotide ] ) were added to a serum - containing solution ( 10 vol % fbs in hbs ) and mixed well for 3 s . [SEP]
[CLS] the fluorescence B-property of the lsnas was monitored with a plate reader ( synergy h4 , excitation at 560 nm , emission at 583 and 672 nm , 9 . 0 nm slit width ) at 37 °c . [SEP]
[CLS] for details on dna loading and concentration dependent serum stability studies , see section s4 ( supporting information ) . [SEP]
[CLS] control experiments consisting of lsnas assembled with 1 mol % fluorescein - and rhodamine - labeled lipids B-material with unlabeled cholesterol - modified dna ( 100 × 10 −9 m final [ dna ] ) were utilized to measure the exchange rate of the lipids B-material in 10 vol % fbs . [SEP]
[CLS] the fluorescence B-property was monitored on a plate reader over 10 h at 30 min intervals ( excitation at 480 × 10 −9 m , emission at 583 nm and 550 nm , 9 nm slit width ) . [SEP]
[CLS] a final reading was performed after 36 h to observe the equilibrium fluorescence B-property . [SEP]
[CLS] confocal imaging and flow B-technique cytometry I-technique were performed on u - 87 mg cells B-material ( epithelial , glioblastoma ) using the recommended culture conditions in complete growth media ( minimum essential medium supplemented with fbs ( 10 vol % ) , penicillin ( 0 . 2 units ml −1 ) , and streptomycin ( 0 . 1 μg ml −1 ) ) . [SEP]
[CLS] raw - blue cells B-material ( invivogen , ca , usa ) , which are derivatives of raw 264 . 7 macrophage cells B-material stably expressing a secreted alkaline phosphatase under a nf - κb promoter , were cultured as recommended by the supplier in complete growth media ( dulbecco ' s modified eagle medium supplemented with 10 vol % heat inactivated fbs , penicillin ( 0 . 2 units ml −1 ) , streptomycin ( 0 . 1 μg ml −1 ) , normocin ( 100 μg ml −1 ) , and l - glutamine ( 2 × 10 −3 m ) ) . [SEP]
[CLS] a comparative cell B-material - uptake study between the two types of lsnas ( cholesterol - tail and lipidtail constructs ) was carried out using u87mg cells B-material . [SEP]
[CLS] cells B-material were plated at 20 000 cells B-material per well in a 96 well plate in complete growth media . [SEP]
[CLS] the cells B-material were placed in the incubator B-technique to recover overnight . [SEP]
[CLS] the following day , the cells B-material were treated with both cholesterol - and lipidtail lsnas , cholesterol - dna , and dbco - dna with a final dna concentration 0 . 5 × 10 −6 m for 1 , 2 , and 4 h . [SEP]
[CLS] after each time point , the cells B-material were triple washed with 1× pbs , trypsinized ( 5 % trypsin , 30 μl for 5 min at 37 °c ) , and fixed in 200 μl of 4 % paraformaldehyde . [SEP]
[CLS] flow B-technique cytometry I-technique was performed on the cells B-material using the red laser and red fluorescence B-property channel on a guava easycyte 8ht instrument ( millipore , billerica , ma , usa ) . [SEP]
[CLS] the distribution of cell B-material fluorescence B-property of the gated - cells B-material was collected and the mfi was calculated . [SEP]
[CLS] error - values were determined using the standard deviation of the median signal from three different wells . [SEP]
[CLS] for the scavenger receptor B-material inhibition studies , the cells B-material were incubated B-technique with fucoidan ( 50 μg ml −1 ) for 30 min prior to the addition of the respective oligonucleotide structures , and flow B-technique cytometry I-technique was performed after 1 h of incubation B-technique with the lsnas using the aforementioned protocol . [SEP]
[CLS] u87mg cells B-material were seeded in an 8 well chamber slide ( german # 1 . 5 , labtek ii , thermo fisher scientific inc . , waltham , ma , usa ) at 10 000 cells B-material per well and incubated B-technique overnight . [SEP]
[CLS] fret reporter lsnas ( 0 . 1 × 10 −6 m by oligonucleotide ) were then incubated B-technique with the cells B-material in complete growth media for 1 and 24 h . [SEP]
[CLS] the media was removed and the cells B-material were rinsed with pbs and fixed with 4 % paraformaldehyde for 10 min at room temperature . [SEP]
[CLS] the paraformaldehyde was removed and the cells B-material were rehydrated in pbs for imaging . [SEP]
[CLS] the cell B-material nuclei were stained with hoechst 3342 ( invitrogen , thermo fisher scientific inc . , carlsbad , ca , usa ) following the manufacturer ' s instructions . [SEP]
[CLS] confocal microscopy B-technique imaging of these cells B-material was carried out on a zeiss lsm 800 inverted laser - scanning confocal microscope ( carl zeiss , inc . , thornwood , ny , usa ) at 40 × and 63 × magnification . [SEP]
[CLS] acceptor photobleaching experiments were performed by zooming into and exciting the cy5 dye in a small region of interest ( roi ) with a 640 nm laser at 100 % power for 40 cycles . [SEP]
[CLS] rhodamine and cy5 fluorescence B-property intensities were measured before and after photobleaching of the cy5 dye . [SEP]
[CLS] the fret efficiencies were determined by comparing the intensity of the rhodamine fluorescence B-property intensity within the roi before and after cy5 photobleaching using imagej software ( available free of charge through https : / / imagej . nih . gov / ij / ) . [SEP]
[CLS] approximately 10 cells B-material per roi were imaged for each condition for calculating fret efficiencies , which were determined using the following equation after subtraction of the background fluorescence B-property : [SEP]
[CLS] hek - blue - mtlr9 cells B-material were plated in 96 well plates at a density of 50 000 cells B-material per well in complete growth media ( see section s9 for details on media , 200 μl of media per well ) . [SEP]
[CLS] immediately after plating , the cells B-material were treated with cholesterol - tail or lipid - tail lsnas and incubated B-technique at 37 °c for 16 h . [SEP]
[CLS] the assay was developed using the manufacturers recommended protocol , which is described in section s6 ( supporting information ) . [SEP]
[CLS] for the pulse - chase experiments , hek - blue - mtlr9 were plated as described above . [SEP]
[CLS] immediately after plating , the cells B-material were treated with the cholesterol - tail or lipid B-material - tail lsnas ( see table s1 of the supporting information ) at 250 × 10 −9 m final dna concentration per well . [SEP]
[CLS] the cells B-material were incubated B-technique with the different lsnas for 15 , 30 , 45 , 60 , 90 , and 180 min . [SEP]
[CLS] after each time point , the media was removed and replaced with fresh media and further incubated B-technique at 37 °c for 16 h . [SEP]
[CLS] the quanti - blue analysis then proceeded as described in section s6 ( supporting information ) . [SEP]
[CLS] two different functionalization strategies for lsnas . [SEP]
[CLS] 1 . dissociation of lsnas in the presence of other liposomal B-nanoparticle templates . [SEP]
[CLS] a ) a schematic representation of the dissolution process for lsnas in the presence of other liposomes B-nanoparticle . [SEP]
[CLS] b ) the fret ratio of the cholesterol - tail and lipid - tail lsnas in the presence of excess dopc liposomes B-nanoparticle . [SEP]
[CLS] c ) the fret ratio of both lsnas incubated B-technique in buffer . [SEP]
[CLS] disassembly of lsnas in serum . [SEP]
[CLS] a ) a schematic representation of the disassembly of fret reporting lsnas synthesized with cy5 - labeled dna and rhodamine labeled lipids B-material upon incubation B-technique in 10 vol % serum solution . [SEP]
[CLS] b ) rhodamine fluorescence B-property measured over time for cholesterol - tail and lipid B-material tail lsnas . [SEP]
[CLS] c ) the fret ratio between rhodamine and cy5 for both lsnas . [SEP]
[CLS] cellular uptake of lsnas . [SEP]
[CLS] histograms of cellular fluorescence B-property intensity and the median fluorescence B-property intensity for a ) u87 - mg cells B-material and b ) raw - blue macrophages incubated B-technique with structures incorporating cy5 - labeled dna ( * * p < 0 . 01 , * * * p < 0 . 001 ; anova with a bonferroni post hoc test ) . [SEP]
[CLS] acceptor photobleaching fret imaging of lsnas . [SEP]
[CLS] confocal images of lsnas synthesized with cy5 - labeled dna ( red ) attached to rhodamine - labeled liposomes B-nanoparticle ( green ) a ) before and b ) after photobleaching of cy5 . [SEP]
[CLS] the cell B-material nuclei are stained with hoechst ( blue ) . [SEP]
[CLS] a dashed box indicates the region of interest ( roi ) where photobleaching occurs . [SEP]
[CLS] enlarged images of the roi for cy5 and rhodamine fluorescence B-property are shown below each image . [SEP]
[CLS] scale bars = 10 μm . [SEP]
[CLS] c ) the fret efficiency of lsnas internalized by cells B-material as determined by measuring the fluorescence B-property intensity of rhodamine - labeled lipids B-material before and after photobleaching of the cy5 - labeled dna ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; anova with a bonferroni post hoc test ) . [SEP]
[CLS] 5 . activation of raw blue macrophages by lsnas . [SEP]
[CLS] a ) tlr9 activation resulting from overnight incubation B-technique with cpg and control ( t25 ) lsnas along with the linear cpg dna . [SEP]
[CLS] b ) tlr9 activation from pulse - chase treatment with cpg lsnas . [SEP]
[CLS] the activation is normalized to that measured after overnight incubation B-technique at the same concentration ( * p < 0 . 01 , anova with bonferroni post hoc test ) . [SEP]
[CLS] number of dna strands per particle when incubated B-technique with different ratios of liposome B-nanoparticle to dna . [SEP]
[CLS] we report a strategy for creating a new class of protein transfection materials composed of a functional protein B-material core B-material chemically modified with a dense shell B-material of oligonucleotides . [SEP]
[CLS] these materials retain the native structure and catalytic ability of the hydrolytic enzyme β - galactosidase , which serves as the protein B-material core B-material , despite the functionalization of its surface with [UNK] dna strands . [SEP]
[CLS] the covalent attachment of a shell B-material of oligonucleotides to the surface of β - galactosidase enhances its cellular uptake of by up to [UNK] - fold and allows for the use of working concentrations as low as 100 pm enzyme . [SEP]
[CLS] dna - functionalized β - galactosidase retains its ability to catalyze the hydrolysis of β - glycosidic linkages B-property once endocytosed , whereas equal concentrations of protein B-material show little to no intracellular catalytic activity . [SEP]
[CLS] proteins B-material represent a highly evolved class of natural nanoparticles B-nanoparticle with an unparalleled degree of structural and compositional homogeneity , as well as diversity of functional applications . [SEP]
[CLS] in particular , the efficient intracellular delivery of functional proteins B-material has widespread applications in medicine and provides a means for engineering cellular functions . [SEP]
[CLS] however , the cellular uptake of functional proteins B-material is impeded by their inherent instability , large sizes , and charged surfaces . [SEP]
[CLS] to address these limitations , a variety of approaches have been developed for stabilizing proteins B-material and enhancing their cellular uptake , which include covalent or noncovalent attachment of polymers B-material , conjugation to cellpenetrating peptides B-material , formulation with lipids B-material , liposomes B-nanoparticle , or nanoparticles B-nanoparticle , and attachment to highly charged natural or engineered proteins B-material . [SEP]
[CLS] while each of these strategies has unique attributes , they often require incubation B-technique of cells B-material with relatively high protein B-material concentrations and in many cases result in formulations with poorly defined compositions or limited stabilities . [SEP]
[CLS] recently , spherical nucleic B-material acid I-material ( sna ) - nanoparticle B-nanoparticle conjugates , which consist of a nanoparticle B-nanoparticle core B-material surrounded by a dense shell B-material of oligonucleotides , have emerged as exciting new architectures with diverse biological applications in gene regulation , immunomodulation B-property , and intracellular detection . [SEP]
[CLS] these applications are possible due to the superior cellular uptake and physiological stability of snas relative to their individual components . [SEP]
[CLS] this enhanced cellular internalization of snas is derived from the 3 - d architecture of the conjugates and its ability to engage scavenger receptors on the surfaces of most cells B-material . [SEP]
[CLS] importantly , the favorable biological properties of snas are independent of their nanoparticle B-nanoparticle cores B-material , which can therefore be chosen based on potential biological applications rather than practical synthetic limitations . [SEP]
[CLS] based on these observations , we hypothesized that proteins B-material could serve as the nanoparticle B-nanoparticle core B-material of snas and the dense shell B-material of oligonucleotides as a biocompatible B-property polymer B-material shell B-material that promotes cellular uptake . [SEP]
[CLS] these supramolecular structures , termed prosnas ( figure 1a ) , are distinct from previous examples of protein - dna conjugates that contain only a few conjugated oligonucleotides and lack protein - based functionalities , those based on virus capsids , or those that employ the attached oligonucleotides as a polyanionic structure to which cationic B-material polymers B-material noncovalently associate , but that alone are ineffective in promoting cellular uptake . [SEP]
[CLS] in contrast to more traditional nanoparticles B-nanoparticle , proteins B-material are characterized by surfaces with a nonuniform distribution of chemically reactive sites and core B-material structures that are held together by a complex network of relatively weak interactions . [SEP]
[CLS] an essential consideration for the implementation of our approach is therefore whether a dense shell B-material of oligonucleotides can be conjugated to the surface of a protein B-material while retaining its native structure and catalytic functionality . [SEP]
[CLS] to demonstrate that this is the case , the large ( 464 kda ) homotetrameric enzyme β - galactosidase ( β - gal ) ( figure 1a , ( 1 ) ) was chosen as a model system . [SEP]
[CLS] due to its large size ( 17 . 5 × 14 × 8 . 5 nm ) , β - gal does not efficiently traverse cellular membranes and has therefore served as an ideal model system for testing the feasibility of various strategies for the intracellular delivery of functional enzymes . [SEP]
[CLS] additionally , β - gal catalyzes the hydrolysis of β - glycosidic bonds between galactose and a diverse range of organic moieties , allowing one to determine the effects of substrate size and chemical composition on catalysis . [SEP]
[CLS] the surface of β - gal was modified with fluorophores to allow quantification of the protein B-material concentration after modification with oligonucleotides and to provide a handle for monitoring cellular uptake via flow B-technique cytometry I-technique and confocal microscopy B-technique ( vida infra ) . [SEP]
[CLS] addition of a 5 - fold excess of a thiol - reactive fluorochrome ( af647 maleimide , figure 1b , i ) yielded a tetramer with [UNK] fluorophore modifications ( af 4 - β - gal ) , as determined by uvvis absorbance spectroscopy B-technique ( figure 2a ) . [SEP]
[CLS] the surface of af 4 - β - gal was then functionalized with dna essentially as previously described . [SEP]
[CLS] briefly , surface lysine B-material amines B-material were reacted with small polyethylene glycol ( peg ) polymers B-material with an azide and an amine - reactive nhydroxy succinimide moiety ( figure 1b , ii ) at opposing termini . [SEP]
[CLS] the covalently attached azides were then reacted with dna strands containing the strained cyclooctyne , dibenzocyclooctyne ( dbco ) at the 5 ′ - terminus via copper - free click chemistry ( figure 1b , iii ) . [SEP]
[CLS] the sequence used here ( dggt ) 10 was chosen based on previous work that showed enhanced cellular uptake of snas with g - rich shells B-material relative to poly dt shells B-material . [SEP]
[CLS] this strategy yielded the conjugate prosna β - gal ( figure 1a , ( 2 ) ) , with 25 ± 1 strands of dna per tetrameric enzyme . [SEP]
[CLS] the level of dna B-event modification I-event was determined by comparing the difference in absorbance at 260 nm between af 4 - β - gal and prosna β - gal ( figure 2a ) . [SEP]
[CLS] agarose B-technique gel I-technique electrophoresis I-technique of prosna β - gal showed an increase in its electrophoretic B-property mobility I-property relative to af 4 - β - gal due to the introduction of a large number of negatively charged phosphate groups within the dna shell B-material ( figure s2 ) . [SEP]
[CLS] an additional staining step for dna revealed that the protein B-material and dna ran with the same electrophoretic B-property mobility I-property for prosna β - gal , whereas a mixture of af 4 - β - gal and free dna ran as two distinct bands , demonstrating the covalent attachment of oligonucleotides rather than nonspecific association with its surface . [SEP]
[CLS] circular dichroism spectroscopy B-technique was employed to determine whether the β - gal that composes the prosna core B-material retains its native structure , which is essential for preserving its catalytic functionality . [SEP]
[CLS] as shown in figure 2b , the observed spectrum of prosna β - gal agrees well with a theoretical spectrum obtained by summing the spectra of af 4 - β - gal and an equivalent concentration of the free oligonucleotides . [SEP]
[CLS] this agreement indicates that the enzyme retains its secondary structure after attachment of a shell B-material of oligonucleotides . [SEP]
[CLS] to determine whether the attached oligonucleotides provide a steric barrier B-property to or alter the enzyme active site and impair its catalytic functionality , we performed a fluorescence B-property assay utilizing the substrate , 7 - hydroxycoumarin - 3 - carboxylic acid ( cug ) ( figure 2c , d ) , which releases a fluorescent B-property coumarin derivative upon hydrolysis of the β - glycosidic linkage B-property with galactose . [SEP]
[CLS] despite the high degree of surface modification of the protein B-material and the relatively large size of the cug substrate , prosna β - gal retained catalytic activity at all enzyme concentrations tested . [SEP]
[CLS] we next tested whether prosna β - gal shows enhanced cellular uptake relative to af 4 - β - gal in multiple mammalian cell B-material lines ( hacat , human keratinocytes ; c166 , mouse epithelial cells B-material ; and skov3 , human ovarian adenocarcinoma cells B-material ) . [SEP]
[CLS] cells B-material were incubated B-technique with 0 . 1 or 1 nm protein B-material for 1 . 5 - 12 h , and their uptake was determined by flow B-technique cytometry I-technique ( figures 3 and s3 ) . [SEP]
[CLS] compared to cells B-material incubated B-technique with af 4 - β - gal , prosna β - gal showed an [UNK] - 280 - fold increase in cellular uptake . [SEP]
[CLS] at the lowest concentration tested ( 0 . 1 nm ) , prosna β - gal showed nearly 2 orders of magnitude greater cellular uptake than af 4 - β - gal . [SEP]
[CLS] we have previously shown that the route for enhancements in the cellular uptake of conventional sna conjugates involves engagement of cell - surface scavenger receptors followed by caveolae - mediated endocytosis B-event . [SEP]
[CLS] to evaluate whether prosna β - gal is uptaken by a similar mechanism , cells B-material were treated with fucoidan , a scavenger receptor B-material ligand , after which cellular uptake of prosna β - gal was monitored by flow B-technique cytometry I-technique . [SEP]
[CLS] as shown in figure s4 , treatment with fucoidan resulted in an 80 - 90 % decrease in the cellular uptake of prosna β - gal , which is consistent with the conclusion that it is internalized by a similar mechanism as traditional sna conjugates . [SEP]
[CLS] importantly , prosna β - gal showed no apparent toxicity B-property toward any of the cell B-material lines tested here , as demonstrated by measuring cellular metabolic redox activity using the commonly employed cell B-property viability I-property indicator B-property resazurin ( figure s5 ) . [SEP]
[CLS] to ensure that the transfected enzymes remained functional within the cellular milieu , we employed two catalytic assays based on distinct substrates . [SEP]
[CLS] for both assays , hacat , skov3 , and c166 cells B-material were incubated B-technique with 1 nm af 4 - or prosna β - gal for 12 h . [SEP]
[CLS] after washing the cells B-material with phosphate buffered saline ( pbs ) to remove any enzyme bound to the cell B-material surface , a 1 mg ml −1 solution of the membrane permeable β - gal substrate 5 - bromo - 4 - chloro - 3indolyl - β - d - galactopyranoside ( xgal , figure s6 ) was added , and the cells B-material were incubated B-technique for 3 h at 37 °c . [SEP]
[CLS] hydrolysis of the β - glycosidic linkage B-property of xgal results in the release of an indole derivative that , upon oxidation , dimerizes and forms an insoluble blue precipitate that can be visualized by light microscopy B-technique ( figure s6a ) . [SEP]
[CLS] as shown in figure 4a , essentially all cells B-material treated with 1 nm prosna β - gal showed accumulation of the blue product throughout the entire cell B-material volume , demonstrating that the transfected protein B-material remains active intracellularly . [SEP]
[CLS] in the case of hacat cells B-material , prosna β - gal treatments as low as 0 . 1 nm still produced a visible response ( figure s7 ) . [SEP]
[CLS] in contrast , untreated cells B-material or cells B-material treated with up to 10 nm af 4 - β - gal showed minimal substrate hydrolysis , demonstrating that product formation is catalyzed by transfected enzymes and not endogenous β - gal and that the shell B-material of dna is necessary for cellular uptake of functional enzymes ( figure s8 ) . [SEP]
[CLS] a separate assay based on the fluorogenic substrate , c 12 - fluorescein di ( β - dgalactopyranoside ) ( c 12 - fdg , figure s6b ) , was used to further demonstrate that catalysis originates from transfected β - gal . [SEP]
[CLS] c 12 - fdg is not fluorescent B-property , but upon hydrolysis of the glycosidic bonds between fluorescein and galactose produces a fluorescent B-property signal upon excitation by 488 nm light . [SEP]
[CLS] this assay allows for simultaneous visualization of the cellular uptake of native or prosna β - gal and the reaction product , c 12 - fluorescein . [SEP]
[CLS] cells B-material were transfected with either 1 nm af 4 - or prosna β - gal and then incubated B-technique with c 12 - fdg for 1 h at 37 °c , washed , and examined by confocal fluorescence B-technique microscopy I-technique for intracellular fluorescence B-property ( figures 4b and s9 - 10 ) . [SEP]
[CLS] for the prosna , both the enzyme ( af647 channel ) and c 12 - fdg reaction product ( fitc channel ) were observed within the cells B-material at 1 nm treatments ( figure s11 ) . [SEP]
[CLS] conversely , cells B-material treated with the native enzyme exhibited no signal for either the product or the enzyme . [SEP]
[CLS] in conclusion , we have developed a chemical strategy for transforming cell B-material membraneimpermeable proteins B-material into prosnas that enter cells B-material at low concentrations . [SEP]
[CLS] the results presented here show that these architectures are highly uptaken by cells B-material and function as intracellular enzymes . [SEP]
[CLS] this work is an initial proof - of - concept that lays the foundation for creating a new class of biologically active materials from a nearly limitless library of protein B-material nanoparticles B-nanoparticle that can serve as the prosna core B-material [SEP]
[CLS] future design iterations will allow tuning of both the dna shell B-material , which compared to other polymers B-material is highly monodisperse and sequence specific , as well as the protein B-material core B-material , which presents multiple orthogonal functional groups B-nanoparticle on I-nanoparticle its surface that should allow for the attachment of several distinct functionalities such as imaging agents , targeting moieties , and functional oligonucleotides . [SEP]
[CLS] refer to web version on pubmed central for supplementary material B-material . [SEP]
[CLS] ( a ) cartoon representation of β - gal before ( left ) and after ( right ) functionalization with dna . [SEP]
[CLS] the representation was adapted from pdb id 1bgl . [SEP]
[CLS] 40 surface lysines B-material and cysteines B-material are represented as blue and yellow sticks , respectively . [SEP]
[CLS] af fluorophores covalently attached to the surface of the protein B-material are shown as magenta sticks . [SEP]
[CLS] ( b ) molecules used for modification of β - gal with fluorophores ( i ) , azides ( ii ) , and dna ( iii ) . [SEP]
[CLS] characterization of the structure and catalytic functionality of native ( solid black traces ) and prosna ( dashed red traces ) β - gal . [SEP]
[CLS] ( a ) uv - vis absorbance spectra used to quantitate the functionalization of β - gal with alexafluor 647 and dna . [SEP]
[CLS] ( b ) cd spectra demonstrating the retention of the secondary structure of β - gal after functionalization with dna . [SEP]
[CLS] ( c ) fluorescence B-property assay for determining the catalytic activity of β - gal variants based on the reaction depicted in ( d ) . [SEP]
[CLS] 3 . cellular uptake of native ( blue ) and prosna ( green ) β - gal , as determined by flow B-technique cytometry I-technique . [SEP]
[CLS] fluorescence B-property was measured in hacat , skov3 , and c166 cells B-material 12 h after treatment with either 0 . 1 nm ( top ) or 1 nm ( bottom ) enzyme . [SEP]
[CLS] the fold increase over untreated cells B-material ( red ) for each sample is listed below the graph with the standard deviation for n = 3 trials in parentheses . [SEP]
[CLS] the y - axis is cell B-material count for a single trial of > 5000 cell B-material events . [SEP]
[CLS] 4 . intracellular catalytic activity of native and prosna β - gal . [SEP]
[CLS] ( a ) light micrographs of hacat ( left ) , skov3 ( middle ) , and c166 ( right ) cells B-material after incubation B-technique with the β - gal substrate , xgal . [SEP]
[CLS] the blue color apparent in cells B-material pretreated with prosna β - gal results from the hydrolysis of xgal and formation of an insoluble reaction product . [SEP]
[CLS] scale bar = 100 μm . [SEP]
[CLS] ( b ) confocal fluorescence B-property micrographs of c166 cells B-material to simultaneously monitor the intracellular location of prosna β - gal ( af647 channel ) and the presence of fluorescein ( fitc channel ) , which is the product of the intracellular reaction between c 12 - fdg and prosna β - gal . [SEP]
[CLS] nuclei were stained with hoechst stain to approximate the location within the cell B-material . [SEP]
[CLS] scale bar = 20 μm . [SEP]
[CLS] dna nanostructures assembled on living cell B-material membranes have become powerful research tools . [SEP]
[CLS] synthetic lipid B-material membranes I-material have been used as a membrane model to study the dynamic behavior of dna nanostructures on fluid soft lipid B-material bilayers I-material , but without the inherent complexity of natural membranes . [SEP]
[CLS] herein , we report the assembly and disassembly of dna nanoprisms on cellmimicking micrometer - scale giant membrane vesicles derived from living mammalian cells B-material . [SEP]
[CLS] three - dimensional dna nanoprisms with a dna arm and a cholesterol anchor were efficiently localized on the membrane surface . [SEP]
[CLS] the assembly and disassembly of dna nanoprisms were dynamically manipulated by dna strand hybridization and toehold - mediated strand displacement . [SEP]
[CLS] furthermore , the heterogeneity of reversible assembly / disassembly of dna nanoprisms was monitored by forster resonance energy transfer . [SEP]
[CLS] this study suggests the feasibility of dnamediated functional biomolecular assembly on cell B-material membranes for biomimetics studies and delivery systems . [SEP]
[CLS] oligonucleotides have emerged as programmable building blocks to create dynamic molecular devices 1 from built - in dna nanostructures . [SEP]
[CLS] these dynamic dna functional units exhibit good performance and have been used in the construction of dna walkers , tweezers , logic circuits , gene regulators and smart therapeutics [SEP]
[CLS] most recently , bioengineering has focused on dna nanostructures on the cell B-material surface , resulting in the * corresponding authors : tan @ chem . ufl . edu , qlliu @ iccas . ac . cn . [SEP]
[CLS] orcid qiaoling liu : 0000 - 0001 - 9487 - 0944 weihong tan : 0000 - 0002 - 8066 - 1524 notes design of many nanostructures and devices interacting with the cell B-material membrane . [SEP]
[CLS] hence , most such dna nanostructures built on synthetic lipid B-material membranes I-material have served as biomimetic membrane proteins B-material , such as ion B-material channels , membrane - sculpting protein B-material and " snap ( soluble B-property nsf attachment protein B-material ) receptor B-material " ( snare ) protein . [SEP]
[CLS] to further manipulate dna nanostructures at the mesoscale , strategies combining dynamic dna nanotechnologies have been developed to study the complexities of cell B-material membranes , such as cell - surface recognition and membrane receptor B-material studies . [SEP]
[CLS] however , such studies with dna nano - structures are often confounded by dynamic cell B-material behavior , observed , for example , in internalization and " flip - flop " leaflet movements . [SEP]
[CLS] to solve this problem , researchers have turned to fluid soft lipid B-material bilayers I-material as materials to build an ideal model for cell - surface studies of dna nanostructures . [SEP]
[CLS] to date , different studies have reported the dynamic behavior of tethered dna nanostructures on various lipid B-material membranes I-material . [SEP]
[CLS] however , all of these strategies have overlooked an obvious , but powerful , obstacle to the cell - mimicking membrane environment . [SEP]
[CLS] as we know , membrane physico - chemical properties have a significant impact in dynamic behavior of dna nanostructures and their assemblies . [SEP]
[CLS] this means that dna nanostructures on these synthetic model membranes would be remarkably different from their dynamic behavior on the natural membrane . [SEP]
[CLS] thus , it is necessary to study the dynamic behavior of dna nanostructures on cell - mimicking giant vesicles model which would further advance our understanding on behavior of dna nanostructures on biological interfaces . [SEP]
[CLS] recently , giant membrane vesciles derived from live cells B-material was proved to be an idea cell B-material / membrane model . [SEP]
[CLS] because of their highly similarity to cell B-material membrane structure , these membrane vesicles possess the unique superiority on a broad spectrum of studies such as biomembrane physics and biomimetic chemistry , such as phase separation and functional lipid B-material raft domains , as well as membrane protein interactions . [SEP]
[CLS] however , the assembly of dna nanostructures in such cell - mimicking giant membrane vesicles has not been carried out , even though they have been proved to be an idea platform as cell B-material membrane model . [SEP]
[CLS] herein , as a proof - of - concept , we report manipulating the assembly / disassembly of 3d dna nanostructures on the cell - mimicking surfaces of micrometer - scale giant membrane vesicles ( mvs ) . [SEP]
[CLS] dna triangular prisms ( tps ) were selected as a feasible and efficient 3d dna nanostructural model to study their dynamic behavior on lipid B-material membrane I-material . [SEP]
[CLS] utilizing dna hybridization and dna strand displacement reaction , the dynamic assembly / disassembly processes of tps on the mvs surface were studied , followed the monitoring by forster resonance energy transfer ( fret ) between the fluorophore pairs , and results also indicated the heterogeneity of dna assembly on mvs . [SEP]
[CLS] as a starting point , a box - like nanoprism scaffold was initially fabricated by three long single - stranded dnas ( ssdna ) after annealing . [SEP]
[CLS] because of 3d dna nanostructures consists of at least three dna strands , dna tps scaffolds can be created with the minimum number of dna strands to meet the requirement of economy in the use of dna materials . [SEP]
[CLS] one ssdna segment elongates from its top face , farthest from the vesicle surface , and serves as an arm ( arm strand ) , whereas a cholesterol - labeled ssdna segment elongates from its bottom face , as an anchor ( anchor strand ) ( figure 1b ) in order to immoblize the dna nanoprism onto the surface of mvs . [SEP]
[CLS] this nanoprism binds to the surface via hydrophobic B-property cholesterol anchorage to the lipid B-material bilayer I-material of mvs , thus facilitating dna - mediated dynamic regulation of the functional nanoprisms on the cell - mimicking membrane . [SEP]
[CLS] initially , we employed the native polyacrylamide B-technique gel I-technique electrophoresis I-technique ( n - page ) to confirm the formation of the nanoprism scaffold in buffer solution ( see supporting information ( si ) , figure s1 ) . [SEP]
[CLS] then the 3d dna scaffold was transformed into a functional unit by loading an arm and an anchor strand ( figure 1b and si , figure s2 ) . [SEP]
[CLS] after successfully demonstrating the feasibility of constructing these 3d dna nanostructures , we investigated their plasticity in terms of ready assembly and disassembly . [SEP]
[CLS] to accomplish this , the assembly / disassembly process of dna nanoprisms was studied in buffer solution to verify their capacity for dynamic reversible manipulation ( figure 1a ) . [SEP]
[CLS] two different dna nanostructures , tp - a and tp - b , were first constructed by loading alternative arm a and arm b strands on the top face , respectively . [SEP]
[CLS] assembly of tp - a and tp - b was achieved by hybridizing with a dna linker . [SEP]
[CLS] the optimized experiment also showed the high assembly efficiency of dna nanoprisms ( si , figure s3a , c ) . [SEP]
[CLS] then , to disassemble the dimeric dna nanoprism , a toehold - mediated dna strand displacement reaction was induced by a displacement strand , the disassembly efficiency was also optimozed ( si , figure s3b , d ) . [SEP]
[CLS] next , n - page results verified the dynamic assembly / disassembly process of tp - a and tp - b achieved by dna hybridization and dna strand displacement reaction ( figure 1c ) . [SEP]
[CLS] fluorescence B-property spectral and kinetics results also demonstrated this efficient reversible manipulation , which in agreement with the gel B-technique electrophoresis I-technique results ( si , figure s4 ) . [SEP]
[CLS] different from dna nanoprisms assembled in buffer solution , where dna strands are able to diffuse freely , the kinetics of dna nanostructures on the surface of cell - mimicking giant vesicles was expected to be much more complex because they were derived from living cells B-material and contain cell membrane constituents , such as proteins B-material and glycocalyx , likely impacting the kinetics of assembly and disassembly . [SEP]
[CLS] previously , we developed a facile strategy to prepare micrometer - scale giant membrane vesicles . [SEP]
[CLS] they showed excellent cell - mimicking properties , thus providing an ideal dynamic soft platform at the mesoscale to study the behavior and kinetics of dna nanostructures . [SEP]
[CLS] herein , cell - mimicking mvs detached from hela cells B-material were initially obtained with good quality and high yields . [SEP]
[CLS] to confirm the successful insertion of dna nanostructures on the biological surface , multicolor fluorescence B-property colocalization of nanoprisms was observed . [SEP]
[CLS] first , three different dna - fluorophore sequences were assembled with two nanoprism scaffolds in the test tube , in which a cholesterol - dna - alexa fluor 488 sequence ( af 488 - labeled anchor strand ) was loaded on the bottom face of all nanoprisms . [SEP]
[CLS] afterward , fluorophore - modified nanoprisms tp - cy3 and tp - cy5 were created , in which cy3 - and cy5 - labeled arm strands were loaded on each respective top face . [SEP]
[CLS] to anchor nanoprisms on mvs , 22 . 2 μl mixtures of 9 μm cholesterol - labeled tp - cy3 and tp - cy5 were added to 400 μl mvs with 1640 medium containing 5 mm mg 2 + . [SEP]
[CLS] the divalent cation B-material contributes to the binding B-event of I-event dna I-event nanostructures to the negatively charged lipid B-material bilayers I-material , and it also maintains the stability and integrity of dna nanostructures . [SEP]
[CLS] after incubation B-technique with mvs at 37 °c for 30 min , laser confocal B-technique scanning I-technique microscopy B-technique ( lcsm ) was employed to verify that the modified nanoprisms were readily localized on the cell - mimicking membrane of mvs in group 1 ( figure 2a , the first panel ) , followed by quantification of fluorescence B-property colocalization ( si , figure s5 ) . [SEP]
[CLS] in the absence of dna scaffolds , the anchor strand could immobilize onto the membrane via cholesterol insertion ( group 2 ) , while the arm strands without cholesterol mainly distributed outside of the giant vesicles ( group 3 ) . [SEP]
[CLS] this result demonstrated that dna tps could bind to the lipid B-material bilayer I-material though cholesterol insertion , thus providing a facile strategy for anchoring dna nanostructures to our cell - mimicking giant vesicles ( si , figure s6 ) . [SEP]
[CLS] moreover , the fluorescence B-property intensity of the overlay images ( 488 + cy3 + cy5 , figure 2a , last line ) was measured ( figure 2b ) and also demonstrated that these dna nanostructures could successfully anchor onto the membrane of mvs . [SEP]
[CLS] although the preference of dna nanoprisms localized on the surface of mvs promised their reversible assembly and disassembly , synthetic giant vesicles made from phospholipid was not an idea platform for the surface manipulation , since nanoprisms are easily enriched in the chamber of mvs ( si , figure s7 ) . [SEP]
[CLS] it indicate the behavior of dna nanostructures on synthetic model membranes would be remarkably different from theirs on cell - mimicking surface . [SEP]
[CLS] to characterize the assembly / disassembly of cholesterol - labeled nanoprisms on the cellmimicking membrane of mvs , we used a fret configuration composed of tp - cy3 and tp - cy5 . [SEP]
[CLS] thus , the dynamic assembly / disassembly of nanoprisms on the mvs could be visualized via the changes of fret efficiency between cy3 and cy5 fluorescent B-property dyes ( figure 3a ) . [SEP]
[CLS] initially , these nanoprisms ( tp - cy3 : tp - cy5 = 1 : 1 . 2 ) were mixed with mvs in solution and incubated B-technique at 37 °c for 10 min ( panel 1 ) . [SEP]
[CLS] the assembly of tp - cy3 and tp - cy5 was performed by adding 1 . 5 - fold excess dna linker and incubating B-technique for 50 min at 4 °c ( panel 2 ) . [SEP]
[CLS] interestingly , we found that low temperature favored the assembly of tp - cy3 and tp - cy5 compared to 37 °c ( si , figure s8 ) , possibly due to the formation of a liquid - order phase in the lipid B-material membrane I-material . [SEP]
[CLS] we speculated that the cholesterol component of anchor strands prefers to exist in this phase , which may increase the opportunity for molecular collision and thus facilitate the assembly of nanoprisms . [SEP]
[CLS] to monitor the disassembly , 2 - fold excess displacement strand was added into solution of mvs and the mixture was incubated B-technique for another 1 h . [SEP]
[CLS] as shown in panel 3 , decreased fret efficiency can be observed by separation of the cy3 - and cy5 - labeled nanoprisms on the cell - mimicking membrane driven by toehold - mediated dna strand displacement . [SEP]
[CLS] when a random strand was added to assemble tp - cy3 with tp - cy5 on the mvs surface , little effect could be detected , demonstrating that the assembly process was not driven by temperature , but rather by strand hybridization based on specific watson - crick dna base pairing ( figure 3a , panel 4 ) . [SEP]
[CLS] furthermore , quantitative assay of fret efficiency between heteronanoprisms demonstrated the feasibility of nanoprism assembly / disassembly on the mvs surface ( figure 3b ) . [SEP]
[CLS] more interestingly , dna assembly on the mvs surface revealed heterogeneous fret efficacy . [SEP]
[CLS] the calculated ratiometric fluorescence B-property from a randomly selected giant vesicle via acceptor photobleaching showed the heterogeneous fret efficiency of dimeric nanoprism assembly on the surface of our giant vesicles ( figure 3c ) . [SEP]
[CLS] normally , lipid B-material structure in a biomembrane ( e . g . , plasma membrane and organelle membrane ) exists in the form of separated microdomains , indicating the heterogeneity of the biomembrane . [SEP]
[CLS] the membrane of mvs derived from mammalian cells B-material represents one such biomembrane . [SEP]
[CLS] thus , we speculated that our giant vesicles would also exhibit membrane heterogeneity and that the observed heterogeneous fret efficiency was likely caused by localization of cholesterol - labeled nanoprisms in microdomains . [SEP]
[CLS] along with developing nanotechnology , oligonucleotides not only play an important biological role in living systems but also have emerged as building blocks for self - assembly nano - fabrication . [SEP]
[CLS] here , the dna nanoprism served as the simplest 3d dna nanostructure model and was thought to find a wide applications in logic computation , bioanalysis and biomimetics . [SEP]
[CLS] besides this nanostructural aspect , the method is also generally applicable to other dna nanostructures based on watson - crick base pairing and the anchorage of hydrophobic B-property components to membrane . [SEP]
[CLS] combining with cell - mimicking surface , manipution of these dna nanofabrications provide a method to functionalize cell B-material surface , which have to overcome dynamic cell B-material surface movements , and suggest the long - term feasiblitity to regulate the membrane structures . [SEP]
[CLS] in summary , using dna strand hybridization and toehold - mediated strand displacement , we have engineered the respective assembly and disassembly of dna nanostructures on cellmimicking membranes of mvs . [SEP]
[CLS] the status of a single nanoprism and its dimeric assembly were monitored by fret , and heterogeneous dna assembly on mvs was observed . [SEP]
[CLS] on the basis of the reported dynamic programmability , predictability and addressability of dna nanostructures , the manipulation of membrane - anchored dna nanostructures can serve as a new strategy for engineering artificial cells B-material and combining dna nanotechnology with biomimetics . [SEP]
[CLS] 1 . dynamic assembly / disassembly of dna triangular nano - prism in reaction buffer . [SEP]
[CLS] ( a ) schematic representation of dna - mediated assembly / disassembly of tp - a and tp - b by dna hybridization and dna strand displacement reaction . [SEP]
[CLS] ( b ) schematic representation of cholesterol - labeled dna triangular nanoprism anchored on the bilayer of mvs . [SEP]
[CLS] ( c ) native polyacrylamide B-technique gel I-technique electrophoresis I-technique ( 5 % ) analysis of dynamic assembly / disassembly of dna nanoprisms in reaction buffer . [SEP]
[CLS] lane 1 : tp scaffold . [SEP]
[CLS] lane 2 : tp - a . [SEP]
[CLS] lane 3 : tp - b . [SEP]
[CLS] lane 4 : hybridizing tp - a and tp - b by linker strand to assemble nanoprisms . [SEP]
[CLS] lane 5 : 1 . 2 - fold excess displacement strand to disassemble the dimeric nanoprism . [SEP]
[CLS] l : 20 - bp ladder consisting of double strands of dna with length increasing in 20 - bp steps . [SEP]
[CLS] all dna bands were stained by stains - all and then imaged using the bio - rad chemidoc xrs system . [SEP]
[CLS] anchoring dna nanoprisms on cell - mimicking giant vesicles . [SEP]
[CLS] ( a ) confocal B-technique laser I-technique scanning I-technique microscopy I-technique imaging of colocalization of 500 nm nanoprisms on mvs . [SEP]
[CLS] panel 1 : tp - cy3 and tp - cy5 anchored on lipid B-material bilayer I-material through cholesterol - insertion . [SEP]
[CLS] panel 2 : single strand of chol - dna - alexa fluor 488 . [SEP]
[CLS] panel 3 : single strand of chol - dna - alexa fluor 488 and free arm strands of cy3 - dna and cy5 - dna . [SEP]
[CLS] all samples were incubated B-technique with 400 μl solution of mvs at 37 °c for 30 min . [SEP]
[CLS] scale bar : 5 μm . [SEP]
[CLS] ( b ) cross section of fluorescence B-property intensities ( white solid line ) in groups 1 , 2 and 3 , respectively . [SEP]
[CLS] 3 . assembly / disassembly of 3d dna nanostructures on cell - mimicking membrane of mvs . [SEP]
[CLS] ( a ) confocal B-technique laser I-technique scanning I-technique microscopy I-technique imaging of manipulation of dna nanoprisms on mvs in 1640 medium containing 5 mm mg 2 + at 4 °c . [SEP]
[CLS] ( 1 ) 500 nm tp - cy3 and tp - cy5 . [SEP]
[CLS] ( 2 ) linker strand added to form dimeric nanoprism assembly of two separate tps . [SEP]
[CLS] ( 3 ) displacement strand added to disassemble the dimeric nanoprism . [SEP]
[CLS] ( 4 ) random linker strand added to group ( 1 ) showed little effect on assembly . [SEP]
[CLS] ( b ) normalized fluorescence B-property intensity measurements for panels 1 to 3 from panel a . [SEP]
[CLS] each column represents the statistical sample population of 15 mvs . [SEP]
[CLS] p values were calculated by newman - keuls multiple comparison test , * p < 0 . 05 . [SEP]
[CLS] ( c ) acceptor bleaching experiment to study fret efficiency on an individual mv . [SEP]
[CLS] scale bar : 5 μm . [SEP]
[CLS] dna hybridization onto dna - functionalized nanoparticle B-nanoparticle surfaces ( e . g . , in the form of a spherical nucleic B-material acid I-material ( sna ) ) is known to be enhanced relative to hybridization free in solution . [SEP]
[CLS] surprisingly , via isothermal titration calorimetry , we reveal that this enhancement is enthalpically , as opposed to entropically , dominated by ~ 20 kcal / mol . [SEP]
[CLS] coarse - grained molecular dynamics simulations suggest that the observed enthalpic enhancement results from structurally confining the dna on the nanoparticle B-nanoparticle surface and preventing it from adopting enthalpically unfavorable conformations like those observed in the solution case . [SEP]
[CLS] the idea that structural confinement leads to the formation of energetically more stable duplexes is evaluated by decreasing the degree of confinement a duplex experiences on the nanoparticle B-nanoparticle surface . [SEP]
[CLS] both experiment and simulation confirm that when the surface - bound duplex is less confined , i . e . , at lower dna surface density or at greater distance from the nanoparticle B-nanoparticle surface , its enthalpy of formation approaches the less * [SEP]
[CLS] favorable enthalpy of duplex formation for the linear strand in solution . [SEP]
[CLS] this work provides insight into one of the most important and enabling properties of snas and will inform the design of materials that rely on the thermodynamics of hybridization onto dna - functionalized surfaces , including diagnostic probes and therapeutic agents . [SEP]
[CLS] spherical nucleic B-material acids I-material ( snas ) are a class of structures typically made by arranging linear nucleic B-material acids I-material at high density around a nanoparticle B-nanoparticle core B-material . [SEP]
[CLS] snas have become important entities in the development of medical diagnostic probes , intracellular small - molecule detection agents , rna tracking agents , and building blocks for colloidal crystal engineering [SEP]
[CLS] their unique properties , which are highly differentiated from linear structures , make them very attractive for such uses . [SEP]
[CLS] one of these properties is a higher affinity constant for complementary nucleic B-material acids I-material . [SEP]
[CLS] depending on the sequence , snas can bind complements orders of magnitude more tightly than linear forms of the same sequence . [SEP]
[CLS] despite the importance of this enhanced binding for many of the sna applications , its origin remains unknown . [SEP]
[CLS] given the restricted nature and preorientation of the short strands that define snas , enhancement of hybridization could be attributed to entropic contributions . [SEP]
[CLS] however , here we demonstrate that complement binding on snas carries a higher entropic penalty than binding in the linear form . [SEP]
[CLS] we show that the binding enhancement is instead enthalpically driven ( scheme 1 ) and explain its thermodynamic origin . [SEP]
[CLS] we use temperature - dependent fluorescence B-property melting studies , isothermal titration calorimetry ( itc ) , and coarse - grained molecular dynamics ( md ) simulations to determine the entropy and enthalpy of hybridization for linear dna B-event binding I-event as well as binding of a complement to an sna . [SEP]
[CLS] via a combination of experiment and simulation , we explain that structural confinement on a nanoparticle B-nanoparticle surface prevents dna from adopting unfavorable binding conformations that ultimately account for the observed enthalpically dominated binding enhancement on snas . [SEP]
[CLS] to determine the entropies and enthalpies of binding , we performed concentrationdependent fluorescence B-property hybridization experiments ( figure 1a , b ) . [SEP]
[CLS] we studied , under identical conditions , a linear 12 - mer dna system and 5 . 9 nm gold B-nanoparticle nanoparticle I-nanoparticle snas functionalized with ~ 46 dna strands of the same sequence . [SEP]
[CLS] both systems were prepared in a 1 : 1 stoichiometry of either sna or linear dna to a complementary strand , and a dna helix - coil transition temperature was measured over a range of concentrations ( figures 1a , s1 , and s2 ) . [SEP]
[CLS] the concentration dependence of the helix - coil transition temperature reflects the thermodynamics of hybridization through the van ' t hoff relationship , 20 [SEP]
[CLS] here t m is the transition temperature , c t is the combined concentration of sna ( or linear dna ) and complement , r is the gas constant , and δh° and δs° are the enthalpy and entropy of hybridization , respectively . [SEP]
[CLS] importantly , this analysis treats the sna as a single molecular entity and concentrations are adjusted to ensure 1 : 1 binding of complementary strand to sna ( see si for details ) . [SEP]
[CLS] this is the case for many of the sna ' s uses as probes for high - sensitivity detection or as antisense gene - regulation agents , where target concentration is relatively low with respect to probe . [SEP]
[CLS] the less steep slope for the sna system reveals that the binding enthalpy on snas , δh° = −91 . 3 ± 5 . 5 kcal / mol , is far more favorable than for linear dna , δh° = −40 . 6 ± 2 . 4 kcal / mol ( figure 1a , b and table s1 ) . [SEP]
[CLS] remarkably , the sna system also exhibits a higher entropic loss upon hybridization , with tδs° = −75 . 8 ± 5 . 3 kcal / mol at 298 k vs tδs° = −27 . 2 ± 2 . 3 kcal / mol for linear dna . [SEP]
[CLS] since the increased enthalpic gain in the sna system more than compensates for the larger entropic cost , the free energy of hybridization is lower for snas than for linear dna ( δg°s na = −15 . 5 ± 0 . 2 kcal / mol vs δg°l inear = −13 . 4 ± 0 . 1 kcal / mol ) , and the association constant is correspondingly higher , k eq sna = ( 2 . 3 ± 0 . 8 ) × 10 11 m −1 vs k eq linear = ( 6 . 8 ± 1 . 1 ) × 10 s m −1 . [SEP]
[CLS] whereas the enhanced binding confirms prior observations , the larger entropic penalty for sna binding and the increased enthalpic gain are puzzling given the conformational constraints of the nanoparticle - bound dna , and the view that the dense packing of dna on the sna is likely to result in destabilizing steric and electrostatic interactions . [SEP]
[CLS] indeed , we have observed such thermodynamic trends before , but refrained from commenting on their origin because of the lack of a suitable explanation . [SEP]
[CLS] the van ' t hoff analysis assumes that dna hybridization proceeds in the dilute limit in a two - state manner and that the enthalpy of this process is independent of temperature . [SEP]
[CLS] since these assumptions have been shown to significantly affect van ' t hoff - derived enthalpies of linear dna hybridization , we sought to corroborate our findings with a model - independent technique . [SEP]
[CLS] specifically , to confirm the larger enthalpy of hybridization for snas , we performed itc experiments on the same systems . [SEP]
[CLS] the itc curve shapes ( figures 1c , d and s3 - s5 ) indicate that dna hybridization on the sna differs significantly from hybridization free in solution . [SEP]
[CLS] the linear - dna system shows a sigmoidal binding isotherm with an inflection point at a molar ratio of 1 , reflecting 1 : 1 binding stoichiometry ( figure 1c ) . [SEP]
[CLS] in contrast , the sna system exhibits double - sigmoidal behavior with inflection points at molar ratios of 4 and 15 strands per particle ( figure 1d ) . [SEP]
[CLS] this shape implies that snas exhibit a type of negative cooperativity , where binding of the first four strands is enthalpically more favorable than subsequent hybridization events . [SEP]
[CLS] such negative cooperativity is consistent with prior observations . [SEP]
[CLS] the itc curves also directly yield the hybridization enthalpies from the released heat q , showing an enthalpy gain that is 20 . 7 ± 2 . 2 kcal / mol higher for binding on snas ( figure 1c , d and table s2 ) . [SEP]
[CLS] the qualitative agreement between these data and the fluorescence B-property data suggests that the relative entropic and enthalpic contributions determined from the van ' t hoff analysis are qualitatively reliable , despite the assumptions of the model . [SEP]
[CLS] discrepancies in the absolute values of hybridization enthalpies derived from calorimetry and the van ' t hoff analysis have been previously observed in linear dna systems . [SEP]
[CLS] differences can be explained by deviations from two - state behavior and changes in heat capacity associated with dna melting . [SEP]
[CLS] additionally , itc experiments are conducted at much higher concentrations than van ' t hoff experiments and are therefore more susceptible to excludedvolume effects . [SEP]
[CLS] we suspect that all of these factors play a role in the systems under study and , if considered in the van ' t hoff analysis , may lead to better agreement with calorimetric values . [SEP]
[CLS] with strong experimental evidence to support that binding to the sna is enthalpically more favorable than binding to linear dna , we turned to coarse - grained md simulations to understand the origin of this enhancement . [SEP]
[CLS] simulations of the hybridized and the unhybridized state were performed for both the linear and the sna systems using the 3spn . 2 model ( see si for computational details ) . [SEP]
[CLS] this model separates the dna into three sites per nucleotide , one each for the phosphate , sugar , and base , and has been parametrized to reproduce correct structural , thermodynamic , mechanical , and kinetic properties of dna . [SEP]
[CLS] assuming incompressibility , we computed the enthalpy of duplex formation as [SEP]
[CLS] where e h is the internal energy of the system with a hybridized duplex , e u is the internal energy of the unhybridized strands , and e s is the energy of a single complementary dna strand in solution ( figure 2a ) . [SEP]
[CLS] the simulations confirmed the experimentally observed trend for the enthalpy of hybridization , with an enhancement of ~ 5 . 3 kcal / mol associated with hybridization on the sna relative to free in solution ( figure 2b ) . [SEP]
[CLS] to achieve high statistical accuracy , the simulations were performed with implicit ions B-material , using the debye - husckel approximation , but we confirmed that the same trends are obtained when using explicit salt B-material and counterions ( details in si ) . [SEP]
[CLS] owing to the lack of explicit solvent , δh° differs quantitatively from the experimental values . [SEP]
[CLS] however , the simulations make it possible to separate the inter - and intramolecular contributions . [SEP]
[CLS] to identify the primary origin of the observed enthalpic enhancement , we broke down the hybridization enthalpies of the linear duplex and the sna duplex into ( i ) interstrand base - pairing , ( ii ) cross - stacking , ( iii ) electrostatic , and ( iv ) intrastrand structural energies ( figure 2c and table s3 ) . [SEP]
[CLS] for each of these contributions , we defined δδh as the difference between the enthalpy of hybridization on the sna ( 3h sna ) and the enthalpy of hybridization for linear dna ( δh linear ) . [SEP]
[CLS] we found that the main contribution to the enhancement of δh° on the sna was the change in structural energy , which comprises covalent - bond , angle , dihedral , and base - stacking energies , and was most pronounced for the t 10 - linker region of 10 thymine bases that connects the duplex to the nanoparticle B-nanoparticle surface ( figure s7 ) . [SEP]
[CLS] since experimentally the presence of the t 10 linker did not affect the enthalpy of hybridization for the linear dna ( figure s4 ) , we conclude that the sna architecture must give rise to the change δδh in the structural hybridization energy . [SEP]
[CLS] confinement due to surface attachment and molecular crowding prevents hybridized dna on the nanoparticle B-nanoparticle from adopting energetically unfavorable conformations that cause distortions in the bond angles , dihedrals , and intrastrand base stacking away from the minimum - energy conformation , as would occur in the unconfined linear dna case . [SEP]
[CLS] it has been observed that molecular crowding or excluded - volume effects increase local dna concentration and as a result stabilize duplex formation . [SEP]
[CLS] yet , excluded volume also restricts the degrees of freedom of hybridizing molecules and biases the dna toward more enthalpically stable conformations . [SEP]
[CLS] this effect is reminiscent of the stability observed for locked nucleic B-material acid I-material ( lna ) hybridization . [SEP]
[CLS] lna is a synthetic rna analog for which the ribose moiety is structurally constrained by a 2 ' oxygen to 4 ' carbon methylene bridge . [SEP]
[CLS] incorporation of lna bases into dna oligomers has led to a demonstrated enhancement of the thermodynamic stability of duplexes . [SEP]
[CLS] this effect is enthalpically dominated , as shown by calorimetry and is thought to result from the conformational restriction of base - stacking and hydrogen B-material bonding interactions . [SEP]
[CLS] to test if dna confinement on the surface of the nanoparticles B-nanoparticle indeed resulted in an enhanced enthalpy of hybridization , as suggested by the md simulations , we performed itc on two sna systems with less confined dna . [SEP]
[CLS] we hypothesized that if conformational restriction of duplexes on the sna resulted in an enhanced enthalpy of hybridization , then less confined sna duplexes should have a less favorable enthalpy of hybridization . [SEP]
[CLS] first , we decreased the dna surface density by functionalizing nanoparticles B-nanoparticle with only 30 dna strands , to obtain a density 33 % lower than that of the original snas . [SEP]
[CLS] in support of our hypothesis , the hybridization enthalpy of the first four dna strands on these low - density snas was 6 . 4 ± 2 . 7 kcal / mol less favorable ( figures 3a and s6a ) . [SEP]
[CLS] to further explore the degree to which confinement on snas could be tuned , we tested an sna architecture with an even lower degree of confinement . [SEP]
[CLS] we moved the duplex - forming region of the dna further away from the nanoparticle B-nanoparticle surface by replacing the t 10 - linker region with a linker region composed of 30 thymine bases ( t 30 ) . [SEP]
[CLS] for this design , we maintained the high dna density of ~ 46 strands per particle . [SEP]
[CLS] the increased distance from the nanoparticle B-nanoparticle surface caused a striking decrease in the enthalpy of duplex formation of 16 . 0 ± 2 . 8 kcal / mol ( figures 3b and s6b ) . [SEP]
[CLS] the effect was so strong that nearly all enhancement of the enthalpy disappeared , with the enthalpy of hybridization on the t 30 - sna nearly identical to the enthalpy of hybridization of linear dna . [SEP]
[CLS] the combined density and linker data were corroborated by simulation ( figure s8 ) and demonstrated that the enthalpy of complementary dna hybridization onto snas can be tuned by as much as 20 kcal / mol simply by varying the degree of confinement of a surface - bound strand . [SEP]
[CLS] structural confinement also helps explain the considerable entropic cost of complement hybridization on an sna . [SEP]
[CLS] in simulations , we found that upon hybridization single - stranded dna surrounding the duplex on the surface became structurally more ordered ( figure s9 ) . [SEP]
[CLS] while this effect was minor on a per - strand basis , collectively these contributions significantly reduced the ensemble degrees of freedom . [SEP]
[CLS] this entropic cost counteracts hybridization of the first dna strand on an sna but has been shown to reduce the entropic cost for subsequent hybridization events . [SEP]
[CLS] in conclusion , we have demonstrated that , relative to linear dna , the enthalpy of complement hybridization is more favorable on spherical nucleic B-material acids I-material and results in an enhanced free energy of binding . [SEP]
[CLS] while one could make intuitive arguments that the observed binding enhancement on snas is entropically driven , experimental and computational data show that it is an enthalpically driven process . [SEP]
[CLS] this new insight can inform future engineering of dna - functionalized surfaces . [SEP]
[CLS] the surface architecture of snas can be modified to increase or decrease the enthalpic contributions to hybridization and consequently influence therapeutically and diagnostically relevant association constants . [SEP]
[CLS] a ) van ' t hoff plots from which thermodynamic constants are extracted for binding between complementary linear strands and either linear 12 - mer dna ( blue ) or snas ( red ) . [SEP]
[CLS] ( b ) comparison of enthalpic gain and entropic cost derived from the van ' t hoff plots . [SEP]
[CLS] ( c ) isothermal titration calorimetry of 12 - mer dna duplex hybridization free in solution and ( d ) 12 - mer dna duplex hybridization on snas functionalized with ~ 46 strands per particle . [SEP]
[CLS] upper panel : differential heating power δp vs time . [SEP]
[CLS] lower panel : integrated heats of reaction q vs molar ratio . [SEP]
[CLS] ( a ) coarse - grained md simulations of 12 - mer dna before ( e u ) and after ( e h ) duplex formation free in solution and on an sna functionalized with 46 strands . [SEP]
[CLS] ( b ) comparison of simulation - derived enthalpies of hybridization for a duplex formed free in solution and one formed on an sna . [SEP]
[CLS] ( c ) breakdown of contributions to the enthalpy of hybridization . [SEP]
[CLS] 3 . enthalpy of hybridization onto snas for the first four complementary strands as a function of ( a ) dna surface density and ( b ) linker length . [SEP]
[CLS] linear dna hybridization enthalpy ( blue ) is provided for comparison . [SEP]
[CLS] a new class of polymer B-material spherical nucleic B-material acid I-material ( sna ) conjugates comprised of poly ( lactic - coglycolic acid ) ( plga ) nanoparticle B-nanoparticle ( np B-nanoparticle ) cores B-material is reported . [SEP]
[CLS] the nucleic B-material acid I-material shell B-material that defines the plga - sna exhibits a half - life of more than 2 h in fetal bovine serum . [SEP]
[CLS] importantly , the plga - snas can be utilized to encapsulate a hydrophobic B-property model drug , coumarin 6 , which can then be released in a polymer B-material composition - dependent tunable manner , while the dissociation rate of the nucleic B-material acid I-material shell B-material remains relatively constant , regardless of core B-material composition . [SEP]
[CLS] like prototypical gold B-material np B-nanoparticle conjugate snas , plga - snas freely enter raw - blue cells B-material and can be used to activate toll - like receptor B-material 9 in a sequence - and dose - dependent manner . [SEP]
[CLS] taken together , the data show that this novel nanoconstruct provides a means for controlling the release kinetics of encapsulated cargos in the context of the sna platform , which may be useful for developing combination therapeutics . [SEP]
[CLS] spherical nucleic B-material acids I-material ( snas ) are a class of nanoconjugates that are overcoming challenges that face current nucleic B-material acid I-material therapies . [SEP]
[CLS] they provide privileged access at both the cellular and tissue levels . [SEP]
[CLS] for example , snas are actively transported across cell B-material membranes by engaging class a scavenger receptors while unmodified linear nucleic B-material acids I-material do not enter cells B-material in significant amounts without the use of transfection agents . [SEP]
[CLS] in addition , the polyvalent , densely functionalized nucleic B-material acid I-material shell B-material that defines an sna can act as a high affinity binder B-property for different classes of ligands , including certain receptor B-material proteins B-material and complementary nucleic B-material acid I-material sequences . [SEP]
[CLS] consequently , snas have emerged as a powerful platform for developing molecular diagnostic probes and as lead compounds in gene regulation and immunomodulation B-property therapies [SEP]
[CLS] the 3d architecture of the sna , rather than the chemical composition of the nanoparticle B-nanoparticle ( np B-nanoparticle ) core B-material , is the origin of many of the biochemical properties that make them exceedingly useful in the life sciences and medicine . [SEP]
[CLS] therefore , to realize the full clinical potential of the sna construct , recent efforts have focused on designing novel constructs from biocompatible B-property , organic B-material np B-nanoparticle cores I-material , including liposomes B-nanoparticle , proteins B-material , and polymeric micelles B-material that add additional functionality and lead to therapeutic advantages . [SEP]
[CLS] for many combination therapies , the ideal sna should exhibit the following properties . [SEP]
[CLS] first , it should consist of a biocompatible B-property , biodegradable B-property organic B-material core I-material capable of carrying and temporally releasing drugs in a tunable fashion . [SEP]
[CLS] second , it should contribute to the longterm stability of the nucleic B-material acid I-material shell B-material so that the chemical and biophysical properties of such a construct can be retained under challenging physiological conditions . [SEP]
[CLS] finally , it should be straightforward to synthesize in a scalable manner . [SEP]
[CLS] with these considerations in mind , poly ( lactic - co - glycolic acid ) ( plga ) is an attractive material B-material as the core B-material for snas . [SEP]
[CLS] it is biocompatible B-property and biodegradable B-property , relatively stable , and exhibits composition - dependent and therefore tunable release kinetics for encapsulated cargos . [SEP]
[CLS] herein , we report a facile synthetic strategy for the preparation ( scheme 1 ) of plga - snas from 50 nm diameter plga particle cores B-material terminated with azides and oligonucleotides terminated with the dibenzocyclooctyne ( dbco ) group via cufree click chemistry . [SEP]
[CLS] note that others have made nucleic acid - plga conjugates ( an aptamer - plga nanoconstruct ) for targeting purposes , prior to the understanding of the sna architecture ( not simply nucleic - nanoparticle B-nanoparticle conjugates ) and its important role in facilitating scavenger receptor a mediated cellular uptake . [SEP]
[CLS] in a typical particle preparation procedure , plga - peg - n 3 ( poly ( lactide - co - glycolide ) - b - poly ( ethylene glycol ) - azide ) / plga ( m w = 38 000 - 42 000 da , polylactic acid ( pla ) : polyglycolic acid ( pga ) = 50 : 50 ) ( 20 % w / w ) was dissolved in acetonitrile and injected dropwise into water B-material under rapid mixing , resulting in 45 - 150 nm diameter plga - peg - n 3 nps B-nanoparticle , depending upon the polymer B-material concentration in acetonitrile ( figure s1 , supporting information ) . [SEP]
[CLS] since nanostructures with diameters less than 100 nm are believed to reach tumor B-material sites , penetrate tumor B-material tissues , and enter cancer cells B-material via the enhanced permeability and retention effect , we used synthetic conditions that yielded nps B-nanoparticle with a hydrodynamic diameter of ≈50 nm for all studies described herein . [SEP]
[CLS] next , five mole equivalents of dbco - modified oligonucleotides were added to plga - peg - n 3 nps B-nanoparticle in a buffer solution containing 0 . 5 m nacl with 0 . 3 % ( v / v ) poloxamer 188 in 1x phosphate buffered saline . [SEP]
[CLS] the reaction mixture was then incubated B-technique at 25 °c for 48 h . [SEP]
[CLS] the unreacted nucleic B-material acids I-material were separated from the target plga - snas by passing them through an amicon filter ( size cutoff = 100 k ) . [SEP]
[CLS] the purified sna product was collected from the filter and analyzed by 1 % agarose B-technique gel I-technique electrophoresis I-technique ( figure s2a , supporting information ) . [SEP]
[CLS] the linear nucleic B-material acids I-material and plga - snas exhibit different electrophoretic B-property mobility I-property shifts due to size and charge . [SEP]
[CLS] in addition , consistent with dna functionalization of the plga np B-nanoparticle core B-material , the average sna diameter , determined by dynamic B-technique light I-technique scattering I-technique ( dls ) , is ≈15 nm larger than the unfunctionalized core B-material ( 50 nm for plga - peg - n 3 , polydispersity index ( pdi ) = 0 . 088 ; 65 nm for plga - snas , pdi = 0 . 131 , figure 1b ) . [SEP]
[CLS] the plga - peg - n 3 nps B-nanoparticle and plga - snas were imaged by atomic B-technique force I-technique microscopy I-technique ( afm ) in the liquid phase , allowing us to visualize the solution - phase nps B-nanoparticle and obtain quantitative size distributions ( figure 1a , c ) . [SEP]
[CLS] nps B-nanoparticle deposited on a modified si substrate show that plga - snas retain their spherical shape upon surface functionalization ( figure 1a , c and figure s2b , supporting information ) . [SEP]
[CLS] since the surface density of oligonucleotides on snas affects many of their biochemical properties , the average number of strands per sna conjugate was determined by making plga - snas with cy5 - tagged t 20 oligonucleotides and measuring both particle size and absolute number of dna strands by using fluorescence B-technique spectroscopy I-technique methods . [SEP]
[CLS] after np B-nanoparticle isolation and purification , the nps B-nanoparticle were redispersed in buffer , diluted in water B-material , and the concentration was measured with a nanoparticle B-nanoparticle tracking analysis system ( figure s3a , supporting information ) . [SEP]
[CLS] the oligonucleotide concentration was measured by first dissolving the plga core B-material of the sna , which results in release of the oligonucleotides that define the shell B-material , and quantifying and comparing the cy5 fluorescence B-property against a standard curve ( figure s3b , supporting information ) . [SEP]
[CLS] each ≈65 nm ( 50 nm core B-material ) plga - sna has an average of 199 ± 16 strands or a surface density of 5 . 2 pmole cm −2 . [SEP]
[CLS] we note that this surface density is lower than a typical 13 nm au sna , where the surface density is ≈30 pmol cm −2 , but greater than liposomal B-nanoparticle snas ( ≈3 . 2 pmol cm −2 ) , which also have been taken into the clinical trials . [SEP]
[CLS] the cooperative binding of snas is a direct consequence of their high nucleic B-material acid I-material surface coverage . [SEP]
[CLS] indeed , when plga - snas are incubated B-technique with a batch of 13 nm au snas bearing a complementary sequence , a red precipitate forms , which is due to polymerization via dna hybridization ( figure 1d ) . [SEP]
[CLS] consistent with this observation , this precipitate exhibits a sharp melting transition when heated above the melting temperature of the dna ( full - width at half - maximum ≈4 °c , figure s4 , supporting information ) , characteristic of snas . [SEP]
[CLS] nps B-nanoparticle made of soft materials have been utilized to encapsulate a wide range of drugs , such as chemotherapy agents and nucleic B-material acids I-material . [SEP]
[CLS] furthermore , codelivery of such agents within one nanoscale entity has been shown to enhance therapeutic efficacy in certain cases . [SEP]
[CLS] however , a challenge with such combination therapy strategies pertains to how one can precisely control the temporal release of both drugs independently so that therapeutic effects can be maximized . [SEP]
[CLS] additionally , encapsulating both hydrophobic B-property and hydrophilic B-property drugs in one entity often results in poor drug - loading efficiencies , involves complicated preparation processes , and can lead to a significant increase in np B-nanoparticle size . [SEP]
[CLS] the plga - sna construct can spatially compartmentalize a chemotherapy drug and therapeutic nucleic B-material acids I-material in a single entity , so the loading and release of surface - conjugated nucleic B-material acids I-material and encapsulated drugs can be independently controlled . [SEP]
[CLS] we hypothesize that by varying the chemical composition of the plga polymer B-material , the release kinetics of the encapsulated drugs can be tuned , while the release rates of the nucleic B-material acid I-material shell B-material remain relatively constant . [SEP]
[CLS] to investigate the release profiles of the nucleic B-material acids I-material on the surface of plga - snas , we synthesized three batches of plga - snas with different polymer B-material core B-material compositions based on different molecular weights and molar ratios of pla to pga , namely rg 502 , rg 504 , and rg 756 s ( table 1 ) . [SEP]
[CLS] the sizes of plga - snas prepared from such polymers B-material are relatively close ( ± 10 nm ) , suggesting that the np B-nanoparticle size attained during nanoprecipitation does not depend significantly on the chemical composition of the polymer B-material ( figure s5a , supporting information ) . [SEP]
[CLS] the release profiles of the nucleic B-material acids I-material on the plga - snas were investigated by utilizing a fluorescence B-property turn - on experiment where the plga - sna core B-material and the 15 - mer oligonucleotides were labeled with rhodamine and cy5 ( figure 2a ) , creating a forster resonance energy transfer ( fret ) plga - sna . [SEP]
[CLS] the dissociation of the nucleic B-material acid I-material shell B-material was evaluated in 10 % fetal bovine serum ( fbs ) . [SEP]
[CLS] when the nucleic B-material acids I-material are released from the surface of plga - snas , the rhodamine fluorescence B-property ( λ em = 573 nm ) increases , while the cy5 fluorescence B-property ( λ em = 670 nm ) decreases ( figure 2b ) . [SEP]
[CLS] the release profiles of the nucleic B-material acid I-material shell B-material for all three plga - snas are similar , exhibiting half - lives of more than 2 h with k ob ranging from 7 . 4 × 10 −5 to 8 . 8 × 10 −5 s −1 ( figure 2c , table 1 ) . [SEP]
[CLS] these k obs values suggest that plga - snas are nearly 100 - fold more stable than the most clinically advanced liposomal B-nanoparticle snas and three times more stable than the lipid B-material - tail snas ( k obs = 7 . 9 × 10 −3 s −1 and 2 . 8 × 10 −4 s −1 for liposomal B-nanoparticle sna and lipid - tail lsnas , respectively ) . [SEP]
[CLS] the increased stability of the plga - snas are likely due to the covalent bond utilized to immobilize the nucleic B-material acids I-material on the nps B-nanoparticle and the intrinsically higher stability of polymer B-material nps B-nanoparticle ( as compared with liposomes B-nanoparticle ) . [SEP]
[CLS] additionally , nucleic B-material acids I-material conjugated to the plga np B-nanoparticle also exhibited increased stability against dnase i degradation , as compared to the linear counterparts ( figure s6 , supporting information ) . [SEP]
[CLS] the enhanced stability of these snas will likely make them last longer under physiological conditions , potentially leading to increased therapeutic efficacies in certain settings ( e . g . , systemic use ) . [SEP]
[CLS] next , we investigated the release kinetics of coumarin 6 , a commonly used fluorescent B-property , hydrophobic B-property model drug , encapsulated within the plga - snas . [SEP]
[CLS] to load coumarin 6 into the polymer B-material matrix , it ( 0 . 5 % ( w / w ) ) was codissolved with plga in acetonitrile ( figure s5b , supporting information ) , and the mixture was injected into water B-material to form the particles via the precipitation method . [SEP]
[CLS] these structures were converted into snas via the procedure described above . [SEP]
[CLS] the percent release is determined relative to the initial amount of coumarin 6 loaded into the polymer B-material matrix . [SEP]
[CLS] rg 502 has markedly higher release at each time interval and a faster release rate at early time points ( figure 3 ) . [SEP]
[CLS] this is likely because these particles consist of a polymer B-material with a smaller molecular weight that results in faster release rate . [SEP]
[CLS] mechanistically , drug release from the plga nps B-nanoparticle is a complicated process involving diffusion , hydrolysis , bulk erosion , and surface erosion , giving rise to different degradation and release rates achieved by changing the end group or molecular weight of the plga . [SEP]
[CLS] in contrast , nucleic B-material acids I-material are immobilized on the surface of the snas using the same attachment chemistry for each formulation and the dissociation rate of nucleic B-material acids I-material remains relatively constant for all formulations studied , which suggests that the governing mechanism of nucleic B-material acid I-material shell B-material dissociation is the hydrolysis of the ester backbone defining the plga polymer B-material . [SEP]
[CLS] to investigate the potential of the plga - sna construct as a therapeutic platform , we synthesized plga - snas with cy5 - tagged t 20 oligonucleotides and quantified their cellular uptake in a raw - blue macrophage reporter cell B-material line . [SEP]
[CLS] as expected , the flow B-technique cytometry I-technique results showed that plga - snas readily enter the raw - blue cells B-material ( [ dna ] = 100 × 10 −9 m , 2 h ) without the use of toxic B-property transfection agents ( figure 4a ) . [SEP]
[CLS] the cellular uptake of plga - snas into raw - blue cells B-material is both time and dose dependent ( figure 4b ) , and at shorter times such as 0 . 5 h , the uptake of plga - snas is tenfold greater than their linear counterparts ( figure 4b ) . [SEP]
[CLS] the biodegradable B-property and bio - compatible nature of plga confers no toxicity B-property at concentrations ranging from 10 × 10 −9 m to 2 × 10 −6 m ( figure 4c ) . [SEP]
[CLS] last , to evaluate the ability of the plga - snas to act as a potential immunotherapy platform , we synthesized plga - snas bearing cpg motifs , a known agonist capable of engaging and activating tlr9 . [SEP]
[CLS] significantly , plga - snas activate tlr9 in raw - blue cells B-material in a dose - dependent manner , outperforming their linear counterparts throughout the concentration range studied ( figure 4d ) . [SEP]
[CLS] taken together , we have demonstrated an extremely facile strategy for preparing a new class of snas under mild conditions that add different properties and functionalities to the existing sna constructs utilizing click chemistry for surface functionalization ( see table s2 in the supporting information ) . [SEP]
[CLS] this two - step method yields highly monodisperse plga - snas without the aggressive extrusion ( often > 10 rounds ) , time - consuming lyophilization of the lipid B-material components , and freeze - thaw cycling ( often > 5 rounds ) that are currently employed during liposomal B-nanoparticle sna preparation . [SEP]
[CLS] the drug - release kinetics of drug - encapsulated plga - snas can be independently tuned without significantly changing the half - life of nucleic B-material acids I-material on the nps B-nanoparticle . [SEP]
[CLS] moreover , this system provides another handle to further test our hypothesis that the unique properties of snas are in large part due to their 3d arrangement of linear nucleic B-material acids I-material and are core independent . [SEP]
[CLS] synthesis of plga - snas utilizing nanoprecipitation and cu - free click chemistry . [SEP]
[CLS] 1 . a , c ) ( insets ) images of plga - peg - n 3 nps B-nanoparticle and plga - snas acquired by afm . [SEP]
[CLS] histograms were fit to gaussian distributions with an average height of 49 ± 13 nm for plga - peg - n 3 nps B-nanoparticle and 66 ± 19 nm for plga - snas . [SEP]
[CLS] b ) dls histograms of plga - peg - n 3 np B-nanoparticle ( black ) and plga - snas ( red ) after functionalization with nucleic B-material acids I-material . d ) cooperative melting profile of plga - snas . [SEP]
[CLS] green particles : 13 nm au snas synthesized with complementary sequences . [SEP]
[CLS] inset : optical image of red precipitates after au snas were incubated B-technique with plga - snas that bear a complementary sequence . [SEP]
[CLS] 2 . a ) schematic representation of a fret plga - sna and fret turn - on experiment . [SEP]
[CLS] rhodamine was excited at 530 nm and the emission spectrum was recorded from 550 to 700 nm . [SEP]
[CLS] b ) representative fluorescence B-property kinetics landscape of fret plga - snas . c ) release profiles of nucleic B-material acids I-material on the surface of the snas in 10 % fbs . [SEP]
[CLS] a ) coumarin 6 was utilized as a fluorescent B-property model drug encapsulated inside the plga matrix for the evaluation of drug - release kinetics . b ) release kinetics of coumarin 6 from plga - snas prepared from the three polymer B-material compositions in 10 % fbs . [SEP]
[CLS] a ) confocal image of raw - blue cells B-material treated with 200 × 10 −9 m cy5 - tagged plga - snas ( red ) for 1 h . [SEP]
[CLS] scale bar = 10 × 10 −6 m . b ) cellular uptake kinetics of plga - snas and linear nucleic B-material acids I-material in raw - blue cells B-material at 100 and 200 × 10 −9 m . [SEP]
[CLS] c ) cytotoxicity B-property of plga - snas evaluated using an 3 - ( 4 , 5 - dimethylthiazol - 2 - yl - ) - 2 , 5 - diphenyltetrazolium bromide B-material ( mtt ) assay . [SEP]
[CLS] d ) plga - snas activating tlr9 assayed by quanti - blue . [SEP]
[CLS] cpg : tlr9 activating motifs ; gpc : control sequence . [SEP]
[CLS] we describe a new , and vastly superior approach for labeling spherical nucleic B-material acid I-material conjugates ( snas ) with diagnostic probes . [SEP]
[CLS] snas have been shown to provide the unique ability to traverse the cell B-material membrane and deliver surface conjugated dna into cells B-material while preserving the dna from nuclease degradation . [SEP]
[CLS] our previous work on preparing diagnostically labeled snas was labor intensive , relatively low yielding , and costly . [SEP]
[CLS] here , we describe a straightforward and facile preparation for labeling snas with optical and mr imaging probes with significantly improved physical properties . [SEP]
[CLS] the synthesis of gd ( iii ) labeled dna au nanoparticle B-nanoparticle conjugates is achieved by sequential conjugation of 3 ′ - thiol - modified oligonucleotides and cofunctionalization of the particle surface with the subsequent addition of 1 , 2 diothiolate modified chelates of gd ( iii ) ( abbreviated : dna - gd iii @ aunp B-nanoparticle ) . [SEP]
[CLS] this new generation of sna conjugates has a 2 - fold increase of dna labeling and a 1 . 4 - fold increase in gd ( iii ) loading compared to published constructs . [SEP]
[CLS] furthermore , the relaxivity ( r 1 ) is observed to increase 4 . 5 - fold compared to the molecular dithiolane - gd ( iii ) complex , and 1 . 4 - fold increase relative to previous particle constructs where the gd ( iii ) complexes were conjugated to the oligonucleotides rather than directly to the au particle . [SEP]
[CLS] importantly , this simplified approach ( 2 steps ) exploits the advantages of previous gd ( iii ) labeled sna platforms ; however , this new approach is scalable and eliminates modification B-event of I-event dna I-event for attaching the contrast B-technique agent I-technique , and the particles exhibit improved cell B-material labeling . [SEP]
[CLS] for nearly two decades spherical nucleic B-material acid I-material conjugates ( snas ) have been prepared for applications ranging from the engineering of supra - molecular lattices , monitoring gene expression and regulation , biodetection , as cell B-material transfection agents , and more recently for cancer therapy . [SEP]
[CLS] these nanoparticle B-nanoparticle conjugates have been thoroughly investigated with respect to their in vivo stability and toxicity B-property profiles , hybridization thermodynamics , kinetics , and nuclease resistance in vitro . [SEP]
[CLS] as the clinical community has become more focused on understanding diseases at the molecular and cellular level , a fundamental need for noninvasive methods of studying in vivo biochemical processes has emerged . [SEP]
[CLS] traditional methods such as western blotting and immunohistochemistry require sample destruction that prevents longitudinal studies . [SEP]
[CLS] to overcome these limitations a variety of molecular imaging B-technique techniques I-technique have been developed to investigate in vivo events , making use of such modalities as optical , mri , pet , spect , and ultrasound . [SEP]
[CLS] each of these modalities has unique advantages and disadvantages regarding sensitivity , spatial / temporal resolution , and the type of radiation or lack thereof . [SEP]
[CLS] the in vivo detection limits of each modality can be augmented by using contrast B-technique agents I-technique and reporter probes and are used in applications such as cell B-material tracking , reporting gene expression , and monitoring therapeutic paradigms . [SEP]
[CLS] mr imaging is a staple of experimental and clinical diagnostic radiology due to excellent soft - tissue contrast , high imaging resolution , and the absence of ionizing B-property radiation . [SEP]
[CLS] mri is capable of 3d - imaging of biological structures , and in research settings at very high field , processes can be imaged at near - cellular resolution ( ~ 10 um ) . [SEP]
[CLS] unlike fluorescence B-property and other light - based imaging B-technique techniques I-technique , mri does not require optically transparent samples . [SEP]
[CLS] detailed structural information can be obtained in minutes , and single slices in seconds . [SEP]
[CLS] due to its established position as a clinical diagnostic tool , mr imaging is a major focus of translational imaging research . [SEP]
[CLS] two fundamental challenges in the development mr contrast B-technique agents I-technique ( ca ' s ) that must be overcome for use in experimental preclinical imaging are i . , amplification of the contrast B-technique agent I-technique signal ; ii . , specific agent delivery to cells B-material and tissues of interest . [SEP]
[CLS] the majority of ca ' s are based on either paramagnetic B-property or superparamagnetic B-property metal B-material ions B-material . [SEP]
[CLS] these species act as potent relaxation agents due to the presence of unpaired electrons . [SEP]
[CLS] in general , paramagnetic B-property ( t 1 ) agents , such as gd ( iii ) , decrease t 1 with a lesser effect on t 2 , resulting in increased signal in the vicinity of the agent . [SEP]
[CLS] conversely , superparamagnetic B-property agents primarily decrease the t 2 with a lesser effect on t 1 , resulting in a signal void in the vicinity of the agent . [SEP]
[CLS] gd ( iii ) is the most widely used metal B-material ion B-material for t 1 ca ' s due to its high magnetic B-property moment and long electron relaxation time . [SEP]
[CLS] to overcome these challenges we have developed a number of nanoparticle B-nanoparticle conjugates that significantly enhance the observed mr signal ( 1000 - fold ) over molecular complexes [SEP]
[CLS] this synthetic strategy consisted of covalently attaching gd ( iii ) complexes to thiol - modified oligonucleotides ( via click chemistry ) and conjugating this derivative to 13 . 1 nm gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) . [SEP]
[CLS] because of the increased local concentration of gd ( iii ) per particle , there is a significant increase in the observed r 1 ( relaxivity mm −1 s −1 , particularly at low magnetic B-property field strengths ) . [SEP]
[CLS] as a result of this mr signal amplification we have successfully detected mr reporter genes , 40 imaged the in vivo biodistribution of glioblastoma and fate mapped human neural stem cells B-material . [SEP]
[CLS] these promising results stimulated the design of the next generation of gd ( iii ) - modified snas while maintaining the modularity and utility of these important nanoparticles B-nanoparticle . [SEP]
[CLS] our goal in this study was to significantly improve two important features : i . , further increase the gd ( iii ) payload through direct surface conjugation to the aunp B-nanoparticle ; ii . , greatly simplify the synthesis by eliminating the gd ( iii ) attachment to the sh - modified oligonucleotides . [SEP]
[CLS] here , we describe a straightforward and scalable approach to the synthesis of a 1 , 2 dithiolane modified gd ( iii ) - macrocycle ( 4 : see supporting information for details ) , conjugated to a sna to form dt - gd ( iii ) - sna ( figure 1 ) . [SEP]
[CLS] we characterized these new constructs by measuring the proton relaxation properties and particle stability , and quantifying cell B-material - labeling efficiency . [SEP]
[CLS] in a systematic study by mirkin et al . to determine the maximum achievable density of thiolated oligonucleotides that could be loaded onto a aunp B-nanoparticle surface , 15 nm spherical particles were functionalized with densities up to 19 pmol / cm 2 when salt - aged with 1 m sodium B-material chloride I-material ( corresponding to 0 . 11 dna / nm 2 , or 78 dna / aunp B-nanoparticle ) . [SEP]
[CLS] it is known that the density of 3 ′ - thiolated dna packing on a spherical aunp B-nanoparticle surface is limited by the negative charge present on the polyanionic backbone of immobilized oligonucleotides . [SEP]
[CLS] by comparison , organic ligand densities have been reported to reach as high as 6 . 3 ± 0 . 6 monothiolated ligands per nm 2 for aunps B-nanoparticle of comparable diameter ( 3 - mecaptopropionic acid ) . [SEP]
[CLS] given the greater than 50 - fold difference between oligonucleotide and organic ligand densities achievable on the particle surface , we estimated that the surface area remaining unmodified ( after saturation of the particle with thiolated oligonucleotides ) could support a densely packed layer of gd ( iii ) complexes ( an approach that we refer to as backfilling because the gd ( iii ) is added post dna addition ) . [SEP]
[CLS] importantly , dt - gd ( iii ) - snas have demonstrated the ability to bind previously synthesized snas at high surface densities , and can be modified ( i . e . , backfilled with 4 ) onto the surface of these particles without the need for reducing B-property agents I-property . [SEP]
[CLS] in addition , this nanoconjugate design is observed to increase r 1 relaxivity per gd ( iii ) and particle payload by greater than 3 - fold when compared to the previously reported dna - gd iii @ aunps B-nanoparticle , while maintaining high stability , biocompatibility B-property , and cellular uptake of previous generations . [SEP]
[CLS] this new dt - gd ( iii ) - sna design leverages numerous beneficial features of previous gd ( iii ) nanoconjugate cas , while providing an extremely facile synthesis ( and scalability ) of other gd ( iii ) - labeled particle conjugates . [SEP]
[CLS] the 1 , 2 - dithiolane anchor was chosen for this work because it has been found that the cyclic disulfide functionality is an excellent surface binding ligand for gold B-material . [SEP]
[CLS] significantly , studies have described an enhancement of colloidal stability using dna modified with lipoic acid as compared to monothiolated dnas in the presence of high concentrations of dithiothreitol . [SEP]
[CLS] the dt - gd ( iii ) complex used here ( 4 , figure 1 ) was synthesized by using the cu ( i ) - catalyzed huisgen azide - alkyne cycloaddition of an azide derivative of ( ± ) - lipoic acid , and a previously published alkyne bearing , τ m - optimized gd ( iii ) ( complex 3 , see si ) . [SEP]
[CLS] purification of dt - gd ( iii ) was performed using hplc and characterization confirmed by high resolution esi - tof mass spectroscopy B-technique ( schemes s1 - 3 , figure s1 ) . [SEP]
[CLS] to determine the maximum density of gd ( iii ) complexes on the surface of the particle , 13 nm aunps B-nanoparticle were functionalized with only 4 for quantification of surface loading and r 1 relaxivity . [SEP]
[CLS] particle conjugation was achieved by mixing an excess of 4 into a stock of freshly prepared solution of 13 . 0 ± 1 . 6 nm citrate stabilized aunps B-nanoparticle with 0 . 01 % tween with shaking for 24 h . after labeling , the particles were washed and concentrated by centrifugation . [SEP]
[CLS] the pure 4 particle conjugates have a r 1 of 17 . 1 mm −1 s −1 at 37 °c ( 1 . 41 t ) , and a total surface loading of 1125 ± 17 gd ( iii ) complexes per particle ( determined by inductively coupled plasma mass spectrometry icp - ms ) . [SEP]
[CLS] to test the efficacy of this approach , poly dt - snas were synthesized using the same batch of 13 nm citrate - stabilized aunps B-nanoparticle as used to prepare the pure 4 conjugates . [SEP]
[CLS] specifically , snas were made by salt - aging 3 ′ - thiolated 24 - mer poly dt dna bearing a 5 ′ cy3 fluorophore . [SEP]
[CLS] upon completion of the conjugation process , particles were concentrated and purified from excess dna , citrate , and sodium B-material chloride I-material by successive rounds of centrifugation . [SEP]
[CLS] to assess the dna conjugation efficiency , snas were incubated B-technique with potassium B-material cyanide to digest the gold B-material cores B-material , and uv / vis measurement of cy3 was used to quantify dna loading . [SEP]
[CLS] results indicated a total surface loading of 199 ± 0 . 6 strands per particle . [SEP]
[CLS] using the purified poly dt snas concentrated to ten times their initial concentration , 50 , 000 equiv of dt - gd ( iii ) were added directly , and functionalization was achieved with stirring over 24 h . [SEP]
[CLS] this " backfilling " approach to prepare dt - gd ( iii ) - snas proceeded without the use of any reducing B-property agents I-property , and were purified by centrifugation with an r 1 of 22 . 0 mm −1 s −1 at 37 °c ( 1 . 41 t ) , with a particle payload of 730 ± 11 gd ( iii ) complexes per particle ( table 1 ) . [SEP]
[CLS] relative to the pure dt - gd ( iii ) conjugates , this represents a 23 % improvement in r 1 , sacrificing only 35 % of the gd ( iii ) loading . [SEP]
[CLS] having removed a portion of the initial poly dt conjugation mixture , dt - gd ( iii ) was added directly ( without purification of particles from excess dna ) to test the capacity of performing dna conjugation and backfilling is a single vessel . [SEP]
[CLS] after a further 24 h to complete the addition of 4 , particles were purified by centrifugation . [SEP]
[CLS] the cofunctionalization process produced dt - gd ( iii ) - snas loaded with 691 dt - gd ( iii ) per particle , which is in good agreement with previous preparations of dt - gd ( iii ) - snas and confirms the effectiveness of combining these conjugation steps . [SEP]
[CLS] furthermore , the kinetics of the backfilling procedure was examined . [SEP]
[CLS] a purified batch of poly dt snas were mixed with dt - gd ( iii ) , and aliquots were removed at 1 , 2 , 4 , 8 , and 24 h . [SEP]
[CLS] particles from each time point were purified and results indicated that 76 % of particle loading was complete within 60 min and completed after 24 h ( see figure s10 ) . [SEP]
[CLS] to compare how the new dt - gd ( iii ) - snas perform relative to the previous generations of gd ( iii ) - dnas aunp B-nanoparticle conjugates ( in which the gd ( iii ) is covalently attached to the dna ) , gd - dna @ spheres were synthesized as recently described . [SEP]
[CLS] analysis of these conjugates showed increased particle loading compared with all previously reported 13 nm nanoconjugates of this design : totaling 515 ± 15 gd ( iii ) complexes per particle and an r 1 of 12 . 8 mm −1 s −1 at 37 °c ( 1 . 41 t ) . [SEP]
[CLS] despite the remarkably high gd ( iii ) loading observed for the gd - dna @ spheres , the density of gd ( iii ) was still limited to only 0 . 97 complexes per nm 2 resulting from 103 ± 3 dna per particle ( corresponding to a dna density of 0 . 19 dna per nm 2 ) . [SEP]
[CLS] the limitations which prevented further loading result from density restrictions supplied by the negative charges on the dna backbone , and the steric crowding imposed by the covalent linker from the modified dna bases to gd ( iii ) complexes . [SEP]
[CLS] in comparison , the loading densities of gd ( iii ) and dna for dt - gd ( iii ) - snas were 1 . 4 - and 2 - fold greater , respectively ( table s16 ) . [SEP]
[CLS] importantly , the observed increase in density of oligonucleotide loading may provide both increased utility for sequences tailored to specific applications , and enhanced nuclease stability ( relative to less densely functionalized snas ) . [SEP]
[CLS] to investigate the stability of the 1 , 2 - dithiolane conjugates on the au particle , the loss of 4 from both dt - gd ( iii ) - sna and gd - dna @ sphere constructs in cell B-material culture conditions were compared using icp - ms . [SEP]
[CLS] specifically , concentrated solutions of both conjugates were diluted to equimolar concentrations in pbs , dmem , 10 % fbs in dmem and 100 % fbs , and incubated B-technique at 37 °c under sterile conditions for 2 weeks . [SEP]
[CLS] particle concentration and gd ( iii ) loading was quantified at time zero for each solution , and each condition was run in duplicate . [SEP]
[CLS] time points were recorded at 24 , 48 , 120 , 168 , and 336 h by removal of an aliquot from the incubated B-technique solution followed by centrifugation and icp - ms analysis of supernatant gd ( iii ) . [SEP]
[CLS] in addition , the colloidal stability was assessed by observation of the plasmon resonance peak at time points of 1 , 7 , and 14 days for both constructs under all conditions . [SEP]
[CLS] after 24 h , both nanoconjugates had visibly precipitated out of dmem , while the remaining solutions remained visibly red and colloidally stable throughout all time points ( figure 2a ) . [SEP]
[CLS] analysis of the surface plasmon resonance peak for each construct indicated the maximum value at 527 and 521 nm for backfilled and gd ( iii ) dna conjugated particles in water B-material for time zero , and all solutions showed a variable increase in this value over 14 days , particularly solutions of fbs ( figure s11 ) . [SEP]
[CLS] results of the supernatant analysis were plotted as a percentage of loss from the starting gd ( iii ) concentration ( figure 2b ) . [SEP]
[CLS] these data indicate that under these stringent conditions , both constructs were vulnerable to degradation under all conditions except pbs , despite the different presentation and number of thiols B-material present on the monothiolated gd ( iii ) dnas and the backfilled dt - gd ( iii ) complexes . [SEP]
[CLS] however , under commonly used cell B-material culture conditions of 10 % fbs with an incubation B-technique time of 24 h , the new dt - gd ( iii ) - snas were 2 - times more stable than the gd - dna @ spheres with a gd ( iii ) loss of 12 . 9 % . [SEP]
[CLS] given the similar trend of the degradation observed between the two constructs , the nature of coordination of 4 to the gold B-material surface of the particle and its role in stability remain unclear , relative to monothiolated dna . [SEP]
[CLS] to answer this question , experiments are underway to verify the degree in which the 1 , 2dithiolane is bound in a bidentate fashion to the au surface . [SEP]
[CLS] to test the biocompatibility B-property and cellular uptake efficiency of dt - gd ( iii ) - snas and gd - dna @ spheres , hela cells B-material were prepared and incubated B-technique using various dilutions of nanoconjugates in 10 % fbs in dmem . [SEP]
[CLS] after 24 h cells B-material were washed , trypsinized , and counted . [SEP]
[CLS] cell B-material counts were as expected and no cell B-material toxicity B-property was observed for any of the incubation B-technique concentrations . [SEP]
[CLS] counted cells B-material were digested in 1 : 1 hno 3 and hcl and gd ( iii ) and gold B-material content was examined using icp - ms . [SEP]
[CLS] per concentration of nanoconjugates observed , the dt - gd ( iii ) - snas provided improved gd ( iii ) uptake , particularly at lower incubation B-technique concentrations ( figure 3 ) . [SEP]
[CLS] interestingly , the improved gd ( iii ) payload was not the only cause of the improved uptake , as cells B-material dosed with the dt - gd ( iii ) - snas appeared to take up more of the nanoconjugates at each of the concentrations examined ( figure s13 ) . [SEP]
[CLS] this result can be explained by the higher density loading of dna present on the dt - gd ( iii ) - snas relative to the dna - gd @ spheres , which are limited in the densities of dna which can be achieved due to the steric crowding of the particle surface resultant from the long linker arm of the covalently attached gd ( iii ) complex . [SEP]
[CLS] we have discovered a facile and scalable methodology for the functionalization of sna ' s with optical and gd ( iii ) mr imaging probes . [SEP]
[CLS] the nanoconjugates consist of an optical probe ( cy3 - labled - dna , 5 ) and hundreds of mr contrast B-technique agents I-technique ( dithiolane functionalized gd ( iii ) complexes , 4 ) to create a powerful multimodal imaging conjugate . [SEP]
[CLS] the new class of particles exhibits increased r 1 relaxivity , improved gd ( iii ) loading / particle , increased cellular uptake at lower incubations B-technique concentrations , with comparable conjugate stability relative to the previous generations of our multimodal nanoparticles B-nanoparticle . [SEP]
[CLS] the observed increase in lowfield magnetic B-property relaxivity , improved gd ( iii ) loading , and significantly higher cellular uptake of the particles will allow the use of far less contrast B-technique agent I-technique per cell labeling experiments . [SEP]
[CLS] over the last two decades we have routinely discovered that there is no " one imaging probe fits " for different cell B-material types . [SEP]
[CLS] due to the modular synthesis of the snas , many different nucleotide sequences can be tested and targeting groups can replace the cy3 dye on the 5 ′ end of the dna . [SEP]
[CLS] this vastly simplified synthetic approach is facilitating the creation of new gd ( iii ) labeled nanoconjugates in our laboratory that are tailored to each cell B-material type . [SEP]
[CLS] by the use of this the new " backfilling " approach for the preparation of multimodal imaging probes we can quickly learn which probe ( s ) are best suited to each individual cell B-material type . [SEP]
[CLS] synthetic strategy for the preparation of dt - gd ( iii ) - sna nanoparticles B-nanoparticle : synthetic scheme of dt - gd ( iii ) particle conjugates is achieved without the presence of reducing B-property agents I-property . [SEP]
[CLS] the synthesis and characterization of 4 and 5 can be found in supporting information . [SEP]
[CLS] standard salt aging conjugation protocol using 5 ′ cy3 poly dt snas starting from citrate - stabilized aunps B-nanoparticle . [SEP]
[CLS] once the 5 ′ cy3 poly dt snas are conjugated to the aunp B-nanoparticle , complex 4 is added to form the final product dt - gd ( iii ) - sna . [SEP]
[CLS] 2 . stability of dt - gd ( iii ) - snas measured under various cell culture related conditions over 14 days at 37 °c . [SEP]
[CLS] ( a ) dt - gd ( iii ) - snas were examined for colloidal stability in pbs , 10 % fbs , and fbs . [SEP]
[CLS] ( b ) supernatant analysis of gd ( iii ) loss under similar conditions in 10 % fbs and 100 % fbs . [SEP]
[CLS] gd ( iii ) loss during long - term storage in pbs was minimal ( figures12 ) . [SEP]
[CLS] 3 . twenty - four hour cell B-material uptake experiment comparing dt - gd ( iii ) - snas and dna - gd @ spheres described by ( a ) gd ( iii ) dosing , or ( b ) aunp B-nanoparticle dosing concentrations . [SEP]
[CLS] relaxivity r 1 and particle loading for the gd ( iii ) nanoconjugates reported a ionic relaxivity describes the relaxivity per gd ( iii ) ion B-material . [SEP]
[CLS] molecular relaxivity refers to the sum of relaxivities / particle . [SEP]
[CLS] table 1 . 11 ab 60 mhz , 37 °c in water B-material with 0 . 01 % tween 20 . [SEP]
[CLS] c prepared as as previously described . [SEP]
[CLS] 42 na = not applicable . [SEP]
[CLS] oligonucleotides ( ons ) are considered a form of informational drug , where drug - like properties ( pharmacophore ) and target information ( dianophore ) are independent of each other . [SEP]
[CLS] therefore , on therapeutics promises to dramatically reduce the cost of new drug development , as a change in disease target in principle requires only a change in the on sequence . [SEP]
[CLS] [ 8 ] [ 9 ] however , direct utilization of ons as a drug is hampered by enzymatic degradation , poor cellular uptake , rapid liver clearance , unwanted activation of the immune system , and overall low biochemical efficacy . [SEP]
[CLS] various chemical modifications of the on or vectors ( of viral , polycationic , and liposomal B-nanoparticle formulations ) have been utilized to improve the bioavailability B-property of the nucleic B-material acid I-material . [SEP]
[CLS] [ 14 ] [ 16 ] despite exhaustive efforts , however , these strategies remain subject to several longstanding drawbacks , including toxicity B-property , immunogenicity B-property , on instability , and off - target side effects . [SEP]
[CLS] [ 17 ] [ 18 ] [ 19 ] [SEP]
[CLS] recently , we reported a novel form of brush polymer - dna conjugate termed pacdna ( polymer - assisted compaction of dna ) , which consists of ons covalently attached to the backbone of a sterically congested brush polymer B-material having polyethylene glycol ( peg ) side chains . [SEP]
[CLS] the compaction of dna by the densely packed peg side chains impart the dna with selective accessibility , shielding dna from proteins B-material while maintaining the hybridization with a complementary dna strand , both kinetically and thermodynamically . [SEP]
[CLS] the steric selectivity of the pacdna greatly lowers various side effects associated with dna - protein interactions , including degradation , toll - like receptor B-material 9 ( tlr9 ) activation , coagulopathy ( dna B-event binding I-event to thrombin ) , and hepatic capture . [SEP]
[CLS] interestingly , the pacdna can enter cells B-material via endocytosis B-event and effectively knock down target genes through an antisense mechanism . [SEP]
[CLS] while this brush - architectured peg has been proven useful for the delivery of oligonucleotides , a deeper understanding of how various structural parameters affect hybridization kinetics , nuclease resistance , cellular uptake , and ultimately gene regulation efficacy is of great value for the further development of peg - based on vectors . [SEP]
[CLS] it is , however , difficult to explore these parameters using brush - type architectures due to the nontrivial design and synthesis involved . [SEP]
[CLS] moreover , the backbone of the brush polymer B-material is nondegradable . [SEP]
[CLS] for biopharmaceutical use , it is desirable to adopt materials regarded as generally safe for drug formulations . [SEP]
[CLS] in this context , a simple , efficient , and robust method to diversify the structure of peg - based vectors is of significance in advancing the rational design of effective noncationic on vectors . [SEP]
[CLS] herein , we created a library of micelles B-material with tunable peg length and density to investigate the structure - dependent steric selectivity and on bioactivity ( scheme 1 ) . [SEP]
[CLS] the micelles B-material are coassembled from two amphiphilic B-property diblock copolymers , dna - b - poly ( ε - caprolactone ) ( dna - b - pcl ) and peg - b - pcl , as well as the pcl homopolymer ( m n = 10 . 5 kda ) . [SEP]
[CLS] a 21base dna sequence and three peg lengths ( m n 2 , 5 , and 10 kda ) are used to create the diblock copolymers . [SEP]
[CLS] the pcl homopolymer contributes to the micellar core B-material volume but not the shell B-material , thereby modulating the densities of micelle B-material surface moieties . [SEP]
[CLS] the dna - b - pcl amphiphile B-property is synthesized in two steps . [SEP]
[CLS] first , azide - terminated pcl is prepared via ring - opening polymerization ( rop ) of ε - cl using o - ( 2 - azidoethyl ) heptaethylene glycol as the initiator ( for 1 h / c nmr and infrared spectra , see figures s1 - s3 ) . [SEP]
[CLS] subsequently , an on with a 5 ′ dibenzocyclooctyl ( dbco ) group and a 3 ′ cy3 reporter ( table s1 ) is coupled to the azide - capped pcl through copper - free click chemistry in dimethyl sulfoxide ( dmso ) : water B-material mixture ( 9 : 1 v : v ) . [SEP]
[CLS] unreacted pcl and dna are removed by dialysis and reverse - phase hplc , respectively , to yield pure conjugates ( ~ 85 % conjugation yield ) as determined by agarose B-technique gel I-technique electrophoresis I-technique ( figures 1 , s4 , and s5 ) . [SEP]
[CLS] for proof - of - concept , we choose an antisense dna sequence ( g3139 by genta inc . ) that targets the antiapoptotic b - cell lymphoma 2 ( bcl - 2 ) family proteins B-material as the hydrophilic B-property block . [SEP]
[CLS] bcl - 2 is an important biomarker B-property for many cancers , including several types of breast and ovarian cancers , for which only one small molecule inhibitor has obtained regulatory approval . [SEP]
[CLS] the peg - b - pcl amphiphiles B-property are synthesized by using hydroxy terminated peg to initiate the rop of ε - cl . the degree of polymerization of the pcl is controlled via initiator : monomer B-material stoichiometry to be 100 , and successful synthesis was confirmed by 1 h nmr and dimethylformamide ( dmf ) gel permeation chromatography B-technique ( gpc ) ( figure s6 and table s2 ) . [SEP]
[CLS] coassembly of the block copolymers and the pcl homopolymer is achieved via nanoprecipitation ( gradual solvent exchange from dmso to nanopure water B-material ) . [SEP]
[CLS] to systematically probe the relationship between the structural parameters of dna - peg nanoparticles B-nanoparticle and their steric selectivity , two series of nanostructures were prepared : ( 1 ) micelles B-material containing dna - b - pcl and peg - b - pcl copolymers having varying peg lengths and ( 2 ) micelles B-material containing dna - b - pcl , peg 10k - b - pcl , and varying amounts of pcl homopolymer . [SEP]
[CLS] the first series emphasizes the effect of the relative lengths of the peg and the dna , while the second series enables the study of the surface peg density . [SEP]
[CLS] to study the ability of the dna component within the coassembled micelles B-material to hybridize with a complementary ( sense ) sequence , we adopted a fluorescence B-property quenching assay in which a quencher ( dabcyl ) - modified sense strand is mixed with cy3 - labeled particles . [SEP]
[CLS] upon hybridization , the fluorophore - quencher pair is brought to proximity , leading to a reduction in the fluorescence B-property signals ( figure 2a ) . [SEP]
[CLS] the rate of fluorescence B-property reduction is therefore an indicator B-property of the hybridization kinetics . [SEP]
[CLS] a scrambled strand is used as a control to rule out the possibility of nonspecific interactions . [SEP]
[CLS] as shown in figure 2b , both free dna and micellar nanoparticles B-nanoparticle can hybridize with the sense strand , while the scrambled sequence causes no change in the fluorescent B-property signals . [SEP]
[CLS] the micelles B-material , however , exhibit a two - population behavior : a population with rapid hybridization kinetics similar to that of free dna , and a slowhybridizing population , which increases with increasing peg content ( figures 2b and c ; see supporting information section 3 . 6 for calculation of peg density ) . [SEP]
[CLS] notably , micelles B-material containing no peg - b - pcl ( spherical nucleic B-material acid I-material - like micelles B-material , see figure s7 ) comprise almost entirely the fast - hybridization population . [SEP]
[CLS] we attribute the slow - hybridizing population to the submicellar microstructure , which causes excessive hindrance around the dna and is affected by the density of the peg . [SEP]
[CLS] indeed , by diluting the micelle B-material core B-material with pcl homopolymer ( thus a decrease in surface peg density ) , the fast - hybridizing population can be almost fully restored ( figures 2d and 2e ) . [SEP]
[CLS] we next probed the enzyme accessibility of the dna component within the micelles B-material using bovine pancreas dnase i as a model enzyme . [SEP]
[CLS] cy3 - labeled micelles B-material are prehybridized with quencher - labeled sense strands . [SEP]
[CLS] upon introduction of dnase i , the dsdna is degraded , resulting in an increase in fluorescence B-property ( figure 3a ) . [SEP]
[CLS] as shown in figure 3b , naked dsdna is readily accessed and degraded in the presence dnase i , with a half - life ( t 0 . 5 ) of 12 . 0 ± 0 . 5 min . [SEP]
[CLS] in contrast , micelles B-material consisting of pure dna - b - pcl show slightly enhanced nuclease resistance ( t 0 . 5 : 36 ± 1 min ) due to increased steric hindrance and local high salt B-material concentrations , which is consistent with previous reports . [SEP]
[CLS] interestingly , adding peg 2kb - pcl to the micelle B-material ( 10 : 1 m : m peg : dna amphiphile B-property ratio ) results in an increase in degradation rate ( t 0 . 5 : 22 ± 2 min ) . [SEP]
[CLS] amphiphiles B-property of higher peg molecular weight ( 5 and 10 kda ) , on the other hand , enhance dna stability under identical conditions . [SEP]
[CLS] the highest stability is achieved with peg 10k - b - pcl , which gives a t 0 . 5 of 142 ± 15 min at 10 : 1 peg : dna amphiphile B-property ratio , and 240 ± 18 min at 20 : 1 . [SEP]
[CLS] one interpretation of these results is that longer peg chains can more effectively shield the underlying dna strands from enzymatic access ( figure 3c and table s3 ) . [SEP]
[CLS] when the peg block is too short , the diluting effect of the pcl block becomes dominant , which serves to increase the spacing of dna strands , leading to more facile enzyme access and degradation . [SEP]
[CLS] although higher molecular weight peg - b - pcl amphiphiles B-property can better protect the dna strands from degradation , they also increase the proportion of the slow - hybridizing population . [SEP]
[CLS] for biopharmaceutical applications , it is desirable to maximally retain the hybridization capability while minimizing protein B-material access . [SEP]
[CLS] to this end , we systematically investigated how a homopolymer pcl in the nanoparticle B-material formulation I-material can be used to tune the spacing of particle surface moieties and maximize dna B-event binding I-event selectivity . [SEP]
[CLS] micelles B-material consisting of peg 10k - b - pcl and dna - b - pcl ( 20 : 1 m : m ) are used as a base composition , to which different molar ratios of pcl ( 10 - 200 , relative to dna - b - pcl ) are added . [SEP]
[CLS] it is found that pcl significantly increases particle core B-material size from 62 ± 3 nm ( base composition , estimated from tem images ) to 155 ± 5 nm ( with 200 equiv pcl ) , which correlates with a drop of surface peg density from 6 . 3 × 10 13 to 1 . 5 × 10 13 peg / cm 2 ( see table s4 ) . [SEP]
[CLS] the reduction in peg density results in acceleration in both hybridization and enzymatic access , with the change in hybridization being faster . [SEP]
[CLS] with 20 equiv of pcl ( density : 5 . 0 × 10 13 peg / cm 2 ) , hybridization can be mostly restored ( > 85 % hybridized in 5 min ) , while protein B-material shielding remains nearly unaffected ( t 0 . 5 of 238 min vs . 240 min for base composition , figures 3d and e ) . [SEP]
[CLS] in contrast , excessive of pcl ( 200 equiv ) results in incremental improvements in hybridization but substantial loss in enzyme stability , showing a t 0 . 5 of ~ 40 min . [SEP]
[CLS] these results are consistent with our hypothesis that pcl homopolymer can be used to tune the peg density of the micelles B-material , thereby opening a window in which dna B-event binding I-event selectivity can be maximized . [SEP]
[CLS] in addition to nuclease stability , efficient cellular uptake is another important aspect in high antisense efficacy . [SEP]
[CLS] to examine the extent of endocytosis B-event , skov3 cells B-material are incubated B-technique with a library of cy3 - labeled micelles B-material and free dna having the same dna concentration ( 1 μm ) for 4 h . [SEP]
[CLS] flow B-technique cytometry I-technique shows that , compared with free dna , coassembled micelles B-material have enhanced cell B-material uptake ( 20 - 90× relative to that of free dna , figure 4 ) , with shorter peg and lower peg densities leading to more uptake ( figure 4a ) . [SEP]
[CLS] micelles B-material with no surface peg ( dna - b - pcl only ) show the highest uptake at ~ 150× that of free dna . [SEP]
[CLS] these peg - free micelles B-material are structurally analogous to spherical nucleic B-material acids I-material ( snas ) , which have been shown to exhibit high , nonspecific cell B-material uptake due to recognition by class a scavenger receptors and endocytosis B-event via a lipid - raft - dependent , caveolae - mediated pathway . [SEP]
[CLS] with the addition of moderate amounts of pcl homopolymer in the micellar core B-material , cellular uptake is increased , but only to a small extent ( figure 4b ) . [SEP]
[CLS] however , when 200 equiv of pcl is added , cell B-material uptake is augmented to sna - like levels . [SEP]
[CLS] the unusually high cellular uptake is likely associated with the exposed hydrophobic B-property regions of the micelle B-material surface caused by excessive loading of pcl , which interacts with cells B-material in a different mechanism than pegand dna - dominated surfaces . [SEP]
[CLS] these results are corroborated by confocal B-technique laser I-technique scanning I-technique microscopy I-technique . [SEP]
[CLS] while cells B-material treated with free dna show no or very weak fluorescence B-property , dna micelles B-material give much stronger signals under the identical imaging settings , with increasing peg contents reducing the intensities of fluorescent B-property signals ( figures 4c and s10 ) . [SEP]
[CLS] the same experiments were also performed with hela cells B-material , which show a similar general trend ( figure s11 ) . [SEP]
[CLS] collectively , the results here indicate that the uptake of dna - peg - pcl micelles B-material can be tuned by adjusting the surface composition exposed to the cell B-material from being peg - like to sna - like and hydrophobic B-property . [SEP]
[CLS] next , we examined how different micelle B-material structures affect antisense gene silencing efficacy . [SEP]
[CLS] because cellular endosomes are associated with digestive environments that can degrade the antisense sequence , higher stability may contribute to greater overall efficacy . skov3 cells B-material were treated with the two series of micelles B-material at an equal dose of dna ( 1 μm ) for 24 h , followed by culturing for 48 h in fresh media . lipofectamine - complexed dna and free dna were used as positive and negative controls , respectively . the levels of bcl - 2 were analyzed by western blotting . as shown in figures 5a and s12a , the bcl - 2 levels are significantly reduced by micelles B-material having 10 kda peg amphiphiles B-property . the best of these is peg 10k - b - pcl : dna - b - pcl : pcl ( 20 : 1 : 20 ) , showing 92 % reduction in expression ( band densitometry analysis , normalized to β - actin ) . these micelles B-material coincide with those showing the highest nuclease stability but moderate cellular uptake and hybridization readiness , suggesting that protein B-material inhibition is a key factor in achieving high efficacy . [SEP]
[CLS] indeed , lowstability micelles B-material having 5 kda peg show considerably lower efficacy ( 22 % knockdown for peg 5k - b - pcl : dna - b - pcl , 10 : 1 ) even though they exhibit better cell B-material uptake and hybridization . [SEP]
[CLS] interestingly , although pure dna - b - pcl micelle B-material ( sna - like ) only shows a small level of nuclease resistance , its antisense activity is significant ( 75 % knockdown ) . [SEP]
[CLS] this phenomenon is attributed to the unusually high cellular uptake associated with the snas . [SEP]
[CLS] we also performed a dose - dependent study for the two - component system , peg 10k - b - pcl : dna - b - pcl ( 20 : 1 ) and the corresponding three - component system with 20 equiv of pcl . [SEP]
[CLS] it is found that both systems are effective at high concentrations ( > 500 nm ) , but at a lower concentration ( 100 nm ) , the three - component system shows higher gene knockdown efficacy ( 84 vs 18 % , figures 5b and s12b ) , signifying that increased hybridization availability ( figure 2d ) of the antisense on is important because these two systems show roughly the same nuclease stability . [SEP]
[CLS] because the micelles B-material consist primarily of dna , peg , and pcl , components regarded by the united states food and drug administration as generally safe for pharmaceuticals , we anticipate that these coassembled nanoparticles B-nanoparticle are noncytotoxic . [SEP]
[CLS] indeed , 3 - ( 4 , 5 - dimethyl - thiazol - 2 - yl ) - 2 , 5 - diphenyl tetrazolium bromide B-material ( mtt ) assay of skov3 cells B-material treated with the micelles B-material show that viability after 48 h of incubation B-technique remains at nearly 100 % . [SEP]
[CLS] in contrast , dna complexed with lipofectamine exhibits significant cytotoxicity B-property ( > 50 % cell B-event death I-event at 100 nm of dna ) , as expected from typical polycationic carrier systems ( figure s13 ) . [SEP]
[CLS] in summary , we demonstrated a simple yet novel form of nucleic acid - based block copolymer micelles B-material that can be used for effective antisense gene regulation . [SEP]
[CLS] by screening a library of micelle B-material compositions , we revealed a series of structure - property relationships . [SEP]
[CLS] it was found that dna hybridization availability and enzyme shielding are affected by the molecular weight of the peg block and the surface peg density . [SEP]
[CLS] these parameters also affect cellular uptake with peg - dominated surfaces showing moderate uptake and dnadominated surfaces showing high uptake . [SEP]
[CLS] correlations were also established between antisense activity and the degree of protein B-material shielding , hybridization availability , and cellular uptake . [SEP]
[CLS] among these , protein B-material shielding appears to have the strongest influence on antisense activity . [SEP]
[CLS] consisting of biodegradable B-property and biocompatible B-property components , these dna block copolymer micelles B-material represent an important departure from cationic B-material systems which have been exhaustively investigated as nucleic B-material acid I-material delivery vectors . [SEP]
[CLS] the general strategy employed here can also be expanded to include other types of biomolecules such as sirna , microrna , and peptides B-material . [SEP]
[CLS] agarose gel ( 1 % ) electrophoresis B-technique of free dna and various micelle B-material compositions . [SEP]
[CLS] certain samples with high 10 kda peg contents migrate in the opposite direction of free dna due to the transient interactions between peg and the passing cations B-material . [SEP]
[CLS] ( a ) schematics of the fluorescence B-property assay used for quantifying dna hybridization . [SEP]
[CLS] ( b and d ) hybridization kinetics for free dna vs two - and three - component nanoparticles B-nanoparticle . [SEP]
[CLS] ( c and e ) relationship between peg density and the percentage of the fast - hybridizing dna population . [SEP]
[CLS] ( a ) schematics of the fluorescence B-property assay used for monitoring the kinetics of dna degradation by dnase i . ( b and d ) dna degradation kinetics for free dna vs two - and three - component nanoparticles B-nanoparticle . [SEP]
[CLS] ( c and e ) relationship between peg density and the dna nuclease half - life . [SEP]
[CLS] 4 . ( a and b ) flow B-technique cytometry I-technique measurements of skov3 cells B-material treated with cy3 - labeled free dna and micellar nanoparticles B-nanoparticle ( conc : 1 μm of dna ) . [SEP]
[CLS] ( c ) corresponding confocal fluorescence B-property images . [SEP]
[CLS] scale bar : 20 μm . [SEP]
[CLS] ( a ) efficacy for antisense gene knockdown using coassembled micelles B-material and controls ( 1 μm dna ) . [SEP]
[CLS] ( b ) dose response of two coassembled micelles B-material with high efficacy . [SEP]
[CLS] only a tiny fraction of the nanomedicine - design space has been explored , owing to the structural complexity of nanomedicines and the lack of relevant high - throughput synthesis and analysis methods . [SEP]
[CLS] here , we report a methodology for determining structure - activity relationships and design rules for spherical nucleic B-material acids I-material ( snas ) functioning as cancer - vaccine candidates . [SEP]
[CLS] first , we identified ~ 1 , 000 candidate snas on the basis of reasonable ranges for 11 design parameters that can be systematically and independently varied to optimize sna performance . [SEP]
[CLS] second , we developed a high - throughput method for making snas at the picomolar scale in a 384 - well reprints and permissions information is available at www . nature . com / reprints . [SEP]
[CLS] format , [SEP]
[CLS] and [SEP]
[CLS] used a mass spectrometry assay to rapidly measure sna immune activation . [SEP]
[CLS] third , we used machine learning to quantitatively model sna immune activation and identify the minimum number of snas needed to capture optimum structure - activity relationships for a given sna library . [SEP]
[CLS] our methodology is general , can reduce the number of nanoparticles B-nanoparticle that need to be tested by an order of magnitude , and could serve as a screening tool for the development of nanoparticle B-nanoparticle therapeutics . [SEP]
[CLS] nanotechnology is beginning to play a major role in the development of new therapeutic modalities . [SEP]
[CLS] currently , over 100 drugs based on nanomaterials B-material are in clinical trials or approved for therapeutic use [SEP]
[CLS] these structures are promising because of their multifunctionality , which directly relates to their relatively large size and often complex architectures when compared with conventional small molecules or biologics . [SEP]
[CLS] however , due to this complexity , little attention has been paid to how structural changes inform biological activity . [SEP]
[CLS] consider , for example , spherical nucleic B-material acids I-material ( snas ) , which are made by chemically arranging short sequences of dna or rna around a nanoparticle B-nanoparticle core B-material ( fig . 1a ) [SEP]
[CLS] snas exhibit properties that are substantively different from the short , linear oligonucleotides that comprise them , including the ability to actively cross mammalian cell B-material membranes without the need for transfection reagents , a resistance to nuclease degradation , and the ability to carry large and complex cargo ( such as oligonucleotides and peptides B-material ) into many cell B-material types . [SEP]
[CLS] these properties make snas an attractive candidate in cancer immunotherapy , as structures with dual functionality can be rapidly prepared from lipids B-material , oligonucleotide adjuvants and peptide B-material antigens . [SEP]
[CLS] when delivered to antigen presenting cells B-material ( apcs ) , snas activate the immune system and , in a lymphoma model , show superior activity compared with the same free antigen and linear oligonucleotides [SEP]
[CLS] however , the modularity of an sna allows for a large number of possible designs and compositions , and identifying the nanoparticle B-nanoparticle architectures best for inducing multiple aspects of cellular immune responses , such as potency , selectivity and efficacy , remains a challenge . [SEP]
[CLS] furthermore , understanding how variations in sna structure influence any individual step in generating immune responses at the cellular level ( for example , toll - like receptor B-material ( tlr ) activation and antigen presentation ) is also challenging , particularly if the dependence on activity is nonlinear across multiple variables . [SEP]
[CLS] here , we describe a new approach for synthesizing a library of snas that are qualitatively similar but structurally distinct , in conjunction with a mass spectrometry - based screening protocol that can rapidly and quantitatively determine the ability of an sna structure to activate the tlr9 pathway . [SEP]
[CLS] first , we show how this methodology can be used to make and screen ~ 1 , 000 sna architectures ( 800 of which are unique ) . [SEP]
[CLS] in addition , we describe how machine learning models can be trained with this data and subsequently used to accurately predict the tlr9 stimulatory activity of snas based on structural features . [SEP]
[CLS] significantly , these models provide a ranking of the order of importance of 11 structural parameters , as well as sna drug concentration . [SEP]
[CLS] the library screen and analysis by machine learning revealed several non - intuitive and nonlinear consequences of structural variation on tlr9 activation ; identification of these relationships was made possible only by the parallel examination of multiple variables . [SEP]
[CLS] collectively , these insights have important implications in the design of sna - based therapeutics . [SEP]
[CLS] additionally , since this methodology can be extended to other nanotherapeutics , this work points towards a new way of designing and optimizing nanomedicines for a wide variety of uses . [SEP]
[CLS] modular design of snas . [SEP]
[CLS] immunostimulatory snas consist of three modular components - the nanoparticle B-nanoparticle core B-material , oligonucleotide shell B-material and peptide B-material antigen - each of which can be arranged in a variety of configurations [SEP]
[CLS] to establish an appropriate library for high - throughput evaluation , we focused on 11 properties across these components ( fig . 1b ) . [SEP]
[CLS] we used 1 , 2 - dioleoyl - snglycero - 3 - phosphocholine ( dopc ) and 1 , 2 - dioleoyl - sn - glycero - 3 - phosphoethanolamine ( dope ) to form liposomes B-nanoparticle that are biocompatible B-property , straightforward to synthesize and capable of encapsulating the antigen [SEP]
[CLS] we focused on two liposome B-nanoparticle core B-material sizes with average diameters of ~ 70 and ~ 100 nm that were made from dopc or a mixture of 80 % dopc and 20 % dope , respectively . [SEP]
[CLS] the size of the sna can influence its rate of cellular uptake , and inclusion of dope in the liposomes B-nanoparticle is believed to affect the peptide B-material release rate and endosomal escape , which is important for peptide B-material processing . [SEP]
[CLS] the oligonucleotide shell B-material serves two roles . [SEP]
[CLS] it facilitates cellular uptake and serves as the adjuvant , which activates the innate immune system in a sequence - specific manner . [SEP]
[CLS] the oligonucleotides used in the design of snas in the library varied in five ways : sequence , backbone chemistry , conjugation chemistry to the liposome B-nanoparticle , site of lipid B-material functionalization and surface density of presentation by snas ( albeit over a narrow range ) . [SEP]
[CLS] we chose a cpg dna oligonucleotide ( odn1826 ) known to activate mouse tlr9 , as well as an inactive control where the cpg motif is inverted to gpc . [SEP]
[CLS] tlr9 is an endosomal protein B-material that recognizes unmethylated cpg oligonucleotides associated with bacteria and viruses . [SEP]
[CLS] to explore the importance of backbone composition , we synthesized linear oligonucleotides with either phosphodiester ( po ) or phosphorothioate ( ps ) backbones , since phosphorothioate oligonucleotides are known to induce higher immune activation , but snas comprising phosphodiester backbones present activities comparable to phosphorothioate structures [SEP]
[CLS] we evaluated distinct strategies for conjugating oligonucleotides to the nanoparticles B-nanoparticle by preparing structures with cholesterol or dope , both of which insert into the liposomal B-nanoparticle cores B-material and can be chemically attached to the 3 ′ or 5 ′ ends of the oligonucleotides . [SEP]
[CLS] finally , since oligonucleotide density is known to influence cellular uptake and protein B-event binding I-event of snas , we evaluated the oligonucleotide surface density at 0 . 5 , 1 and 2 pmol cm −2 ( referred to as 1× , 2× and 4× , respectively ) . [SEP]
[CLS] the 4× structure represents the upper limit of what is synthetically viable via our high - throughput procedures at present . [SEP]
[CLS] as our test case , we chose the ova 257 - 264 peptide B-material from ovalbumin - a well - studied model antigen . [SEP]
[CLS] since peptide B-material properties can vary dramatically with amino B-material acid I-material composition , we also tested a peptide B-material antigen from the e7 protein B-material of human papillomavirus17 . [SEP]
[CLS] to study how the release rate of the antigen influences nuclear factor kappa light chain enhancer of activated b cells B-material ( nf - κb ) activation , we evaluated snas wherein the antigen was either encapsulated within the sna architecture or chemically conjugated to oligonucleotides complementary to the cpg oligonucleotides and associated with snas through nucleic B-material acid I-material hybridization . [SEP]
[CLS] as a control , we investigated how the addition of a complement affects tlr9 stimulation . [SEP]
[CLS] we synthesized and tested three subsets of snas ( ova - encapsulated snas , e7encapsulated snas and surface - presented ova snas ) representing the key possible combinations of the parameters , with a few synthesis - limited exceptions noted below regarding lipid B-material composition , oligonucleotide surface density and surface - conjugated peptide B-material antigen ( see methods and table 1 ) . [SEP]
[CLS] variation across the 11 structural features - spanning the nanoparticle B-nanoparticle core B-material , oligonucleotide chemistry , surface presentation of oligonucleotides and incorporation of antigen - led to the design of a library with 960 total snas , 800 of which are unique . [SEP]
[CLS] to enable the screening of sna libraries , we developed a high - throughput assay for the rapid and quantitative measurement of cellular responses to the snas ( fig . 2a ) . [SEP]
[CLS] we cultured raw - blue macrophages in 384 - well plates and treated each well with a distinct sna at 4 oligonucleotide concentrations between 1 nm and 1 μm ( each separated by a factor of 10 ) . [SEP]
[CLS] raw - blue cells B-material are engineered to secrete secreted embryonic alkaline phosphatase ( seap ) on activation of nf - κb ( a major transcription B-event factor that is activated by tlr9 signalling ) , as well as other signals , to regulate the immune response . [SEP]
[CLS] we collected the culture media and determined the concentration of seap using samdi ( self - assembled monolayers for maldi , where maldi stands for matrix - assisted laser desorption / ionization B-property ) mass spectrometry - a label - free assay for high - throughput , quantitative analysis of enzymatic activity [SEP]
[CLS] samdi uses monolayers presenting a selective capture chemistry against a background of non - binding tri ( ethylene glycol ) groups to isolate substrates and products from a complex mixture . [SEP]
[CLS] subsequent analysis of the monolayers by maldi mass spectrometry ( maldi - ms ) quantitates the amount of substrate and product , which is a direct measure of the enzyme concentration ( fig . 2b , c ) . [SEP]
[CLS] here , we mixed the media containing seap with a phosphorylated peptide B-material substrate , captured the substrate and dephosphorylated product on monolayers and then analysed the samples by samdi ( see methods for experimental details ) . [SEP]
[CLS] we chose this technology for its ability to quantify enzyme activities at high throughput , without dependence on the common optical methods , which can be negatively affected by the light scattering and absorbance of the nanoparticles B-nanoparticle . [SEP]
[CLS] these artefacts are difficult to correct because of their dependence on nanoparticle B-nanoparticle properties such as size , concentration and aggregation . [SEP]
[CLS] furthermore , samdi is compatible with small sample volumes for analysis , thereby reducing the amounts of snas , cells B-material and reagents necessary for evaluation ( by around sixfold compared with the amounts used in optical assays ) . [SEP]
[CLS] with this assay , we measured the responses to 960 snas at 4 concentrations and with 2 biological replicates , and acquired 2 samdi spectra for each sample . [SEP]
[CLS] along with standards and controls , more than 8 , 500 cell B-material culture wells were used , and more than 17 , 000 samdi spectra were analysed . [SEP]
[CLS] these data revealed many insights into the importance of each structural feature , and how the combinations of features impact immune activation . [SEP]
[CLS] below , we highlight some of the most prominent trends . [SEP]
[CLS] varying the design parameters of snas induced a broad range of immune activation ( figs . 3a , d shows the encapsulated ova subset with the active cpg oligonucleotide sequence , and supplementary fig . 1 shows the encapsulated e7 subset ) . [SEP]
[CLS] almost all of the snas with the active oligonucleotide sequence outperformed the linear phosphodiester oligonucleotide . [SEP]
[CLS] additionally , many snas , including those with a phosphodiester backbone , were more potent than the linear oligonucleotide with the phosphorothioate backbone . [SEP]
[CLS] with 11 design parameters under investigation , we sought to identify the relative importance of design choices on immune activation . [SEP]
[CLS] multifactor analysis of variance ( anova ) ( supplementary table 1 ) revealed , unsurprisingly , that oligonucleotide concentration and oligonucleotide sequence ( that is , active or control ) heavily influenced activation . [SEP]
[CLS] after sequence , the feature that had the greatest impact on immune activation was the lipid B-material moiety conjugated to the oligonucleotide for liposome B-nanoparticle attachment . [SEP]
[CLS] cholesterol conjugation resulted in higher levels of immune activation than dope conjugation ( p = 4 . 8 × 10 −16 ) . [SEP]
[CLS] however , snas with cholesterol - conjugated oligonucleotides without cpg motifs also induced similarly high levels of activation at the 1 μm oligonucleotide concentration ( [ seap ] : 798 and 747 ng ml −1 for active and inactive , respectively ; fig . 3b ) , indicating a sequenceindependent activation of tlr9 . [SEP]
[CLS] the linear oligonucleotide does not activate tlr9 ; therefore , these results indicate that these snas may activate nf - κb via another mechanism . [SEP]
[CLS] one possible explanation is that cholesterol groups delivered to cells B-material on the sna induce additional activation . [SEP]
[CLS] our cholesterol conjugation chemistry utilizes carbamates , which can be cleaved by esterases , including sterol o - acyltransferases [SEP]
[CLS] any potentially released cholesterol , which is known to activate the unfolded protein B-material response pathway in macrophages , may also induce nf - κb activation . [SEP]
[CLS] in contrast , snas without cpg - containing oligonucleotides conjugated to dope ( instead of cholesterol ) lead to dramatically lower secretion of seap compared with their cholesterolconjugated counterparts ( p < 1 × 10 −16 ; fig . 3c ) . [SEP]
[CLS] we conclude that dope conjugation provides a way to synthesize snas that trigger an innate immune response exclusively through activation of tlr9 . [SEP]
[CLS] however , the combination of tlr9 stimulation and nonspecific activation by snas with cholesterol - conjugated oligonucleotides may be advantageous for inducing a greater overall immune response . [SEP]
[CLS] because of the dominant effects of conjugation chemistry , we analysed the remaining sna properties separately for snas with cholesterol - and dope - conjugated oligonucleotides . [SEP]
[CLS] interestingly , we observed differences in the preferred conjugation terminus when different conjugation chemistries were used ( fig . 3e , f ) . [SEP]
[CLS] with cholesterol conjugation , 5 ′ - conjugated snas showed significantly higher activity than 3 ′ - conjugated snas ( ova subset : p < 2 . 2 × 10 −16 for all concentrations ; mean [ seap ] : 566 and 439 ng ml −1 at 100 nm for 5 ′ and 3 ′ conjugation , respectively ) ; however , dope - conjugated snas did not show a difference with conjugation terminus ( ova subset : p = 1 for all concentrations ; mean [ seap ] : 324 and 330 ng ml −1 at 100 nm for 5 ′ and 3 ′ conjugation , respectively ) . [SEP]
[CLS] furthermore , conjugation from the 5 ′ terminus did not lead to loss of immune activation for either conjugation chemistry , which contradicts reports that modifications at the 5 ′ end inactivate the tlr9 activity of linear cpg oligonucleotides [SEP]
[CLS] similar to well - known trends with linear oligonucleotides , the oligonucleotide backbone also influenced the immunostimulatory activity of the snas ( supplementary table 1 and fig . 3g , h ) [SEP]
[CLS] snas with phosphorothioate backbones generally outperformed their phosphodiester counterparts ( p = 5 × 10 −9 for dope - and p = 2 . 7 × 10 −4 for cholesterolconjugated snas ) . [SEP]
[CLS] however , a more pronounced dependence on oligonucleotide backbone was observed with dope - conjugated snas than with cholesterol - conjugated snas . [SEP]
[CLS] for dope - conjugated snas , the mean seap concentrations were 191 and 463 ng ml −1 for phosphodiester and phosphorothioate backbones , respectively , whereas for cholesterolconjugated snas , they were 431 and 573 ng ml −1 ( all at 100 nm ) . [SEP]
[CLS] in contrast , at the highest concentration of 1 μm , snas with phosphodiester oligonucleotides outperformed their phosphorothioate counterparts . [SEP]
[CLS] notably , the activity induced by dope - conjugated snas with phosphorothioate oligonucleotides consistently decreased when the oligonucleotide concentration increased from 100 nm to 1 μm . the dope - conjugated phosphorothioate linear oligonucleotide , but not the phosphodiester backbone , showed a similar reduction in activity at 1 μm ( fig . 3h ) , suggesting that this behaviour is due to the specific stimulatory properties of the dope - conjugated oligonucleotide . [SEP]
[CLS] these results lead us to conclude that dope - conjugated oligonucleotides with phosphorothioate backbones provide an advantage if greater potency is desired . [SEP]
[CLS] phosphorothioate backbones have the added benefit of resistance to nuclease degradation in vivo [SEP]
[CLS] however , these results also show that snas with oligonucleotides composed of phosphodiester backbones can achieve similar levels of activation when present at higher concentrations . [SEP]
[CLS] while class b cpg oligonucleotides are less effective with phosphodiester backbones , using snas with phosphodiester oligonucleotides may be worth the loss in potency because of the reduction in toxicity B-property and cost , since the sna structure may provide sufficient resistance to nuclease activity [SEP]
[CLS] oligonucleotide density on the surface of the nanoparticle B-nanoparticle has a small and variable impact on immune activation . [SEP]
[CLS] surprisingly , there was not a strong or consistent trend in how oligonucleotide density affected activity , with neither the highest nor lowest densities showing the best activity . [SEP]
[CLS] in previous studies , snas with higher oligonucleotide densities led to higher biological activity in cellular uptake and rnase h - mediated degradation of messenger rna ; however , the nanoparticle B-nanoparticle designs in those studies were limited to gold B-material cores B-material , and used different core sizes and oligonucleotide densities compared with this study [SEP]
[CLS] from these observations , we conclude that the choice of oligonucleotide density for these constructs over this narrow density range should be based on other considerations , such as stability in vivo , which is inextricably linked to potency . [SEP]
[CLS] core B-material diameter and lipid B-material composition influence the immune activation of snas in an encapsulated peptide B-material - specific manner . [SEP]
[CLS] in both encapsulated sna subsets , the lipid B-material composition generally did not have a significant impact on activity , as determined by anova ( supplementary table 1 ) , except in one particular context discussed below . [SEP]
[CLS] additionally , core B-material diameter was not a significant parameter in the encapsulated ova subset , whereas it had a significant impact with encapsulated e7 group . [SEP]
[CLS] since all combinations of parameters evaluated were both with and without peptide B-material , we were able to isolate the effects of peptide B-material encapsulation by comparing pairs of snas with identical properties except for the amount of peptide B-material encapsulation . [SEP]
[CLS] we subtracted the seap concentration of the sna without peptide B-material from the sna with the highest peptide B-material concentration ( fig . 4a ) . [SEP]
[CLS] this analysis revealed that core B-material diameter and lipid B-material composition were influential when e7 , but not ova , was encapsulated . [SEP]
[CLS] specifically , for the e7 subset , snas with 100 nm cores B-material containing peptide B-material induced higher levels of nf - κb activation ( p = 5 . 7 × 10 −5 ) , and the magnitude of this effect also depended on lipid B-material composition ( fig . 4b ) . [SEP]
[CLS] within the subset of snas with cholesterol - conjugated oligonucleotides on 100 nm cores B-material , the snas with 100 % dopc cores B-material showed higher immune activation than those with 80 % dopc and 20 % dope cores B-material ( p = 0 . 0011 ; fig . 4b ) . [SEP]
[CLS] we observed no dependence between the presence of antigen and immune activation when the antigen was ova ( fig . 4c ) . [SEP]
[CLS] these results clearly illustrate that peptide B-material encapsulation can impact the ability of snas to activate tlr9 and reveal crosstalk between the molecular components of snas intended to induce innate or adaptive immunity . [SEP]
[CLS] unlike oligonucleotides , the physicochemical properties of peptides B-material vary dramatically with sequence , which can affect their interaction with the rest of the sna structure . [SEP]
[CLS] for example , the differences in the isoelectric points of the peptides B-material , which are 5 . 7 and 8 . 8 for the e7 and ova peptides B-material , respectively , result in different net charges for the peptides B-material , which could affect their interaction with the positively charged liposome B-nanoparticle core B-material . [SEP]
[CLS] we conclude that the interactions between liposomes B-nanoparticle and peptides B-material must be taken into account when designing and evaluating nanomedicines , as they can lead to large shifts in the immune activation of snas , especially at high levels of peptide B-material encapsulation . [SEP]
[CLS] the versatility of the sna architecture allows for alternative methods of incorporating the antigen into the structure , apart from loading in the lipid B-material core B-material . [SEP]
[CLS] we investigated one such alternative - conjugation of the antigen to a complementary oligonucleotide , which is then hybridized to a lipid - anchored oligonucleotide . [SEP]
[CLS] as a control , we also synthesized snas with the complementary oligonucleotide but without peptide B-material conjugation . [SEP]
[CLS] in these snas , the cpg - containing oligonucleotide is double stranded , and thus is differentiated from snas with only single - stranded oligonucleotides . [SEP]
[CLS] in this conjugated ova subset , we used dopeconjugated oligonucleotides to prevent the non - specific nf - κb activation by cholesterolconjugated snas described above . [SEP]
[CLS] our results show that snas synthesized with this strategy shared some trends with their single - stranded counterparts . [SEP]
[CLS] after oligonucleotide sequence , the most influential property on immune activation was backbone chemistry , with phosphorothioate backbones outperforming phosphodiester versions ( fig . 5a ) . [SEP]
[CLS] again , we found that the core B-material properties of lipid B-material composition and core B-material diameter were not significant . [SEP]
[CLS] interestingly , for the snas with phosphorothioate oligonucleotides , addition of the complement oligonucleotide ( either to half or all of the anchored oligonucleotides ) did not change immune activation at concentrations of 100 nm or 1 μm , respectively ( fig . 5b ) . [SEP]
[CLS] furthermore , there was no difference between snas composed of the complement with and without conjugated peptide B-material . [SEP]
[CLS] however , at low concentrations ( 10 nm ) , higher complement densities led to higher immune activation ( fig . 5c ) . [SEP]
[CLS] this effect may be a function of sna uptake , where higher complement densities create higher charge densities on the surface and increase the uptake of snas , which in turn leads to higher immune activation . [SEP]
[CLS] in contrast , complementation strongly reduced the activity of phosphodiester - backbone snas at the highest concentration tested ( fig . 5d ) . [SEP]
[CLS] a possible explanation for the decreased activity in duplexed snas is that the duplexing interferes with the oligonucleotide interaction with tlr9 ; however , it is not clear why the interaction with tlr9 would be different with phosphodiester and phosphorothioate backbones . [SEP]
[CLS] these results suggest that the strategy of including antigens by duplexing antigen - conjugated complementary oligonucleotides is effective with phosphorothioate snas , without concern for losing activation of tlr9 . [SEP]
[CLS] because many of the parameters studied were interdependent , we utilized supervised machine learning models and evaluated their performance to better understand sna properties . [SEP]
[CLS] we applied supervised models to automatically predict immune activity from sna properties with the expectation that properties relevant to immune activation would improve a model ' s predictive capability . [SEP]
[CLS] these models differ from traditional data analysis in that instead of explicitly programming them with formulas , they ' learn ' from the data on their own . [SEP]
[CLS] specifically , we employed multiple linear regression , logistic regression and nonlinear xgboost to fit training data , and cross - validation of test data was conducted using the q 2 statistic . [SEP]
[CLS] q 2 quantifies the accuracy of the predicted seap concentrations against measured values , and ranges from −∞ to 1 , where 0 indicates no predictive power ( equivalent to predicting the mean ) and 1 indicates perfect prediction [SEP]
[CLS] we trained each model with all combinations of properties ( that is , two properties at a time , three properties at a time , and so on ) and analysed their q 2 performance . [SEP]
[CLS] as additional properties were added to the models , the q 2 performance increased , plateauing for most models and decreasing in the xgboost model for the surface - presented ova subset ( fig . 6a , b ) . [SEP]
[CLS] since clear nonlinear trends were observed in the data , as described above , the model performance increased with the nonlinearity of the model in both subsets ( mean increase from 0 . 53 for the linear model to 0 . 83 for xgboost ) . [SEP]
[CLS] analysis of the most predictive sna property combinations demonstrates that highly predictive properties remain significant and informative as more properties are introduced into the model ( supplementary fig . 2a , b ) . [SEP]
[CLS] in addition , the order of importance of the properties was largely consistent between the encapsulated ova and surface - conjugated ova subsets , suggesting that the ordering is robust regardless of peptide B-material localization . [SEP]
[CLS] to ensure that these trends are not artefacts , we repeated this analysis with randomized data . [SEP]
[CLS] q 2 values for all of these models were zero or below , indicating that the predictions are specific to our data ( supplementary fig . 3 ) . [SEP]
[CLS] in addition , we calculated the standard deviations of the error between the predicted and actual values for the xgboost to further validate the model ( supplementary fig . 4 ) . [SEP]
[CLS] the standard deviations were in the range of 10 - 30 ng ml −1 , which is very small compared with the range of activities that exceeds 1 , 000 ng ml −1 . [SEP]
[CLS] for the encapsulated ova and surface - presented ova subsets , the q 2 value stopped increasing beyond five and four properties , respectively ( fig . 6a ) . [SEP]
[CLS] at first glance , one might conclude that only these highly predictive properties are relevant ; however , when repeating this analysis with fixed values for sequence and concentration ( the two features with the greatest impact ) , the q 2 values stopped increasing after another five properties were added ( fig . 6a ) , indicating that formerly seemingly non - predictive properties do , in fact , influence immune activation ( supplementary fig . 2c ) . [SEP]
[CLS] taken together , these properties , which appear non - influential in a global context , become impactful in a restricted design space . [SEP]
[CLS] capturing the maximum structure - activity relationship with minimum sna synthesis and evaluation . [SEP]
[CLS] next , we investigated whether a similar q2 level is attainable with fewer , randomly selected sna designs . [SEP]
[CLS] this question is particularly relevant when synthesis and evaluation of full libraries are impractical , but when exploration of a large design space is desired . [SEP]
[CLS] in this case , one could synthesize a random subset that would capture the most important trends and then suggest additional candidates to evaluate . [SEP]
[CLS] to this end , we simulated this process by training an xgboost model on a random selection of snas and testing predictions on the remaining , unselected snas within the three subsets ( fig . 6c ) . [SEP]
[CLS] we identified the points of diminishing returns , which balance the minimum number of snas with maximum q 2 , by calculating the sample size closest to training size 1 and q 2 = 1 . [SEP]
[CLS] this point is 90 , 20 and 31 snas ( out of 336 , 336 and 288 snas ) with q 2 = 0 . 67 , 0 . 88 and 0 . 66 for the encapsulated e7 , encapsulated ova and surface - presented ova subsets , respectively . [SEP]
[CLS] these points represent a mean of 16 % of the total number of snas , suggesting that a small number of randomly selected snas can predict structure - activity relationships of a relatively large sna library . [SEP]
[CLS] in practice , this external q 2 ( prediction of non - synthesized snas ) cannot be measured with a randomized subsample , but an internal q 2 can be measured by cross - validating within the randomized subsample . [SEP]
[CLS] we show that the internal and external q 2 values are highly correlated ( fig . 6d and supplementary fig . 5 ) , suggesting that we can identify points of diminishing returns as we continually synthesize random snas from an arbitrary library size . [SEP]
[CLS] combined with the high - throughput sna synthesis and characterization approach described above , the machine learning analysis shows that a combined experimental and computational method can probe and predict the structure - activity relationships of tens of thousands of snas with a much smaller subset ( order of thousands ) of structures . [SEP]
[CLS] this work , as well as other approaches , makes clear the need to consider the full range of structure - activity relationships when designing nanomedicines by high - throughput processes . [SEP]
[CLS] although high - throughput techniques are industry standards in the combinatorial screening of small - molecule drugs , such approaches are just beginning to be implemented to define structure - activity relationships for therapeutic nanoconstructs . [SEP]
[CLS] the data presented here show that such properties can be strongly interrelated in non - obvious ways , and emphasize the risks of using limited datasets to make global conclusions about one structural consideration being more critical than others . [SEP]
[CLS] our results show that predictions about sna activity simply based on what is known about the individual components of an sna ( that is , cpg , ova , e7 and phospholipids ) are inaccurate in many cases . [SEP]
[CLS] this interdependence and non - linearity are underscored when applying the nonlinear machine learning models , as opposed to linear ones , in predicting the biological response of snas . [SEP]
[CLS] indeed , to realize rational approaches to vaccinology , this work makes a strong case for the combination of high - throughput experimentation and computational analysis in determining the structureactivity relationships of nanomedicines in general and snas in particular . [SEP]
[CLS] note that this study did not directly pursue the identification or optimization of a candidate immunotherapeutic for a specific disease . [SEP]
[CLS] rather , this effort has examined how a single key biochemical step ( tlr9 activation ) in the generation of an immune response can be activated by nearly 1 , 000 variations in sna structure . [SEP]
[CLS] a key finding from our use of supervised machine learning to analyse the data generated for ~ 1 , 000 sna structures is the accuracy of predicting activities when using data obtained only from relatively small sublibraries . [SEP]
[CLS] however , a broader use of this approach will require the careful design of structures in the random subsets to ensure the validity of predictions , and follow - up with experimental confirmation of predicted activities . [SEP]
[CLS] in our study , we applied the machine learning independently for libraries defined by the selection of antigen and position of the antigen , with the benefit of knowing that the selection of antigen influences activity . [SEP]
[CLS] this type of approach to structure - activity relationships is limited to predictions based on properties included in the training set . [SEP]
[CLS] in addition to the unavoidable potential of missing unique combinatorial effects that are not captured in the sampled space , the accuracy and scalability of prediction that can be accomplished by supervised machine learning may differ for libraries consisting of other types of nanostructures . [SEP]
[CLS] our constraint to a single readout and its dependence on a wide range of variables has led to a lesson in the design and development of a type of nanomedicine that could not have been extracted by the alternative and more conventional approach of evaluating a small number of candidate structures and analysing multiple immune system readouts ( for example , cytokine expression or cellular proliferation ) in vivo ; such an approach is severely limited in throughput by the small number of structures that can be prepared and evaluated in parallel . [SEP]
[CLS] conversely , we note that tlr9 activation in a model cell B-material line reports on one key step among many involved in raising cellular immune responses ( for example , biodistribution , antigen delivery and presentation by different types of apcs ) , and that structures optimized for a single output may not be the best therapeutic candidates when examined in vivo . [SEP]
[CLS] the pursuit of identifying and ultimately arriving at sna ( or other nanoparticle B-nanoparticle ) immunotherapeutic agents will be well served by the combination of using high - throughput library screens in cellular assays , and in vivo examination of structures whose selection is informed by the library screen . [SEP]
[CLS] dope and dopc were purchased from avanti polar lipids B-material . [SEP]
[CLS] phosphoramidites for dna synthesis were purchased from glen research . [SEP]
[CLS] peptide B-material antigens were custom ordered from genscript . [SEP]
[CLS] 2 , 2 ' - dipyridyldisulfide , hexadecylphosphonic acid , tris ( 2carboxyethyl ) phosphine hydrochloride , maleimide and 2 , 4 , 6 - trihydroxyacetophenone were purchased from sigma - aldrich . [SEP]
[CLS] monolayer disulfides were purchased from chemtos . [SEP]
[CLS] peptide B-material synthesis reagents were purchased from anaspec and milliporesigma . [SEP]
[CLS] dna was synthesized with a mermade 12 synthesizer . [SEP]
[CLS] cholesterol modification was done on the column in the synthesizer using 3 ′ - cholesteryl - teg cpg ( glen research ) for 3 ′ modifications and cholesteryl - teg phosphoramidite ( glen research ) for 5 ′ modifications . [SEP]
[CLS] for dope - modified oligonucleotides , a thiol - modified oligonucleotide was synthesized . [SEP]
[CLS] dna sequences are shown in supplementary table 2 . [SEP]
[CLS] to 1 mol equivalent of succinimidyl 4 - ( p - maleimidophenyl ) butyrate ( smpb ; thermo fisher scientific ) and 1 mol equivalent of n , n - diisopropylethylamine was added 1 ml of dope as received from avanti polar lipids B-material ( 25 mg ml −1 in chloroform ) . [SEP]
[CLS] the reaction was incubated B-technique for 24 h at room temperature . [SEP]
[CLS] the reaction was checked for completion with thin - layer chromatography B-technique using 20 % methanol in dichloromethane as the mobile B-property phase . [SEP]
[CLS] on disappearance of the dope band in thin - layer chromatography B-technique , the reaction was washed three times with water B-material , and the organic phase was dried under vacuum . [SEP]
[CLS] the thiol B-material - modified oligonucleotide was reduced with 200 mm dithiothreitol ( dtt ) in 100 mm phosphate buffer ( ph 8 . 0 ) for 2 h at 40 °c . [SEP]
[CLS] the oligonucleotide was purified away from dtt with nap - 10 columns using water B-material as the mobile B-property phase ( ge healthcare ) . [SEP]
[CLS] the reduced oligonucleotide was immediately reacted with dope - smpb as follows : dope - smpb ( 50 mol equivalents ) was dissolved in ethanol in the same volume as the oligonucleotide . [SEP]
[CLS] the two solutions were mixed together and incubated B-technique at room temperature for 48 h . [SEP]
[CLS] the reaction mixture was washed with chloroform three times to remove excess lipid B-material . [SEP]
[CLS] the interface and the aqueous phase was lyophilized . [SEP]
[CLS] the reaction yield and purity were determined by 20 % denaturing polyacrylamide B-technique gel I-technique electrophoresis I-technique gels I-technique . [SEP]
[CLS] typically , yields were greater than 90 % and no further cleanup was performed . [SEP]
[CLS] dopc ( 25 mg in chloroform ) was transferred to a glass vial and dried overnight into a thin film , first under an n 2 stream followed by high vacuum . [SEP]
[CLS] for dopc - dope mixture liposomes B-nanoparticle , 20 mol % dope was added to the 25 mg of dopc before drying . [SEP]
[CLS] the lipid B-material film was rehydrated with 1 ml of 1× phosphate buffered saline ( pbs ) and vortexed until no more clumps were visible . [SEP]
[CLS] for encapsulated peptides B-material , the peptide B-material was dissolved into the pbs at 0 . 1 and 1 mg ml −1 . [SEP]
[CLS] the lipid B-material suspensions were frozen in liquid nitrogen B-material and thawed in a bath sonicator with sonication . [SEP]
[CLS] the freeze - thaw was repeated three times . [SEP]
[CLS] the solution was then extruded through 200 , 100 , 80 and 50 nm filters . [SEP]
[CLS] two filters were used for each extrusion , and the solution was passed through these filters 11 times . [SEP]
[CLS] the liposomes B-nanoparticle were split into 2 after the 80 nm extrusion . [SEP]
[CLS] half of the solution was saved , and the remainder was extruded through a 50 nm filter . [SEP]
[CLS] the liposomes B-nanoparticle were dialysed against 1× pbs overnight to remove non - encapsulated peptide B-material . [SEP]
[CLS] the liposomes B-nanoparticle were characterized by dynamic B-technique light I-technique scattering I-technique for size ( z - average reported ) , and phosphatidylcholine assay for concentration ( milliporesigma ) . [SEP]
[CLS] the liposomes B-nanoparticle extruded through the 50 and 80 nm filters had z - averages of ~ 70 and ~ 100 nm , respectively . [SEP]
[CLS] for dopc - dope mixture snas , dope did not interfere with the phosphatidylcholine assay , so we assumed that the dopc - to - dope ratio remained 80 : 20 . [SEP]
[CLS] the liposome B-nanoparticle concentrations were calculated from the diameter and the lipid B-material concentration , as described by banga et al . . [SEP]
[CLS] the complementary oligonucleotides were reduced with dtt as described above and mixed with 55 equivalents of 2 , 2 ' - dipyridyldisulfide in 100 mm phosphate buffer ( ph 8 . 0 ) . [SEP]
[CLS] the reaction was incubated B-technique at 40 °c for 24 h . [SEP]
[CLS] the reaction process was monitored by absorption of pyridinethione at 343 nm . [SEP]
[CLS] on completion , the modified oligonucleotide was washed 3 times with water B-material in a 3k mwco spin filter . [SEP]
[CLS] the oligonucleotide was then mixed with 1 equivalent of cysteine - modified ova ( csiinfekl ) and incubated B-technique at 40 °c overnight . [SEP]
[CLS] the process was again monitored at 343 nm and washed with a spin filter as described above . [SEP]
[CLS] the purified peptide B-material - oligonucleotide conjugate and 1 equivalent of the lipid - conjugated oligonucleotide were mixed in duplex buffer ( 30 mm hepes ( ph 7 . 4 ) , 100 mm potassium B-material acetate and 2 mm magnesium B-material acetate ) . [SEP]
[CLS] the mixture was heated to 65 °c for 10 min and slow - cooled to room temperature by turning off the heat block and allowing the temperature to equilibrate . [SEP]
[CLS] lipid - modified oligonucleotides or duplexes were mixed with liposomes B-nanoparticle in a 384 - well plate in a 40 µl final volume . [SEP]
[CLS] the final concentration of lipid - modified oligonucleotide or duplex in each well was 10 µm . [SEP]
[CLS] the concentration of liposomes B-nanoparticle was adjusted to accommodate snas of various oligonucleotide densities . [SEP]
[CLS] after mixing , the plate was sealed and incubated B-technique at room temperature for 24 h . [SEP]
[CLS] the crpy - nh2 peptide B-material substrate was synthesized using standard fluorenylmethoxycarbonyl solid - phase peptide B-material synthesis methods on a rink amide B-material resin . [SEP]
[CLS] the amino terminus was acetylated . [SEP]
[CLS] the peptide B-material was purified by reverse - phase high - performance liquid B-technique chromatography I-technique on a c - 18 column in a gradient from water B-material to acetonitrile , and fractions were checked for the correct mass by maldi - ms . [SEP]
[CLS] the peptide B-material was lyophilized and stored as a solid until use . [SEP]
[CLS] stainless steel plates custom designed for use in maldi instruments were cleaned and used to evaporate a 1 , 536 - spot pattern of 5 nm titanium B-material ( 0 . 02 nm s −1 ) , then 35 nm gold B-material ( 0 . 05 nm s −1 ) , using an aluminium mask . [SEP]
[CLS] the gold B-material array plates were incubated B-technique overnight at 4 °c in an ethanolic solution containing a 1 : 4 ratio of an asymmetric disulfide terminated with a maleimide B-material group I-material and a tri ( ethylene glycol ) group , and a symmetric disulfide terminated with tri ( ethylene glycol ) groups , with a 0 . 5 mm total disulfide concentration . [SEP]
[CLS] the plates were then rinsed with ethanol , dried and placed in a solution of 10 mm hexadecylphosphonic acid in ethanol for 10 min at room temperature . [SEP]
[CLS] plates were then rinsed with ethanol , dried and then used for the seap assay . [SEP]
[CLS] raw - blue cells B-material ( invivogen ) were cultured as described by the manufacturer . [SEP]
[CLS] the cells B-material were collected and suspended at 550 , 000 cells B-material ml −1 , and 17 , 000 cells B-material were distributed into 384well culture plates with a thermo fisher scientific multidrop combi . [SEP]
[CLS] next , 10× sna solutions were added to the cell B-material culture plates with a tecan liquid handler , then cultured at 37 °c under 5 % co2 . [SEP]
[CLS] after ~ 16 h , the cell B-material culture plates were centrifuged at 300 rcf for 1 min , then 10 μl of media was transferred to a 384 - well reaction plate . [SEP]
[CLS] recombinant seap ( 0 - 1 , 600 ng ml −1 ) was prepared in media from untreated cells B-material and then added to empty wells . [SEP]
[CLS] this was used as the standard curve . [SEP]
[CLS] to minimize free thiol B-material in the media , which competes with substrate immobilization , 1 μl of 11 mm tris ( 2 - carboxyethyl ) phosphine hydrochloride in water B-material was added to the plates and they were incubated B-technique for 15 min at 60 °c to first reduce cystine to cysteine B-material . [SEP]
[CLS] the 60 °c incubation B-technique also inactivates any potential phosphatases other than seap , which is stable at 60 °c . [SEP]
[CLS] next , 1 μl of 12 mm maleimide was added to react with free cysteines B-material for 1 h at 37 °c . [SEP]
[CLS] some 8 μl of 75 μm crpy peptide B-material substrate in reaction buffer ( 300 mm tris , ph 8 . 5 and 2 . 5 mm mgcl2 ) was added to the reaction plate , then incubated B-technique for 1 h at 37 °c . [SEP]
[CLS] to this , 2 μl of 11 mm pridoxal 5 ′ - phosphate hydrate in reaction buffer was added . [SEP]
[CLS] next , 0 . 75 μl of the reaction solutions were transferred to 1 , 536 - spot samdi array plates and incubated B-technique for 1 h at 37 °c . [SEP]
[CLS] the plates were rinsed with water B-material and ethanol , then dried with air . [SEP]
[CLS] matrix ( 15 mg ml −1 2 , 4 , 6trihydroxyacetophenone in acetone ) was applied to the samdi plates and they were analysed by maldi using an applied biosystems sciex tof / tof 5800 maldi instrument in positive reflector mode . [SEP]
[CLS] the spectra were analysed by calculating the areas under the curves for the [ m + h ] + and [ m + na ] + disulfide peaks corresponding to the substrate and product masses . [SEP]
[CLS] each sna subset was tested in two wells ( biological a , assay used to evaluate the structure - activity relationships between sna properties and tlr9 activation of apcs . [SEP]
[CLS] libraries of snas are incubated B-technique with raw - blue macrophages in 384 - well plates . [SEP]
[CLS] the macrophages have been engineered to secrete seap into the media . [SEP]
[CLS] after ~ 16 h , the media is transferred , processed and mixed with a phosphorylated substrate . [SEP]
[CLS] the solution is transferred to samdi plates with 1 , 536 spot arrays of monolayers presenting maleimides to selectively capture the substrate and product by a maleimide - thiol reaction . [SEP]
[CLS] seap assay . b , an example samdi spectrum showing the immobilized substrate and product . [SEP]
[CLS] performing maldi - ms on the self - assembled monolayers ( that is , samdi ) results in mass spectra containing quantitative information on the relative amounts of substrate and product ( that is , the extent of dephosphorylation ) . [SEP]
[CLS] c , an example standard curve used to convert the samdi spectral data for the library into seap concentration . [SEP]
[CLS] sna design space : the total design space investigated in this study , divided into three subsets [SEP]
[CLS] sna architecture and design properties . [SEP]
[CLS] fig . 1 | a , the three components of immunostimulatory snas . [SEP]
[CLS] b , the parameters investigated for each of the sna design properties , organized by core B-material , antigen and oligonucleotide property categories . [SEP]
[CLS] the potential design space has 3 , 072 variants . [SEP]
[CLS] po , phosphodiester ; ps , phosphorothioate . [SEP]
[CLS] 2 | . samdi assay workflow [SEP]
[CLS] fig . 3 | trends in immune activation due to changes in the oligonucleotide properties of snas . [SEP]
[CLS] a , seap concentrations observed for all of the active - sequence snas in the encapsulated ova subset ( all data in this figure are from this subset ) , compared with the po and ps versions of linear oligonucleotides with the same active sequence . [SEP]
[CLS] b , c , comparison of snas with the active and control sequences , for groups of snas with cholesterol - conjugated oligonucleotides ( b ) and those with dope - conjugated oligonucleotides ( c ) . [SEP]
[CLS] d , dimension stacking plot of the active - sequence snas , showing the seap concentration for each combination of design properties . [SEP]
[CLS] larger and darker circles indicate greater seap concentration . [SEP]
[CLS] for core B-material lipid B-material composition : c , 100 % dopc lipid B-material composition ; c / e , mixture [SEP]
[CLS] fig . 4 | trends in immune activation due to changes in the peptide B-material encapsulation of snas . [SEP]
[CLS] a , difference in seap concentration between each sna with the highest peptide B-material concentration ( 10× ) and the corresponding sna without any peptide B-material ( 0× ) for the two encapsulated peptide B-material subsets . [SEP]
[CLS] b , c , average difference in seap concentration between snas with 10× and 0× e7 peptide B-material concentrations , grouped by core B-material diameter and the combination of conjugation chemistry ( cholesterol ( chol ) versus dope ) and lipid B-material composition , for the e7 ( b ) and ova subsets ( c ) , at 100 nm oligonucleotide concentration ( n = 16 for chol , c / e and chol , c ; n = 24 for dope , c ) . [SEP]
[CLS] bar heights and error bars represent means ± s . e . m . [SEP]
[CLS] p values were calculated by two - way anova with sidak ' s post - hoc test . [SEP]
[CLS] all differences between means with p < 0 . 05 are indicated ( * * * * p < 0 . 0001 ) . [SEP]
[CLS] fig . 5 | trends in immune activation due to hybridization . [SEP]
[CLS] a , seap concentrations for all active - sequence snas in the surface - presented ova subset . [SEP]
[CLS] b , mean seap concentration of ps - backbone , active - sequence snas , grouped by the combinations of complement density ( comp . ) and surface antigen density ( pep . ) ( n = 12 ) . [SEP]
[CLS] shaded areas indicate the response at 10 nm , 100 nm and 1 , 000 nm oligonucleotide concentration , from left to right . [SEP]
[CLS] c , mean seap concentration of snas with a phosphorothioate backbone , 0 % peptide B-material and active sequence , as a function of complement density , at a 10 nm oligonucleotide concentration ( n = 12 ) . [SEP]
[CLS] d , mean seap concentration of snas with a phosphodiester backbone and active sequence , as a function of complement density , at a 1 μm oligonucleotide concentration ( 100 % : n = 36 , 50 % : n = 24 , 0 % : n = 12 ) . [SEP]
[CLS] bar heights and error bars represent means ± s . e . m . [SEP]
[CLS] p values were calculated by one - way anova with tukey ' s post - hoc test . [SEP]
[CLS] all differences between means with p < 0 . 05 are indicated ( * * * * p < 0 . 0001 ) . [SEP]
[CLS] analysis by machine learning . [SEP]
[CLS] fig . 6 | a , b , q 2 values of the highest - performing sna property combinations are shown across different numbers of properties for encapsulated ova ( a ) and surface - presented ova subsets ( b ) . [SEP]
[CLS] in a , for the xgboost model , the active sequence and 100 nm subset is shown , in addition to both active and inactive sequences for all concentrations . [SEP]
[CLS] c , d , xgboost q 2 performance when selecting and training on a random sna subsample and testing predictions on the unselected snas ( c ) or cross - validating within the selected subsample ( d ) . [SEP]
[CLS] the shading represents 90 % confidence intervals . [SEP]
[CLS] the arrows point to the points of diminishing returns . [SEP]
[CLS] and 100 % na , not applicable ; po , phosphodiester ; ps , phosphorothioate . [SEP]
[CLS] nat biomed eng . author manuscript ; available in pmc 2019 april 08 . [SEP]
[CLS] glioblastoma ( gbm ) is the most common and lethal primary brain tumor B-material in adults , with nearly 100 % of patients ultimately succumbing to the disease . [SEP]
[CLS] median patient survival is 15 months , and no standard of care currently exists for recurrent cases . [SEP]
[CLS] glioma stem cells B-material ( gscs ) , a rare and highly aggressive subpopulation of cells B-material within these tumors B-material , have recently emerged as drivers of tumor B-material initiation and recurrence , and a growing body of evidence suggests that they must be completely eradicated to prevent relapse . [SEP]
[CLS] toward this goal , we have developed polyethyleniminewrapped spherical nucleic B-material acid I-material nanoparticles B-nanoparticle ( pei - snas ) targeting gli1 , a transcription B-event factor within the hedgehog signaling pathway that is crucial for the maintenance of gscs . [SEP]
[CLS] here , we demonstrate that gli1 pei - snas bind scavenger receptors on gbm cells B-material to undergo endocytosis B-event in a caveolae / lipid raft / dynamin - dependent manner . [SEP]
[CLS] they further achieve ~ 30 % silencing of tumor B-material - promoting hedgehog pathway genes and downstream target genes that promote the aggressive , chemoresistant phenotype of gbm . [SEP]
[CLS] this produces a 30 % decrease in proliferation that correlates with a robust onset of gbm cell B-material senescence as well as an ~ 60 % decrease in metabolic activity with or without cotreatment with temozolomide ( tmz ) , the frontline chemotherapy for gbm . [SEP]
[CLS] most importantly , gli1 pei - snas impair the self - renewal capacity of gbm cells B-material as indicated by a 30 - 40 % reduction in the expression of stemness genes and further impair the formation of stem - like neurospheres . [SEP]
[CLS] they also substantially improve neurosphere chemosensitivity as demonstrated by a 2 - fold increase in the fraction of cells B-material undergoing apoptosis B-event in response to low doses of tmz . [SEP]
[CLS] these results underscore the potential for sirna therapeutics * [SEP]
[CLS] rna interference ( rnai ) therapeutics have received tremendous attention recently for their potential to revolutionize the management of diseases with a known genetic basis . [SEP]
[CLS] in a clinical setting , rnai offers the ability to silence the expression of genes that promote disease progression with greater potency and specificity than small molecule drugs . [SEP]
[CLS] however , one unresolved challenge toward recognizing the clinical potential of rnai is maximally delivering sirna to the targeted disease site . [SEP]
[CLS] this is because sirna is highly susceptible to degradation by nucleases present ubiquitously in physiological conditions , is cleared rapidly from circulation , and cannot cross cellular membranes due its large size and negative charge . [SEP]
[CLS] as a result , there is a need to develop carriers to protect sirna and efficiently deliver it to sites of disease . [SEP]
[CLS] one of the greatest challenges in developing clinically translatable sirna carriers is achieving a balance between efficacy and toxicity B-property ; many strongly cationic B-material carriers that are highly effective for intra - cellular sirna delivery are also toxic B-property due to their tendencies to destabilize cellular membranes and trigger immune responses . [SEP]
[CLS] the reverse is also true : many materials with more favorable biocompatibility B-property profiles are less effective as transfection agents . [SEP]
[CLS] toward the goal of identifying carriers to maximize sirna delivery efficacy and minimize toxicity B-property , we have recently developed a hybrid delivery vehicle B-material consisting of a spherical nucleic B-material acid I-material ( sna ) core B-material and a polyethylenimine ( pei ) shell B-material that provides greater cellular uptake and endosomal escape as compared to highly biocompatible B-property snas and greater cytocompatibility and transfection B-property efficiency I-property as compared to pei - sirna polyplexes . [SEP]
[CLS] snas , which consist of radially oriented , densely arranged sirna stabilized on a gold B-nanoparticle nanoparticle I-nanoparticle core B-material , exhibit a controlled sirna architecture that imparts unique properties favoring sirna delivery to biological systems . [SEP]
[CLS] most notably , snas are rapidly taken up by > 50 cell B-material types , provide steric and electrostatic hindrances against endonucleases , and do not induce an immune response in animal models . [SEP]
[CLS] further , their successful delivery of sirna and mirna to glioblastoma tumors B-material has prompted the first clinical trial evaluating their use as therapeutics for glioblastoma and gliosarcoma . [SEP]
[CLS] however , their tendency to accumulate within late endosomes limits their transfection B-property efficiency I-property . [SEP]
[CLS] simultaneously , polycationic materials such as pei have been widely investigated as sirna carriers for their ability to encapsulate and protect nucleic B-material acids I-material and rapidly enter cells B-material . [SEP]
[CLS] in particular , pei is strongly cationic B-material due to its high amine B-material content , which enables pei - sirna polyplexes to overcome a major bottleneck to their intracellular delivery : achieving endosomal escape . [SEP]
[CLS] however , toxicity B-property common to polycationic materials has limited its clinical translation . [SEP]
[CLS] interestingly , we found that pei - wrapped snas afford enhanced cellular uptake and endosomal escape to improve gene silencing efficacy while dramatically reducing the cytotoxicity B-property of pei , warranting continued investigation of these constructs as therapeutic gene regulatory agents for diseases with a genetic basis . [SEP]
[CLS] one devastating disease that might benefit from the continued development and application of rnai therapeutics is glioblastoma multiforme ( gbm ) . [SEP]
[CLS] gbm is the most common and lethal neurological tumor B-material in adults , representing nearly 50 % of all malignant primary brain tumors B-material . [SEP]
[CLS] the three main treatment strategies , surgery , radiation , and chemotherapy , often fail to completely eradicate the disease , in part due to intrinsic or acquired resistance to therapy characteristic of gbm tumors B-material . [SEP]
[CLS] consequently , tumor B-material recurrence is inevitable , and nearly 100 % of patients eventually succumb to disease . [SEP]
[CLS] recently , a growing body of work has identified a role of developmental pathways in gbm progression . [SEP]
[CLS] one such pathway is the hedgehog ( hh ) signaling pathway . [SEP]
[CLS] during development , hh signaling plays a key regulatory role in tissue patterning and stem cell B-material maintenance . [SEP]
[CLS] it is subsequently inactivated in most differentiated adult tissues , during which the ptch1 transmembrane receptor B-material represses smo , a g - protein coupled receptor B-material ( gpcr ) - like protein B-material . [SEP]
[CLS] however , aberrant pathway activation is implicated in many cancers , including gbm . [SEP]
[CLS] in gbm , this aberrant activation is most commonly initiated when extracellular sonic hh ligand binds to ptch1 , which relieves its suppression of smo to drive an intracellular signaling cascade that ultimately results in the translocation of the gli1 transcription B-event factor to the nucleus , where it transcriptionally B-event regulates the expression of genes that promote gbm progression . [SEP]
[CLS] when activated in gbm , hh / gli1 signaling induces proliferation and survival signaling and also maintains an aggressive subpopulation of gbm cells B-material called glioblastoma stem cells ( gscs ) . [SEP]
[CLS] gscs are pluripotent , highly tumorigenic cells B-material that resist therapy and generate the bulk of gbm tumors B-material . [SEP]
[CLS] this is enabled by their ability to divide asymmetrically ; they can divide to produce additional gscs by self - renewal , or they can divide and differentiate into nontumorigenic progenitor and differentiated gbm cells B-material . [SEP]
[CLS] further , gscs cycle slowly and tend to reside within the hypoxic tumor B-material core B-material , rendering them highly refractory to radiation and chemotherapies that target rapidly dividing cells B-material . [SEP]
[CLS] as a result , traditional therapies fail to eliminate gscs , which in turn drive tumor B-material recurrence . [SEP]
[CLS] due to their highly aggressive characteristics , gscs must be eradicated to achieve complete tumor B-material regression . [SEP]
[CLS] gscs may be eliminated by targeting the developmental pathways that maintain them , such as hh signaling . [SEP]
[CLS] therefore , we hypothesized that gbm progression could be halted by targeting gli1 with rnai therapeutics . [SEP]
[CLS] much research has previously been dedicated to targeting a diverse selection of genes toward the goal of reducing gbm chemoresistance , stemness , and ultimately disease progression . [SEP]
[CLS] for example , nanocarriers delivering sirna against genes that promote survival , drug resistance , and dna repair such as bcl2 , bcl2l12 , 8 survivin , cyclin d1 , 26 mdr1 , egfr and egfrviii , and mgmt , have been previously demonstrated to improve gbm therapeutic response in preclinical testing . [SEP]
[CLS] importantly , many of these genes are known transcriptional B-event targets of gli1 ; therefore , we hypothesized that targeting gli1 upstream of these genes might have broad effects toward halting gbm progression . [SEP]
[CLS] in this work , we developed gli1 - targeted pei - snas , characterized their cellular uptake and intracellular trafficking mechanisms , and demonstrated that they can reduce the chemoresistance and stemness of gbm cells B-material ( figure 1 ) . [SEP]
[CLS] our results demonstrate that pei - snas bind cells B-material via scavenger receptors and undergo both dynamin - dependent , caveolae - mediated endocytosis B-event and macropinocytosis ( figure 2 ) . [SEP]
[CLS] following endocytosis B-event , the majority of pei - snas are routed to late endosomes and lysosomes . [SEP]
[CLS] despite this , gli1 pei - snas can successfully reduce the expression of the hh signaling components gli1 and smo by ~ 30 % , and this corresponds to an ~ 30 % reduction in hh transcriptional B-event target genes that promote gbm progression including cyclind1 , c - myc , bcl2 , and abcg2 ( figure 4 ) . [SEP]
[CLS] gene regulation by gli1 pei - snas also mediates a 30 % reduction in gbm cell B-material proliferation ( figure 5a ) and a distinguishable onset of cellular senescence ( figure 5b ) . [SEP]
[CLS] further , gli1 pei - snas reduce the metabolic activity of gbm cells B-material by ~ 60 % alone or in combination with tmz ( temozolomide , the frontline chemotherapy for gbm ) ( figure 5c ) . [SEP]
[CLS] importantly , gli1 pei - snas impair the self - renewal capacity of gbm cells B-material as indicated by a 30 - 40 % reduction in the expression of stemness genes ( figure 6a , b ) and by impairing the formation of neurospheres ( figure 6a , c , d ) . [SEP]
[CLS] this translates to a substantial improvement in neurosphere chemosensitivity as demonstrated by a 2 - fold increase in the fraction of cells B-material undergoing apoptosis B-event in response to low doses of tmz ( figure 7 ) . [SEP]
[CLS] these findings underscore the potential for sirna therapeutics targeting gli1 to reduce gbm resistance to therapy and warrant the continued development of polycation - sna hybrid sirna carriers for potent gene regulation . [SEP]
[CLS] citrate - stabilized gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle , 15 nm ) were prepared using the frens method and treated with 0 . 1 % diethyl pyrocarbonate ( depc ) to inactivate rnases . [SEP]
[CLS] snas were synthesized and characterized for sirna loading as previously reported . [SEP]
[CLS] briefly , rnase - free aunps B-nanoparticle were suspended in 0 . 2 % tween - 20 and 350 mm nacl and subsequently functionalized with thiolated sirna ( 1 nmol per ml of 10 nm aunps B-nanoparticle ; integrated dna technologies , coralville , ia ) . [SEP]
[CLS] the nacl concentration was slowly increased to 500 mm and incubated B-technique overnight prior to passivation with 2 kda methoxy - polyethylene glycol - thiol B-material ( mpeg - sh ; laysan bio , arab , al ) to increase stability . [SEP]
[CLS] pei - snas were synthesized by incubating B-technique purified snas suspended in water B-material at 10 nm with 1 mg / ml 25 kda branched pei ( sigma - aldrich , st . louis , mo ) for 15 min under sonication to prevent aggregation , and then , the pei - snas were purified by centrifugation to remove unbound pei . [SEP]
[CLS] experimental section nanoparticle B-nanoparticle synthesis and characterization . sirna sequences used are as follows : scr , 5 ′ - ugauaagucguuggugcacdt - 3 ′ ; gli1 , 5 ′ - uugggagucaaauuccuggcdt - 3 ′ . [SEP]
[CLS] using an oligreen assay to measure sirna loading , scr - snas contained 53 . 3 ± 6 . 5 duplexes , and gli1 - snas contained 58 . 7 ± 11 . 2 duplexes . [SEP]
[CLS] all loading was measured prior to coating B-material snas with pei . [SEP]
[CLS] u87 - mg cells B-material were purchased from the american type culture collection ( atcc , manassas , va ) , cultured in dulbecco ' s modified eagle ' s medium ( dmem ; vwr , radnor , pa ) supplemented with 10 % fetal bovine serum ( fbs ; gemini bio - products , west sacramento , ca ) , and maintained in a humidified incubator B-technique at 37 °c , 5 % co 2 . [SEP]
[CLS] for neurosphere experiments , u87 - mg cells B-material were seeded as a single - cell suspension in lowadhesion plates cultured in neurocult nsa ( stemcell technologies , vancouver , bc , canada ) medium supplemented with recombinant human epidermal growth factor ( egf , 20 ng / ml ) , recombinant human basic fibroblast growth factor ( bfgf , 10 ng / ml ) , and heparin ( 2 μg / ml ) . [SEP]
[CLS] to study the intracellular trafficking of pei - snas , u87 - mg cells B-material were stably transformed with gfp - tagged endosomal markers using standard lentiviral transduction procedures . [SEP]
[CLS] briefly , rab5 - gfp ( addgene # 56530 ) , rab7 - gfp ( addgene # 12605 ) , rab11 - gfp ( addgene # 12674 ) , or lamp1 - gfp ( addgene # 34831 ) were cloned into a lentiviral transfer vector ( system biosciences , palo alto , ca ) by restriction cloning . [SEP]
[CLS] lentiviral particles were produced by triple - transfecting ( transit - lenti transfection reagent ; mirus bio , madison , wi ) 293tn cells B-material ( system biosciences , palo alto , ca ) with either transfer vector and lentiviral packaging and envelope plasmids ( addgene # 12260 , 12259 ) . [SEP]
[CLS] lentivirus was harvested , filtered , and diluted in cell B-material culture medium to transform u87 - mg cells B-material . [SEP]
[CLS] cells B-material stably expressing the desired protein B-material were selected with 1 mg / ml puromycin ( vwr , radnor , pa ) . [SEP]
[CLS] endocytosis B-event pathways responsible for the cellular uptake of pei - snas were investigated using flow B-technique cytometry I-technique and fluorescence B-technique microscopy I-technique . [SEP]
[CLS] u87 - mg cells B-material were seeded in 24 - well culture plates to 60 - 70 % confluence and treated with inhibitors to various components of endocytic pathways according to the conditions detailed in table s1 . [SEP]
[CLS] treated cells B-material were incubated B-technique with cy5 - labeled pei - snas ( 40 nm sirna ) for 4 h and either trypsinized for flow B-technique cytometry I-technique analysis using a novocyte flow cytometer ( acea biosciences , san diego , ca ) or fixed in 4 % formaldehyde , counterstained with dapi and dylighttm488 - phalloidin ( cell B-material signaling technology , danvers , ma ) , and mounted on slides using gelvatol mounting medium for fluorescence B-technique microscopy I-technique using a zeiss axioob - server . z1 microscope ( zeiss , thornwood , ny ) . [SEP]
[CLS] pei - sna distribution into neurospheres was conducted similarly by culturing neurospheres for 1 week prior to treatment with endocytosis B-event inhibitors and pei - snas and subsequently dissociating them for flow B-technique cytometry I-technique analysis or imaging them intact using a zeiss lsm880 confocal microscope to collect z - stacks of whole spheres . [SEP]
[CLS] u87 - mg cells B-material stably expressing rab5 - gfp , rab7 - gfp , rab11 - gfp , or lamp1 - gfp were seeded in 35 mm glass bottom dishes to 60 - 70 % confluence . [SEP]
[CLS] cells B-material were incubated B-technique with cy5 - labeled pei - snas for 24 h , counterstained with cellmask orange ( thermo fisher scientific , waltham , ma ) , and fed with fluorobrite dmem media ( thermo fisher scientific , waltham , ma ) . [SEP]
[CLS] cells B-material were imaged live using a zeiss lsm880 confocal microscope equipped with an incubated B-technique stage . [SEP]
[CLS] z - stacks were acquired to analyze pei - sna / endosomal colocalization throughout the entire volume of cells B-material . [SEP]
[CLS] quantitative colocalization analysis was performed as previously described to calculate mander ' s colocalization coefficients for each endosomal marker . [SEP]
[CLS] briefly , image analysis was performed using data from three independent experiments . [SEP]
[CLS] regions of interest ( rois ) were identified by manually tracing individual cells B-material based on the cellmask orange channel . [SEP]
[CLS] both the cy5 and gfp channels were median filtered with a 3 - by - 3 - by - 3 neighborhood and top - hat filtered using a 2 μm disk element . [SEP]
[CLS] mccs were calculated for each roi in each image stack . [SEP]
[CLS] the gene regulation potency of gli1 pei - snas was evaluated using qpcr . [SEP]
[CLS] u87 - mg cells B-material were seeded at 100 000 cells B-material / well in a 12 - well culture plate and grown overnight . [SEP]
[CLS] cells B-material were incubated B-technique with pei - snas ( 50 nm sirna ) for 24 h in complete medium , fed with fresh medium , and incubated B-technique a further 48 h at 37 °c , 5 % co 2 . [SEP]
[CLS] rna was isolated using an isolate ii rna mini kit ( bioline , taunton , ma ) , and qpcr was performed using sensifast sybr one - step master mix on a lightcycler 96 ( roche diagnostics corporation , indianapolis , in ) . [SEP]
[CLS] gene expression was normalized to that of gapdh . [SEP]
[CLS] primer sequences are listed in table s2 . [SEP]
[CLS] proliferation , senescence , and viability . [SEP]
[CLS] gli1 pei - snas were evaluated for their ability to reduce gbm cell B-material proliferation , senescence , and viability in combination with tmz . [SEP]
[CLS] to measure proliferation , u87 - mg cells B-material were treated with gli1 pei - snas as described for qpcr analysis and then assessed using an edu assay ( thermo fisher scientific , waltham , ma ) . [SEP]
[CLS] briefly , treated u87 - mg cells B-material were incubated B-technique with 10 μm edu for 16 h , then trypsinized , fixed in 4 % formaldehyde , and permeabilized with 0 . 05 % saponin prior to staining according to the manufacturer ' s protocol . [SEP]
[CLS] edu incorporation was detected by an alexafluor488 - azide and measured by flow B-technique cytometry I-technique ( ex 488 nm / em 530 / 30 nm ) using a novocyte flow cytometer . [SEP]
[CLS] senescence was evaluated in cells B-material treated with pei - snas using a senescence - associated β - galactosidase ( saβgal ) kit ( cell B-material signaling technology , danvers , ma ) according to the manufacturer ' s protocol . [SEP]
[CLS] stained cells B-material were imaged using a zeiss axioobserver . [SEP]
[CLS] z1 microscope equipped with a color camera . [SEP]
[CLS] u87 - mg cell B-material metabolic activity ( taken to correlate with viability ) was assessed following cotreatment with gli1 pei - snas and tmz . [SEP]
[CLS] cells B-material were seeded in 96well plates at a density of 2500 cells B-material / well and treated with scr or gli1 pei - snas as described above . [SEP]
[CLS] treated cells B-material were then exposed to tmz concentrations ranging from 0 - 1000 μm for an additional 72 h and then evaluated using an mtt B-technique assay I-technique ( thermo fisher scientific , waltham , ma ) according to the manufacturer ' s protocol . [SEP]
[CLS] each treatment group was performed in triplicate , and separate cells B-material treated with equivalent volumes of dmso ( used to reconstitute and store tmz ) were used to ensure that toxicity B-property was due to tmz treatment rather than the dmso vehicle B-material . [SEP]
[CLS] a neurosphere culture model was used to assess the impact of gli1 pei - snas on the selfrenewal capacity of u87 - mg cells B-material . [SEP]
[CLS] first , u87 - mg cells B-material were seeded in standard adherent culture at a density of 25 000 cells B-material / ml in dulbecco ' s modified eagle ' s medium ( dmem ) supplemented with 10 % fetal bovine serum ( fbs ) . [SEP]
[CLS] cells B-material were treated with gli1 pei - snas or scr pei - snas for 24 h at 37 °c , 5 % co 2 , fed with fresh medium , and incubated B-technique a further 48 h at 37 °c , 5 % co 2 . [SEP]
[CLS] cells B-material were then trypsinized and seeded in suspension at a density of 10 000 cells B-material / ml to grow as neurospheres as described above for 1 week at 37 °c , 5 % co 2 . [SEP]
[CLS] after 7 days , the entirety of each well was imaged using a zeiss axioobserver . [SEP]
[CLS] z1 microscope equipped with automated stage control . [SEP]
[CLS] images were stitched using zeiss efficient navigation software ( zen 2 . 0 ; zeiss ) and exported for analysis , and then , spheres were counted and measured in imagej . [SEP]
[CLS] the sphere size reported is the diameter of the projected area imaged by bright - field microscopy B-technique . [SEP]
[CLS] to assess the effect of gli1 pei - snas on neurosphere response to tmz , we used a similar neurosphere model . [SEP]
[CLS] cells B-material were pretreated with pei - snas in adherent culture and then seeded as neurospheres as described above . [SEP]
[CLS] at 72 h postseeding of the neurospheres , cells B-material were exposed to pei - snas for an additional 24 h and then resuspended in fresh medium containing tmz . [SEP]
[CLS] spheres were incubated B-technique in tmz for 72 h and subsequently assessed for apoptosis B-event using an annexin - v - fitc / pi assay ( cayman chemical company , ann arbor , mi ) . [SEP]
[CLS] annexin - v - fitc / pi staining was analyzed on a novocyte flow cytometer . [SEP]
[CLS] all experiments were performed in triplicate , and data represent means ± standard deviations from three independent replicates unless otherwise indicated . [SEP]
[CLS] groups with significant differences were identified using oneway anova with a post hoc tukey test ( or student ' s t - test when only two groups were compared ) , and differences were considered significant at p < 0 . 05 . [SEP]
[CLS] statistical tests were performed in matlab software ( mathworks , natick , ma ) , and flow B-technique cytometry I-technique data was analyzed using novoexpress software ( acea biosciences , san diego , ca ) . [SEP]
[CLS] to begin our evaluation of gli1 pei - snas , we were interested in understanding the mechanism by which pei - snas are taken up by cells B-material . [SEP]
[CLS] importantly , the mechanism of endocytosis B-event can determine the intracellular fate of the sirna cargo , which must reach the cytosol to facilitate gene silencing . [SEP]
[CLS] based on previous studies that have separately demonstrated that both pei - based polyplexes and snas 34 undergo clathrin - independent , caveolae - mediated endocytosis B-event , we expected to observe similar results . [SEP]
[CLS] we further anticipated that pei - snas would bind to cells B-material via class a scavenger receptors , which has been previously reported for snas . [SEP]
[CLS] for our studies , we used chemical inhibitors to different endocytosis B-event mechanisms ( table s1 ) to determine which pathways were necessary for cellular uptake of pei - snas fluorescently B-property labeled with cy5 - sirna . [SEP]
[CLS] cells B-material were subsequently analyzed by flow B-technique cytometry I-technique and fluorescence B-technique microscopy I-technique to identify changes in both net cellular association and in cellular localization of pei - snas . [SEP]
[CLS] as expected , we found that fucoidan ( fcd ) , a potent scavenger receptor B-material inhibitor , significantly decreases the net cellular uptake of pei - snas by up to 75 % , and little association of pei - snas with cells B-material was detected by fluorescence B-technique microscopy I-technique ( figure 2 ) . [SEP]
[CLS] similarly , we found that methyl - βcyclodextrin ( mβcd ) , a caveolae / lipid B-material raft inhibitor , but not chlorpromazine ( cpz ) , a clathrin inhibitor , significantly reduces the uptake of pei - snas by up to 50 % ( figure 2 ) . [SEP]
[CLS] while cargo internalized by clathrin or caveolae - dependent mechanisms can ultimately be routed to lysosomes , caveolae can also fuse with intermediate B-property vesicles called caveosomes , which do not acidify and can avoid lysosomal trafficking in some cases . [SEP]
[CLS] interestingly , one study found that polyplexes taken up by clathrin - mediated endocytosis B-event are routed to lysosomes for degradation , but polyplexes taken up by caveolae - dependent mechanisms were more likely to evade lysosomes and induce efficient transfection . [SEP]
[CLS] we additionally found that the cellular uptake of pei - snas occurs in a dynamin - dependent manner , indicated by a significant 50 % reduction in pei - sna uptake in cells B-material treated with dynasore ( figure 2 ) . [SEP]
[CLS] cytochalasin d , which disrupts actin polymerization to inhibit phagocytosis B-event , does not significantly reduce pei - sna uptake ( figure 2 ) . [SEP]
[CLS] having demonstrated that pei - snas undergo both dynamin - dependent , caveolae - mediated endocytosis B-event and macro - pinocytosis , we next sought to understand the consequences of this uptake mechanism on the intracellular trafficking of pei - snas . [SEP]
[CLS] while our previous research demonstrates that pei - snas are visible in early endosomes within 1 h and undergo reduced lysosomal accumulation relative to snas and pei - sirna polyplexes , we were further interested in determining whether pei - snas accumulate within other endosomal compartments , including early endosomes , late endosomes , recycling endosomes , and lysosomes . [SEP]
[CLS] in these studies , we used u87 - mg cells B-material engineered to stably express fluorescently B-property tagged endosomal compartments , including rab5 - gfp , rab7 - gfp , rab11 - gfp , and lamp1 - gfp , which label early endosomes , late endosomes , recycling endosomes , and lysosomes , respectively , to determine the subcellular localization of pei - snas by confocal microscopy B-technique . [SEP]
[CLS] cells B-material were incubated B-technique with cy5 - pei - snas for 24 h and imaged by confocal microscopy B-technique to capture z - stacks containing the entire volume of the cells B-material . [SEP]
[CLS] to quantitatively assess colocalization between pei - snas and endosomal compartments , manders ' colocalization coefficients ( mccs ) were calculated for each endosomal compartment . [SEP]
[CLS] mccs measure the fractional overlap of fluorescent B-property signals and range from 0 - 1 , where mcc = 0 indicates that no colocalization is present , and mcc = 1 indicates that the two signals colocalize perfectly . [SEP]
[CLS] here , we have calculated both the fractional overlap of cy5 - pei - snas and gfp - endosomes ( mcc 1 ) to evaluate the fraction of sirna signal within endosomes as well as the fractional overlap of gfp - endosomes and cy5 - pei - snas ( mcc 2 ) to evaluate the fraction of endosomes containing sirna . [SEP]
[CLS] we found that cy5 - pei - snas colocalize to the greatest extent with lamp1 - gfp ( mcc 1 = 0 . 87 ± 0 . 04 ) and rab7 - gfp ( mcc 1 = 0 . 84 ± 0 . 04 ) , which was significantly greater than colocalization with rab11 - gfp ( mcc 1 = 0 . 73 ± 0 . 06 ) or rab5 - gfp ( mcc 1 = 0 . 65 ±0 . 04 , figure 3 a , b ) . [SEP]
[CLS] additionally , lamp1 - gfp colocalizes with cy5 - pei - snas ( mcc 2 = 0 . 61 ± 0 . 002 ) to a significantly greater extent than other endosomal markers ( mcc 2 all ~ 0 . 45 , figure 3a , b ) . [SEP]
[CLS] therefore , we conclude that pei - snas accumulate to the greatest extent within rab7 + late endosomes and lamp1 + lysosomes . [SEP]
[CLS] this is consistent with previous studies , which demonstrate that avoiding retention within the endolysosomal network remains a challenge for rnai therapeutics . [SEP]
[CLS] these results demonstrate that future research should seek to reduce the lysosomal entrapment of these constructs possibly through the incorporation of cell B-material penetrating peptides B-material or ionizable B-property lipids B-material to provide additional mechanisms of achieving endosomal escape . [SEP]
[CLS] additionally , the relatively strong colocalization of pei - snas with rab5 + early endosomes ( mcc > 0 . 5 ) suggests that pei - snas are continually endocytosed through 24 h incubation B-technique . [SEP]
[CLS] unexpectedly , we also observed relatively high colocalization with rab11 + recycling endosomes , suggesting a mechanism by which pei - snas may be exocytosed . [SEP]
[CLS] while it is unusual for nanoparticles B-nanoparticle to exhibit colocalization with rab11 + vesicles , 39 previous studies investigating the intracellular fate of snas have demonstrated that while the gold B-material core B-material is retained within lysosomes , the oligonucleotide shell B-material is gradually cleared from cells B-material over a period of 24 h , likely due to nuclease degradation and oligonucleotide exocytosis . [SEP]
[CLS] a similar process may explain our results , which show that cy5 - sirna can colocalize with rab11 + recycling endosomes . [SEP]
[CLS] next , we evaluated the gene regulation potency of gli1 pei - snas using qpcr to measure mrna expression of hh signaling components and downstream target genes known to promote gbm progression . [SEP]
[CLS] gli1 pei - snas significantly reduce gli1 expression by 30 % relative to pei - snas carrying a scrambled control sirna sequence ( scr pei - snas ; figure 4 ) . [SEP]
[CLS] we also observed a significant 30 % decrease in the expression of the gpcr - like protein B-material smo ( figure 4 ) , which is also considered an oncogene that can induce aberrant hh signaling to further drive cancer progression . [SEP]
[CLS] mutations to smo are common to many cancers , and therapeutics targeting smo often fail in the clinic , because acquired smo mutations render tumor B-material cells B-material refractory to smo - targeted therapy . [SEP]
[CLS] our results demonstrate that gli1 - targeted rnai can reduce the expression of both oncogenes . [SEP]
[CLS] interestingly , we did not observe significant differences in the expression of the transmembrane receptor B-material ptch1 . [SEP]
[CLS] ptch1 normally inhibits smo to suppress hh activity and is thus considered the tumor B-material suppressor of the hh pathway , so we were encouraged that gli1 pei - snas did not reduce the expression of this gene . [SEP]
[CLS] we also observed ~ 30 % decreases in the pro - gbm gli1 transcriptional B-event target genes cyclin d1 , c - myc , bcl - 2 , and abcg2 ( figure 4 ) . [SEP]
[CLS] cyclin d1 promotes cell B-material cycle progression through the g1 / s transition , and its overexpression in gbm correlates with poor prognosis . [SEP]
[CLS] further , silencing cyclin d1 inhibits proliferation , induces apoptosis B-event , and reduces the invasive capacity of gbm cells B-material . [SEP]
[CLS] while c - myc also promotes proliferation in numerous cancers , it has been further recognized as a key regulator of glioma cancer stem cells B-material . [SEP]
[CLS] bcl - 2 opposes apoptosis B-event and contributes substantially to gbm therapy resistance , and inhibiting bcl - 2 can enhance the response of glioma cells B-material to tmz . [SEP]
[CLS] abcg2 is an abc transporter known for its drug efflux activity to promote cell B-material survival and also regulates selfrenewal and the expression of stemness genes in glioma cells B-material . [SEP]
[CLS] taken together , these results demonstrate that gli1 pei - snas can downregulate multiple genes that contribute to gbm progression , chemoresistance , and stemness , and we expect the observed gene regulation to exert tumor B-material suppressive effects . [SEP]
[CLS] to test whether the gene regulation capacity of gli1 pei - snas is sufficient to induce a tumor B-material suppressive response against gbm cells B-material , we investigated the impact of gli1 pei - snas on proliferation , senescence , and response to tmz . [SEP]
[CLS] using a 5 - ethynyl - 2 ′ deoxyuridine ( edu ) assay to measure proliferation , we found that gli1 pei - snas significantly reduce the fraction of proliferative ( edu + ) u87 - mg cells B-material by ~ 30 % relative to cells B-material treated with scr pei - snas ( figure 5a ) . [SEP]
[CLS] this is consistent with our measured decreases in cyclin d1 and c - myc expression ( figure 4 ) and with previous reports demonstrating that suppressing gli1 reduces gbm proliferation . [SEP]
[CLS] in parallel with our observed decrease in proliferation , we recently reported that silencing gli1 can induce senescence in pten - deficient u87 - mg cells B-material . [SEP]
[CLS] to determine whether gli1 pei - snas could also elicit this effect , we employed a senescence - associated β - galactosidase ( saβ - gal ) assay to visually identify cells B-material undergoing senescence . [SEP]
[CLS] saβgal staining demonstrated that u87 - mg cells B-material treated with gli1 pei - snas broadly undergo senescence , as indicated by teal saβgal staining , while cells B-material treated with scr pei - snas do not ( figure 5b ) . [SEP]
[CLS] during senescence , cells B-material enter a stable state of cell B-material cycle arrest but remain metabolically active . [SEP]
[CLS] while much remains unknown regarding the anticancer B-property implications of senescence , a growing body of research has demonstrated that senescence can induce tumor B-material suppressive effects and even compensate for apoptosis B-event in some contexts , such as when expression of the tumor B-material suppressor pten is lost . [SEP]
[CLS] interestingly , senescent cells B-material acquire a senescenceassociated secretory phenotype ( sasp ) , which can trigger immune responses to either promote tumor B-material progression or promote immune clearance of tumor B-material cells B-material . [SEP]
[CLS] in aggregate , our data suggests that gli1 pei - sna - mediated senescence exerts tumor B-material suppressive effects , though future research should continue to investigate the effects of the sasp induced by gli1 silencing on gbm progression . [SEP]
[CLS] next , we were interested in investigating the effects of gli1 pei - snas on gbm cell B-material sensitivity to the frontline chemo - therapy , tmz . [SEP]
[CLS] one way we could envision using such a gli1 - targeted therapy is to first administer our gli1 rnai therapeutic to downregulate cellular resistance mechanisms and subsequently treat with tmz . [SEP]
[CLS] to model this schedule , we first treated u87 - mg cells B-material with gli1 pei - snas , incubated B-technique the cells B-material for 72 h , and then treated cells B-material with tmz doses ranging from 0 - 1000 μm for an additional 72 h , after which we measured cellular metabolic activity using a 3 - ( 4 , 5 - dimethylthiazol - 2 - yl ) - 2 , 5diphenyltetrazolium bromide B-material ( mtt ) assay . [SEP]
[CLS] our results demonstrate that gli1 pei - snas significantly reduce u87 - mg metabolic activity by ~ 60 % alone and in combination with low doses of tmz ( figure 5c ) , suggesting that gli1 pei - snas might reduce the required dose of tmz to achieve a therapeutic effect . [SEP]
[CLS] notably , the observed decrease in metabolic activity appears to be largely due to gli1 pei - snas alone rather than tmz , which may reflect the fact that u87 - mg cells B-material grown in adherent culture are highly refractory to tmz , as shown by our own previous studies and studies conducted by others . [SEP]
[CLS] this is supported by the observation that tmz failed to reduce metabolic activity in cells B-material treated with scr pei - snas except at the highest dose tested ( 1000 μm ) , which is 20 - fold higher than the maximum clinically feasible tmz dose at the tumor B-material site . [SEP]
[CLS] at this dose of tmz , scr pei - snas reduced metabolic activity by ~ 40 % , while we observed a similar but slightly larger decrease of ~ 57 % in cells B-material treated with gli1 pei - snas . [SEP]
[CLS] in addition to supporting prior knowledge that adherent - cultured u87 - mg cells B-material are tmz - insensitive , our results are also consistent with our finding that gli1 pei - snas induce senescence in adherent - cultured u87 - mg cells B-material . [SEP]
[CLS] because tmz is an alkylating agent and therefore exhibits selective toxicity B-property to actively dividing cells B-material , the onset of senescence could explain why tmz is ineffective toward reducing metabolic activity in this context . [SEP]
[CLS] the results from these intriguing experiments prompted us to next evaluate combination therapy using tmz and gli1 pei - snas in gbm neurospheres , which exhibit greater sensitivity to clinically relevant tmz doses . [SEP]
[CLS] 50gli1 pei - snas reduce stemness and impair self - renewal of gbm cells B-material . [SEP]
[CLS] we were next interested in whether gli1 pei - snas could reverse the stemness of gbm cells B-material to impair the self - renewal capacity of gscs . [SEP]
[CLS] first , we determined the extent to which gli1 pei - snas could reduce the expression of stemness genes in adherent - cultured cells B-material ( figure 6a , b ) . [SEP]
[CLS] by qpcr , we found that gli1 pei - snas significantly reduce the expression of cd133 and nanog by 38 and 25 % , respectively ( figure 6b ) . [SEP]
[CLS] we also observed a slight but insignificant decrease in sox2 expression ( figure 6b ) . [SEP]
[CLS] to determine whether these changes are sufficient to impair self - renewal , we used a sphere - formation assay , in which u87 - mg cells B-material are seeded in suspension to form multicellular neuro - spheres that exhibit increased stemness relative to adherent - cultured cells B-material [SEP]
[CLS] in these experiments , u87 - mg cells B-material were pretreated with pei - snas for 72 h and then dissociated and suspended in serum - free medium supplemented with growth factors to form neurospheres over a period of 7 days ( figure 6a ) . [SEP]
[CLS] bright - field images show that neurospheres grown from cells B-material pretreated with gli1 pei - snas are significantly smaller than those grown from cells B-material pretreated with scr pei - snas , exhibiting an ~ 40 μm decrease in average projected diameter ( figure 6c , d ) . [SEP]
[CLS] further , we found that treatment with gli1 pei - snas reduced the total number of neurospheres formed by 26 % ( figure 6d ) , though this result was not statistically significant . [SEP]
[CLS] notably , we detected this reduction in size and number of neurospheres 10 days after gli1 pei - sna treatment , whereas previous work demonstrates that a single transfection with snas can silence gene expression for up to 96 h . [SEP]
[CLS] here , we demonstrate that pei - snas are a useful vehicle B-material for achieving sustained effects of gli1 - targeted therapy . [SEP]
[CLS] consistent with our other results , this suggests that gli1 pei - snas can impair the stemness of gbm cells B-material to reduce the formation of neurospheres through self - renewal . [SEP]
[CLS] our findings are corroborated by prior studies , which reported that either transient transfection of sigli1 or treatment with a smo inhibitor into gbm neurospheres significantly hinder neuro - sphere growth . [SEP]
[CLS] similar results have been obtained using the pharmacological gli inhibitor , gant61 , encapsulated within plga B-nanoparticle nanoparticles I-nanoparticle . plga - gant61 reduced tumor B-material - sphere formation in colon and breast cancer cell B-material lines . [SEP]
[CLS] the ability of hh inhibitors to decrease the number of cancer stem cells B-material ( cscs ) has been demonstrated in vivo as well ; one study reported that plga - peg nanoparticles B-nanoparticle delivering another pharmacological gli inhibitor , hpi - 1 , reduced the number of aldh + cscs in a murine orthotopic pancreatic cancer xenograft . [SEP]
[CLS] in totality , these results suggest that gli1 pei - snas can impair the self - renewal capacity of u87 - mg cells B-material and may be useful to eliminate the aggressive gsc subpopulation . [SEP]
[CLS] gli1 pei - snas potentiate the neurosphere response to tmz chemotherapy . [SEP]
[CLS] finally , we were interested in whether treating neurospheres with gli1 pei - snas could improve neurosphere response to tmz . [SEP]
[CLS] we first confirmed that pei - snas could enter neurospheres by confocal microscopy B-technique and flow B-technique cytometry I-technique . [SEP]
[CLS] confocal microscopy B-technique demonstrates that cy5 - labeled pei - snas can easily penetrate small neurospheres ( < 100 μm ) but accumulate to the greatest extent within the periphery of larger neurospheres ( figure 7a ) within 24 h . [SEP]
[CLS] flow B-technique cytometry I-technique analysis of neurospheres dissociated after treatment demonstrates that cy5 - labeled pei - snas can enter 91 . 3 % of cells B-material grown as neurospheres ( figure 7b ) . [SEP]
[CLS] taken together with our microscopy B-technique data , this suggests that pei - snas can penetrate neurospheres greater than 100 μm in diameter but show greater accumulation near the periphery than in the center . [SEP]
[CLS] this is consistent with previous work demonstrating that snas can successfully transfect human tumor B-material neurospheres , 8 so we were encouraged that our pei - snas retain this property . [SEP]
[CLS] we were further interested in examining the mechanism by which pei - snas penetrate neurospheres . [SEP]
[CLS] we found that the scavenger receptor B-material inhibitor fcd reduces the uptake of pei - snas by neuro - sphere - cultured cells B-material by 43 % , and the caveolae / lipid B-material raft inhibitor mβcd reduces uptake by 64 % ( figure 7c ) , demonstrating that these are mechanisms that are required for pei - sna distribution through neurospheres . [SEP]
[CLS] to investigate the capacity for gli1 pei - snas to potentiate the neurosphere response to tmz , we treated cells B-material according to the timeline in figure 7d . [SEP]
[CLS] adherent - cultured cells B-material were pretreated with pei - snas for 72 h , seeded as neurospheres , and incubated B-technique a further 72 h . [SEP]
[CLS] spheres were treated with a second pei - sna pulse for 24 h , resuspended in tmzcontaining medium , and incubated B-technique for 72 h . [SEP]
[CLS] spheres were dissociated and analyzed for apoptosis B-event using an annexin - v - fitc / propidium iodide B-material ( pi ) assay . [SEP]
[CLS] we found that spheres primed with gli1 pei - snas prior to low - dose tmz treatment ( 50 μm ) exhibited a nearly 2fold increase in the fraction of apoptotic cells B-material relative to cells primed with scr pei - snas ( figure 7e , f ) . [SEP]
[CLS] gli1 pei - snas also increased the fraction of apoptotic cells B-material in response to 100 and 200 μm tmz by 62 and 50 % , respectively . [SEP]
[CLS] this is consistent with previous research , which demonstrated that the pharmacological hedgehog pathway inhibitor cyclopamine can increase the fraction of caspase3 + apoptotic gbm stem cell B-material cultures in combination with tmz . [SEP]
[CLS] an additional report found that cyclopamine could potentiate the cytotoxic B-property effects of tmz in cd133 + glioma stem cells B-material . [SEP]
[CLS] however , the clinical translation of cyclopamine has been deterred by severe toxicity B-property and rapid clearance from circulation . [SEP]
[CLS] further , cyclopamine targets smo , upstream of gli1 in the hedgehog signaling pathway , and a growing body of work has demonstrated that cancer cells B-material frequently acquire resistance to smo antagonists , potentially limiting their utility . [SEP]
[CLS] 55 among gli1 inhibitors , furthest along in development is gant61 , which , despite its potency in preclinical models , is poorly stable at physiological conditions . [SEP]
[CLS] further , the ability of gant61 to cross the blood - brain barrier B-property remains poorly understood . [SEP]
[CLS] because snas have previously demonstrated sufficient stability in physiological conditions and can accumulate within glioma xenografts and improve tumor B-material response to tmz , we are hopeful that a gli1 - targeted construct might behave similarly . [SEP]
[CLS] based on our results and the results of others , we hypothesize that gli1 pei - snas may also accumulate within gbm tumors B-material in vivo to exert antitumor effects that also extend to gscs , though this remains to be evaluated in future work . [SEP]
[CLS] in this work , we have demonstrated that gli1 pei - snas can improve the response of gbm cells B-material and aggressive gbm neurospheres to tmz chemotherapy . [SEP]
[CLS] we found that gli1 pei - snas bind scavenger receptors on gbm cells B-material to undergo endocytosis B-event in a caveolae / lipid B-material raft / dynamin - dependent manner and that this leads to trafficking through the classical endolysosomal pathway . [SEP]
[CLS] despite many of the nanoparticles B-nanoparticle accumulating within late endosomes and lysosomes , gli1 pei - snas could silence the expression of genes associated with the hedgehog signaling pathway that promote the aggressive , chemoresistant phenotype of gbm . [SEP]
[CLS] further , this leads to a decrease in proliferation that correlates with an onset of gbm cell B-material senescence as well as a decrease in metabolic activity with or without tmz cotreatment . [SEP]
[CLS] importantly , gli1 pei - snas reduced the growth of stem - like neurospheres and sensitized neurospheres to low doses of tmz chemotherapy . [SEP]
[CLS] these results warrant further development of pei - snas and gli1 - targeted therapies to alleviate drug resistance and recurrence for gbm patients . [SEP]
[CLS] refer to web version on pubmed central for supplementary material B-material . [SEP]
[CLS] gli1 - targeted pei - snas were developed to suppress gbm - promoting hedgehog signaling and mitigate the chemoresistance and stemness of gbm cells B-material . [SEP]
[CLS] gli1 pei - snas reduce the mrna expression of gli1 and downstream target genes by qpcr . [SEP]
[CLS] gene expression is normalized to that of gapdh , and data shown are means ± sem ; * p < 0 . 05 relative to scr pei - sna control by student ' s t - test . [SEP]
[CLS] abbreviationsgbm glioblastoma pei polyethylenimine sna spherical nucleic B-material acid I-material tmz temozolomide rnai rna interference hh hedgehog gpcr g - protein coupled receptor B-material gsc glioblastoma stem cell B-material mβcd methyl - β - cyclodextrin cpz chlorpromazine fcd fucoidan cytd cytochalasin d mcc manders ' colocalization coefficient edu 5 - ethynyl - 2 ′ - deoxyuridine saβgal senescence - associated β - galactosidase sasp senescence - associated secretory phenotype qpcr quantitative polymerase chain reaction pi propidium iodide B-material aunp B-nanoparticle gold nanoparticle B-nanoparticle depc diethyl pyrocarbonate [SEP]
[CLS] assessment of the uptake mechanism for pei - snas , as determined by flow B-technique cytometry I-technique ( top ) or visualized by fluorescence B-technique microscopy I-technique ( bottom ) . [SEP]
[CLS] data are average geometric means ± standard deviations . [SEP]
[CLS] figure 2 . p < 0 . 05 and * * p < 0 . 005 relative to control cells B-material by one - way anova with post hoc tukey . [SEP]
[CLS] for fluorescence B-technique microscopy I-technique images , scale = 50 μm . [SEP]
[CLS] intracellular trafficking of pei - snas . [SEP]
[CLS] ( a ) confocal microscopy B-technique was used to visualize cy5 - pei - sna localization to endocytic compartments after 24 h incubation B-technique with cells B-material . [SEP]
[CLS] endocytic compartments were labeled by stably expressing gfp - tagged markers for early endosomes ( rab5 + ) , recycling endosomes ( rab11 + ) , late endosomes ( rab7 + ) , or lysosomes ( lamp1 + ) . [SEP]
[CLS] scale bar = 20 μm . [SEP]
[CLS] ( b ) results from quantitative colocalization analysis to calculate the fractional overlap of cy5 - pei - snas with gfp - endosomal markers and vice versa . [SEP]
[CLS] mcc = manders ' colocalization coefficient ; * p < 0 . 05 by one - way anova with post hoc tukey . [SEP]
[CLS] 5 . gli1 pei - snas reduce proliferation and chemoresistance . [SEP]
[CLS] ( a ) by edu assay , gli1 pei - snas reduce u87 proliferation by ~ 30 % . [SEP]
[CLS] flow cytometric histograms ( left ) and quantification of edu + cells B-material ( right ) . [SEP]
[CLS] data are means ± stds , * p = 0 . 02 . [SEP]
[CLS] ( b ) saβgal staining ( teal ) demonstrating that gli1pei - snas induce senescence in u87 cells B-material . [SEP]
[CLS] scale = 100 μm . [SEP]
[CLS] ( c ) by mtt B-technique assay I-technique , gli1 pei - snas reduce u87 metabolic activity alone and in combination with tmz , * p < 0 . 01 relative to scr pei - sna control with equivalent tmz dose by one - way anova with post hoc tukey . [SEP]
[CLS] gli1 pei - snas reduce stemness and impair self - renewal of u87 cells B-material . [SEP]
[CLS] ( a ) schematic depicting the neurosphere culture model and experimental design ; red cells B-material illustrate gscs . [SEP]
[CLS] ( b ) qpcr showing expression of genes associated with stemness following exposure to pei - snas . [SEP]
[CLS] gene expression is normalized to that of gapdh . [SEP]
[CLS] data are means ± stds ; * p < 0 . 001 relative to scr pei - sna . [SEP]
[CLS] ( c ) representative bright - field images of neurospheres cultured from u87 cells B-material after exposure to pei - snas . [SEP]
[CLS] scale = 200 μm . [SEP]
[CLS] ( d ) gli1 pei - snas reduce the size and number of neurospheres formed , as measured from 25 tiled bright - field images per treatment group per experiment ; * p = 0 . 03 by student ' s t - test . [SEP]
[CLS] gli1 pei - snas potentiate neurosphere response to tmz chemotherapy . [SEP]
[CLS] ( a ) confocal microscopy B-technique visualizing gli1 pei - sna distribution into small ( top ) and large ( bottom ) neurospheres . [SEP]
[CLS] scale = 100 μm . [SEP]
[CLS] ( b ) flow cytometric histogram of cy5 - pei - sna uptake by cells B-material grown as neurosperes . [SEP]
[CLS] ( c ) flow cytometric analysis of the mechanism by which gli1 pei - snas distribute throughout neurospheres . [SEP]
[CLS] data are geometric mean fluorescence B-property intensity ( gmfi ) ± std normalized to control cells B-material , * p < 0 . 05 by one - way anova with post hoc tukey . [SEP]
[CLS] ( d ) experimental timeline for determining effect of cotreating neurospheres with gli1 pei - snas and tmz . [SEP]
[CLS] ( e ) flow cytometric density plots of annexin - v / pi apoptosis B-event analysis of neurospheres cotreated with gli1 pei - snas and tmz . [SEP]
[CLS] ( f ) summary of annexin - v / pi apoptosis B-event analysis . [SEP]
[CLS] data are means ± stds from n = 2 replicates . [SEP]
[CLS] * p < 0 . 05 by one - way anova with post hoc fisher ' s least significant difference test . [SEP]
[CLS] nanomedicine is a discipline that applies nanoscience and nanotechnology principles to the prevention , diagnosis , and treatment of human diseases . [SEP]
[CLS] self - assembly of molecular components is becoming a common strategy in the design and syntheses of nanomaterials B-material for biomedical applications . [SEP]
[CLS] in both natural and synthetic self - assembled nanostructures , molecular cooperativity is emerging as an important hallmark . [SEP]
[CLS] in many cases , interplay of many types of noncovalent interactions leads to dynamic nanosystems with emergent properties where the whole is bigger than the sum of the parts . [SEP]
[CLS] in this review , we provide a comprehensive analysis of the cooperativity principles in multiple self - assembled nanostructures . [SEP]
[CLS] we discuss the molecular origin and quantitative modeling of cooperative behaviors . [SEP]
[CLS] in selected systems , we describe the examples on how to leverage molecular cooperativity to design nanomedicine with improved diagnostic precision and therapeutic efficacy in medicine . [SEP]
[CLS] nanomaterials B-material are rapidly evolving and impact a broad range of applications in photonics , electronics , and medicine . [SEP]
[CLS] in particular , they play an increasingly important role in medicine , where numerous nanosystems have been developed for biochemical sensing , molecular imaging , disease diagnosis , and treatment . [SEP]
[CLS] various nanoplatforms have been extensively investigated to address challenges in medicine to overcome deficiencies in conventional small molecular sensors and drugs , resulting in the rapid growth of nanomedicine as a new discipline . [SEP]
[CLS] in contrast to " top - down " methods like lithography , a " bottom - up " approach allows the formation of nanoscopic architectures driven by noncovalent self - assembly of molecular components . [SEP]
[CLS] self - assembly , which bridges the structures of individual building blocks and the function of the obtained nanocomplex , is an essential part of nanotechnology . [SEP]
[CLS] the underlying supramolecular chemistry principles were described by lehn and whitesides over two decades ago . [SEP]
[CLS] compared to covalent chemistry , noncovalent self - assembly employs weak and polyvalent interactions to achieve a thermodynamically stable nanostructure . [SEP]
[CLS] this strategy can produce nanoscopic structures ( 10 4 −10 10 da ) that are not easily synthesizable by covalent chemistry . [SEP]
[CLS] the resulting system often has a faster temporal response to environmental stimuli due to the lower energy barrier B-property ( e . g . , dissociation of noncovalent complexes requires lower energy than breaking of covalent bonds ) . [SEP]
[CLS] a hallmark of self - assembled systems is molecular cooperativity , where the system behaves quite differently as a whole from the sum of parts acting in isolation . [SEP]
[CLS] positive cooperativity has been identified in many biological and physiological processes ( e . g . , oxygen B-material transport by hemoglobin ) . [SEP]
[CLS] mechanistic investigations on several established selfassembled nanosystems also suggest that positive cooperativity contributes to enhanced detection sensitivity and specificity in chemical and biological sensing . [SEP]
[CLS] understanding the supramolecular self - assembly process and associated cooperativity offers a new paradigm for the design and development of nanomaterials B-material in medicine . [SEP]
[CLS] in this article , we highlight the recent advances in the investigation of cooperativity principles underlying the design of self - assembled nanomedicine ( figure 1 ) . [SEP]
[CLS] the current review focuses on the bottom - up chemistry and material B-material science considerations of nanomedicine . [SEP]
[CLS] implementation of a top - down method for nanomedicine development is beyond the scope of the current review . [SEP]
[CLS] cooperativity is frequently employed in biology to modulate molecular recognition through sequential binding events , usually operated by the conformational changes of the macromolecules . [SEP]
[CLS] the binding may display either positive or negative cooperativity . [SEP]
[CLS] positive cooperativity is described as synergistic ( whole is bigger than the sum of the parts ) and negative cooperativity as interfering . [SEP]
[CLS] in this section , we begin the discussion of biological cooperativity using well - established protein / rna folding and allosteric examples ( e . g . , hemoglobin - o 2 interactions ) , then move on to more complex multivalent cell surface interactions , and finally present the emerging microphase separations of large protein signaling complexes . [SEP]
[CLS] the woodson group reported that cooperative assembly of rna helices reduces the misfolding of tetrahymena group i ribozyme . [SEP]
[CLS] disruption of the tetraloop structure destabilizes the free energy of rna folding by 2−3 kcal / mol . [SEP]
[CLS] the same group also reported that the cooperative tertiary interaction guides rna folding ( figure 2 ) . [SEP]
[CLS] interaction between tertiary structures increases the free energy gap between the native state and the intermediate B-property state , thereby facilitating the rna folding to the native state . [SEP]
[CLS] daniel and coworkers further quantified tertiary contact interactions in rna folding using single - molecule forster resonance energy transfer method . [SEP]
[CLS] allosteric cooperativity is extensively investigated and describes the process where ligand binding at one site regulates the binding or function at another site . [SEP]
[CLS] goodey suggests that the conformational mobility B-property is a common mechanism that underlies allosteric regulation and catalysis in biological systems . [SEP]
[CLS] intrinsic flexibility of proteins B-material contributes to multiple conformations that can interconvert at different time scales . [SEP]
[CLS] the binding of an allosteric effector may lead to the conformational change with modulated binding site geometries and activity . [SEP]
[CLS] as a result , allostery is used by nature to regulate the catalytic function of proteins B-material . [SEP]
[CLS] 38 - 40 2 . 2 . 1 . hemoglobin - oxygen B-material [SEP]
[CLS] binding . - all cells B-material in our body use oxygen B-material to make atp , which provides the energy for many physiological functions . [SEP]
[CLS] oxygen B-material molecules are transported from the lung to individual cells B-material . [SEP]
[CLS] after oxygen B-material is breathed into the lung , it first diffuses to the blood . [SEP]
[CLS] its low solubility B-property in I-property water I-property ( 40 mg / l ) makes it impossible to meet the metabolic needs of our tissues and cells B-material . [SEP]
[CLS] hemoglobin , a tetrameric protein B-material residing in the red blood cells B-material , serves as a carrier for the transportation of oxygen B-material . [SEP]
[CLS] allosteric oxygen B-material binding is associated with conformational changes of hemoglobin triggered by the oxygen−iron ( ii ) interactions . [SEP]
[CLS] perutz first reported the structure of hemoglobin in various forms . [SEP]
[CLS] each hemoglobin molecule consists of two α subunits and two β subunits with similar 3d structures . [SEP]
[CLS] the binding affinity of hemoglobin to oxygen B-material molecules depends on the heme cofactor , responsible for the red color of blood . [SEP]
[CLS] each heme group I-material has a central iron B-material atom I-material chelated by protoporphyrin . [SEP]
[CLS] each iron B-material within the heme group can serve as a single binding site to an oxygen B-material molecule , and one hemoglobin protein B-material can bind to four oxygen B-material molecules . [SEP]
[CLS] under normal physiology , the iron B-material is in the ferrous ( fe 2 + ) oxidation state . [SEP]
[CLS] the binding of the oxygen B-material molecule to the ferrous ion B-material results in a smaller ferrous ion B-material , allowing it to move into the plane of the porphyrin . [SEP]
[CLS] such oxygenationdriven conformation change leads to a transition from deoxy t state to oxy r state of the quaternary structure of hemoglobin , where one pair of αβ subunits rotates relative to the other by 15 degrees . [SEP]
[CLS] the structural alteration in hemoglobin significantly changes the oxygen B-material binding affinity to hemoglobin . [SEP]
[CLS] initial oxygen B-material binding to hemoglobin facilitates the binding of the second and ensuing oxygen B-material molecules ( figure 3 ) . [SEP]
[CLS] when three binding sites of hemoglobin are occupied , the binding affinity of the last free site for oxygen B-material is 20 - fold higher than that for the first oxygen B-material molecule . [SEP]
[CLS] the cooperative binding improves the oxygen B-material transport efficiency . [SEP]
[CLS] the oxygen−hemoglobin saturation curve displays a sigmoid shape , typical for a cooperative binding process . [SEP]
[CLS] catalysis . - enzymes can dramatically accelerate the rate of biochemical reactions by reduction of activation energy barriers B-property . [SEP]
[CLS] many enzymes function as oligomeric complexes of multiple subunits , and each subunit contains an active site for ligand binding and / or catalysis . [SEP]
[CLS] 4 summarizes representative cooperative activation processes of enzymes . [SEP]
[CLS] an " induced fit model " has often been used to describe the enzyme−substrate interactions . [SEP]
[CLS] the initial interaction is capable of inducing conformational changes of enzymes to increase the strength of subsequent binding events . [SEP]
[CLS] the conformational changes are described as a key mechanism of enzyme catalysis . [SEP]
[CLS] it is worth pointing out that the initiation of the conformation change is usually the rate - limiting step instead of the ensuing steps . [SEP]
[CLS] another cooperativity example resides in the sequential assembly of weak binding components into a stable multi - molecular complex . [SEP]
[CLS] one such example is the nucleosomemediated cooperativity between transcription B-event factors . [SEP]
[CLS] sequential binding B-event of I-event different I-event transcription B-event factor I-event proteins I-event to the promoter region is critical for precise control of gene expression . [SEP]
[CLS] dna regions depending on histone binding status can be classified as nucleosomal ( n ) such multicomponent cooperativity is also seen in the formation of interferon - β ( ifn - β ) " enhanceosome " complex , a multiprotein complex that binds to the ifn - β enhancer site on the dna . [SEP]
[CLS] this multiprotein complex contains more than five proteins B-material , and these proteins B-material assemble cooperatively on a chromatin template with the help of an architectural factor . [SEP]
[CLS] preorganization of some proteins B-material generates a new binding site for others with additional stabilization . [SEP]
[CLS] the absence of any individual component will destabilize the eventual nanocomplex , which suggests strong multivalent cooperativity among individual components . [SEP]
[CLS] biological macromolecules are spatially organized within the cells B-material . [SEP]
[CLS] membrane - bound subcellular organelles offer the physical separation needed for biochemical reactions in optimized compartments within a cell B-material . [SEP]
[CLS] hyman and co - workers first reported subcellular structures consisting of heterogeneous mixtures of proteins B-material and nucleic B-material acids I-material in membraneless organelles . [SEP]
[CLS] the formation of these nonmembraned organelles is driven by phase separation similar to polymer B-material condensation . [SEP]
[CLS] living cells B-material contain many such types of nanoscopic droplet - like structures from different compositions of biological molecules ( figure 6 ) . [SEP]
[CLS] phase separation and condensation of biomacromolecules also display supramolecular cooperativity . [SEP]
[CLS] one example is the phase separation of proteins B-material with intrinsically disordered regions . [SEP]
[CLS] intrinsically disordered proteins B-material ( idps ) are crucial components of the cellular signaling machinery . they participate in the dynamic assembly of signaling complexes and membrane - less nuclear and cytoplasmic organelles . [SEP]
[CLS] idps are found in many biomolecular condensates such as stress granules , germ granules , and nuclear ultrastructures . [SEP]
[CLS] many intrinsically disordered proteins B-material undergo similar phase separation in vitro under solution conditions . [SEP]
[CLS] idps display complex allosteric cooperativity that is responsible for their tunable regulatory interactions . [SEP]
[CLS] another example is the formation of micrometer - sized droplets from multivalent protein B-material complexes ( e . g . , 2 + 3 systems ) . [SEP]
[CLS] rosen and co - workers reported the nephrin / nck / n - wasp system constituting a three - component interaction with the formation of phase separated liquid droplets ( figure 7 ) . [SEP]
[CLS] the cooperative association is controlled by the phosphorylation status of the nephrin protein B-material and consequently shifted the phase boundary of the complex . [SEP]
[CLS] noncovalent self - assembly of molecular modules can form thermodynamically stable nanocomplexes in biological systems . [SEP]
[CLS] they determine the higher order structures of proteins B-material , dna , and rna as well as molecular recognition between biomacromolecules . [SEP]
[CLS] through multivalent interactions , molecules or groups of molecules associate into organized structures with increasing complexity . [SEP]
[CLS] a hallmark of these nanoscale structures and architectures is positive cooperativity , which arises from subtle interplay of two or more noncovalent interactions . [SEP]
[CLS] compared to noncovalent interactions , the length of a covalent bond is short with an average distance less than 0 . 2 nm between pairing atoms B-material . [SEP]
[CLS] the strength of the covalent bond is strong varying from 149 kj / mol for breaking an i−i bond to 411 kj / mol for a c−h bond . [SEP]
[CLS] covalent synthesis alone is incapable of generating well - defined , functional structures with dimensions from tens of nanometers to hundreds of nanometers in size , which covers biological structures from protein B-material complexes to viruses to subcellular organelles . [SEP]
[CLS] noncovalent interactions can occur at longer distances than covalent bonds . [SEP]
[CLS] interaction of hydrophobic B-property surfaces or electrostatic interactions between charged species can happen over tens of nanometers . [SEP]
[CLS] compared to covalent bonds , noncovalent bonds are 10−100 times weaker ( table 1 ) . [SEP]
[CLS] polyvalent interactions involving multiple types of noncovalent bonds through contact of large surface areas compensate for the weaker bond strengths , while allowing the formation , disintegration , and reformation of large scale structures that are not easily attainable by covalent chemistry . [SEP]
[CLS] numerous reviews have discussed the nature and strengths of noncovalent interactions . [SEP]
[CLS] in this section , we offer a brief summary of several key types of noncovalent interactions that impact molecular cooperativity in biological environments . [SEP]
[CLS] the mixture of oil and water B-material tends to segregate into two independent phases : an aqueous phase and an oil phase with well - defined boundaries . [SEP]
[CLS] the noncovalent interactions that are responsible for aggregation of hydrophobic B-property structures are termed the hydrophobic B-property effect . [SEP]
[CLS] hydrophobic B-property interactions I-property tend to minimize the energy penalty in order to insert a nonpolar molecule into water B-material . [SEP]
[CLS] solvation of nonpolar substances in water B-material can disrupt the hydrogen B-material bonding network of water B-material . [SEP]
[CLS] a large hydrophobic B-property solute is able to force the water B-material into a rigid cage . [SEP]
[CLS] the cages restrict the motion and increase the structural organization of water B-material molecules , which facilitates hydrogen B-material bonding interactions and gains in enthalpy . [SEP]
[CLS] meanwhile , the randomness ( entropy ) of the water B-material molecules decreases and causes an overall penalty in free energy . [SEP]
[CLS] to minimize such penalty , nonpolar molecules tend to come together and aggregate in aqueous solution to exclude water B-material molecules ( figure 8 ) . [SEP]
[CLS] hydrophobic B-property interaction I-property contributes to a multitude of biological structures and processes such as cell B-material membranes , protein B-material folding , formation of subcellular vesicles , and insertion of membrane proteins B-material into the nonpolar lipid B-material environment . [SEP]
[CLS] chemists have learned to use the hydrophobic B-property effect as a strategy to generate well - defined structures . [SEP]
[CLS] numerous drug delivery carriers such as polymer - or lipid B-material - based nanoparticles B-nanoparticle have been developed to improve the pharmacological properties of encapsulated drugs . [SEP]
[CLS] amphiphilic B-property block copolymers have been synthesized to form micellar nanoparticles B-nanoparticle for the delivery of hydrophobic B-property therapeutics . [SEP]
[CLS] liposome B-nanoparticle was first demonstrated in the 1960s and is one of the few nanoparticle - based drug carriers that were translated into the clinic successfully . [SEP]
[CLS] a hydrogen B-material bond describes attractive interactions between a hydrogen B-material donor and an acceptor ( most often an electron rich atom B-material such as oxygen B-material or nitrogen B-material ) . [SEP]
[CLS] although the interaction is relatively weak , multiplication of hydrogen B-material bonds can drive the self - assembly of individual building blocks to well - defined nano or macrostructures . [SEP]
[CLS] formation of a protein B-material α - helix and dna base pairs are well - known examples of hydrogen B-material bondmediated complexes . [SEP]
[CLS] noncovalent hydrogen B-material bond interactions have also been used to form higher order complexes from synthetic molecules . [SEP]
[CLS] whitesides and co - workers reported a stable supramolecular complex from cyanuric acid ( ca ) and melamine ( m ) based on the hydrogen B-material bond interactions . [SEP]
[CLS] the rotello group reported a polymer - mediated ` bricks and mortar ' strategy to order surface functionalized gold B-material particles into aggregated assemblies via intermolecular B-property hydrogen I-property bonding I-property interactions I-property . [SEP]
[CLS] breaking of ionic bonds in vacuum requires higher energy ( e . g . , 788 kj / mol for separating na + cl − ion B-material pairs ) than the breaking of covalent bonds ( e . g . , 411 kj / mol for c−h bonds ) . [SEP]
[CLS] in aqueous environments , solvation of ions B-material by water B-material molecules dramatically reduces the energy cost to separate oppositely charged species . [SEP]
[CLS] coulomb interactions between two pointcharges are shielded by a factor of relative permittivity ( ε r , also known as dielectric constant ) of the medium . [SEP]
[CLS] for water B-material , the value of ε r , is 78 . 3 at 25°c , which places the electrostatic interactions at the same energy scale as other noncovalent interactions ( table 1 ) . [SEP]
[CLS] in biological systems , electrostatic interactions between charged macromolecules are important in nucleic B-material acid I-material condensation , ligand−receptor binding , and cell−cell interactions [SEP]
[CLS] layer - by - layer self - assembly represents a common strategy to construct nanoparticles B-nanoparticle based on electrostatic interactions . [SEP]
[CLS] the film architecture and composition can be precisely controlled at the nanoscale . [SEP]
[CLS] this capability has spawned the development of artificial cells B-material and drug delivery systems . [SEP]
[CLS] electrostatic interaction - mediated condensation between polycations and the phosphate backbone of nucleic B-material acids I-material has been investigated for the development of gene delivery systems over the past several decades . [SEP]
[CLS] 147 - 152 [SEP]
[CLS] in chemistry , π−π stacking describes the noncovalent , attractive interactions between neighboring aromatic residues . [SEP]
[CLS] the stacking effect is critical in multiple biological processes , such as protein B-material folding , 28 molecular recognition and template - directed synthesis . [SEP]
[CLS] 154 many groups have reported noncovalent complexes based on π−π interactions . [SEP]
[CLS] stoddart and co - workers have designed several generations of rotaxanes and catenanes functionalized with electron - rich and electron - deficient aromatic units . [SEP]
[CLS] meijer and co - workers have developed a hierarchical self - assembly strategy to produce molecular nanostructures . [SEP]
[CLS] a nucleation−growth strategy is conceptualized that yields a high degree of cooperativity from π−π stacking interactions between adjacent repeating B-material units I-material . [SEP]
[CLS] in macromolecular self - assembly systems , multiple types of noncovalent interactions as described above can simultaneously occur , with compensating energetics leading to highly complex architectures and interacting dynamics . [SEP]
[CLS] such examples include the association of hydrophobic B-property side chains with h - bonding of polypeptide B-material backbones and saltbridge formation during protein B-material folding ; the interplay of electrostatic and hydrophobic B-property interactions I-property in the chaotropic anion - induced micelle B-material self - assembly ; and predominantly hbonding and hydrophobic B-property interactions I-property in the thermosensitive properties of elastin - like polymers B-material . [SEP]
[CLS] a hallmark of complex and dynamic systems is the emergence of cooperativity ( figure 9 ) . [SEP]
[CLS] below we summarize a few well - established cooperative systems , with the hope of deciphering the underlying mechanism to help predict and program new systems in the future . [SEP]
[CLS] spherical nucleic B-material acids I-material ( snas ) are three - dimensional nanostructures with densely packed nucleic B-material acids I-material covalently conjugated to the nanoparticle B-nanoparticle surface ( figure 10 ) . [SEP]
[CLS] these constructs were originally created using gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) by the mirkin group . [SEP]
[CLS] the unique three - dimensional framework introduces new physical , chemical , and biological properties over one - dimensional linear nucleic B-material acids I-material , which found broad uses in biological sensing , molecular diagnostics , and intracellular gene regulation . [SEP]
[CLS] the linear nucleic B-material acid I-material chains are typically functionalized with a headgroup to improve the stability of the nanocomplex in aqueous environments . [SEP]
[CLS] the first sna conjugates were prepared by covalent attachment of the alkanethiol - terminated , single - stranded oligonucleotides to the surface of gold B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] a dense layer of nucleic B-material acids I-material can be achieved through salt B-material additions , where positively charged counterions are necessary to minimize electrostatic repulsion between adjacent negatively charged dna strands . [SEP]
[CLS] in living systems , the nucleic B-material acids I-material usually exist in the hybridized duplex structure . [SEP]
[CLS] in contrast , the snas adopt their morphology to the shape of the inorganic B-material cores I-material . [SEP]
[CLS] besides sna - nps B-nanoparticle , several nucleic acid−based assemblies have been developed for biological sensing or catalysis applications . [SEP]
[CLS] the willner group reported improved specificity in the sensing of dna or selected sequence of aptamers . [SEP]
[CLS] the nucleic B-material acid I-material structures activated the dnazyme cascades that catalyzed the oxidation of abts 2− by h 2 o 2 . [SEP]
[CLS] the kolpashchikov group developed a binary dna probe for nucleic B-material acid I-material detection . [SEP]
[CLS] two short dna hairpin cooperativities to the targeted sequence enabled the molecular recognition with high sensitivity and selectivity . [SEP]
[CLS] nanoparticles B-nanoparticle . - the hybridization of complementary nucleic B-material acid I-material sequences enables the binding interactions between sna particles with matched dna sequences . [SEP]
[CLS] these interactions lead to the aggregation of gold B-nanoparticle - I-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] the sna nanoparticles B-nanoparticle can be released from the aggregates through dehybridization upon heating that disrupts noncovalent base pairing interactions . [SEP]
[CLS] dna duplexes and snas have characteristic melting temperatures ( t m ) when dehybridization occurs . [SEP]
[CLS] jin and co - workers reported a striking sharp melting curve for the dehybridization of sna - np B-nanoparticle aggregates . 171 typically , the melting of the linear dna duplex happens over a broad temperature range ( ~ 20 °c ) . [SEP]
[CLS] in contrast , the thermal transition of the sna−au nps B-nanoparticle from the aggregate state to the individual particle state occurs over a narrower temperature range of 2−8°c . [SEP]
[CLS] in addition , the phase transition temperature of sna is higher than that of the corresponding free dna duplex . [SEP]
[CLS] the sharp phase transition was observed in both sna - nps B-nanoparticle and a chip - based assay . [SEP]
[CLS] importantly , a single oligonucleotide base - pair mismatch can be differentiated by melting behavior from those with fully complementary sequences . [SEP]
[CLS] mechanistic investigation suggests that high surface density of oligonucleotides on sna - nps B-nanoparticle contributes to polyvalent interparticle connections that are collectively stronger in binding compared to free dna duplexes in aqueous solution . [SEP]
[CLS] high salt B-material concentration is necessary to achieve the melting cooperativity and sharp phase transition . [SEP]
[CLS] the schatz group proposed a " shared ion B-material cloud " model to describe the cooperative melting transition in sna - nps B-nanoparticle , which is supported by experimental evidence ( figure 11 ) . [SEP]
[CLS] the established thermodynamic model also enabled the quantitative assessment of the contributions from the neighboring - duplex effect . [SEP]
[CLS] nguyen and co - workers reported as little as two dna duplexes were necessary to elicit cooperative melting behavior . [SEP]
[CLS] the experimental data fit well into a coarse - grain dynamic stimulation - based model . [SEP]
[CLS] the oligonucleotides assumed an orientation that enabled the sharing of counterions for the cooperative response . [SEP]
[CLS] temperature . - in contrast to natural biomacromolecules , the physicochemical properties of synthetic nanomaterials B-material can be easily modified by tailoring their structure and composition . [SEP]
[CLS] for example , controlling the surface nucleic B-material acid I-material density of aunps B-nanoparticle can affect the hybridization efficiency and cooperative melting response ( figure 12 ) . [SEP]
[CLS] the thermal transition temperature was found proportional to the surface dna density while keeping nanoparticle B-nanoparticle and target concentration unaltered . [SEP]
[CLS] one unique feature of nanomaterials B-material is the large surface - to - volume ratio due to the small nanoparticle B-nanoparticle size . [SEP]
[CLS] the nanoparticle B-nanoparticle size is expected to affect the phase transition behaviors of snas . [SEP]
[CLS] the melting transition temperature decreased from 50 to 47 °c when the size of gold B-material particles increased from 13 to 50 nm , respectively . [SEP]
[CLS] interestingly , larger snas generally exhibited sharper melting transitions compared to smaller ones . [SEP]
[CLS] the melting curves of natural single strand dna exhibit a salt B-material concentration dependence . [SEP]
[CLS] the transition temperature of snas increased from 41 to 61 . 5 °c when the nacl concentration went from 0 . 05 to 1 . 0 m . [SEP]
[CLS] in addition , the increase in salt B-material concentration also led to the formation of larger aggregates . [SEP]
[CLS] this can be attributed to a charge shielding effect by the salt B-material , which can reduce electrostatic repulsions between the oligonucleotidemodified gold B-nanoparticle nanoparticles I-nanoparticle and permit further hybridization between nanoparticles B-nanoparticle . [SEP]
[CLS] aggregation of snas in dna sensing can result in a distinct color change from red to purple by visual inspection . [SEP]
[CLS] the electromagnetic coupling between nanoparticles B-nanoparticle that affects the surface plasmon resonance is distance dependent , which also impacts the van der waals and electrostatic interactions between particles . [SEP]
[CLS] the melting analysis showed that longer interparticle distance resulted in higher transition temperature of snas . [SEP]
[CLS] further mechanistic investigation suggested that the electrostatic interaction was expected to be the dominant factor in regulating distance - dependent melting behaviors . [SEP]
[CLS] besides dnas , ribonucleic B-material acids I-material ( rnas ) have also shown a promising therapeutic effect . [SEP]
[CLS] 177 rnas were also introduced onto aunps B-nanoparticle surface to generate the rna snas . [SEP]
[CLS] in a recent study , barnaby et al . reported a systematic investigation on the structure−function relationships in rna snas , which would help elucidate the interactions of rnas with a specific type of serum nucleases . [SEP]
[CLS] a combined experimental and theoretical study investigated the impact of several key parameters ( i . e . , rna sequence , density , linker , etc . ) of rna - snas for rational design of snas in biomedical applications . [SEP]
[CLS] stimuli - responsive polymers B-material often display a sharp change in physical or chemical properties upon a small perturbation in environmental conditions , which is used for the design of " smart " nanomaterials B-material for the controlled release of therapeutics . [SEP]
[CLS] thermoresponsiveness is usually measured as a change of light transmittance or solubility B-property of polymeric materials . [SEP]
[CLS] thermoresponsive nanomaterials B-material are among the most investigated systems in drug delivery and cancer therapy . [SEP]
[CLS] the sharp thermal response was exploited for the triggered - release of drugs in response to change in the surrounding temperature . [SEP]
[CLS] for biomedical applications , thermosensitive nanocarriers are expected to retain their therapeutic load at normal physiological temperature ( i . e . , 37 °c ) ; upon local heating by an external source , the nanocarriers can rapidly release the drug in the desired location . [SEP]
[CLS] thermoresponsive systems include liposomes B-nanoparticle or polymeric micelles B-material that undergo phase transitions at specific temperatures . [SEP]
[CLS] poly ( n - isopropylacrylamide ) ( pnipam ) was first synthesized in the 1950s , and it is widely adopted for use as a thermosensitive polymeric drug carrier . [SEP]
[CLS] it is typically prepared by polymerization of commercially available n - isopropylacrylamide monomer B-material . [SEP]
[CLS] when heated above 32 °c in water B-material , pnipam undergoes conformation changes from a hydrated gel to an aggregated solid across the lower critical solution temperature ( lcst ) . [SEP]
[CLS] the gel will lose about 90 % of its original volume . [SEP]
[CLS] this lcst temperature has close proximity to physiological temperatures that can trigger a reversible phase transition without causing damage to surrounding tissues . [SEP]
[CLS] considerable efforts have been dedicated to the design of pnipam - based thermosensitive nanomaterials B-material as delivery vehicles B-material for controlled drug release . [SEP]
[CLS] pnipam polymer B-material stays in the gel state below the lcst , where water B-material molecules form a hydrated cage around the hydrophobic B-property moieties along the polymer B-material chain . [SEP]
[CLS] when temperatures are raised above the phase transition temperature , hydrogen B-material bonds between the polymers B-material and water B-material molecules become more favorable in comparison to polymer−polymer or water−water interactions . [SEP]
[CLS] such destabilization results in the desolvation of the hydrophobic B-property groups of polymer B-material chains . [SEP]
[CLS] the increase in entropy of the released water B-material molecules and hydrophobic B-property interactions I-property drives the collapse of polymer B-material chains . [SEP]
[CLS] the lcst can be controlled by adjusting the hydrophobicity−hydrophilicity ratio of the polymer B-material chains . [SEP]
[CLS] an increase in hydrophilic B-property groups increases the lcst , and an increase in hydrophobic B-property groups has the opposite effect . [SEP]
[CLS] studies show that the concentration or molecular weight of the polymer B-material has little effect on the phase transition temperature of pnipam . [SEP]
[CLS] it is notable that some thermoresponsive systems do display molecular weight or size dependence in lcst transitions . [SEP]
[CLS] 4 . 2 . 1 . cooperativity in gelation of thermoresponsive polymers B-material . - the first detailed study of thermosensitive pnipam in aqueous solution was reported in 1969 by they observed the change in turbidity of a solution upon heating at 32 °c . [SEP]
[CLS] since then , continuous efforts have been made to investigate the phase transition properties of pnipam and its derivatives . [SEP]
[CLS] extensive mechanistic investigation suggests that the driving force for this phase transition is the balance of hydrophilic B-property and hydrophobic B-property moieties . [SEP]
[CLS] pnipam chains carry two types of bound water B-material molecules with one around the hydrophobic B-property isopropyl moiety and the other associated with the amide B-material group I-material . [SEP]
[CLS] change in the hydration status of the hydrophobic B-property side chains results in association of the pnipam chains . [SEP]
[CLS] tanaka and co - workers first reported cooperative dehydration of the pnipam chains in the temperature - induced phase separation . [SEP]
[CLS] they concluded that dehydration of the neighboring water B-material molecules around the polymer B-material chains was responsible for the sharp phase transition with little dependence on molecular weight or chain length . [SEP]
[CLS] the degree of hydration versus temperature by theoretical calculation correlated well with the experimental data reported by fujishige et al . [SEP]
[CLS] the winnik group also investigated the phase transition behavior of cyclic pnipam in aqueous solution . [SEP]
[CLS] they found that the melting curves of cyclic pnipam solutions occurred over a much wider temperature range over the linear counterpart , indicating the importance of side chain geometry on cooperative response . [SEP]
[CLS] a recent study by muller - buschbaum reported how partial dehydration affected the volume changes in the phase separations of pnipam hydrogel . [SEP]
[CLS] 204 [SEP]
[CLS] temperature . - fujishige et al . reported that neither molecular weight ( 5 × 10 4 to 8 . 4 × 10 6 da ) nor concentration ( 0 . 01 to 1 wt % ) greatly impact the thermal transition temperature of pnipam . [SEP]
[CLS] in contrast , many studies show that the lcst is tunable by shifting the hydrophilic B-property / hydrophobic B-property balance . [SEP]
[CLS] different types of n - alkyl - substituted poly ( meth ) acrylamides have been synthesized , and their lcst values were investigated . [SEP]
[CLS] poly ( n - n - propylacrylamide ) ( pnnpam ) had a lcst of 10 °c compared to 32 °c of pnipam , suggesting that hydrophobic B-property geometry affects the transition temperature ( table 2 ) . [SEP]
[CLS] logp is the octanol−water partition coefficient of a molecule , which is commonly used as a quantitative measure of molecular hydrophobicity B-property . [SEP]
[CLS] a higher logp indicates stronger hydrophobicity B-property . [SEP]
[CLS] the lcst of poly ( ncyclopropylacrylamide ) ( pncpam ) , in which iso - propyl of pinpam is replaced by the less hydrophobic B-property cyclo - propyl group , occurs around 53 °c . [SEP]
[CLS] poly ( n , n - diethylacrylamide ) ( pdeam ) displayed a phase transition temperature similar to that of pnipam at 33 °c . [SEP]
[CLS] the lcst of poly ( n , n - ethylmethyl acrylamide ) ( pnemam ) shifted to a much higher temperature of 70 °c . [SEP]
[CLS] it is worth noting that the poly ( n , n - dimethyl acrylamide ) did not show phase transition behavior below the boiling point of water B-material . [SEP]
[CLS] the hydrophobicity B-property of pnipam can also be controlled by incorporating an additional alkyl B-material group I-material in the backbone instead of side chains . poly ( n - isopropyl methacrylamide ) has a lcst at 45 °c , which indicates that restricting the rotation freedom of the polymer B-material backbone can increase the transition temperature and is opposite to that in the side chain . [SEP]
[CLS] salt B-material can greatly impact the solubility B-property of proteins B-material . [SEP]
[CLS] the structure of water B-material in the vicinity of different solute ions B-material has been studied for many decades . [SEP]
[CLS] hofmeister initially observed that different salts B-material have contrasting effects on protein B-material solubilities B-property . [SEP]
[CLS] the ions B-material are divided into kosmotropes or chaotropes depending on their ability to make or break water B-material network structures , respectively . [SEP]
[CLS] kosmotropes decrease protein B-material solubility B-property in I-property water I-property whereas chaotropes increase the solubility B-property . [SEP]
[CLS] salt B-material also critically affects the physicochemical properties of synthetic polymers B-material . [SEP]
[CLS] the cremer group reported the salt B-material effect on the thermoresponsive behavior of pnipam . [SEP]
[CLS] they found that increasing the concentration of nacl led to a decrease of lcst . [SEP]
[CLS] they then expanded the ion B-material effect on pnipam to the entire hofmeister series . [SEP]
[CLS] 214 specific anions B-material ' ability to lower the lcst of pnipam followed the hofmeister trend in protein B-material solubility B-property ( figure 13 ) . [SEP]
[CLS] mechanistic investigation indicates that chaotropic species lowered the lcst via change of surface - tension , which triggers hydrophobic B-property collapse . [SEP]
[CLS] for kosmotropic anions B-material , the surface - tension and polarization of hydrated water B-material molecules are both important in regulating the transition temperature of pinpam . [SEP]
[CLS] in a follow - up study , zhang et al . found that the effect of hofmeister anions B-material on the lcst of pinpam was molecular weight - dependent . [SEP]
[CLS] the promise of pnipam in biomedical applications has inspired further development of other thermoresponsive polymers B-material . [SEP]
[CLS] the gibson group synthesized a series of poly ( acrylamide ) - based polymers B-material with cyclic alkyl B-material groups I-material as n - substituents . [SEP]
[CLS] poly ( nvinylpiperidone ) ( pvpip ) , with a six - member - ring side chain , showed a lcst between 65 and 90°c . [SEP]
[CLS] the phase transition temperature of poly ( n - vinylcaprolactam ) ( pvcap ) shifted to a lower 40 °c with a seven - member - ring side chain . [SEP]
[CLS] although pvpip and pvcap demonstrated similar hydrophobicity - dependent phase transition behavior , they also showed significant molecular weight - dependent lcst shift , which is different from the case of pnipam . [SEP]
[CLS] the zhang group synthesized a series of n - ester - substituted poly ( acrylamide ) s and systematically investigated their lcst behavior . [SEP]
[CLS] in one of their polymer B-material series , poly ( nacryloylglycine methyl ester ) ( pnagme ) , the melting temperature displayed strong molecular weight dependence . [SEP]
[CLS] the lcst decreased from 57 to 42 °c when the polymerization degree increased from 20 to 180 . [SEP]
[CLS] they also observed that increasing the concentration of nacl shifted the lcst to a lower temperature . [SEP]
[CLS] new thermoresponsive nanomaterials B-material have also been developed by coating B-material these polymers B-material onto the surface of different solid nanoparticles B-nanoparticle . [SEP]
[CLS] edwards et al . demonstrated that poly ( ethylene glycol ) methacrylate ( pegma ) coated gold B-nanoparticle nanoparticles I-nanoparticle facilitates their transport across an oil / water B-material interface above the lcst . [SEP]
[CLS] boyer et al . prepared a series of thermoresponsive block copolymers with tunable phase transition temperature by altering monomer B-material compositions . [SEP]
[CLS] the tenhu group reported the development of thermoresponsive nanoparticles B-nanoparticle by grafting pnipam brushes on the surface of gold B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] increasing the molecular weight of pnipam resulted in the decrease of lcst . [SEP]
[CLS] they also found that a decrease in gold B-nanoparticle nanoparticle I-nanoparticle size resulted in a small increase of phase transition temperature . [SEP]
[CLS] klok and co - workers observed similar size - dependent thermotransitions in their system . [SEP]
[CLS] 192 [SEP]
[CLS] elastin is an elastic protein B-material that allows tissues to resume their original shape after stretching or contracting . [SEP]
[CLS] elastin - like polypeptides B-material ( elps ) are synthetic polymers B-material inspired from mammalian elastin . [SEP]
[CLS] early pioneering work by urry and co - workers identified a pentapeptide repeat , vpgxg , where x refers to any natural amino B-material acid I-material except proline for the development of elps . [SEP]
[CLS] these polymers B-material display thermal transitions with an lcst similar to that of pnipam . [SEP]
[CLS] the thermoresponse and biocompatibility B-property make elps ideal materials for different biomedical applications ( figure 14 ) . [SEP]
[CLS] - elastin - like polypeptides B-material can be genetically engineered with precise control of peptide B-material sequence and chain length . [SEP]
[CLS] mechanistic investigation of elps elucidates the key factors that impact their phase transition temperature . [SEP]
[CLS] the elps have also been used to investigate the physical behavior of intrinsically disordered proteins B-material ( idps ) . [SEP]
[CLS] the hinderberger group pioneered the study of the temperature - triggered reversible phase transition behavior of elps ( figure 15 ) . [SEP]
[CLS] they showed that the hydration layers can vary depending on the composition of hydrophobic B-property side chains and amide B-material backbones . a strongly coupled hydration state can lead to a cooperative dehydration of both segments . [SEP]
[CLS] chilkoti and co - workers established an empirical model to predict the transition temperature of elps from amino B-material acid I-material composition , peptide B-material chain length , and concentration in phosphate buffered saline . [SEP]
[CLS] they also performed molecular simulation of the lcst behavior of elps . [SEP]
[CLS] increase of temperature can lead to a gradual conformational change of elps , arising from the formation of more ordered secondary structures . [SEP]
[CLS] higher temperature also exposed the hydrophobic B-property side chains of valine B-material to water B-material , contributing to the collapse of polypeptide B-material chains I-material . [SEP]
[CLS] temperature of elps . - it is generally accepted that the folding and phase transition behavior of proteins B-material is encoded in its amino B-material acid I-material sequence . [SEP]
[CLS] mutation of key residues in a protein B-material can result in dramatic alteration of property and function . [SEP]
[CLS] elastin - like polymers B-material contain a specific amino B-material acid I-material sequence that is critical for their thermosensitivity . [SEP]
[CLS] the temperature - induced phase transition of elps is affected by ph , ionic strength , and polymer B-material molecular weight . [SEP]
[CLS] it is also expected that alteration of key residues may also significantly impact their phase transition behavior . [SEP]
[CLS] the rodriguez - cabello group reported a series of model elastin - like polymers B-material to investigate how amino B-material acid I-material sequence affects solution−gel transition temperature . [SEP]
[CLS] they synthesized three types of elps with the same molecular weight and composition but different sequences . [SEP]
[CLS] the lcst values of the three elp structural isomers ( e100a40 , e50a40e50 , and e50a20e50a20 ) were 43 . 8 , 47 . 1 , and 60 . 1 °c , respectively . [SEP]
[CLS] these data showed that the arrangement of polymer B-material blocks greatly impacted the lcst of elps . [SEP]
[CLS] in another systematic comparison , they found that the transition temperature decreased with the increase in polymer B-material chain length . [SEP]
[CLS] quiroz et al . recently reported a number of elastin - like polymers B-material with variable lower or upper critical solution temperatures . [SEP]
[CLS] they screened a library of elps offering heuristic evidence to identify proteins B-material that may display thermal responsiveness and established the foundation for encoding their phase transition behavior at the sequence level . [SEP]
[CLS] mutation of key residues or insertion of additional amino B-material acids I-material in each repeating B-material unit I-material drastically shifted the phase transition temperature ( left panel , figure 16 ) . [SEP]
[CLS] further examination of different sequence parameters showed the number of repeating B-material units I-material or molecular weight played an important role in controlling the lcst ( right panel , figure 16 ) . [SEP]
[CLS] the phase transition temperature also showed linear correlation with polymer B-material concentration in one specific example . [SEP]
[CLS] a change of environmental conditions such as ph or salt B-material concentration also resulted in the shift of phase transition temperature . [SEP]
[CLS] cho et al . investigated the effect of ion B-material species and concentration on the reversible gelation temperature of elps . [SEP]
[CLS] temperature - triggered hydrophobic B-property self - assembly of elps generally followed the hofmeister trend . [SEP]
[CLS] mechanistic investigation suggested that kosmotropes increased the lcst by polarizing interfacial water B-material molecules in the hydration shell B-material of elps . [SEP]
[CLS] chaotropic anions B-material lowered the gelation−solution transition temperature via reduced surface tension . [SEP]
[CLS] these observations were in agreement with previous conclusions in the poly ( n - isopropylacrylamide ) ( pnipam ) system . [SEP]
[CLS] the holland group reported that the molecular architecture also impacted the thermal phase transition of elps . [SEP]
[CLS] they designed a star - shaped elastin - like polypeptide B-material and compared its lcst behavior with the linear analogs . [SEP]
[CLS] melting curve analysis showed that the transition temperature of both the linear and star elps decreased with the increase in concentration . [SEP]
[CLS] their results also showed that the molecular architecture and morphology also contributed to the folding of elps chains . [SEP]
[CLS] dysregulated ph is considered to be a distinct characteristic of tumors B-material as described by barber and co - workers . [SEP]
[CLS] cancer cells B-material have increased intracellular ph ( ph i ) and decreased extracellular ph ( ph e ) compared to normal tissues . [SEP]
[CLS] the increased ph i protects the cancer cells B-material from apoptotic cell B-event death I-event , facilitates cell B-material proliferation , and is necessary for cell B-material migration . [SEP]
[CLS] the decreased extracellular ph , or tumor B-material acidosis , activates proteases for matrix remodeling and cancer metastasis B-event . highly glycolytic tumors B-material are shown to have an acidic extracellular ph by gillies and others . [SEP]
[CLS] in addition to metabolic abnormality , impaired lymphatic drainage may further contribute to the accumulation of acidic metabolites inside the tumors B-material . [SEP]
[CLS] extensive investigations suggest that regardless of the bioenergetic types of tumors B-material , tumor B-material acidosis is a persistent characteristic of solid cancers . [SEP]
[CLS] targeting tumor B-material extracellular acidity offers a viable strategy for cancer staging and drug delivery . [SEP]
[CLS] these ph - responsive nanocarriers can be classified into two categories : polymers B-material with ionizable B-property moieties and polymers B-material with cleavable covalent linkages B-property . [SEP]
[CLS] ionizable B-property polymers B-material employ a non - covalent strategy to achieve ph sensitivity , where dissociation of carboxylic B-material acids I-material or protonation of amine B-material groups I-material occurs at different ph values . [SEP]
[CLS] carboxylic B-material acid I-material - based hydrogels and their ph - triggered drug release behavior were first reported in the 1950s . [SEP]
[CLS] these hydrogels undergo ph - driven swelling upon ionization B-property in aqueous medium , and the apparent pk a of carboxylic B-material acids I-material in these hydrogels varies with the monomer B-material structure , copolymer composition , and surrounding environment . [SEP]
[CLS] philippova et al . reported the impact of hydrophobic B-property groups on the ph response in the poly ( acrylic acid ) hydrogels . [SEP]
[CLS] 255 hydrophobic B-property n - alkyl acrylates were blended in the poly ( acrylic acid ) network . [SEP]
[CLS] data showed that hydrophobic B-property modification increased the apparent pk a of poly ( acrylic acid ) - based ph - responsive hydrogels . [SEP]
[CLS] the bae group developed polymeric sulfonamides for cytosolic delivery of nucleic B-material acids I-material . [SEP]
[CLS] these polymers B-material had a reversible phase transition at ph 7 . 4 . [SEP]
[CLS] the research group was able to lower the pk a to the endosomal ph range by copolymerization with n , ndimethylacrylamide ( dmaam ) monomers B-material . [SEP]
[CLS] the pk a of the obtained copolymers shifted from 6 . 9 to 6 . 1 as the feeding ratio of hydrophilic B-property dmaam increased from 50 % to 90 % . [SEP]
[CLS] in another study , kang et al . reported that the pk a and buffering effect of oligomeric sulfonamides ( osas ) are influenced by the hydrophobicity B-property of the sulfonamide monomer B-material . [SEP]
[CLS] park et al . also found that the pk a of polymers B-material containing sulfonamide groups showed polymer B-material concentration dependence where higher concentrations led to an increased transition ph . gao and co - workers reported a library of ultra - ph sensitive ( ups ) nanoparticles B-nanoparticle for tumortargeted imaging and drug delivery applications . [SEP]
[CLS] the ups nanoparticles B-nanoparticle are composed of block copolymers of peo - b - pr , where peo is poly ( ethylene oxide B-material ) and pr is a hydrophobic B-property block with multiple ionizable B-property tertiary B-material amines B-material ( figure 17 ) . [SEP]
[CLS] at ph below the apparent pk a , the copolymers with protonated ammonium groups stay in solution as unimers . [SEP]
[CLS] upon ph increase , the pr segments become neutral and associate into core−shell micelles B-material . [SEP]
[CLS] when fluorescent B-property dyes are conjugated onto the hydrophobic B-property pr segment , the ups systems display a sharp ph transition with over a 100 - fold increase in fluorescence B-property intensity within 0 . 25 ph unit , which allows precise imaging of acidification of tumors B-material or endocytic organelles . [SEP]
[CLS] the sharp ph transition , absent in commonly used small molecular and polymeric ph sensors , inspired the mechanistic investigation on the molecular basis of the cooperative response . [SEP]
[CLS] the authors first compared the ph responsive behavior of ph - sensitive small molecules or polymers B-material . [SEP]
[CLS] cooperativity in reversible protonation of ups block copolymers . - ph titration results showed that nh 4 cl ( pk a = 10 . 5 ) and chloroquine ( pk a = 8 . 3 ) , commonly used lysosomotropic agents to manipulate the ph of endocytic organelles , had typical broad ph responses in the range of ph 7 to 11 . [SEP]
[CLS] ph titrations of several extensively investigated ph - sensitive polymers B-material including polyethylenimine ( pei ) , poly ( l - lysine ) ( pll ) , chitosan B-material , and poly ( l - histidine B-material ) ( plh ) showed different degrees of broad ph response compared to small molecular bases . [SEP]
[CLS] in contrast , ph titration of three ups polymers B-material ( pdpa , pdba , and pd5a with propyl , butyl , and pentyl side chains , respectively ) showed initial ph decrease after hcl addition followed by a remarkable plateau , indicating a strong buffer effect and ultra - ph response . [SEP]
[CLS] a plot of ph transition sharpness as a function of the octanol−water partition coefficient ( logp ) of the repeating B-material unit I-material from different polymers B-material suggested that the hydrophobic B-property micellization B-event contributed to the sharp ph transition of ups nanoparticles B-nanoparticle . [SEP]
[CLS] dialysis and 1 h nmr experiments were used to investigate the ph - triggered self - assembly process . [SEP]
[CLS] collective evidence indicated that the micelle B-material phase transition is responsible for the bistable protonation states along the titration coordinate . [SEP]
[CLS] this all - or - nothing divergent proton distribution between the unimer and micelle B-material states is a hallmark of positive cooperativity ( figure 18 ) . [SEP]
[CLS] 270 [SEP]
[CLS] 4 . 4 . 2 . [SEP]
[CLS] tunable pk a and ph transition sharpness . - ma et al . reported a copolymerization strategy to fine - tune the pk a of ups block copolymers ( figure 19 ) . [SEP]
[CLS] a library of ups nanoprobes B-nanoparticle was established to cover a broad physiological ph range from 4 to 7 . 4 , where polymers B-material with more hydrophobic B-property repeating B-material units I-material displayed lower pk a . [SEP]
[CLS] readily tunable pk a may offer exciting opportunities to target endosomes for the cytosolic delivery of diagnostic and therapeutic agents before reaching lysosomes . [SEP]
[CLS] in a recent study , li et al . reported a quantitative correlation between the hydrophobicity B-property of repeating B-material units I-material of ups block copolymers and their pk a values . [SEP]
[CLS] they also expanded the composition of ups nanoprobes B-nanoparticle to polymers B-material with aromatic side chains . [SEP]
[CLS] in the same study , they showed that both anionic species and salt B-material concentration affect the apparent pk a of ups copolymers . [SEP]
[CLS] higher salt B-material concentration led to the increase of apparent pk a ( figure 20 ) . [SEP]
[CLS] when sodium B-material chloride I-material concentration increased from almost zero to 0 . 15 m , the pk a values of a representative polymer B-material increased by 1 . 1 ph unit . [SEP]
[CLS] moreover , chaotropic anions B-material ( clo 4 − ) had the most impact whereas kosmotropic anions B-material ( so 4 2− ) had the least effect on the apparent pk a . [SEP]
[CLS] many proteins B-material in living systems display a unique ph - dependent membrane insertion property . [SEP]
[CLS] phlip peptides B-material consist of about 36 amino B-material acids I-material . in acidic environments , phlips can insert across the cell B-material membrane with increased accumulation in acidic tissues ( figure 21 ) . [SEP]
[CLS] in the acidic tumor B-material environment , the low ph - driven insertion characteristics were exploited for the development of tumor - targeted imaging agents and drug delivery systems . [SEP]
[CLS] in 2007 , engelman and co - workers reported a fluorescently B-property labeled phlip for tumor B-technique imaging I-technique . [SEP]
[CLS] the imaging probe identified solid tumors B-material with good signal - to - noise ratio ( 3−5 times higher in tumors B-material than adjacent normal tissues ) and was stable over 4 days . [SEP]
[CLS] a phlipbased delivery system was also reported for the transport of phalloidin , a cell - impermeable toxin , into the cytoplasm of cancer cells B-material . [SEP]
[CLS] the phlip peptide B-material inserted its c terminus across the cell B-material membrane at lower ph , which allowed for triggered release of toxin through the cleavage of a disulfide bond . [SEP]
[CLS] proliferation of multiple cancer cell B-material types was inhibited . [SEP]
[CLS] nitin and co - workers designed an alexa - 647 labeled phlip for the imaging of variations in extracellular ph in head and neck squamous cell B-material carcinoma . [SEP]
[CLS] the fluorescence B-property intensity is 4−8 - fold higher in cancer tissues over healthy tissues . [SEP]
[CLS] the molecular mechanism and principles of pk a control were extensively studied . [SEP]
[CLS] weerakkody et al . reported the structure−property correlations in altering the transition pk a of phlips by screening a library of 16 rationally designed peptides B-material . [SEP]
[CLS] it was found that the pk a values of phlip variants were sequence - dependent . for example , the variants with asp residue generally displayed a lower pk a while the analogs containing glu residues usually had a higher pk a . [SEP]
[CLS] in another study , they showed that the pk a of phlips shifted to a lower ph as the hydrophobic B-property thickness of the membrane increased . [SEP]
[CLS] both the composition of the peptides B-material and the physical properties of the lipid B-material bilayers I-material impacted the ph - triggered membrane insertion process and ensuing tumor B-material targeting , organ distribution , and blood clearance outcomes . [SEP]
[CLS] it was postulated that phlips can exist in three distinctive states : unstructured and soluble B-property state in aqueous solution , unstructured and binding state to the outer leaflet of the cell B-material membrane , and α - helical state after membrane insertion in response to acidic ph signal . [SEP]
[CLS] the two aspartic B-material acid I-material residues were critical for the observed ph - induced membrane insertion behavior . [SEP]
[CLS] these residues are negatively charged at neutral or basic ph , which prevents insertion into the phospholipid bilayers due to electrostatic repulsion . [SEP]
[CLS] at low ph , the protonated carboxylate B-material groups I-material enable the reduction in polarity leading to the conformation change and membrane insertion . [SEP]
[CLS] previous investigations have shown that the formation of an α - helix is a cooperative process . [SEP]
[CLS] engelman and co - workers reported that increasing the number of ionizable B-property residues can promote the ph - dependent cooperative membrane insertion process ( figure 22 ) . [SEP]
[CLS] protonation of the initial asp allowed peptides B-material to insert into the cell B-material membrane partially . [SEP]
[CLS] consequently , exposure in the membrane environment drives further protonation of the adjacent asp , leading to a positive feedback and complete membrane insertion . [SEP]
[CLS] the cooperative insertion process was further validated by the mutation of conformation - restrained proline residue by glycine B-material . [SEP]
[CLS] data show that the proline at position 20 , midway through the transmembrane region , is crucial for ph - induced insertion activity . replacement of proline - 20 by glycine B-material resulted in variable insertion over a broader ph range , suggesting reduced cooperativity compared to wide type phlips . [SEP]
[CLS] cooperativity is universally found in the nanoscale systems where identical or near - identical components self - assemble into multicomponent structures through a multitude of noncovalent interactions . [SEP]
[CLS] cooperativity can be described as the synergistic process in which individual components interact with each other to accelerate or facilitate the formation of a multicomponent complex , which is usually the most thermodynamically favorable state . [SEP]
[CLS] cooperativity can be manifested in either intramolecular ( e . g . , protein B-material folding ) or intermolecular ( e . g . , micellization B-event ) processes . [SEP]
[CLS] mechanistically , nanoscale cooperativity can be broadly categorized into two types : allostery and preorganization . [SEP]
[CLS] in allosteric cooperativity , binding between a and b induces conformational change of a , which results in increased binding affinity for component c ( figure 23a ) . [SEP]
[CLS] compared to the free state a , the formation of ab complex opens a new binding site on a with enhanced binding affinity for c . in the preorganization model ( figure 23b ) , the initial complexation of a and b decreases the number of nonproductive configurations and thereby reduces the entropic cost of bringing c into the bound state from its free state . [SEP]
[CLS] preorganization promoted cooperativity can further be augmented by the additional interactions ( figure 23c ) . [SEP]
[CLS] for example , initial formation of complex ab not only facilitates the binding between a and c , additional interactions between b and c render gains in free energy of binding that further drive the formation of complex abc . [SEP]
[CLS] it should be noted that these two types of cooperativity are not mutually exclusive and can occur concurrently in the same nanosystem . [SEP]
[CLS] 5 . 1 . 1 . cooperative folding of proteins B-material . - scientists have long studied the impact of amino B-material acid I-material sequence on a protein B-material ' s native structure and the stochastic nature of the folding process . [SEP]
[CLS] a " folding funnel " hypothesis is proposed in an energy landscape model ( figure 24 ) . [SEP]
[CLS] the transition states , the energy barrier B-property that denatured conformations must overcome in order to fold into the native state , are represented by the saddle points on the surface of the above landscape . [SEP]
[CLS] superimposed on the surface are intermediate B-property states that represent different stages of the progressive folding process . [SEP]
[CLS] the folding funnel theory assumes the existence of many non - native local minima of free energy , where partially folded proteins B-material are trapped . [SEP]
[CLS] the folding funnel theory hypothesizes that hydrophobic B-property collapse plays an essential role in the folding of proteins B-material . [SEP]
[CLS] hydrophobic B-property interaction I-property between amino B-material acids I-material ' side chains stabilizes the intermediate B-property states and in the folded domains . [SEP]
[CLS] the free energy of folded structures can be further lowered by the relocation of charged side chains on the surface of proteins B-material or the formation of salt B-material bridges to balance the charges in the core B-material . [SEP]
[CLS] the interplay of many types of noncovalent interactions contributes to the observed positive cooperativity , a hallmark of protein B-material folding . [SEP]
[CLS] the dynamic coupling between the interactions which stabilizes a packed natural state determines the cooperativity of the folding landscape . [SEP]
[CLS] in other words , cooperativity implies a favored protein B-material folding pathway to the native state . [SEP]
[CLS] strong coupling between the stabilization forces will lead to a cooperative two - state transition in protein B-material folding as observed in the self - assembly of small globular proteins B-material . [SEP]
[CLS] 5 . 1 . 2 . cooperative activation of ion B-material channels . - in voltage - gated channels ( e . g . , na + , k + and ca 2 + ) , separate protein B-material domains are responsible for ion B-material conduction and voltage sensing . [SEP]
[CLS] isacoff showed that the two subunits of the human hydrogen B-material voltage - gated channel 1 ( hv1 ) affect one another during gating with positive cooperativity . [SEP]
[CLS] opening of either subunit favors the opening of the other one dramatically . [SEP]
[CLS] this model correlated with the experimental observation that the two pores of hv1 tended to stay either both open or closed ( all or nothing ) ( figure 25 ) . [SEP]
[CLS] 299 [SEP]
[CLS] polymers B-material . - tanaka and co - workers proposed a " pearl - necklace " model to describe the cooperative hydration process in the solvation of pnipam polymers B-material ( figure 26 ) . [SEP]
[CLS] when a water B-material molecule initiates a hydrogen B-material bond with an amide B-material group I-material in the backbone , it results in displacement of the isopropyl group to enable the second water B-material molecule to form another hydrogen B-material bond . [SEP]
[CLS] consecutive hydration of water B-material molecules behaves like a pearl - necklace type along the polymer B-material chain . [SEP]
[CLS] when temperature increases , each sequence can be dehydrated cooperatively , leading to the collective collapse of the polymer B-material chain and observed sharp melting curve . [SEP]
[CLS] in a separate study , wu and co - workers have discovered the presence of a molten globule state along the thermal transition coordinate of a single pnipam chain , which resembles that in protein B-material folding . [SEP]
[CLS] the molten globule state is characterized by a dense core B-material and a molten shell B-material , which suggests a heterogeneous assembly process during phase transition . [SEP]
[CLS] - a polymeric allosteric model was proposed by li et al . to describe the ph - triggered phase transition of ultra - ph sensitive block copolymers . [SEP]
[CLS] the polymer B-material chains with multiple ionizable B-property tertiary amines B-material were considered as a multisite receptor B-material and the protons as monovalent ligands . [SEP]
[CLS] experimental data showed that the copolymers in the micelle B-material state were mostly neutral , whereas the majority of the tertiary B-material amines B-material were protonated in the unimer state in solution . [SEP]
[CLS] in the protonation of ups polymers B-material , the micelles B-material initially created a hydrophobic B-property core B-material to prevent the protons from ionizing B-property the tertiary amines B-material ( figure 27 ) . [SEP]
[CLS] protons cannot break through the hydrophobic B-property barrier B-property until a critical ph threshold ( or a critical proton concentration ) is reached . [SEP]
[CLS] once protonation started , the ionized B-property ammonium groups are hypothesized to expose the hydrophobic B-property chains to the aqueous environment , which facilitates the protonation of the remaining tertiary B-material amines B-material . [SEP]
[CLS] the reversed deprotonation process also displayed strong ph cooperativity following the " loss of protons - increase of hydrophobicity B-property - polymer B-material condensation " cycle . [SEP]
[CLS] the hydrophobic B-property micellization - driven cooperativity leads to a hill coefficient of 51 and shifts the pk a from alkaline ph to acidic ph ( e . g . , 9 to 5 ) . [SEP]
[CLS] hunter and anderson described different kinds of cooperative behaviors in multicomponent complexes . [SEP]
[CLS] among these , allosteric cooperativity is best understood , where binding a ligand to a multisite receptor B-material will affect the binding affinity of the next ligand as a result of conformational changes ( figure 28a , b ) . [SEP]
[CLS] allosteric enzymes change conformation upon the binding of the first substrate , which affects the binding of molecules at other sites . [SEP]
[CLS] cooperativity is also commonly found in bivalent binding processes such as cell B-event adhesion I-event and chelation ( figure 28c , d ) . [SEP]
[CLS] a bivalent ligand may bind to a bivalent receptor B-material at either site . [SEP]
[CLS] after the first binding , subsequent binding becomes an intramolecular event with reduced entropic cost . [SEP]
[CLS] polyvalent ligand may pertain to multiple distinct binding elements , which can be identical or dissimilar . [SEP]
[CLS] for multivalent interactions , valence over [ 3 + 2 ] can lead to physical cross - links and phase condensation as shown previously in the nephrin / nck / n - wasp system ( figure 7 ) . [SEP]
[CLS] the third type of cooperativity is found ( figure 28 e ) in the oligomerization or polymerization of amyloid peptides B-material , actin strands , and other polymer B-material systems . [SEP]
[CLS] similar to the allosteric scenario , initial organization of repeating B-material units I-material such as nucleation makes the subsequent binding more favorable and triggers cooperative self - assembly . [SEP]
[CLS] recently , cheng and co - workers reported a cooperative synthetic polymer B-material system . [SEP]
[CLS] this polymer B-material can catalyze its own chain elongation . [SEP]
[CLS] initial formation of αhelices accelerates the polymerization rate due to cooperative interactions of macrodipoles between neighboring α - helices . [SEP]
[CLS] to determine whether a protein−ligand binding process exhibits any cooperativity , binding parameters of the ligand to the protein B-material are first quantified at varying concentrations of the ligand . [SEP]
[CLS] in a representative case , θ a is defined as the molar fraction of protein binding sites that are occupied by the ligand of interest . [SEP]
[CLS] for a process with no cooperativity , it takes about 100 - fold change in ligand concentration to increase the site - occupancy from 10 % to 90 % . [SEP]
[CLS] if a system displays positive cooperativity , it takes smaller changes in concentration for the same increase in occupation percentage . [SEP]
[CLS] for allosteric systems such as in protein−ligand interactions , a hill plot is often used to quantify cooperativity . [SEP]
[CLS] in practice , the hill plot is obtained by plotting log ( θ / ( 1 − θ ) ) versus logarithmic concentration of ligands ( eq 1 ) . [SEP]
[CLS] n h : hill coefficient θ : total fraction of receptor B-event binding I-event sites bound to ligand l : unbound ligand concentration [SEP]
[CLS] the hill coefficient n h , corresponding to the slope of this plot measured at 50 % saturation , is used to quantify the cooperativity strength experimentally . [SEP]
[CLS] a hill coefficient of one suggests no cooperativity in the binding process . [SEP]
[CLS] a hill coefficient of greater or less than one indicates positive or negativecooperativity , respectively . [SEP]
[CLS] the hill coefficient is widely used in allosteric binding studies . [SEP]
[CLS] many pharmacokinetic −pharmacodynamic models reported the use of the hill equation to quantify the nonlinear drug dose−response relationships . [SEP]
[CLS] other quantification methods have also been developed in different self - assembly systems where a hill plot is not applicable or not very accurate . [SEP]
[CLS] yifrach showed that a modified boltzmann equation can estimate the degree of cooperativity in voltage - dependent ion B-material channels . [SEP]
[CLS] this approach allowed the quantification of the steady - state cooperativity of ion B-material channels and enzymes . [SEP]
[CLS] camara - campos et al . reported the use of double mutant cycles to investigate chelate cooperativity in multiple hydrogenbonded complexes . [SEP]
[CLS] this method allowed for the delineation of the free energy contribution associated with the intramolecular B-property non B-property - I-property covalent I-property interactions I-property . [SEP]
[CLS] ercolani proposed a method to quantitatively evaluate the cooperativity in helicate and porphyrin ladders . [SEP]
[CLS] he defined a new parameter , statistical stability constant , to evaluate the cooperativity . [SEP]
[CLS] a binding isotherm from a receptor−ligand titration study ( e . g . , fluorescence B-property anisotropy 315 and isothermal titration calorimetry316 ) is another common methodology to analyze cooperativity . [SEP]
[CLS] saykally reported the use of far - infrared vibration−rotation tunneling ( vrt ) spectroscopy B-technique to quantify hydrogen B-material bond cooperativity . [SEP]
[CLS] the mariuzza group employed a surface plasmon resonance method to quantify the strength of binding cooperativity in a three - component complex . [SEP]
[CLS] their method for the quantification of cooperativity strength may probably be applicable in modeling more complicated protein B-material assemblies . [SEP]
[CLS] the hill coefficient of oxygen B-material binding to hemoglobin is in the range 1 . 7−3 . 2 . [SEP]
[CLS] berg group ' s investigation in e . coli indicated that assemblies of bacterial chemoreceptors work cooperatively with a hill coefficient ranging from 1 . 4 to 3 . 8 . [SEP]
[CLS] their results were consistent with several previous reports that long - range cooperative interactions can serve as a general mechanism for signal amplification . [SEP]
[CLS] the maturation of xenopus oocytes with hormone progesterone operates in an all - or - nothing manner . [SEP]
[CLS] the cooperative response is generated by the mitogen - activated protein B-material kinase ( mapk ) cascade . [SEP]
[CLS] analysis of individual oocytes suggested that the response of mapk to progesterone was equivalent to that of a cooperative enzyme with a hill coefficient of 35 . [SEP]
[CLS] li et al . quantified the ph cooperativity of ups polymers B-material ( figure 29 ) . [SEP]
[CLS] the hill coefficients of ultra - ph sensitive block copolymers were around 51 , compared to 1 of commonly used small molecular bases . [SEP]
[CLS] they showed that the cooperativity can be further strengthened by increasing the hydrophobic B-property chain length . [SEP]
[CLS] the cooperativities in similar anion - induced self - assembly systems were also investigated . [SEP]
[CLS] the hill coefficient ranged from 5 to 30 depending on the anion B-material species . [SEP]
[CLS] the self - assembly process was driven by a novel micellization B-event process induced by the chaotropic anions B-material . [SEP]
[CLS] nanoscale cooperativity can be exploited in the design of activatable nanomedicine with increased biological precision and specificity . [SEP]
[CLS] these nanostructures can be designed to stay in the inactive state at normal physiological conditions but become activated at the site of disease to achieve diagnostic and therapeutic functions ( figure 30a ) . [SEP]
[CLS] the release of imaging signals or payloads can be triggered by physical ( ultrasound , heat , light ) , chemical ( ph , redox potential ) , or biological ( enzyme , dna ) stimuli . [SEP]
[CLS] compared to noncooperative systems , cooperative nanostructures can respond to stimuli more rapidly and efficiently ( figure 30b ) . [SEP]
[CLS] small changes in the amount / concentration of target signals ( [ t ] ) are able to elicit large signal changes in diagnostic or therapeutic outcomes . [SEP]
[CLS] another benefit of the cooperative system is the ability to fine - tune the threshold of stimuli response , which can be used to target selective oxygen B-material pressure , ph , or temaperature ( figure 30c ) to enlarge the therapeutic window . [SEP]
[CLS] the precise spatiotemporal control of the activation of functionalized nanoparticles B-nanoparticle will be further discussed in this section with selected cooperative nanomedicine systems . [SEP]
[CLS] a fundamental challenge in medicine is the efficient delivery of therapeutic cargos into the targeted cells B-material . [SEP]
[CLS] in cancer , solid tumors B-material usually show anatomical and pathophysiological properties different from those of normal tissues . [SEP]
[CLS] for example , tumors B-material have leaky vasculature and impaired lymphatic systems that can lead to accumulation of nanoparticulates , a phenomenon termed as the enhanced permeability and retention ( epr ) effect . [SEP]
[CLS] although drug B-nanoparticle - I-nanoparticle loaded I-nanoparticle nanoparticles I-nanoparticle with optimized diameter and surface chemistry have been designed to take advantage of the epr effect to accumulate in tumor B-material sites , cancer cells B-material develop drug resistance over time . [SEP]
[CLS] to overcome drug resistance , active targeting strategies have been developed . [SEP]
[CLS] ligands are functionalized onto the surface of nanoparticles B-nanoparticle by various conjugation chemistries . [SEP]
[CLS] these ligand - encoded nanoparticles B-nanoparticle can specifically bind to the receptors on the surfaces of targeted tumor B-material cells B-material after extravasation . [SEP]
[CLS] the bound nanocarriers can be internalized via endocytosis B-event , enabling the intracellular release of drugs . [SEP]
[CLS] such active targeting methods can overcome the efflux - pump mediated drug resistance with increased intracellular drug concentration . [SEP]
[CLS] the internalization of drug B-nanoparticle - I-nanoparticle loaded I-nanoparticle nanoparticles I-nanoparticle is dependent on receptor - mediated endocytosis B-event . [SEP]
[CLS] both the initial binding events and ensuing cell B-material uptake can be enhanced by multivalent binding ( figure 31 ) . [SEP]
[CLS] the multivalent binding process may display positive cooperativity , where binding of one ligand on the nanoparticles B-nanoparticle will facilitate further binding events for the neighboring ligands . [SEP]
[CLS] for example , in multiple folate B-event receptor I-event binding I-event , the nanoparticle - cell B-material association is enhanced by more than 2 , 500 - fold . [SEP]
[CLS] multivalent antiviral B-property and anti - inflammation therapeutics also showed significantly improved potencies compared to corresponding monovalent counterparts . [SEP]
[CLS] the overall binding affinity of ligand - modified nanoparticles B-nanoparticle to targeted cells B-material generally increases with an increasing ligand density . [SEP]
[CLS] however , too high a ligand density may result in the decrease of binding affinity due to unfavorable steric crowding , where the ligand may have limited conformational freedom to effectively bind to the target molecules . [SEP]
[CLS] besides naturally existing cell B-material surface I-material receptors I-material ( e . g . , folate B-material receptor I-material ) , rapid advances in bioorthogonal chemistry have inspired the metabolic labeling of cancer cells B-material for the targeted delivery of nanomedicine . [SEP]
[CLS] metabolic labeling artificially introduces chemical receptors onto the cell B-material surfaces and enables a " two - step " targeting strategy . [SEP]
[CLS] this strategy is especially useful for delivering therapeutics without nascent biomarkers B-property . [SEP]
[CLS] recently , kim and co - workers developed an active targeting strategy through cu 2 + free bioorthogonal chemistry . [SEP]
[CLS] unnatural sialic acids with azide groups were introduced on the cell B-material surface of tumors B-material via metabolic glycoengineering , which effectively enhanced the accumulation of nanoparticles B-nanoparticle by multivalent interactions . [SEP]
[CLS] biosensors usually rely on biological recognition of disease - specific biomarkers B-property where the signals are further processed by a transducer . [SEP]
[CLS] there are different categories of biosensor designs including small molecules , peptides B-material , aptamers , antibodies B-material , proteins B-material , and different types of nanoparticles B-nanoparticle . [SEP]
[CLS] one significant drawback of conventional biosensing technology is that most biosensors are analog sensors , where noise can be propagated without signal amplification , leading to degraded signal - to - noise ratios . use of nanomaterial B-material biosensors has the potential to overcome the deficiencies of commonly used biosensors . [SEP]
[CLS] one such example is spherical nucleic B-material acid I-material ( snas ) - based nanoflares . [SEP]
[CLS] these nanosensors B-nanoparticle have been used in the molecular sensing of a range of analytes including nucleic B-material acids I-material , proteins B-material , small molecules , and metal B-material ions B-material . [SEP]
[CLS] the combination of an inorganic B-material core I-material and polyvalent oligonucleotide shell B-material offers advantages over unimolecular counterparts . [SEP]
[CLS] a target analyte such as nucleic B-material acids I-material can be recognized by two different designs of snas . [SEP]
[CLS] subsequent binding of target sequences will trigger the aggregation of the snas nanoparticles B-nanoparticle , which is accompanied by a visible color transition . [SEP]
[CLS] the aggregates exhibit a narrow melting transition compared to duplex dnas . [SEP]
[CLS] a single base pair mismatch , insertion , or deletion will result in a shift of transition temperature that leads to the detection of target nucleic B-material acids I-material with high specificity . [SEP]
[CLS] moreover , the high extinction coefficient of gold B-nanoparticle nanoparticles I-nanoparticle allows for sensitive detection of target molecules at lower concentrations than with conventional dyes . [SEP]
[CLS] recently , the mirkin team applied the nanoflare technology to detect circulating tumor B-material cells B-material ( ctcs ) in blood ( figure 32 ) . [SEP]
[CLS] detection of ctcs offers early opportunities for metastatic risk assessment . [SEP]
[CLS] the nanoflares were designed to target mrnas ( mrnas ) that code for protein B-material biomarkers B-property for breast cancer cells B-material . [SEP]
[CLS] they were able to detect the genetic markers of ctcs in blood with less than one percent false positive results . [SEP]
[CLS] this technique also successfully detected ctcs in a murine model of metastatic breast cancer . [SEP]
[CLS] this nano cooperativity - enabled approach offers a new paradigm for tumor B-material diagnosis and personalized treatment . [SEP]
[CLS] cancer is a heterogeneous disease that makes it challenging for universal , cancer - specific detection . [SEP]
[CLS] most common strategies in the development of tumor - targeted imaging agents focus on cell B-material surface proteins B-material such as the folate B-material receptor I-material , 351 chlorotoxin , epidermal growth B-material factor I-material receptor I-material , and some tumor - associated antigens . [SEP]
[CLS] although various preclinical studies have shown some success , the ability to detect a broad range of cancer types is often not possible because of genetic or phenotypic variability among different tumors B-material . [SEP]
[CLS] tumor B-material acidosis , which is well recognized as a hallmark of cancer regardless of genotypes and phenotypes , can be used as a universal target for cancer - specific imaging and drug delivery . [SEP]
[CLS] however , commonly used small molecular ph sensors display broad ph response ( 2 ph units ) and are not capable to differentiate subtle ph variation between tumor B-material and surrounding normal tissues . [SEP]
[CLS] to overcome these deficiencies , zhao et al . reported a transistor - like ph threshold sensor for the tumor - specific fluorescent B-property imaging of different types of cancers ( figure 33 ) . [SEP]
[CLS] the fluorescent B-property nanosensor B-nanoparticle amplified the tumor B-material acidosis signals with discretized output while remaining silent in the normal tissue . [SEP]
[CLS] the binary on / off digitization of tumor B-material ph and surrounding normal tissue ph allows for clear tumor B-material margin depiction with high sensitivity and specificity . [SEP]
[CLS] the real - time image - guided surgery of primary tumors B-material and occult nodules ( < 1 mm 3 ) in mice bearing head and neck or breast tumors B-material significantly improved the longterm survival over white light controls . [SEP]
[CLS] in a separate study , wang et al . reported a hybrid ultra - ph - sensitive ( hyups ) nanosensor B-nanoparticle design to digitize the luminal ph of endocytic organelles in live cells B-material ( figure 34 ) . [SEP]
[CLS] the hyups nanosensor B-nanoparticle consisted of a mixture of three different copolymers with each exhibiting a sharp ( < 0 . 25 ph ) response at different ph thresholds ( ph t ) . [SEP]
[CLS] hyups allowed for the quantification of acidification kinetics of endocytic organelles at a single - organelle resolution . [SEP]
[CLS] compared to a conventional analog ph sensor ( e . g . , lysosensor ) , the hyups design does not require a calibration curve before ph measurement and is less sensitive to photobleaching . [SEP]
[CLS] a digital barcode ( e . g . , 000 , 001 , etc . ) can be easily assigned based on the binary on / off signal output in each fluorescence B-property channel for each organelle , whereas [SEP]
[CLS] lysosensor only measures the average endocytic ph from all the acidic organelles ( including golgi ) within each cell B-material . [SEP]
[CLS] this simple analog to digital signal conversion allowed for fast quantification of organelle ph and permitted identification of mutant kras as an oncologic driver for the accelerated acidification of endocytic organelles in cancer cells B-material . [SEP]
[CLS] nanostructures that can be externally triggered to release drugs on demand have the potential to improve therapeutic efficacy with reduced toxicity B-property . [SEP]
[CLS] one major challenge in the design of externally triggered drug release systems is the low sensitivity and poor response . [SEP]
[CLS] long time exposure to external energy sources may result in serious tissue damage and side effects . [SEP]
[CLS] nanosystems with cooperative response to external stimuli can deliver therapeutics more effectively and precisely . [SEP]
[CLS] elastin - like polypeptides B-material ( elps ) have been developed as thermoresponsive micelles B-material and liposomes B-nanoparticle for drug delivery . [SEP]
[CLS] liu et al . reported a local cancer radiotherapy consisting of elps conjugated to a therapeutic radionuclide . [SEP]
[CLS] this injectable depot successfully delayed the tumor B-material progression and showed controlled advanced - stage cancers . [SEP]
[CLS] chilkoti and coworkers also reported " heat activatable " drug - loaded elp nanoparticles B-nanoparticle to target solid tumors B-material . [SEP]
[CLS] the kostarelos group reported a lipid B-material - peptide B-material nanoplatform for sustained release of therapeutics triggered by hyperthermia . [SEP]
[CLS] the lipid B-material - based nanoparticles B-nanoparticle showed extraordinary stability in blood circulation at physiological temperature . [SEP]
[CLS] in vivo data by cdoxorubicin quantitation illustrated significantly increased tumor B-material accumulation at 24 h after intravenous administration with hyperthermia . [SEP]
[CLS] elp - functionalized plasmonic nanoparticles B-nanoparticle , 368 liposomes B-nanoparticle , and dendrimers B-nanoparticle [SEP]
[CLS] were also developed . [SEP]
[CLS] besides cancer , elp drug depot has also been investigated in applications for joint degeneration , neuro - inflammation , and diabetes [SEP]
[CLS] the backbone of poly ( β - amino esters ) ( paes ) can be degraded through hydrolysis under physiological conditions , which improves their safety profiles in biomedical applications . [SEP]
[CLS] paes were employed in the design of gene / drug delivery systems . [SEP]
[CLS] however , the thermoresponsive behavior of these polymers B-material was less known and their phase transition behavior was largely unexplored . [SEP]
[CLS] recently , wang and co - workers reported the temperatureinduced phase transition behavior of hyperbranched paes ( hpaes ) ( figure 35 ) . [SEP]
[CLS] by varying the length of the ethylene glycol spacers B-material and the molecular weight of polymers B-material , the lcst of hpaes was successfully fine - tuned in aqueous environment . [SEP]
[CLS] the sharp melting curves suggest strong cooperativity in the reversible dehydration of these polymers B-material , which makes them another model system for the investigation of molecular cooperativity in aqueous environment . [SEP]
[CLS] nanomedicine is an interdisciplinary field that integrates physics , chemistry , materials science , biology , and pathophysiology principles toward prevention , diagnosis , and treatment of diseases . [SEP]
[CLS] over the past decade , the field has advanced rapidly as a result of the push by the medical needs to improve patient care and the pull of novel science at the nanoscale that is absent in the traditional single molecular arena . [SEP]
[CLS] multiple therapeutic nanomedicines have progressed into the clinical stages , with some successes ( e . g . , doxil ) as well as unsuccessful attempts . [SEP]
[CLS] the lack of success has spurred heated debates on the potential promise of nanomedicine and allowed for a healthy introspection and reality check by many in the field . [SEP]
[CLS] in contrast to small molecule - based diagnostics and therapeutics , nanomedicine represents a new paradigm that employs a system - based approach toward problem solving . [SEP]
[CLS] a clear advantage is the ability to incorporate multiple functions and tools within a small size confinement to address multiple challenges simultaneously . [SEP]
[CLS] one such example is the multilayered liposomal B-nanoparticle carriers with sequential delivery of two therapeutic drugs and surface functionalization by folate and cy5 . 5 dye ( figure 36 ) . [SEP]
[CLS] the elaborate engineering design synergizes cell B-material surface I-material receptor I-material targeting by the folate ligand for cell B-material uptake , combined with therapeutic targeting by erlotinib and doxorubicin B-material to exploit different vulnerable molecular pathways inside cancer cells B-material . [SEP]
[CLS] it also allows for the tracking of the nanoparticles B-nanoparticle in cancer cells B-material by fluorescence B-technique imaging I-technique . [SEP]
[CLS] such system - based combination of multiple therapeutic and imaging modalities is beneficial over single molecular drug therapy where adaptive resistance arises over time in cancer patients . [SEP]
[CLS] despite the therapeutic promise , nanomedicine also introduces exponentially increased complexity inherent to the multiple interacting components within the system . [SEP]
[CLS] although synergistic outcome is desirable , chaos , where subtle changes in the initial conditions can result in widely divergent outcomes ( aka the butterfly effect ) , can also occur and introduce uncertainties in data irreproducibility , increase in the cost of production , and challenges in quality control [SEP]
[CLS] how to achieve robustness in action from complex nanomedicine systems is a paramount but perplexing challenge . [SEP]
[CLS] biology may provide the answer to many of these challenges . [SEP]
[CLS] biological systems are complex , dynamic systems that have held the imprint of evolution for over four billion years of history on earth . [SEP]
[CLS] although chaos does occur ( e . g . , formation of cancer , memory loss ) , life has a plethora of high fidelity processes such as maintenance of dna identity and hereditary traits . [SEP]
[CLS] in aqueous solution , the unique property of water B-material molecules creates an exceptional environment where noncovalent interactions ( electrostatic , hydrogen B-material bonding , hydrophobic B-property interactions I-property , etc . ) can interact and compensate each other at relatively low energy levels ( 5−100 kj / mol , table 1 ) comparable to the random thermal energy ( ~ 5 kj / mol for each degree of freedom ) in the environment . [SEP]
[CLS] this creates a dynamic system with high entropy and low enthalpy exchange processes ( e . g . , in contrast to covalent bond formation or breaking ) . [SEP]
[CLS] supramolecular self - assembly , whether in protein B-material folding , biomolecular condensation , or gene transcription B-event , introduces cooperativity and nonlinear dynamics to amplify signals over background noise to achieve the intended biological specificity . [SEP]
[CLS] cooperativity has the potential to overcome chaos to achieve robustness in function . [SEP]
[CLS] cooperativity principles , which have not yet drawn significant attention and are not necessarily the mainstream concept in nanomedicine design , appear to be critical in several biomedical applications such as tumor B-technique imaging I-technique ( ups nanoparticles B-nanoparticle ) , drug delivery ( elps ) , and dna sensing ( snas ) . [SEP]
[CLS] these systems manifest a " controlled chaos " phenotype where precipitive phase transitions occur that amplify the signal response to an external stimulus . [SEP]
[CLS] the underlying cooperative process resembles that of protein B-material folding ( hydrophobic B-property collapse ) [SEP]
[CLS] or biomolecular condensation in nature [SEP]
[CLS] in the engineering systems , new phenomena are further discovered as in the case of ups nanoparticles B-nanoparticle , where an all - or - nothing proton distribution phenotype was uncovered between the unimer and micelle B-material states of the ups polymers B-material , respectively . [SEP]
[CLS] this bistable state solution along the ph titration coordinate is responsible for the threshold fluorescence B-property response to subtle ph changes ( e . g . , < 0 . 3 ph unit ) in the surrounding environment , which allowed a binary delineation of tumor B-material margins ( figure 33 ) and improved accuracy in image - guided surgery . [SEP]
[CLS] for the elp and sna systems , molecular cooperativity allowed efficient on - demand drug release and ultrasensitive detection of dna strands , respectively . [SEP]
[CLS] in these examples , molecular cooperativity offers a useful strategy to transform random chaotic events into directed , synchronized outcomes for signal amplification and enlargement of therapeutic windows , which are essential to achieve precision in function for medical applications . [SEP]
[CLS] this review article aims to highlight the cooperativity principles and their potential values in self - assembled nanomedicine design . [SEP]
[CLS] although nature might have learned how to engage cooperativity in managing complex biological functions , we are only touching the tip of the iceberg to implement it in medicine . [SEP]
[CLS] moving forward , several questions may warrant future considerations . [SEP]
[CLS] first , the fundamental nature of cooperativity requires mechanistic clarity . [SEP]
[CLS] nanoscale may represent the smallest size scale at which cooperativity manifests itself . [SEP]
[CLS] despite decades of biophysical research , we have not fully understood the molecular mechanism of protein B-material folding or even the hydrophobic B-property effect . [SEP]
[CLS] the energy costs of water B-material molecules solvating extended areas of hydrophobic B-property surfaces , the contribution of hydrogen B-material bonding and / or electrostatic interactions in the changing dielectric environment , and the effects of different ionic species remain to be elucidated . [SEP]
[CLS] related to the first question is how to model cooperative behaviors . [SEP]
[CLS] cooperativity is an emergent property arising from the system as a whole bigger than the sum of the parts . [SEP]
[CLS] by definition , this may call for systembased models over traditional molecular based simulations . [SEP]
[CLS] chaos theory , originally from the study of weather and climate change , applies probability theory to study dynamic and complex systems . [SEP]
[CLS] similar treatment may be warranted to investigate the cooperativity behavior of nanomedicine systems . [SEP]
[CLS] lastly , is rational prediction of cooperative response feasible in nanomedicine ? [SEP]
[CLS] answers to the first two questions may provide insights for the design of cooperative systems in response to any biological stimuli of interest . [SEP]
[CLS] in the short term , attempts can be made toward the understanding of the structure−property relationships of existing cooperative systems . [SEP]
[CLS] such examples include the effect of hydrophobicity B-property of the polymer B-material matrix on the lcst of pnipam and apparent pk a of ups polymers B-material . [SEP]
[CLS] the latter example draws a different molecular strategy ( i . e . , controlling hydrophobicity B-property of polymer B-material segment ) from the use of electron withdrawing / donating groups to modulate pk a in small ionizable B-property molecules . [SEP]
[CLS] additional mechanistic studies are necessary to build a systematic set of independent evidence to help elucidate nonlinear dynamics of interacting components in coordination . [SEP]
[CLS] although many critical questions remain , we anticipate the field of nanomedicine is at an exciting juncture to make a major impact in the implementation of cooperativity principles in medicine . [SEP]
[CLS] yang [SEP]
[CLS] cooperative binding of oxygen B-material to hemoglobin . [SEP]
[CLS] initial oxygen B-material binding to hemoglobin makes it easier for the subsequent binding events . [SEP]
[CLS] the transition of hemoglobin from oxygen - free state ( t state ) to occupied state ( r state ) displays strong allosteric cooperativity . [SEP]
[CLS] supramolecular cooperativity arises from interplay of multiple types of noncovalent interactions acting in coordination . [SEP]
[CLS] cooperative multivalent interactions increase the binding avidity of ligand - conjugated nanoparticles B-nanoparticle to the cell B-material surface . [SEP]
[CLS] with low binding affinity or open ( o ) state with high binding affinity . [SEP]
[CLS] binding of transcription B-event factors evicts a nucleosome and frees up new distant binding sites for transcription B-event factors with significantly increased binding affinity . [SEP]
[CLS] displacement of nucleosome and generation of new open sites contribute to observed strong cooperative binding of transcription B-event factors . [SEP]
[CLS] . 1 . cooperativity in supramolecular self - assembly of elps . [SEP]
[CLS] 1 . supramolecular self - assembly for the development of cooperative nanomedicine . [SEP]
[CLS] cooperative folding of wildtype ribozyme leads to lowered free energy of the native - like i c intermediate B-property and the native state ( n ) . [SEP]
[CLS] reproduced with permission from ref 33 . [SEP]
[CLS] copyright 2012 elsevier ltd . [SEP]
[CLS] 4 . representative types of cooperative activation of enzymes . [SEP]
[CLS] ( a ) the binding of an allosteric effector leads to increased affinity in the adjacent site . [SEP]
[CLS] ( b ) the binding of an allosteric effector introduces a new active site . [SEP]
[CLS] reproduced with permission from ref 37 . [SEP]
[CLS] copyright 2008 nature publishing group [SEP]
[CLS] key ligand pairs and signaling molecules in an immunological synapse . [SEP]
[CLS] this process is mediated by a series of cooperative bindings of a complementary array of adhesion and costimulatory molecules . [SEP]
[CLS] orchestration of ( 1 ) antigen presentation by mhc molecule to the t - cell B-material receptor B-material , ( 2 ) cd80 / 86 costimulation , and ( 3 ) cytokine signals is necessary to achieve antigen - specific t cell B-material activation . [SEP]
[CLS] reprinted with permission from ref 57 . [SEP]
[CLS] copyright 2017 elsevier ltd . [SEP]
[CLS] 6 . schematic illustration of regulated liquid phase separation in cells B-material . [SEP]
[CLS] reprinted from ref 65 . [SEP]
[CLS] copyright 2017 american association for the advancement of science . [SEP]
[CLS] 7 . representative multivalent self - assembly process and microscopic images of liquid droplets ( scale bar = 20 μm ) . [SEP]
[CLS] reproduced with permission from ref 79 . [SEP]
[CLS] copyright 2012 nature publishing group . [SEP]
[CLS] 8 . schematic illustration of the hydrophobic B-property effect , where aggregation of a hydrophobic B-property substance reduces the number of water B-material molecules in the rigid cage surrounding the hydrophobic B-property surface . [SEP]
[CLS] 10 . distinctive structural forms of nucleic B-material acids I-material . [SEP]
[CLS] reproduced with permission from ref 166 . [SEP]
[CLS] copyright 2012 american chemical society . [SEP]
[CLS] 11 . schematic of the " shared ion B-material cloud " model in the cooperative melting behavior of sna - nps B-nanoparticle . [SEP]
[CLS] reprinted with permission from ref 173 . [SEP]
[CLS] copyright 2007 american chemical society [SEP]
[CLS] 12 . cooperative melting response depending on surface dna density of gold B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] b and c refer to data from solution and glass surface , respectively . [SEP]
[CLS] reprinted with permission from ref 171 . [SEP]
[CLS] copyright 2003 american chemical society . [SEP]
[CLS] 13 . lcst values of pnipam over a broad range of anion B-material concentrations in the hofmeister ion B-material series . [SEP]
[CLS] reprinted with permission from ref 215 . [SEP]
[CLS] copyright 2005 american chemical society [SEP]
[CLS] 14 . different methods for drug delivery using elastin - like polypeptides B-material ( elps ) in vivo . [SEP]
[CLS] reprinted with permission from ref 226 . [SEP]
[CLS] copyright 2010 elsevier ltd . [SEP]
[CLS] 15 . [SEP]
[CLS] coupled hydration layers ( left to right ) lead to cooperative dehydration of water B-material molecules surrounding the elp chains . [SEP]
[CLS] reprinted with permission from ref 228 . [SEP]
[CLS] copyright 2013 american chemical society . [SEP]
[CLS] 16 . effect of amino B-material acid I-material composition ( left panel ) and number of repeating B-material units I-material ( right panel ) on the lcst values of elastin - like polymers B-material . [SEP]
[CLS] reproduced from ref 235 . [SEP]
[CLS] copyright 2015 nature publishing group [SEP]
[CLS] 17 . schematic design of ultra - ph sensitive ( ups ) nanoparticles B-nanoparticle . [SEP]
[CLS] different hydrophobic B-property side chains were used to fine - tune the ph transition of the resulting copolymers . [SEP]
[CLS] reprinted with permission from ref 262 . [SEP]
[CLS] copyright 2011 wiley - vch [SEP]
[CLS] 18 . schematic illustration of distinctive deprotonation pathways by two structurally related copolymers , peo - b - pdma and peo - b - pdpa . [SEP]
[CLS] increase in hydrophobicity B-property of the pdpa copolymers led to an " all or nothing " cooperative deprotonation phenotype but not in pdma copolymers . [SEP]
[CLS] reprinted with permission from ref 270 . [SEP]
[CLS] copyright 2016 nature publishing group [SEP]
[CLS] 19 . ups library consisting of sharp ph threshold nanosensors B-nanoparticle spanning a wide physiological ph range from 4 to 7 . 4 . [SEP]
[CLS] reprinted with permission from ref 261 . [SEP]
[CLS] copyright 2014 american chemical society [SEP]
[CLS] 20 . [SEP]
[CLS] chaotropic anions B-material and concentration impacted the ph transition of ups micelles B-material . [SEP]
[CLS] using peo - b - npdpa as an example , the effects of nacl ( a ) , na 2 so 4 ( b ) , and naclo 4 ( c ) at different salt B-material concentrations on ph titration are presented . [SEP]
[CLS] ( d ) the apparent pk a is ion B-material species and ionic strength dependent . [SEP]
[CLS] reprinted with permission from ref 277 . [SEP]
[CLS] copyright 2016 royal society of chemistry . [SEP]
[CLS] 21 . [SEP]
[CLS] schematic illustration of phlip ' s interaction with lipid B-material bilayers I-material at neutral and acidic ph . [SEP]
[CLS] state i refers to the free peptide B-material conformation at high ph . [SEP]
[CLS] state ii describes the adsorption of the unstructured peptide B-material on the membrane surface . [SEP]
[CLS] at state iii , acidification allows the protonation of asp residues with increased hydrophobicity B-property and results in the formation of a transmembrane α - helix . [SEP]
[CLS] reprinted with permission from ref 283 . [SEP]
[CLS] copyright 2010 national academy of sciences . [SEP]
[CLS] 22 . molecular description of membrane insertion by phlips . [SEP]
[CLS] the insertion and folding of peptide B-material chains appear without intermediate B-property states , indicating a positive cooperative process . [SEP]
[CLS] reprinted with permission from ref 293 . [SEP]
[CLS] copy right 2012 elsevier ltd . [SEP]
[CLS] 23 . [SEP]
[CLS] different types of molecular cooperativity . [SEP]
[CLS] ( a ) allosteric cooperativity : binding of component b changes the conformation of component a and opens a new binding site for component c . [SEP]
[CLS] ( b ) preorganization of complex ab renders intramolecular binding between a and c to facilitate the formation of complex abc . [SEP]
[CLS] ( c ) preorganization of complex ab provides additional stabilization between b and c to drive the formation of complex abc . [SEP]
[CLS] 24 . folding funnel model to describe protein B-material folding . [SEP]
[CLS] intermediate B-property structures collapse into the native state mainly driven by hydrophobic B-property interactions I-property . [SEP]
[CLS] reprinted with permission from ref 295 . [SEP]
[CLS] copyright 2003 nature publishing group [SEP]
[CLS] 25 . cooperative gating of the hv1 channel where both subunit channels stay either open or closed . [SEP]
[CLS] reprinted with permission from ref 299 . [SEP]
[CLS] copyright 2010 nature publishing group [SEP]
[CLS] 26 . pearl - necklace model to describe conformation change of polymer B-material chains by cooperative hydration . [SEP]
[CLS] reprinted with permission from ref 199 . [SEP]
[CLS] copyright 2005 american chemical society [SEP]
[CLS] 27 . ( a ) no positive cooperativity in the protonation of hydrophilic B-property polymers B-material . [SEP]
[CLS] ( b ) hydrophobic B-property phase separation ( micellization B-event ) drives cooperative protonation or deprotonation of ionizable B-property groups at a threshold proton concentration . [SEP]
[CLS] free proton concentration remains the same during ph titration . [SEP]
[CLS] 28 . [SEP]
[CLS] cooperative associations in ligands ( pink ) and receptors with multiple binding sites ( blue ) . [SEP]
[CLS] allosteric cooperativity : initial binding of ligand induces conformational change of receptors and increases the binding affinity of the same ligand ( a ) or a secondary ligand ( b ) . [SEP]
[CLS] multivalence cooperativity : anchoring of first ligand brings the unoccupied binding site closer to free ligand and increases binding affinity of ensuing the same ( c ) or different ligand ( d ) . [SEP]
[CLS] ( e ) cooperative oligomerization or polymerization triggered by initial self - organization of several repeating B-material units I-material . [SEP]
[CLS] 29 . ( a ) binding isotherm and ( b ) hill plot of small molecular base dpa ( dipropylaminoethanol ) , polymeric bases of pei ( polyethylenimine ) , peo - b - pdma ( poly ( ethylene oxide ) - b - poly ( 2 - ( dipropylamino ) ethyl methacrylate ) ) , and peo - b - pdpa ( poly ( ethylene oxide ) - b - poly ( 2 - ( dipropylamino ) ethyl methacrylate ) ) . [SEP]
[CLS] dpa and peo - b - pdma showed no cooperativity . [SEP]
[CLS] pei displayed negative ph cooperativity , and peo - b - pdpa showed strong positive cooperativity . [SEP]
[CLS] ( c ) binding isotherm and ( d ) hill plot of peo - b - pdpa copolymers with different numbers of repeating B-material units I-material in the hydrophobic B-property segment . [SEP]
[CLS] increase of hydrophobic B-property chain length led to stronger positive cooperativity and sharper ph response . [SEP]
[CLS] reprinted with permission from ref 270 . [SEP]
[CLS] copyright 2016 nature publishing group [SEP]
[CLS] 30 . [SEP]
[CLS] cooperative nanomedicine with improved precision and specificity . [SEP]
[CLS] ( a ) schematic illustration of stimuli - responsive nanomedicine . [SEP]
[CLS] ( b ) compared to noncooperative systems , small changes in target signals can lead to amplified response in cooperative systems . [SEP]
[CLS] t refers to the target species ( e . g . , proton ) or signals ( e . g . , heat ) . [SEP]
[CLS] ( c ) tunable transition of cooperative systems enables precise control of signal activation at a predetermined threshold to enlarge the therapeutic window . [SEP]
[CLS] 31 . [SEP]
[CLS] 32 . nanoflares for mrna detection in circulating tumor B-material cells B-material . [SEP]
[CLS] reprinted with permission form ref 349 . [SEP]
[CLS] copyright 2007 american chemical society [SEP]
[CLS] 33 . tumor B-material margin delineation by a ph threshold sensor . [SEP]
[CLS] representative frozen section of hn5 tumor B-material with surrounding tissues showed excellent matching of fluorescence B-property signal with h & e tumor B-material histology ; scale bar = 2 mm . [SEP]
[CLS] dashed line indicates the tumor B-material margin . [SEP]
[CLS] reprinted with permission from ref 361 . [SEP]
[CLS] copyright 2017 nature publishing group [SEP]
[CLS] 34 . [SEP]
[CLS] multispectral hybrid ultra - ph sensitive ( hyups ) nanosensor B-nanoparticle to digitize organelle ph after receptor - I-event mediated I-event endocytosis B-event . [SEP]
[CLS] reprinted with permission from ref 362 . [SEP]
[CLS] copyright 2017 wiley - vch [SEP]
[CLS] 35 . thermoresponsive hyperbranched poly ( β - amino ester ) with tunable phase transition temperature . [SEP]
[CLS] reprinted with permission from ref 378 . [SEP]
[CLS] copyright 2017 american chemical society [SEP]
[CLS] 36 . design of a folate - targeted , cy5 . 5 - encoded multilayered liposomal B-nanoparticle system for sequential delivery of hydrophobic B-property erlotinib and hydrophilic B-property doxorubicin B-material drugs . [SEP]
[CLS] reprinted with permission from ref 392 . [SEP]
[CLS] copyright 2014 american association for the advancement of science . [SEP]
[CLS] li obtained his b . s . ( 2011 ) in polymer B-material chemistry from the university of science and technology of china . [SEP]
[CLS] he earned his ph . d . ( 2017 ) in biomedical science from the university of texas southwestern medical center under the supervision of dr . jinming gao . [SEP]
[CLS] currently , he works as a postdoctoral fellow in the research group of dr . daniel kohane at boston children ' s hospital and harvard medical school . [SEP]
[CLS] his research interest focuses on developing stimuli - responsive nanomaterials B-material for biological sensing and drug delivery . [SEP]
[CLS] he is also interested in mechanistic investigation of supramolecular self - assembly . [SEP]
[CLS] yiguang wang received his ph . d . in pharmaceutics from peking university in 2008 under the supervision of dr . qiang zhang . [SEP]
[CLS] then he worked as a postdoctoral fellow in institute of biophysics , chinese academy of sciences . [SEP]
[CLS] in 2010 , he moved to university of texas southwestern medical center at dallas and worked as a postdoctoral fellow under the supervision of dr . jinming gao . [SEP]
[CLS] in 2015 , he joined peking university school of pharmaceutical sciences as a professor . [SEP]
[CLS] his research focuses on targeted drug delivery , stimuli - responsive nanoparticles B-nanoparticle for cancer imaging and therapy , as well as intracellular trafficking of nanodrug delivery systems . [SEP]
[CLS] gang huang is an assistant professor in the simmons comprehensive cancer center and department of pharmacology at ut southwestern medical center . [SEP]
[CLS] he received his ph . d . in chemistry from the university of north texas in 2004 . [SEP]
[CLS] his postgraduate training was performed at university of utah under dr . russell stewart and at ut southwestern medical center under dr . jinming gao . [SEP]
[CLS] his current research interest focuses on the design and development of novel nanomaterials B-material and nanoarchitectures for cancer diagnosis and targeted therapeutic applications . [SEP]
[CLS] jinming gao received his b . s . in chemistry from peking university in 1991 . [SEP]
[CLS] he obtained his ph . d . under dr . george m . whitesides in physical organic chemistry at harvard university in 1996 . [SEP]
[CLS] his postdoctoral training was under dr . robert s . langer in biomedical engineering at mit . [SEP]
[CLS] in 1998 , he started his lab in the department of biomedical engineering at case western reserve university . [SEP]
[CLS] in 2005 , he moved to ut southwestern medical center at dallas , texas . [SEP]
[CLS] his current research interests focus on the understanding of nanoscale cooperativity and its implementation in niche biomedical applications . [SEP]
[CLS] the lab - invented ph transistor nanosensor B-nanoparticle is entering first - in - human trials for image - guided cancer surgery in 2018 . [SEP]
[CLS] structures , hydrophobicity B-property of repeating B-material units I-material and phase transition temperatures of several representative n - alkyl - substituted poly ( acrylamide ) s [SEP]
[CLS] herein , we design and synthesize site - specifically pegylated oligonucleotide hairpins and demonstrate that their ability to undergo hybridization chain reaction is nearly unaffected by the pegylation . [SEP]
[CLS] the resulting dna - backboned bottlebrush polymers B-material with peg side chains exhibit increased resistance against nucleolytic degradation , enhanced thermal stabilities , and elevated blood retention times in vivo , which collectively pave the way for more therapeutically focused dna nanostructure designs . [SEP]
[CLS] rapid renal clearance , which is the primary body elimination pathway for natural oligonucleotides under ~ 50 kda in size . [SEP]
[CLS] still , the realization of dna nanotechnology in medicine is occurring at a much slower pace compared to the development of new structures . [SEP]
[CLS] unmodified dna exhibits poor nucleolytic stability in vivo . [SEP]
[CLS] in addition , the compact dna nanostructures often require high cation B-material concentrations ( na + , mg 2 + , ca , and so forth ) to minimize the repulsive interaction of the negatively charged phosphate on the dna backbone . [SEP]
[CLS] several compatibilizing macromolecules , for example , cationic B-material peptides B-material , polymers B-material , proteins B-material , and nano - particles , have been developed to bind with preformed dna nanostructures via electrostatic complexation to improve their solution and / or biological stabilities . [SEP]
[CLS] nevertheless , challenges exist regarding the control of the location and number of the compatibilizing agents on the dna nanostructure , removal of excess unbound agent , and potential side effects associated with the agent . [SEP]
[CLS] an alternative approach to improving the biopharmaceutical properties of nucleic acid - based materials is through covalent attachment of poly ( ethylene glycol ) ( peg ) , a biologically " stealth " polymer B-material often adopted in pharmaceutical formulations . [SEP]
[CLS] however , a single chain of linear or slightly branched peg ( 40 - 100 kda ) is generally insuffcient to shield oligonucleotides from interaction with proteins B-material and thus cannot provide proper biopharmaceutical characteristics for systemic use . [SEP]
[CLS] we have recently developed a novel form of brush polymer - oligonucleotide conjugate , termed pacdna ( polymer - assisted compaction of dna ) , to address this challenge . [SEP]
[CLS] the bottlebrush - architectured peg has a large number ( typically > 25 ) of shorter ( 5 - 10 kda ) peg side chains emanating from a central backbone , which significantly increases local peg density and thus steric protection of the conjugated oligonucleotide . [SEP]
[CLS] meanwhile , dna hybridization kinetics and thermodynamics with a complementary sequence are not negatively affected . [SEP]
[CLS] the pacdna exhibits high enzymatic stability ( 10 - 20× increase in half - life ) , can act as a single - entity antisense gene regulation agent without the need for a transfection agent , and minimizes side - effects associated with specific and nonspecific protein−dna interactions , for example , unwanted activation of the innate immune system and coagulopathy . [SEP]
[CLS] while brush - architectured peg is highly effective for shielding conjugated oligonucleotides , it would not be feasible for a preassembled dna nanostructure due to its large size ( the level of shielding is inversely corelated to the distance from the brush backbone ) . [SEP]
[CLS] however , findings about the pacdna imply that if one can precisely and evenly position many shorter peg chains alone the surface of a dna nanostructure , achieving similar peg densities to the pacdna , similarly improved biopharmaceutical properties may be realized ( see scheme 1 ) . [SEP]
[CLS] to achieve such uniformly pegylated dna nanostructures , we have devised a " bottom - up " synthesis , wherein pegylated oligonucleotide hairpins ( hps ) are used to form nanostructured dna . [SEP]
[CLS] this process , in principle , will allow for accurate control over the number and position of the peg chains over the entire surface of the nanostructure . [SEP]
[CLS] hybridization chain reaction ( hcr ) is selected as a model self - assembly reaction . [SEP]
[CLS] hcr is a form of living polymerization with the monomers B-material being a mixture of two stable hps . [SEP]
[CLS] the addition of an initiator strand opens up one of the hps to expose a single - stranded region , which opens up the other hp and reveal another single - stranded region that is identical to the original initiator . [SEP]
[CLS] the resulting chain reaction leads to the consumption of the hps and the formation of a nicked double helix ( polymer B-material ) . [SEP]
[CLS] while hcr has been widely used in analytical chemistry owing to its ability for signal amplification , bottle brush - type materials based upon hcr backbones have not been reported , to the best of our knowledge . [SEP]
[CLS] the choice of hcr is based on three factors . [SEP]
[CLS] first , hcr uses only a pair of building blocks , simplifying the preparation of pegylated versions . [SEP]
[CLS] second , hcr can readily generate high molecular weight structures with repetitive subunits , increasing the overall peg density . [SEP]
[CLS] third , the repetitiveness of the structure reduces the complexity when analyzing thermal melting properties and enzymatic stability of the hcr brush polymer B-material , as there are no non - pegylated segments that may interfere with data interpretation . [SEP]
[CLS] the hcr motifs were designed using the software package nupack ( table s1 ) . [SEP]
[CLS] in order to increase the peg density postassembly , a notably short set of hps were used ( 34 bases vs ~ 50 for typical hcr ) . [SEP]
[CLS] each hp consists of three domains : a 12 nucleotide ( nt ) stem , a 5 nt loop , and a 5 nt sticky end . [SEP]
[CLS] hp1 was modified with a fluorescein tag at the 3 ′ to enable fluorescent B-property tracking . [SEP]
[CLS] to synthesize pegylated hps , an aminemodified thymine ( t ) base in the stem domain was introduced during solid - phase oligonucleotide synthesis , which enables subsequent conjugation to n - hydroxysuccinimide ester - terminated peg ( 5 kda ) . [SEP]
[CLS] a 17 nt initiator was used to trigger the hcr cascade , which generated an analogue of a linear alternating copolymer . [SEP]
[CLS] in addition , a 4 - arm initiator was also synthesized by coupling 3 ′ dibenzocyclooctyne - terminated initiator strands to an azide - functionalized 4 - arm 20 kda peg ( each arm 5 kda ) via copper - free click chemistry . [SEP]
[CLS] the ability of the unmodified hps to undergo hcr was first tested using both the linear and the 4 - arm initiators across a series of hp / initiator ratios . [SEP]
[CLS] the successful formation of high mw polymer B-material was confirmed by agarose B-technique gel I-technique electrophoresis I-technique ( age ) ( figure 1a ) . [SEP]
[CLS] the average degree of polymerization ( dp ) was a function of the hp / initiator ratio , and the highest dp was observed with 0 . 5 equiv of the initiator . [SEP]
[CLS] typical reactions converted ~ 80 % of the monomers B-material to the polymer B-material , as determined by gel band densitometry analysis as well as peak integration using aqueous gel permeation chromatography B-technique ( gpc , figure 1b ) . [SEP]
[CLS] the polymerization resulted in dps in the range of 2 - 20 ( figure s4c ) . [SEP]
[CLS] we next investigated the ability of pegylated hps ( hp peg ) to participate in hcr . [SEP]
[CLS] monomer B-material mixtures of both partial pegylation ( hp1p e g + hp2 ) and full pegylation ( hp1 peg + hp2 peg ) resulted in high mw polymer B-material as a function of hp / initiator ratio ( figures 3c and s4 ) . [SEP]
[CLS] aqueous gpc showed ( figure 1d ) distinct polymer B-material peaks but without baseline separation from the monomers B-material , which are themselves polymers B-material . [SEP]
[CLS] by peak deconvolution , we calculated the yields of the polymerization to be ~ 75 % , which is consistent with the yields calculated by gel band densitometry analysis ( dp ≥ 2 population : 75 % - 85 % ) . [SEP]
[CLS] these results suggest that the pegylated monomers B-material are able to incorporate into hcr , but their incorporation kinetics compared to non - pegylated hps remains unclear . [SEP]
[CLS] in order to compare their reactivity ratio , a " copolymerization " experiment was devised ( figure 2a ) , which involves using a mixture of two types of hp1 : unmodified hp1 ( cy5labeled , red ) and pegylated hp1 ( fluorescein - labeled , green ) . [SEP]
[CLS] the two monomers B-material compete with each other for incorporation into the growing polymer B-material , and their ratio in the polymer B-material versus the feed reflects their relative ability to participate in the hcr ( figure 2b ) . [SEP]
[CLS] it was found that the content of hp1 in the polymer B-material was marginally but consistently higher across a range of hp1 peg / hp1 ratios , suggesting slightly greater reactivity . [SEP]
[CLS] analysis using the mayo −lewis equation and linear fitting via the fineman− ross method shows the ratio of incorporation kinetics ( k hp1 / k hp1 - peg ) to be 1 . 32 ( see supporting information for detailed analysis ) . [SEP]
[CLS] these results indicate that pegylated hps can effectively incorporate into hcr nanostructures with only a minor reduction in reactivity despite the steric congestion associated with the peg chains both on the growing polymer B-material and the monomer B-material . [SEP]
[CLS] the dna - backboned brush polymers B-material ( with 0 . 5 equiv initiator ) were studied by dynamic B-technique light I-technique scattering I-technique ( dls ) and transmission B-technique electron I-technique microscopy I-technique ( tem ) . [SEP]
[CLS] the number - average hydrodynamic diameter ( d h ( n ) ) increased from ~ 15 nm ( hp peg ) to ~ 150 nm ( linear polymers B-material ) or ~ 400 nm ( 4 - arm assemblies ) ( figure 2e ) . [SEP]
[CLS] tem of negatively stained samples ( with 1 % uranyl acetate ) reveals worm - like morphologies for the assembled structures with or without pegylation ( figure 2d ) . [SEP]
[CLS] interestingly , there is a transition in persistent length of the worms on a pyrolytic carbon B-material substrate from ~ 9 ± 3 nm for the non - pegylated assembly ( hp1 + hp2 ) to 13 ± 1 nm for the partially pegylated assembly ( hp1 peg + hp2 ) and to 37 ± 9 nm for the fully pegylated structure ( hp1 peg + hp2 peg ) based upon particle end - to - end distance analyses via easyworm . [SEP]
[CLS] the increased persistent lengths can also be seen in the star polymer B-material initiated by the 4 - arm initiator ( figure s7 ) . [SEP]
[CLS] we attribute the difference in persistent length to the steric congestion of the peg side chains , which enhance the stiffness of the dna backbone . [SEP]
[CLS] similar phenomena have been observed with other dna - based materials as well as synthetic brush polymers B-material and also through computation . [SEP]
[CLS] next , we studied the effect of pegylation on the thermal stability of hcr nanostructures . [SEP]
[CLS] the melting curves of three assemblies ( non - , partially , and fully pegylated ) were recorded by monitoring the absorbance at 260 nm as the temperature was slowly ramped up ( 0 . 2 °c / min ) from 25 to 95 °c ( figure 3a , b ) . [SEP]
[CLS] the first - order derivative of the melting transitions revealed that melting temperatures increased from 72 . 2 °c ( non - pegylated ) to 76 . 3 °c ( partially pegylated ) and to 80 . 2 °c ( fully pegylated ) ( buffer : spsc ) . [SEP]
[CLS] the enhanced thermal stability upon pegylation is likely due to the macromolecular excluded volume effect , which favors volume - reducing reactions such as dna hybridization . [SEP]
[CLS] the sharpness of the melting transition as determined by the full width at half - maximum also decreased from 36 . 5 to 22 . 6 °c , suggesting a degree of cooperativity . [SEP]
[CLS] a key aspect regarding the biomedical applications of dna nanostructures is their in vitro and in vivo stability . [SEP]
[CLS] to examine the enzymatic degradation of the hcr nanostructures , we selected the endonuclease , dnase i , as a model enzyme , which acts upon double - stranded dna with little sequence selectivity ( figure 3d ) . [SEP]
[CLS] because the assembled hcr structures were dispersed in spsc buffer , which significantly inhibits dnase i activity , dialysis against a low - salt B-material buffer ( pbs with 2 . 5 mm mgcl 2 and 0 . 5 mm cacl 2 ) was first performed . [SEP]
[CLS] the dna structures remain stable in the low - salt B-material buffer for at least two months ( figure s8 ) . [SEP]
[CLS] thereafter , dnase i ( 0 . 7 unit / ml ) was incubated B-technique with the dna nanostructures for up to 2 h . [SEP]
[CLS] analysis of the amount of remaining high mw fraction by age ( mw > 50 kda , important for avoiding renal clearance ) as a function of time revealed that pegylation can effectively protect the assembled structure with 4 . 3× increase in enzymatic half - life . [SEP]
[CLS] a similar trend was found for the 4 - armed assemblies ( figure s9 ) . [SEP]
[CLS] while these improvements over free dna do not yet exceed that of the pacdna , we anticipate that there is significant room for further optimization ( e . g . , number of peg chains per hp , mw of peg , etc ) . [SEP]
[CLS] the greater availability of the high mw fraction under digestive conditions may lead to reduced renal clearance rate and longer the blood retention times in vivo . [SEP]
[CLS] to test this hypothesis , we performed a pharmacokinetic study in immunocompetent ( c57bl / 6 ) mice by administering cy5 - labeled hcr nanostructures and controls via the tail vein , and subsequent monitoring of the cy5 concentration in the blood ( figure 3f ) . [SEP]
[CLS] the hcr hp precursors ( including pegylated ) and the non - pegylated assembly ( hp1 + hp2 ) were all rapidly cleared from blood circulation with less than 15 % remaining after 0 . 5 h . in contrast , brush - type assemblies of the pegylated hps resulted in increased blood availability . [SEP]
[CLS] the highest area - under - the - curve ( auc ∞ ) was achieved with the 4 - armed pegylated structure , which is 2 . 6× that of the free dna ( figure 3g ) . [SEP]
[CLS] these data suggest that a favorable plasma pharmacokinetics is positively correlated with the fraction of high - mw dna nanostructures in the blood . [SEP]
[CLS] both the assembly and the use of pegylated monomers B-material are important ; dna self - assembly alone does not prolong blood circulation likely due to rapid digestion . [SEP]
[CLS] in summary , we demonstrate a novel method to impart better biopharmaceutical properties to self - assembled dna - backboned bottlebrush polymers B-material by incorporating pegylated building blocks . [SEP]
[CLS] the strategy in principle allows for precise control over the number and position of the peg chains on the surface of the dna nanostructure . [SEP]
[CLS] using hcr as a model system , we show that the pegylated subunits self - assemble nearly as effectively as the non - pegylated versions . [SEP]
[CLS] the multiple peg side chains improve the thermal stability of the dna nanostructure while protecting the dna from enzymatic degradation . [SEP]
[CLS] the increased nuclease resistance allows for a higher fraction of nondigested dna nanostructure to remain in the blood , which leads to a more favorable pharmacokinetics in mice . [SEP]
[CLS] collectively , our strategy reported herein points to an attractive possibility of utilizing complex dna - based materials in nanomedicine . [SEP]
[CLS] 1 . ( a , c ) age ( 4 . 5 % ) analysis of the hcr assemblies as a function of initiator : hp ratio and composition . [SEP]
[CLS] ( b , d ) aqueous gpc analyses of the hcr assemblies and their precursors . [SEP]
[CLS] ( a ) schematic illustration of the competitive incorporation of pegylated and non - pegylated hp1 into the growing polymer B-material with an opened hp2 peg chain - end . [SEP]
[CLS] ( b ) multiplexed age ( 4 . 5 % ) analysis of the incorporation rate for hp1 ( red ) and hp1 peg ( green ) into the growing hcr polymer B-material . [SEP]
[CLS] ( c ) relationship between the feed ratio of monomers B-material ( hp1 peg and hp1 ) and fraction of them in the polymer B-material . [SEP]
[CLS] ( d ) tem images of hcr nanostructures ( negatively stained with 1 % uranyl acetate ) of varying peg content initiated with the linear initiator . [SEP]
[CLS] ( e ) dls number - average size distribution of the precursor ( hp1 peg ) and the assemblies ( linear and 4 - arm ) , showing the increased hydrodynamic diameter after hcr . [SEP]
[CLS] ( a ) schematic representation of dna thermal melting . [SEP]
[CLS] ( b ) uv−vis melting profile of pegylated and unmodified hcr systems . [SEP]
[CLS] ( c ) boltzmann fitting of the first - order derivative of the for each hcr combination . [SEP]
[CLS] ( d ) stability against dnase i as characterized by age ( 4 . 5 % ) . [SEP]
[CLS] ( e ) percentage of assembled hcr structures maintaining high mw ( > 50 kda ) in the presence of dnase i as a function of time , determined by gel band densitometry analysis . [SEP]
[CLS] ( f ) plasma half - life of hcr monomers B-material and assembled nanostructures in mice and ( g ) blood availability as expressed in auc ∞ ( * * p < 0 . 01 ) . [SEP]
[CLS] this paper describes how the ligand shell B-material containing immunostimulatory oligonucleotides surrounding gold B-nanoparticle nanoparticles I-nanoparticle affects the in vitro activation of macrophages . [SEP]
[CLS] nanoconstructs with similar ligand densities but different oligonucleotide compositions ( from 0 to 100 % immuneactive cytosine - phosphate - guanine , cpg ) were compared . [SEP]
[CLS] maximum immunostimulation was achieved with cpg content as low as 5 % ( with total oligonucleotide surface coverage remaining constant ) , correlating to high levels of anti - tumor B-material cytokine release and low levels of cancerpromoting ones . [SEP]
[CLS] independent of cpg content , gold B-nanoparticle nanoparticles I-nanoparticle with low oligonucleotide densities exhibit poor cellular uptake , leading to insignificant immunostimulation and cytokine release . [SEP]
[CLS] by identifying effects of ligand shell B-material composition on macrophage activation , we can inform the design rules of therapeutic nanoconstructs to achieve specific immune responses . [SEP]
[CLS] activation of the immune system has received increased interest in the development of cancer vaccines , 1 where antibodies B-material , proteins B-material and oligodeoxynucleotides ( odns ) are used as immunomodulatory agents . [SEP]
[CLS] odns are attractive because of their sequence - dependent immunostimulatory ( is ) activity . [SEP]
[CLS] for instance , toll - like receptor B-material 9 ( tlr9 ) recognizes odns with unmethylated cytosine - phosphate - guanine ( cpg ) moieties naturally found in microorganisms . [SEP]
[CLS] therefore , the binding between cpg and tlr9 activates macrophages that then release cytokines that recruit and regulate other leukocytes . [SEP]
[CLS] some cytokines , such as tumor B-material necrosis factor α ( tnf - α ) , have anti - tumor B-material activities , and hence controlling their release is necessary for immunotherapy . [SEP]
[CLS] odns in free form , however , exhibit limited is performance because of their poor cellular uptake and low stability in the bloodstream caused by nuclease degradation [SEP]
[CLS] when odns are densely packed onto the surface of nanoparticles B-nanoparticle and liposomes B-nanoparticle , these nanostructures , which are often referred to as spherical nucleic B-material acids I-material ( snas ) , show higher cellular uptake and stability against degradation compared to free nucleic B-material acids I-material . [SEP]
[CLS] gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) can be used as odn carriers because they are easily functionalized and biocompatible B-property . [SEP]
[CLS] certain au nanoconstructs presenting odns with cpg motifs at high surface densities exhibit stronger is activity compared to free nucleic B-material acids I-material because of enhanced cellular uptake and stability . [SEP]
[CLS] the ligand shell B-material plays the key role in the interactions between cell B-material and nanoconstructs , where odn sequences , backbones ( phosphodiester or phosphorothioate ) , and spatial orientations ( chemisorption of the 3 ' or 5 ' - terminus of odns ) can modulate immune activation . [SEP]
[CLS] structural aspects of the au core B-material , such as size and shape , can control the specificity and intensity of tlr9 - mediated is but not types of cytokines released that define macrophage biological responses . [SEP]
[CLS] although odn - aunps B-nanoparticle containing cpg can induce strong immune activation , 14 the role of polyvalency on tlr9 - mediated is remains unclear . [SEP]
[CLS] previous work demonstrated the importance of polyvalency of oligonucleotide presentation to cellular uptake and to overall nanoconstruct function , but the contribution of polyvalency to interactions with tlr9 ( or other targeted receptors ) is not known . [SEP]
[CLS] to differentiate between the effects of polyvalency on is response and uptake , the cpg content of odn - aunps B-nanoparticle needs to be varied while preserving the overall odn density . [SEP]
[CLS] here we show that the immune activation responses of macrophages to odn - aunps B-nanoparticle are influenced by both the odn composition ( % of immune active cpg ) and density of total odn . [SEP]
[CLS] for nanoconstructs prepared with high ligand densities ( ~ 55 odns per particle ) , a cpg content of only 5 % was sufficient to achieve maximal is activity . [SEP]
[CLS] odn - aunps B-nanoparticle with low cpg content shells B-material ( 1 to 10 % ) resulted in release of high levels of anti - tumor B-material cytokines and low levels of cancer - promoting ones . [SEP]
[CLS] aunps B-nanoparticle with low ligand densities ( ~ 3 odns per particle ) did not show significant is activity and cytokine production due to poor cellular uptake . [SEP]
[CLS] these results indicate that the combination of both odn composition and density determine is intensity and type of cytokines released , and that lower cpg content may be a feature useful in the design of nanoconstructs for targeting macrophage activation for cancer immunotherapy . [SEP]
[CLS] aunps B-nanoparticle were functionalized with two different alkylthiolated odns in sodium B-material citrate buffer following our published protocol ( experimental section ) . [SEP]
[CLS] we selected 15 - nm spherical aunps B-nanoparticle as the core B-material size ( figure s1 ) because larger nps B-nanoparticle and cores B-material with anisotropic morphologies , such as au nanostars , induce higher but non - specific immune activation . [SEP]
[CLS] odns had either immune - active cpg or non - active gpc motifs ( figure 1a ) . [SEP]
[CLS] nanoconstructs with different cpg to gpc ratios and odn densities were selected to evaluate independently the effect of odn composition and overall odn density on immune activation ( figure 1b and table s1 ) . [SEP]
[CLS] for odn composition studies , six types of odn - aunps B-nanoparticle with different cpg content ( from 0 to 100 % ) were compared . [SEP]
[CLS] for ligand density effects , nanoconstructs with ~ 55 and ~ 3 odns / aunp B-nanoparticle ( two significantly different ligand loadings previously used in nanomedicine ) were tested . [SEP]
[CLS] all nanoconstructs were backfilled with mercaptoundecyl hexa ( ethylene glycol ) ( muheg ) to prevent np B-nanoparticle aggregation by steric repulsion . [SEP]
[CLS] the localized surface plasmon resonances of all nanoconstructs in both phosphate - buffered saline and cell B-material media were between 523 and 527 nm , which indicates that the particles did not aggregate and that the nps B-nanoparticle were well dispersed in solution ( figure s2 ) . [SEP]
[CLS] murine raw - blue macrophages were used as the model cell B-material line because several tlrs are expressed , including tlr9 . [SEP]
[CLS] the macrophages were transfected with the secreted embryonic alkaline phosphatase ( seap ) reporter gene , which upon activation , triggers the release of seap molecules into the extracellular surroundings . [SEP]
[CLS] immune activation was quantified by seap levels in cell B-material media . odn - aunps B-nanoparticle with high ligand densities ( ~ 55 odns / aunp B-nanoparticle ) exhibited enhanced is activity as the cpg ( % ) increased ( figure 2a ) . [SEP]
[CLS] aunps B-nanoparticle with higher cpg content reached half maximal activity ( ec 50 ) at lower particle concentrations ( table s1 ) , which indicated that is depends on the amount of cpg delivered by the aunps B-nanoparticle to macrophages . [SEP]
[CLS] at higher particle concentrations ( 10 aunps B-nanoparticle ) , all nanoconstructs with at least an average of 3 cpg per particle ( odn - aunps B-nanoparticle with ≥5 % cpg content ) achieved the same maximal is ( figure s3 ) . [SEP]
[CLS] these results indicate that above a ( relatively low ) critical cpg percentage but fixed high odn density , maximum tlr9 activation can be realized ; note the major shift in is activity occurs between 0 and 3 cpg strands per particle and that increasing the number above these values had a much smaller effect . [SEP]
[CLS] in free form , cpg without a np B-nanoparticle core B-material required ca . 1000 higher concentration than cpg - aunp B-nanoparticle to induce similar is activation levels ( figure s4 ) . [SEP]
[CLS] importantly , is behavior was not caused by cell B-event death I-event , as all samples showed > 90 % cell B-property viability I-property ( figure s5 ) . [SEP]
[CLS] since previous work showed that the np B-nanoparticle core B-material affects cpg targeting specificity , we studied whether changes in the ligand shell B-material also influenced nanoconstruct selectivity towards tlr9 ( figure 2b ) . [SEP]
[CLS] the targeting specificity of nanoconstructs on is was studied by pre - incubating B-technique raw - blue cells B-material with tlr 7 / 8 ( odn 2087 ) or tlr7 / 8 and 9 ( odn 2088 ) antagonists before the addition of 5 - nm odn - aunps B-nanoparticle ( experimental section ) . [SEP]
[CLS] the immune activity of all cpg - containing aunps B-nanoparticle decreased only when tlr9 was blocked , indicating that all nanoconstructs targeted tlr9 . [SEP]
[CLS] cell B-property viability I-property experiments showed that neither tlr antagonists nor nanoconstructs caused significant cytotoxicity B-property ( figure s6 ) . [SEP]
[CLS] to determine how ligand density influences the is activity of odn - aunps B-nanoparticle , we compared nanoconstructs with high ( ~ 55 odns / aunp B-nanoparticle ) and low ( ~ 3 odns / aunp B-nanoparticle ) ligand loading at fixed 5 - nm particle concentration . [SEP]
[CLS] at high ligand density , nanoconstructs with higher cpg content induced stronger macrophage immune activation ( figure 3a ) . [SEP]
[CLS] nanoconstructs with low odn loading , however , did not show significant immune activity . [SEP]
[CLS] the difference of is between high and low ligand densities was caused by the different levels of endocytosis B-event of the nanoconstructs ( figure 3b ) , which were quantified by inductively coupled plasma mass spectrometry ( experimental section ) . [SEP]
[CLS] larger amounts of odns on the aunp B-nanoparticle surface increased the nanoconstruct endocytosis B-event and the delivery of cpg into the cells B-material . [SEP]
[CLS] the particles with same odn density and different cpg content had similar uptake levels ( figure 3b ) . [SEP]
[CLS] these results are in agreement with literature that showed higher odn loading enhances nanoconstruct cellular uptake . [SEP]
[CLS] although treatment with 5 - nm odn - aunps B-nanoparticle with 5 % cpg ( at high loading ) or 100 % cpg ( at low loading ) delivered the same total amount of cpg into the solution ( ca . 15 nm ) , they induced high and no is activity , respectively . [SEP]
[CLS] these results confirm that ligand density affects is activity of aunps B-nanoparticle by improving the cellular uptake of the nanoconstructs . [SEP]
[CLS] structural parameters , such as the size and shape of au core B-material , have been reported to affect the sub - cellular localization of nanoconstructs , which also influence down - stream biological effects . [SEP]
[CLS] hence , we studied the intracellular distribution of aunps B-nanoparticle with different odn compositions by transmission B-technique electron I-technique microscopy I-technique ( figure 4 and figure s7 ) . [SEP]
[CLS] macrophages were treated with 5 - nm odn - aunps B-nanoparticle for 24 h and double - stained after fixation and sectioning to increase the contrast of the organelles ( experimental section ) . [SEP]
[CLS] only high - ligand density aunps B-nanoparticle were studied since they are the only constructs that showed significant endocytosis B-event . [SEP]
[CLS] independent of odn composition , the majority of all aunps B-nanoparticle appeared to be located inside well - defined vesicles . [SEP]
[CLS] these results are in agreement with previously reported transmission B-technique electron I-technique microscopy I-technique and confocal B-technique laser I-technique scanning I-technique microscopy I-technique images that show small aunps B-nanoparticle functionalized with oligonucleotides accumulated within endosomes and lysosomes . [SEP]
[CLS] the subcellular location of the particles is important , because tlr9 receptors are expressed in intracellular membrane compartments such as endosomes , and their activation depends on endosomal delivery of the au nanoconstructs . [SEP]
[CLS] to evaluate the effect of the odn shell B-material composition and density on cytokine production , we quantified the six main cytokines released by macrophages upon pathogen activation at fixed odn - aunps B-nanoparticle ( 5 nm ) [SEP]
[CLS] constructs with higher cpg content and ligand loading maximized release of tumor B-material necrosis factor α ( tnf - α ) , rantes , and mip - 2 ( figure 5 ) . [SEP]
[CLS] these three cytokines promote anti - tumor B-material activity through acute pro - inflammatory I-property reaction ( tnf - α ) and intra - tumor B-material infiltration of leukocytes ( rantes and mip - 2 ) [SEP]
[CLS] elevated levels of g - csf and il - 6 were also detected after macrophage incubation B-technique with aunps B-nanoparticle having high cpg content . g - csf enhances the activity of neutrophils ( the most abundant type of white cells B-material that participate in the innate immune system ) associated with tumor B-material rejection . [SEP]
[CLS] il - 6 promotes both pro - and anti B-property - I-property inflammatory I-property responses , and high levels of this cytokine have been linked to disease progression 30 and remission . [SEP]
[CLS] lif , which induces anti B-property - I-property inflammatory I-property response and is associated with cancer development , was the cytokine with the lowest levels in cell B-material media . [SEP]
[CLS] lif was minimal especially upon treatment with odn - aunp B-nanoparticle with low cpg loading and increased with cpg content . [SEP]
[CLS] these results suggest that aunps B-nanoparticle with lower cpg content ( 1 to 10 % ) are better candidates for cancer immunotherapy , since the release of anti - tumor B-material cytokines is maximized and lower levels of anti B-property - I-property inflammatory I-property il - 6 and lif are induced . [SEP]
[CLS] cpg and gpc in free form ( without a np B-nanoparticle core B-material ) did not promote the release of cytokines ( figure s8 ) , which was consistent with the low is activity of free cpg shown in figure s4 . [SEP]
[CLS] finally , nanoconstructs with low odn loading did not induce significant cytokine release , likely because of low cellular uptake . [SEP]
[CLS] in summary , we found that odn shell B-material composition and density are key features in the response of macrophages by au nanoconstructs . [SEP]
[CLS] although nanoconstructs with higher cpg percentages showed lower ec 50 values , only 5 % cpg was necessary to induce maximal immune activation at high particle concentrations , which maximized the release of cytokines related to cancer therapy and minimized the production of disease - promoting ones . [SEP]
[CLS] at a fixed cpg content ( total cpg amount ) , cellular uptake , which correlates with odn shell B-material density , is a primary contributor to immune stimulation . [SEP]
[CLS] therefore , ligand shell B-material tunability will be critical in the development of cancer therapeutic agents to achieve specific immune responses . [SEP]
[CLS] chloroauric acid , tri - sodium B-material citrate dihydrate ( sodium B-material citrate ) , tris ( 2carboxyethyl ) phosphine hydrochloride ( tcep ) , potassium B-material iodide B-material , iodine B-material , 11mercaptoundecyl hexa ( ethylene glycol ) ( muheg ) , sodium B-material borohydride , monosodium phosphate , dithiothreitol , hydrochloric acid ( 37 % ) , and nitric acid ( 70 % ) were bought from sigma aldrich , st . louis , mo . [SEP]
[CLS] 15 - nm aunps B-nanoparticle were synthesized by citrate reduction of chloroauric acid following a previous protocol . [SEP]
[CLS] cpg odn ( 5 ' - tccatgacgttcctgacgtt - ( sp18 ) - disulfide - 3 ' , phosphodiester backbone ) and gpc odn ( 5 ' - tccatgagcttcctgagctt - ( sp18 ) - disulfide - 3 ' , phosphodiester backbone ) were synthesized by solid phase . [SEP]
[CLS] zeocin , primocin , and antagonists for tlr7 / 8 ( odn 2087 ) and tlr7 / 8 and 9 ( odn 2088 ) were obtained from invivogen , san diego , ca . [SEP]
[CLS] fetal bovine serum ( fbs ) , dulbecco ' s phosphate - buffered saline ( dpbs ) and dulbecco ' s modified eagle ' s medium ( dmem ) were purchased from thermo fisher scientific , waltham , ma . [SEP]
[CLS] cpg and gpc odns were functionalized on aunps B-nanoparticle following our previous published method . [SEP]
[CLS] in short , the odn disulfide bonds were reduced to thiol B-material groups with tcep ( 20 mm ) at room temperature . [SEP]
[CLS] in order to achieve different oligonucleotide compositions , the odns containing cpg and gpc were mixed at different ratios before being exposed to the aunps B-nanoparticle . [SEP]
[CLS] the thiol terminated odns were added to 15 - nm aunp B-nanoparticle solutions ( molar ratios of 200 : 1 and 25 : 1 odn : aunp B-nanoparticle for high and low odn loading , respectively ) and left reacting at room temperature under vigorous shaking for 10 min , followed by the addition of sodium B-material citrate buffer ( 100 mm , ph 5 . 8 ) . [SEP]
[CLS] lastly , muheg was added into the solution at a molar ratio of 2000 : 1 muheg : aunp B-nanoparticle . [SEP]
[CLS] the resulting solution was left shaking at room temperature for 5 h , washed three times with a centrifuge and suspended in dpbs . [SEP]
[CLS] in order to quantify the loading of odns on the aunps B-nanoparticle , the au cores B-material were initially digested by sequentially adding aqueous iodine B-material solution ( 160 mm iodine B-material and 1m potassium B-material iodide B-material ) for 10 min , monosodium phosphate for 10 min , and a mixture of 1 : 5 sodium B-material borohydride : dithiothreitol for 5 min . [SEP]
[CLS] the resulting solution was centrifuged at 21 , 000 ×g , the supernatant collected and the number of odns quantified by quant - it oligreen ssdna assay kit ( thermo fisher scientific , waltham , ma ) . [SEP]
[CLS] the loading of odns per particle was calculated by dividing the number of odns by the aunp B-nanoparticle concentration , which was quantified by inductively coupled plasma mass spectrometry ( icp - ms , icap q , thermo fisher scientific , waltham , ma ) . [SEP]
[CLS] for the preparation of odn - aunps B-nanoparticle , we made the assumption that the ratios of cpg to gpc odns presented by the aunps B-nanoparticle were the same as the ratios of the solutions used to functionalize the nanoconstructs . [SEP]
[CLS] the aunp B-nanoparticle samples were digested prior icp - ms analysis in a mixture of hydrochloric acid and nitric acid ( 1 : 1 ) for 30 min and subsequent 20 - fold dilution in milli - q water B-material . [SEP]
[CLS] the aunp B-nanoparticle size was estimated with images captured by a jeol 1230 transmission electron microscope . [SEP]
[CLS] the zeta B-property potential I-property of odn - aunps B-nanoparticle was measured by a zetaplus ( brookhaven instruments , holtsville , ny ) . [SEP]
[CLS] murine raw - blue macrophage ( invivogen , san diego , ca ) was used as cell B-material line in these experiments , and dmem supplemented with fbs ( 10 % ) , zeocin ( 200 μg / ml ) and primocin ( 100 μg / ml ) was employed as growth medium . [SEP]
[CLS] the is activity of au nanoconstructs was tested by quanti - blue assay ( invivogen , san diego , ca ) . [SEP]
[CLS] raw - blue cells B-material were seeded with a concentration of 2 . 5×10 4 cells B-material per well in 96 - well plates and left to adhere for 20 h at 37 ºc with 5 % co 2 . [SEP]
[CLS] the supernatants were removed and replaced by fresh media containing different concentrations of aunps B-nanoparticle ( 0 to 20 nm ) , and the cells B-material were further incubated B-technique for 24 h at 37 ºc with 5 % co 2 . [SEP]
[CLS] the cell B-material media were collected and centrifuged to remove the aunps B-nanoparticle left in solution . [SEP]
[CLS] 20 μl of the supernatants were transferred to new wells , mixed with 200 μl of quanti - blue assay reagent and incubated B-technique at 37 ºc with 5 % co 2 for 12 h . [SEP]
[CLS] finally , the absorbance of the solutions at 635 nm , which correlates with the secrete embryonic alkaline phosphatase levels released by the cells B-material , was recorded . [SEP]
[CLS] for the antagonist experiments raw - blue cells B-material were pre - incubated B-technique with 500 nm odn 2087 or 2088 for 2 h prior the addition of the aunps B-nanoparticle . [SEP]
[CLS] raw - blue cells B-material were seeded with a density of 2×10 5 cells B-material per well in a 12 - well plate , and incubated B-technique for 20 h at 37 ºc with 5 % co 2 . [SEP]
[CLS] the cell B-material medium was replaced by fresh one containing au nanoconstructs ( 5 nm ) . [SEP]
[CLS] after incubating B-technique for 24 h at 37 ºc with 5 % co 2 , the supernatant was removed and the cells B-material were washed with dpbs 3 times . [SEP]
[CLS] cells B-material were detached from the plates with a scrapper and transferred to an eppendorf tube for additional washing ( 250 g × 5 min ) . [SEP]
[CLS] the cells B-material were suspended in dpbs and counted with a hemocytometer . [SEP]
[CLS] lastly , the cells B-material were digested in a solution of 2 % nitric acid and 2 % hydrochloric acid at 70 ºc for 12 h , and the au quantified by icp - ms . [SEP]
[CLS] the macrophages were seeded in 12 - well plates with a density of 2×10 5 cells B-material per well and incubated B-technique for 20 h at 37 ºc with 5 % co 2 . [SEP]
[CLS] the cell B-material medium was replaced by fresh one containing au nanoconstructs ( 5 nm ) . [SEP]
[CLS] after 24 h incubation B-technique at 37 ºc with 5 % co 2 , the supernatant was removed and the cells B-material were washed with dpbs 3 times . [SEP]
[CLS] cells B-material were detached from the plate with a scrapper , transferred to an eppendorf tube for additional washing ( 250 g × 5 min ) , and the pellet transferred to karnovsky ' s fixative solution to be fixed with a pelco biowave microwave following a previously published protocol . [SEP]
[CLS] after fixation , the cells B-material were double - stained with uranyl acetate and lead citrate , and imaged by a jeol 1230 transmission electron microscope . [SEP]
[CLS] to quantify the release of cytokines by macrophages upon immune activation , raw - blue cells B-material were seeded with a concentration of 2 . 5×10 4 cells B-material per well in 96 - well plates and incubated B-technique at 37 ºc with 5 % co 2 . [SEP]
[CLS] after 20 h , the old cell B-material media were replaced by fresh ones containing odn - aunps B-nanoparticle ( 5 nm ) , and the cells B-material were further incubated B-technique for 24 h at 37 ºc with 5 % co 2 . [SEP]
[CLS] the cell B-material media were collected and centrifuged to remove the aunps B-nanoparticle left in solution . [SEP]
[CLS] 25 μl of the supernatants were used to quantify the released cytokines by a luminex® multiplex kit and luminex® 200 instrument ( invitrogen , carlsbad , ca ) following the manufacturer protocol . [SEP]
[CLS] representative tem image of raw - blue cell B-material after treatment with odn - aunps B-nanoparticle ( 100 % cpg ) . [SEP]
[CLS] the box dimensions are 1 μm × 1 μm . [SEP]
[CLS] heat map of cytokines released by raw - blue cells B-material after 24 - h treatment with high and low odn loading aunps B-nanoparticle ( 5 nm ) . [SEP]
[CLS] 1 . composition of the au nanoconstructs . [SEP]
[CLS] ( a ) odn sequences and muheg used to functionalize the aunps B-nanoparticle . [SEP]
[CLS] ( b ) number of odn per particle under high oligonucleotide density . [SEP]
[CLS] the differences of odn loading between samples with different cpg content were not significant ( p < 0 . 05 , one - way anova ) . [SEP]
[CLS] odn quantification experiments were performed in quadruplicate ; error bars represent one standard deviation of the measurements . [SEP]
[CLS] small fraction of cpg is necessary to immune activate macrophages . [SEP]
[CLS] ( a ) relative is activity of raw - blue cells B-material after 24 - h treatment with aunps B-nanoparticle with different cpg content . [SEP]
[CLS] calibration curves have been offset for clarity . [SEP]
[CLS] ( b ) relative is activity of different nanoconstructs ( 5 nm odn - aunps B-nanoparticle ) in the presence of tlr antagonists . [SEP]
[CLS] ( * ) , ( * * ) and ( * * * ) indicate groups that are significantly different from pbs with p < 0 . 001 , p < 0 . 005 and p < 0 . 01 , respectively ( one - way anova with post hoc tukey hsd test ) . [SEP]
[CLS] all experiments were performed in triplicate ; error bars represent one standard deviation of the measurements . [SEP]
[CLS] odn density of aunps B-nanoparticle strongly affects their is performance and endocytosis B-event . [SEP]
[CLS] raw - blue cells B-material were treated with 5 nm aunps B-nanoparticle with high or low oligonucleotide density and different cpg content for 24 h and ( a ) relative is activity and ( b ) cellular uptake were measured . [SEP]
[CLS] arrows highlight nanoconstructs with 5 % ( high odn density ) and 100 % cpg ( low odn density ) , which contained the same amount of total cpg but showed different is performance . [SEP]
[CLS] the differences of endocytosis B-event between samples with same odn density were not significant ( p < 0 . 05 , one - way anova ) . [SEP]
[CLS] all experiments were performed in triplicate ; error bars represent one standard deviation of the measurements . [SEP]
[CLS] 4 . aunps B-nanoparticle are located inside vesicles independently of their cpg content . [SEP]
[CLS] nanoconstructs with high cpg / odn ratio promote the release of large concentrations ( > 5×10 3 pg / ml ) of pro - I-property inflammatory I-property and chemotactic cytokines . [SEP]
[CLS] the discovery and elucidation of genetic codes have profoundly changed not only biology but also many fields of science and engineering . [SEP]
[CLS] the fundamental building blocks of life comprises of four simple deoxyribonucleotides and yet their combinations serve as the carrier of genetic information that encodes for proteins B-material that can carry out many biological functions due to their unique functionalities . [SEP]
[CLS] inspired by nature , the functionalities of dna molecules have been used as a capping ligand for controlling morphology of nanomaterials B-material and such a control is sequence dependent , which translates into distinct physical and chemical properties of resulting nanoparticles B-nanoparticle . [SEP]
[CLS] herein , we provide an overview on the use of dna as engineered codes for controlling the morphology of metal B-nanoparticle nanoparticles I-nanoparticle , such as gold B-material , silver B-material and pd - au bimetallic B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] fundamental insights into rules governing dna controlled growth mechanisms are also summarized , based on understanding of the affinity of the dna nucleobases to various metals B-material , the effect of combination of nucleobases , functional modification B-event of I-event dna I-event , the secondary structures of dna and the properties of the seed employed . [SEP]
[CLS] the resulting physical and chemical properties of these dna encoded nanomaterials B-material are also reviewed , while perspectives into the future directions of dna mediated nanoparticle B-nanoparticle synthesis are provided . [SEP]
[CLS] the synthesis and characterization of colloidal metal B-nanoparticle nanoparticles I-nanoparticle is an area that has garnered much interest over the past years , because the properties that these nanoparticles B-nanoparticle exhibit at the nanoscale have demonstrated enormous potential in applications that span many diverse areas such as medicine , photonics , catalysis , sensing and electronics . [SEP]
[CLS] the arrangement of atoms B-material in such a nanoscale - confined environment has played a major role in determining the physical and chemical properties of these metal B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] hence , the ability to precisely control their morphologies , such as shape and surface structures is a major avenue to realize their full potential in a myriad of applications . [SEP]
[CLS] among the methods to control the nanoparticle B-nanoparticle morphologies , solution - based synthesis methods involve regulation of kinetic and thermodynamic parameters . [SEP]
[CLS] these parameters are often interdependent and can be meticulously tuned by the selection of different precursors , temperatures , solvents , ph , additives , [ 21 , 23 , 25 , 38 , 39 ] and capping ligands . [SEP]
[CLS] among all those parameters , the capping agent has been the center of interest . [SEP]
[CLS] the capping ligand is often involved in a range of functions , including binding to a specific facet of the nanocrystals , influencing precursor reduction and diffusion , and providing overall colloidal stability . [SEP]
[CLS] for example , commonly used capping ligands , such as cetyltrimethylammonium bromide B-material / chloride B-material ( ctab / ctac ) , citrate , polyvinylpyrrolidone ( pvp ) and polyols , have shown the ability to bind specific facets of noble metals B-material such as { 100 } or { 111 } independently or when complemented with other ligands such as halides ions ( for example , br − is known to stabilize the { 100 } facet ) . [SEP]
[CLS] while these capping ligands have been successful in controlling morphologies of metal B-nanoparticle nanoparticles I-nanoparticle , the lack of systematic variations and fine tuning of their structures , charges or functional groups largely limits their ability to accurately control nanoparticle B-nanoparticle morphology . [SEP]
[CLS] to overcome the above limitation , materials scientists and engineers have resort to biomolecules that have been perfected through evolution , such as amino B-material acids I-material , peptides B-material , protein B-material , virus capsids and nucleic B-material acids I-material to be used in the synthesis of nanomaterials B-material . [SEP]
[CLS] among them , the unique structure of dna showcases several tunable properties such as charge , length , affinity and functional groups , and these properties have been exploited extensively in the field of self - assembly , sensing and imaging . [SEP]
[CLS] recently , they have also been utilized as capping ligands to precisely control the morphologies of nanomaterials B-material during the synthesis . [SEP]
[CLS] this review aims to summarize the recent progress in sequence specific dna - mediated control of metal B-nanoparticle nanoparticle I-nanoparticle morphologies . [SEP]
[CLS] we will briefly discuss the dna - mediated synthesis of metal B-nanoparticle nanoparticles I-nanoparticle , and focus on highlighting the importance of sequence dependent " dna - encoded " morphological control of metals B-material at the nanoscale . [SEP]
[CLS] the process by which the dna of different sequences employed determines the final shape of metal B-nanoparticle nanoparticles I-nanoparticle is quite complex and yet very elegant . [SEP]
[CLS] the challenges thus faced in understanding such a system , along with solutions and promising approaches will be discussed . [SEP]
[CLS] finally , the functional properties of these dna encoded metal B-nanoparticle nanoparticles I-nanoparticle will be presented to show promises of this class of materials for many applications . [SEP]
[CLS] dna has traditionally been considered as a storehouse of genetic information , which consists of different combinations of four deoxyribonucleotides ( g , a , t and c ) . [SEP]
[CLS] the elucidation of genetic codes is a major milestone in biology that allows the dna to encode biological functions . [SEP]
[CLS] from a material B-material chemist ' s perspective , the structure of dna consisting of the sugar - phosphate backbone and the nitrogenous bases can be considered as a welldesigned blend of hydrophobic B-property and hydrophilic B-property functional groups . [SEP]
[CLS] the property of single stranded dna ( ssdna ) to be able to bind metal B-material ions B-material was used extensively in the synthesis of ag nanoclusters . [SEP]
[CLS] shortly after , studies proved that the fluorescence B-property emission from these clusters can be tuned by altering the dna sequence used for the nanocluster synthesis , [ 66 , 67 ] highlighting the importance of the dna sequences in material B-material synthesis . [SEP]
[CLS] the methodology was then quickly extended to other noble metal B-material particles and adopted for different applications , such as in sensing . [SEP]
[CLS] functional modifications , such as amine B-material , biotin and thiol B-material , can be incorporated into the dna sequence without effecting the base pairing ability of that particular sequence . [SEP]
[CLS] such modifications are especially useful in integrating dna with other materials such as gold B-nanoparticle nanoparticles I-nanoparticle and silica particles [SEP]
[CLS] the backbone can also be modified to phosphorothioate ( ps ) to maintain the negative charge or to peptide B-material nucleic B-material acids I-material ( pna ) that exhibits a neutral backbone . [SEP]
[CLS] the four canonical nucleobases can also be replaced with modified bases , with either one of functional groups in the canonical nucleobases deleted , substituted , or replaced with a new functional B-material group I-material . [SEP]
[CLS] for example , hda , ida , hdc , mdc , adt and adg are among many nucleobases analogues that can be synthesized ( figure 1 ) . [SEP]
[CLS] while certain organic polymers B-material used in metal B-nanoparticle nanoparticle I-nanoparticle synthesis can be designed to have charges and functional groups distributed on their backbone , the length or molecular weight of such polymers B-material cannot be controlled as precisely as with dna . [SEP]
[CLS] the superior control over the structure of dna makes it a powerful candidate in directing the shape of the nanomaterials B-material , and can possibly lead to generating novel morphologies and properties with very fine control . [SEP]
[CLS] the process by which the dna genetic code holds accurate information about the translation of dna into rna and then into amino B-material acids I-material for proteins B-material is highly sophisticated . [SEP]
[CLS] it is known that the sequence plays a pivotal role in determining the end product ; even a single mutation can result in a missing , misfolded or malfunctioning protein B-material . [SEP]
[CLS] by the virtue of intricate functionality of dna and its transcribed products , molecules interact specifically , making possible the synthesis of highly complex molecules . [SEP]
[CLS] inspired by this process in nature , we have explored how a specific sequence of dna can influence the final morphologies and surface structure of a particular nanomaterial B-material when present in the synthesis . [SEP]
[CLS] in other words , we aimed to discover and elucidate dna " codes " for morphologies of abiological nanomaterials B-material . [SEP]
[CLS] the ultimate goal is to be able to predict the final morphology of the nanomaterials B-material simply based on the specific dna sequences and nanomaterial B-material precursors used , exactly like how we are now able to predict protein B-material structures with genetic codes . [SEP]
[CLS] while we are far from being able to predict the final nanostructures at this point , considerable progress has been made to understand such synthetic systems . [SEP]
[CLS] a dna - mediated synthesis involves the dna directed deposition of metal B-material on a presynthesized seed . [SEP]
[CLS] to provide information regarding the morphology transition , growth mechanisms and properties , electron microscopy - based imaging B-technique techniques I-technique such as sem and tem ( scanning electron B-technique microscopy I-technique and transmission B-technique electron I-technique microscopy I-technique ) have been used to help characterize the final shape of the metal B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] furthermore , timebased electron B-technique microscopy I-technique studies have shed light on how the nucleation and growth of the seed takes place in the presence of dna . [SEP]
[CLS] in addition , absorption spectra of the system has allowed not only the monitoring of the spr properties of the metal B-nanoparticle nanoparticles I-nanoparticle but also analysis of the structure - function relationship between the morphology of the nanoparticle B-nanoparticle and the spr during the course of the growth . [SEP]
[CLS] how each of these techniques provide information on the dna encoded nanoparticles B-nanoparticle will be described in details in the later sections that discuss the growth mechanisms . [SEP]
[CLS] in this section , we will describe these systems , classified by the seed species employed in the synthesis . [SEP]
[CLS] spherical aunps B-nanoparticle are easily among the most well studied au - based nanomaterials B-material in terms of synthesis and characterization . [SEP]
[CLS] the surface of these aunps B-nanoparticle have been modified extensively with ligands , including dna , for self - assembly and other applications . [SEP]
[CLS] the affinities of dna to the surface of spherical aunps B-nanoparticle have also been studied to reveal that dt has a lower binding affinity as compared to da , dc or dg . the translation of the dna affinity into the final morphology of particles when oligomers of dna with different sequences were used , showed remarkable differences . [SEP]
[CLS] while 30 - mer of dt resulted in spherical aunps B-nanoparticle slightly larger in size , 30 - mer of da and dc resulted in nano - flower ( aunf ) ( figure 2a ) . [SEP]
[CLS] the uneven growth characteristic of the aunf that da and dc imposed can be attributed to the higher binding affinity of the nitrogenous nucleobases to the surface of the spherical gold B-material seed . [SEP]
[CLS] in contrast , a sequence like oligo - t with low binding affinity only manages to colloidally stabilize the final nanoparticle B-nanoparticle allowing just a simple overgrowth to a bigger spherical B-nanoparticle nanoparticles I-nanoparticle , without influencing the shape . [SEP]
[CLS] these uneven features had a direct influence on the plasmonic properties of the nanoparticles B-nanoparticle that were red shifted , where the aunfs formed by oligo - a had a more red shifted peak compared to oligo - c capped particles . [SEP]
[CLS] as a result , the aunf colloidal solutions were blue in color . [SEP]
[CLS] while the affinity of the dna did make a difference in the final shape , the adsorbed and implanted dna on the particle surface contribute to the colloidal stability of the aunps B-nanoparticle . [SEP]
[CLS] the previous study with spherical aunps B-nanoparticle established that dna - mediated shape control was indeed possible . [SEP]
[CLS] as an extension to demonstrate the precise level of nanoparticle B-nanoparticle shape tunability , aunpr was used as the seed , because the seed presented different facets than spherical aunps B-nanoparticle . [SEP]
[CLS] the aunpr was synthesized using a previous protocol and their overgrowth was monitored in the presence of 30 - mer of da , dc , dt and a 20 - mer of dg . [SEP]
[CLS] the anisotropic nature of the seed , makes the monitoring of shape evolution easier from the surface and the edges . [SEP]
[CLS] the presence of each oligonucleotide resulted in a unique shape . [SEP]
[CLS] specifically , rough - round plates , six - pointed stars , round plates and hexagonal plates were formed in the presence of a30 , t30 , c30 and g20 , respectively ( figure 2b and 3b ) . [SEP]
[CLS] the { 111 } surface was preserved in each case except in the formation of the roughing of the surface in the presence of a30 . [SEP]
[CLS] in addition , the growth in the presence of different lengths of the oligonucleotides ( 5 , 10 and 20 - mer of g and 30 - mer of other oligonucleotides ) yielded similar morphologies , suggesting that the sequence of the oligonucleotides , rather than the length , plays an important role in morphology control . [SEP]
[CLS] however , the synthesis in the presence of monomeric deoxyribonucleotides , i . e . damp , dtmp , dgmp and dcmp led to formation of unstable structures that aggregated due to lack of colloidal stability . [SEP]
[CLS] therefore , the length of the oligonucleotides should be long enough ( generally above 5 nucleotides ) to maintain colloidal stability of the nanostructure of desired morphology . [SEP]
[CLS] aunrs are anisotropic nanoparticles B-nanoparticle that exhibit unique absorption and scattering properties in the near infrared ( nir ) region . [SEP]
[CLS] owing to the anisotropic nature , aunrs exhibit both longitudinal and transverse localized surface plasmon resonance ( referred to as l - lspr and t - lspr , respectively ) characterized by the aunr aspect ratio and these anisotropic optical properties have been utilized for optical and biomedical applications . [SEP]
[CLS] the overgrowth of the aunrs in the presence of homo - oligomeric 20 - mer of dna not only lead to different geometries , but also result in plasmonic spectral tuning . [SEP]
[CLS] in the case of a20 , the aunr overgrowth yielded a dumbbell shaped particle ( figure 2c ) . [SEP]
[CLS] the tips of the nanorod B-nanoparticle had more deposition resulting in the red shift of the l - lspr . [SEP]
[CLS] the increase in the thickness of the rod slightly shifts the t - lspr wavelength band . [SEP]
[CLS] c20 and t20 both resulted in the formation a cracked octahedron structure with difference in the gap between the two pointed features . [SEP]
[CLS] while the l - lspr bands for structures formed by both oligonucleotides were blue - shifted , this particular difference in gap allows the l - lspr peaks to be different . [SEP]
[CLS] specifically , the shorter the gap , as observed in the presence of c20 ( 17±3 nm gap as compared to 24±3 nm in the case of t20 ) , the more is the blue shift . [SEP]
[CLS] when the gap is close to zero , the l - lspr is blue - shifted to the extent that the l - lspr merges with the t - lspr to give a single unified lspr peak , which is observed in the presence of g20 . [SEP]
[CLS] the system highlights how subtle changes in the deposition profile can directly influence not only the geometric but also physical properties . [SEP]
[CLS] the interaction of dna with metal B-material nps B-nanoparticle is expected to change depending on the identity of the metal B-material . [SEP]
[CLS] while most studies have been focused on gold B-material - based systems , it is important to be able to expand the metal B-material nps B-nanoparticle to make the above approach more generalizable . [SEP]
[CLS] dnamediated ag overgrowth on agncs as seeds , was the first system to demonstrate the dna sequence dependent nature of ag nanoparticle B-nanoparticle growth . [SEP]
[CLS] not surprisingly , the trend of affinity of the different oligonucleotides to ag was different from that of au . [SEP]
[CLS] a previous study has reported the trend of the affinity to agnps to be in the order , c > g > a > t . the presence of a10 and t10 resulted in formation of stellated structures , specifically truncated octahedra with varying degrees of truncations depending on the sequence . the presence of t10 induced smaller particles with larger truncations , while a10 induces larger particles with smaller edge - truncations . [SEP]
[CLS] the overgrowth of agncs in the presence of c10 formed truncated tetrahedra and the presence of g10 interestingly , did not allow any overgrowth of the seed ( figure 2d ) . [SEP]
[CLS] the stabilization of the { 111 } facet by the dna containing t , a and c resulted in the gradual disappearance of the { 100 } facet that was initially the dominant facet on the agnc seed . [SEP]
[CLS] further investigations suggested the involvement of formation or destruction of secondary structures formed by the dna sequences during the course of the reaction , indicating the structurally dynamic nature of the dna strands in the growth solution . [SEP]
[CLS] the importance of having to consider the substantial role of secondary structure of dna molecules in control of np B-nanoparticle morphologies was demonstrated in this study . [SEP]
[CLS] most recently , yang and coworkers have reported dna - mediated shape evolution of relatively small spherical agnps , in the presence of citrate ligand , to anisotropic morphologies such as nanoprisms . [SEP]
[CLS] oligo - g and oligo - c mediated the formation of nanoprisms , while oligo - t and oligo - a resulted in formation of nanodiscs and 2 - d nano flower bouquets , respectively ( figure 2e ) . [SEP]
[CLS] the spherical agnp seed is generally known to have multiple { 111 } and { 100 } facets . [SEP]
[CLS] the presence of citrate allows the spherical seed to form multiple twinned particles . [SEP]
[CLS] similar to the case of agncs which are enclosed mainly by { 100 } facets and { 111 } , { 110 } sites , the dna had a tendency to bind to the { 111 } facet of the spherical seed and further stabilize it . [SEP]
[CLS] hence , the { 111 } facet dominates the final 2 - d structures . [SEP]
[CLS] although the oligo - c would be expected to have a dominant effect on the overgrowth of the ag spherical B-nanoparticle nanoparticles I-nanoparticle , the most dominant effect was produced by oligo - a sequence , due to the stronger binding affinity of oligo - a with higher energy facets ( twinned surface ) and the lower mobility B-property of the sequence on { 111 } crystal face , leading to the growth of 2d triangular silver B-material bouquets . [SEP]
[CLS] the generalizability of the dna - encoded nanomaterials B-material synthetic approach was further demonstrated , when a10 , t10 , g10 and c10 was used to control the morphology of bimetallic B-nanoparticle nanoparticles I-nanoparticle , specifically pd - au nanoparticles B-nanoparticle . [SEP]
[CLS] pd nanocubes were used as the seed , and in the presence of dna a co - reduction of pd and au precursors was performed . [SEP]
[CLS] the nanocube seed is characterized mainly by six { 100 } faces and additionally { 110 } and { 111 } sites , i . e . the truncation of edges and the corners , respectively . [SEP]
[CLS] the higher surface energy of the { 110 } and { 111 } sites directs both binding of oligonucleotides sequences and deposition of incoming metal B-material . [SEP]
[CLS] those sequences with lower affinity to the seed such as t10 cannot effectively passivated the high energy sites which allows deposition on the high energy sites to form a pd - au core - frame structure , i . e . the metal B-material deposits along the corners and the edges . [SEP]
[CLS] those with higher binding affinity , such as a10 bind to the higher energy sites and passivate the surface to lower their energy , resulting in a complete coverage of the seed . [SEP]
[CLS] therefore rhombi cuboctahedron , cuboctahedron and undulated pd - au shell B-material over the seed were formed in the presence of a10 , c10 and g10 , respectively ( figure 2f ) . [SEP]
[CLS] metal B-material atom B-material diffusion on the seed also plays an important role in directing the morphology and is also simultaneously influenced by the dna B-event binding I-event affinities . [SEP]
[CLS] interestingly , a10 , the sequence with highest binding affinity to gold B-material , can stabilize au surface with higher energy , to allow the formation of smaller nanocrystallites that eventually coalesce into bigger particles that deposit onto the seed . [SEP]
[CLS] this phenomenon is not observed in the case of the other dna bases . [SEP]
[CLS] the ability of dna to control the morphology of pd - au nanoparticles B-nanoparticle was further tested when the seed was changed from a low - index faceted nanocube to a high - indexed concave nanocube . [SEP]
[CLS] the concave cube was different in that it had extended edges and corners in the < 111 > and the < 110 > direction while the { 100 } facets were retained at the center . [SEP]
[CLS] dna was ultimately successful in controlling the final morphology of the pd @ au core - shell nanocrystals but the effect of the high - energy faceted seed cannot be ignored . [SEP]
[CLS] the presence of t10 caused the formation of a gold B-material shell B-material with least deposition of gold B-material along the { 100 } facet and more deposition in the < 111 > and < 110 > directions . [SEP]
[CLS] the g10 - encoded structures had rounded vertices , while the edges of c10 - encoded structure formed octapods with octahedron - like structures consisting of prominent { 111 } surfaces . [SEP]
[CLS] finally , a10 resulted in the formation of a gold B-material shell B-material with an overall rough surface . [SEP]
[CLS] the initial metal B-material deposition is dictated almost entirely by the seed ' s high surface energy . [SEP]
[CLS] after an initial deposition , dna controls the morphology in a sequence dependent manner . [SEP]
[CLS] similar to the cubic system , a10 enable the formation of smaller nanocrytsallites before the deposition onto the seed . [SEP]
[CLS] the fine control displayed in tuning the morphology of the shell B-material demonstrates the immense potential that dna has in shaping hybrid materials that possess disparate properties . [SEP]
[CLS] it is evident , from the above examples , that different sequences of the dna , reflected by their different chemical composition , and hence the structural properties , play an important role in defining the final morphology of the metal B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] the interaction between the dna molecules and the metal B-material atoms B-material can be elucidated to a large extent using chemical and physical methods . [SEP]
[CLS] understanding the complexity of the dna structure and their interaction with metal B-nanoparticle nanoparticles I-nanoparticle on a molecular level allows for deeper insights into dna - mediated nanoparticle B-nanoparticle growth mechanisms . [SEP]
[CLS] as mentioned earlier , the sugar - phosphate backbone and the nucleobases constitute to the framework of dna . [SEP]
[CLS] the nucleotide formed by sugar - phosphate group and one of the four nucleobases is the recurring unit in a given sequence ( figure 1 ) . [SEP]
[CLS] each nucleotide unit changes with respect to the nucleobase . [SEP]
[CLS] the majority of the studies that involve a dnaencoded synthesis use dna molecules that are homo - oligomeric in nature , i . e . the nucleobase of the recurring nucleotide unit is kept the same for each individual study . [SEP]
[CLS] choosing these homo - oligomeric dna aids deciphering the structural elements of each nucleobase involved in stabilizing a particular facet . [SEP]
[CLS] although naturally derived , the ssdna structurally has many aspects that easily make it qualify as a capping agent . [SEP]
[CLS] first , the phosphodiester backbone contributes to the negative charge and thus the hydrophilicity B-property . [SEP]
[CLS] the negative charge is expected to induce charge repulsion between different nanoparticles B-nanoparticle thus providing colloidal stability . [SEP]
[CLS] therefore , the extended oligomeric form of dna is indeed necessary for the stability of metal B-nanoparticle nanoparticle I-nanoparticle . [SEP]
[CLS] nanoparticles B-nanoparticle synthesized with just mononucleotides tended to aggregate . [SEP]
[CLS] additionally , the minimum length of dna required for imparting both colloidal stability and shape control was determine to be 5 bases , beyond which the length of dna does not produce length - specific effects . [SEP]
[CLS] second , in comparison to the backbone , the aromatic rings of purines and pyrimidines in the nucleobases are more hydrophobic B-property . [SEP]
[CLS] depending on the pka values of the nucleobases , the ph and composition of the working solution , the nucleobases are either protonated or deprotonated . [SEP]
[CLS] the potential for metal B-material binding stems from the structure of the dna molecules . [SEP]
[CLS] the nucleobase ring n and the keto o atoms B-material that flank the purines and pyrimidines are involved in metal B-material complexation . [SEP]
[CLS] the exocyclic amine B-material groups I-material also help in metal B-material interaction , ostensibly through coordination . [SEP]
[CLS] in the most general structural form of double - stranded dna ( dsdna ) , the nucleobases on both strands intertwine in an anti - parallel manner , through watson - crick hydrogen B-material bonding to form a helical structure . [SEP]
[CLS] the base - pairing interaction is specific in that , adenine and guanine ( the purines ) form hydrogen B-material - bonds with thymine and cytosine ( the pyrimidines ) , respectively . [SEP]
[CLS] in this way , the functional B-material group I-material - rich nucleobases in dsdna are mostly masked from interactions with the surrounding environment . [SEP]
[CLS] as a result , the dsdna ' s sequence has little role to play in a dna - encoded control of np B-nanoparticle morphologies and almost all dna - encoded studies reported so far use ssdna instead . [SEP]
[CLS] dsdna mediated nanoparticle B-nanoparticle synthesis has been studied separately , wherein the sequence of dna plays no role . [SEP]
[CLS] 3 . 1 . 1 . affinity of the dna bases to various metals B-material : the interaction of dna with metal B-material ions B-material has been extensively studied [SEP]
[CLS] while this interaction is important , what carries more weight in a dna - encoded control np B-nanoparticle morphology is the interaction of dna with metal B-material surfaces . [SEP]
[CLS] a variety of techniques have been used in the past to probe the nucleobase affinity for gold based surfaces . [SEP]
[CLS] among the first examples , mirkin and coworkers reported a temperature - programmed desorption ( tpd ) and reflection−absorption infrared ( rair ) spectroscopic study to calculate the heats of desorption ( δh des ) of the individual nucleobases and nucleosides on a gold B-material surface . [SEP]
[CLS] the general trend for desorption revealed that pyrimidines tend to desorb more easily as compared to purines . [SEP]
[CLS] more specifically , the trend observed with respect to the δh des was guanine ( g ) > adenine ( a ) > cytosine ( c ) > thymine ( t ) , which was in agreement with theoretical studies performed later . [SEP]
[CLS] an isothermal titration calorimetry ( itc ) investigation of pna - based monomer B-material analogue binding to aunps B-nanoparticle showed the affinity order c > g > a > t . [SEP]
[CLS] relevant to dna - encoded synthesis of nanoparticles B-nanoparticle where single stranded homooligomeric dna is used , whitman and coworkers performed more detailed studies on the interaction of ssdna on gold B-material using fourier transform infrared ( ftir ) spectroscopy B-technique and xray photoelectron spectroscopy B-technique ( xps ) . [SEP]
[CLS] competitive adsorption experiments on au surfaces showed relative adsorption affinity of dna nucleobases to be a > c ≥ g > t . [SEP]
[CLS] this trend of affinity has been observed in investigating the underlying mechanisms of growth of aunps B-nanoparticle in presence of ssdna sequences and shall be discussed in a later section . [SEP]
[CLS] a more recent study by liu et al . involving cyclic voltammetry as a method to probe the adsorption of unmodified ssdna at low ph showed that a and c have more effective adsorption to the gold B-material surface compared to t and g due to the protonation of functional residues and higher affinity to gold B-material surface . [SEP]
[CLS] reports of interaction of dna nucleobases with other noble metal B-material surfaces or nanoparticles B-nanoparticle are rare . [SEP]
[CLS] besides gold B-material , only a few reports of interaction of dna with agnps have been reported . [SEP]
[CLS] the earlier study probed the nucleobase affinities based on surface plasmon resonance spectroscopy B-technique ( sprs ) and the signal intensity of surface - enhanced raman scattering ( sers ) and determined the trend to be c > g > a > t . [SEP]
[CLS] the second study used solely colorimetric method to investigate the aggregation kinetics of agnps in the presences of nucleobases to obtain a different trend of a > t > c ≥ g . [SEP]
[CLS] the trends in binding strengths varied greatly between the two studies , probably due to the differences in experimental conditions and properties of agnps , such as their geometries , pre - existing capping ligands , and sizes ) . [SEP]
[CLS] thus , the study of dna interaction with metal B-material surfaces as opposed to nanoparticles B-nanoparticle is capable of providing more consistent trend in binding affinities , as demonstrated in the case of dna interaction with gold B-material surfaces . [SEP]
[CLS] despite the differences , what these studies highlight is that the conditions of the growth can potentially affect the trend of nucleobase affinities for metal B-nanoparticle nanoparticles I-nanoparticle and thus the outcome of a dnamediated growth . [SEP]
[CLS] additionally , to make dna - mediated synthesis of noble - metal nanoparticles B-nanoparticle a more generalizable or predictable protocol , it is imperative to understand and study the affinities of the nucleotides to various surfaces of different metals B-material . [SEP]
[CLS] a few studies have reported dna adsorption studies onto metal B-material oxide I-material particles , a class of materials that are slightly different from metal B-material particles in that the metals B-material exist in a non - zero oxidation state . [SEP]
[CLS] clearly , the interaction of dna depends on the nucleobases that are present in the sequence , which is a result of the interacting functional groups on the nucleobases present . [SEP]
[CLS] while the use of homo - oligomers of dna does provide for a very fine control of nanoparticle B-nanoparticle morphology , systematical programming the binding affinities of dna can be achieved by combining nucleotides , which can further expand the tunable range of nanoparticle B-nanoparticle morphology . [SEP]
[CLS] for a given length of a dna , there is sufficient diversity available with respect to just nucleotide arrangement . [SEP]
[CLS] a classic case is the used of combination sequences in the overgrowth of au nanoprism . [SEP]
[CLS] the use of homo - oligomers result in shapes that are very disparate . [SEP]
[CLS] the use of nucleotide combinations help access morphologies whose features lie in between the morphologies resulting from any two homo - oligomers . [SEP]
[CLS] as shown in figure 3b , surface effects by nucleotides includes roughening by c and a , edge thickening by g and flattening by t . shape effects are more obviously observed when the homo - oligomers were used , i . e . round plates ( a , c ) , hexagonal ( g ) and six pointed star ( t ) . [SEP]
[CLS] a different , yet important aspect of tuning optical properties via nanoparticle B-nanoparticle shape control is evidenced when sequence combinations are used in the over - growth of au nanorods B-nanoparticle . [SEP]
[CLS] the study reveals a and g produce dominant effects with respect to shape - control in combination with c and t . [SEP]
[CLS] the oligomeric g and a gave a blue - shifted l - lspr at 551 nm and red - shifted l - lspr at 856 nm , respectively . [SEP]
[CLS] increasing the a base in the combination sequence ( starting from g20 , a10g20 , a15g15 , a20g10 to a20 ) involving the two dominating bases produces particles that have l - lspr peak red - shifted from 551 nm to 856 nm ( figure 3c ) . [SEP]
[CLS] this implies that the l - lspr can be precisely tuned between a given wavelength range by using the right sequence combination . [SEP]
[CLS] specific modification of a functional B-material group I-material in a dna can bring about more predictable changes . [SEP]
[CLS] for example , the thiol - containing capping ligands including alkyl - thiolated dna are widely used to functionalize gold B-material nanomaterials B-material to impart special functionalities given the soft nature of the thiol B-material ligands that have a higher affinity for the soft gold B-material metal B-material . [SEP]
[CLS] similarly , incorporation of a phosphorothioate ( ps ) modification to the phosphodiester backbone can increase its affinity for aunps B-nanoparticle . [SEP]
[CLS] in the case of aunr overgrowth in the presence of homo - oligomeric dna , the use of a20 induces a red - shift to the l - lspr of the final nano - structures as mentioned above . [SEP]
[CLS] further increase of the dna affinity by incorporating ps modifications ( 1psa20 , 2psa20 , 4psa20 ) resulted in particles with l - lspr red - shifted dramatically from 856 nm ( only a20 ) to 1011 nm ( 4psa20 ) with increasing number of ps modifications . [SEP]
[CLS] structurally , the gap of the final dumb - bell shaped particles increased from 8±2 nm ( only a20 ) to 28±6 nm ( 4psa20 ) which accordingly red - shifted the l - lspr to the nir - ii region ( figure 4 ) . [SEP]
[CLS] the ps modification is one of the wide variety of functionalities that can be incorporated into a dna sequence . [SEP]
[CLS] the incorporation of functional groups can be through the backbone , just like the ps modification or via the nucleobase itself . [SEP]
[CLS] functional modifications may also be employed to elucidate mechanisms in a dna mediated synthesis . [SEP]
[CLS] for example , the amine B-material and carbonyl groups present on the nucleobases play a significant role in binding to metal B-material surfaces . [SEP]
[CLS] removal of these groups would help account for the exact role of the base in the growth . [SEP]
[CLS] similarly , addition of new functional groups such as amine B-material or hydroxyl B-material group I-material can help tune the nucleobase affinity and thus the morphology of the particle . [SEP]
[CLS] with the ps modification being able to influence morphologies of nanoparticles B-nanoparticle , it is clear that the backbone can also play a significant role in engaging with the metal B-material surface . [SEP]
[CLS] the highly charged phosphodiester backbone can be replaced by a neutrally charged backbone , such as using pna , that simply connects the nucleobases but keeps the sequence soluble B-property in aqueous medium . [SEP]
[CLS] the incorporation of modified dna will not only help elucidate the roles of each functional B-material group I-material but will further enrich the dna code library for generating nanoparticles B-nanoparticle with new morphologies . [SEP]
[CLS] single stranded dna of a particular sequence has the ability to fold into structures that involves the intra - molecular or inter B-property - I-property molecular I-property interactions I-property such as hydrogen B-material bonding , between the nucleobases present in the sequence . [SEP]
[CLS] the secondary structures arising from homo - oligomers of dna , i . e . , formed by the interaction between identical nucleobases are more relevant to the present discussion . [SEP]
[CLS] dna that are rich in g or c have the ability to form unusual secondary structures under certain conditions . [SEP]
[CLS] for example , the g - rich dna forms g - quadraplexes which involves g - quartets that are planar in nature and generally assemble in presence of a stabilizing cation B-material such as k + . [SEP]
[CLS] the c - rich dna forms the i - motif of c . c + base pairs involving one or multiple nucleic B-material acid I-material strands . [SEP]
[CLS] the protonation of the n3 of c plays a crucial role in the structure formation , thus making it ph dependent . [SEP]
[CLS] specifically , the i - motif is more stable at lower ph values , such as below physiological ph . [SEP]
[CLS] the dependence of secondary structures formation on the species present in the surrounding environment has led to the design of many switchable dna - based sensors . [SEP]
[CLS] a typical dna - mediated nanoparticle B-nanoparticle growth solution would provide for an acidic environment which is simultaneously populated with metal B-material cations I-material , owing to the metal precursor in solution . [SEP]
[CLS] hence , the formation of secondary structures in solution is a possibility and may be of potential importance . [SEP]
[CLS] the nature of a folded ssdna or a structure that comes together by inter - strand interactions is bulkier when compared to a single unfolded ssdna . [SEP]
[CLS] although the nucleobases are masked due to their supramolecular preoccupation , the as - formed secondary structure may still show potential to interact with the nanoparticle B-nanoparticle seed ' s surface . [SEP]
[CLS] thus far , the formation of g - quadraplex and i - motif has been observed in growth solutions where the overgrowth of silver B-material nanocubes was studied in the presence of short oligomers of dna ( a10 , t10 , g10 and c10 ) ( figure 3a ) . [SEP]
[CLS] the formation of g - quadruplex occurs only after having added the silver B-material precursor and reductant , indicating that the ag + ions B-material played a vital role in structure assembly . [SEP]
[CLS] the ag + ion B-material sequestration by the secondary structure consequently rendered the precursor unavailable for further reduction on the cubic seed surface , thus the morphology of the seed remains unchanged . [SEP]
[CLS] the bulky secondary structure additionally may be involved in the surface passivation of the seed preventing any ag 0 deposition . [SEP]
[CLS] on the contrary , the i - motif structure was formed in solution before the addition of the precursor or reductant . [SEP]
[CLS] owing to the initial slightly acidic environment , the formation of c - h + - c base pairing is favored . [SEP]
[CLS] on addition of precursor and reductant , the high binding affinity of cytosine to ag + disrupts the c - h + - c base pairing to form a c - ag + - c pairing . [SEP]
[CLS] the ag + is still susceptible to reduction and thus destabilization of the secondary structure takes place . [SEP]
[CLS] the formation and destabilization of these structures in growth solution can be readily monitored using circular dichroism ( cd ) spectroscopy B-technique . [SEP]
[CLS] all the dna - encoded synthetic protocols for morphology control involve a seed - mediated synthesis . [SEP]
[CLS] using a seed is known not only to increase monodispersity and uniformity of the final nanostructures , but also influence the metal B-material deposition both in monometallic and bimetallic nanomaterials B-material . [SEP]
[CLS] although dna with different sequences by themselves are able to direct morphologies very differently from each other irrespective of the nanoparticle B-nanoparticle seed , the function of the seed cannot be overlooked . [SEP]
[CLS] the most important step in a dna - mediated synthesis is the choice of seed , which determines what surfaces the dna will potentially interact with . [SEP]
[CLS] by rationally choosing the seed , one would be able to obtain desired information regarding the exact influence of the seed and the dna on the final morphology . [SEP]
[CLS] in this respect , there are two main factors that need to be considered while studying the interaction of the seed ; 1 ) the facets that the seed encloses and its composition and 2 ) the capping ligands that pre - exist on the surface of the seed . [SEP]
[CLS] it is indeed challenging to predict the interaction between dna and a certain metal B-material . [SEP]
[CLS] however , it is even more challenging to predict the affinities of dna to different facets of a nanoparticle B-nanoparticle of the same metal B-material . [SEP]
[CLS] the noble metal B-nanoparticle nanoparticle I-nanoparticle systems that have been studied for dna - mediated growth crystallize in the face - centered cubic ( fcc ) lattice . [SEP]
[CLS] the most common facets that the seeds employed enclose are the lowindexed facets , i . e . { 111 } , { 100 } and the { 110 } facets , the surface energies of which increase in the same order . [SEP]
[CLS] most of the systems that have been studied contain only these three surface facets . [SEP]
[CLS] for example , in the over growth of aunr , the growth is determined by the fact that strong binding affinity dna will bind to { 110 } and { 100 } facets on the aunr surface ( figure 5a ) . [SEP]
[CLS] for nucleobases with strong affinity such as adenine oligomers , the dna binds to the sides of the rods , which has { 110 } and { 100 } facets , resulting in preferential growth at the ends and thus formation of dumbbell shape . [SEP]
[CLS] for nucleobases with relatively weaker binding affinity ( t20 , c20 and g20 ) , the growth initiates from the ends and will grow to favor the formation of { 111 } facet , resulting in the formation of sharp tips at the ends of the nanorods B-nanoparticle . [SEP]
[CLS] thus , the binding affinity of dna to { 100 } and { 110 } will determine how much the sides will continue to grow . [SEP]
[CLS] the weaker binding affinity of the dna results in more growth on the sides , transiting through a cracked octahedron shape and eventually form an octahedron as observed for g20 . [SEP]
[CLS] the c20 and t20 have slightly stronger binding affinity , probably owing to the secondary structure formation in case of g20 , resulting in termination of the growth at cracked octahedron shapes . [SEP]
[CLS] the aunpr consists of two planar { 111 } surfaces and a twinned plane in - between . [SEP]
[CLS] the selected area electron diffraction ( saed ) patterns and high resolution tem studies taken in the [ 111 ] zone axis of the particle contained the forbidden 1 / 3 ( 422 ) reflections , which confirmed the presence of twin defect ( figure 5b ) . [SEP]
[CLS] the defects increase the surface energy of the edge of the prism , allowing the preferential binding of high affinity dna molecules such as a30 . [SEP]
[CLS] the growth in the presence of t10 proceeds with the deposition on this twinned edge due to the sequence ' s low binding affinity . [SEP]
[CLS] thus , during the growth progression , the { 111 } surface is retained at every phase . [SEP]
[CLS] it is therefore important to characterize the seed , as the relative surface energies of the facets plays an important role in determining the extent of dna B-event binding I-event . [SEP]
[CLS] a more interesting area is to study how dna interacts with high - indexed facets . [SEP]
[CLS] 3 . 2 . 2 surface functionality of the seed : before the dna can have access to the facets that the nanoparticle B-nanoparticle seed displays , it has to be able to surpass the shell B-material of pre - existing capping ligands on the seed ' s surface . [SEP]
[CLS] common ligands that stabilize seeds include ctab , ctac , pvp , and citrate . [SEP]
[CLS] each of these ligands is capable of individually interacting with dna . [SEP]
[CLS] for example , ctab as a positively charged molecule tends to have electrostatic interaction with negatively charged dna molecules to form polyplexes . [SEP]
[CLS] this property has been extensively used in dna isolation protocols in biology . [SEP]
[CLS] in the case of citrate , the negative charge of the ligand has the potential to repel dna from the nanoparticle B-nanoparticle surface . [SEP]
[CLS] agncs have pre - existing pvp on the surface . [SEP]
[CLS] although the ligand is neutrally charged , its extended polymeric nature makes the ag surface inaccessible to dna . [SEP]
[CLS] it is hence important to remove the excess ligand that exists in solution and on the surface of the seed to ensure proper interaction between dna and the nanoparticle B-nanoparticle surface . [SEP]
[CLS] however , it should be noted that it is important to maintain a balance where there is enough capping ligand to stabilize the seed in solution , but not in excess to disrupt the dna ' s interaction with the seed . [SEP]
[CLS] addition of dna proceeding the washing of excess ligand ensures the particle stability upon dna B-event binding I-event , similar to a ligand replacement mechanism . [SEP]
[CLS] nanoparticles B-nanoparticle containing ligands that are covalently or very tightly bound as in the case of thiol - based ligands to au based particles , would be expected to make dna B-event binding I-event to the nanoparticle B-nanoparticle surface quite unfavorable . [SEP]
[CLS] from the discussions thus far , it is clear that dna - mediated nanoparticle B-nanoparticle growth involves many different factors that affect the final outcome . [SEP]
[CLS] the factors range from a simple dna sequence to the more complicated surface properties of the seed . [SEP]
[CLS] in order to elucidate the mechanism , tan et al . chose to study the morphological evolution of aunpr to different shapes in the presence of dna molecules of different sequences . [SEP]
[CLS] arresting the growth of the nanoparticles B-nanoparticle at different time points to understand the morphological evolution can give us plenty of information about the intermediates B-property of the reaction and potentially help us elucidate the role of dna . [SEP]
[CLS] the growth of the aunpr in the presence of dna was arrested at different time points by 3 - mercaptopropanoic acid ( mpa ) , which is known to quench the reduction of gold B-material precursor . [SEP]
[CLS] the intermediates B-property of morphological evolution of the prism in the presence of t30 were observed to be the final morphologies formed in the presence of the remaining 3 homo - oligonucleotides ( a30 , g20 and c30 ) . [SEP]
[CLS] initially , the prism growth took place from the sides and evolved from a nonagon shape to a hexagonal shape to a final sixpointed star . [SEP]
[CLS] this observation implied that the dna was capable of binding to the sides of the aunpr and kinetically trapping the intermediates B-property , the extent of which depended on the dna B-event binding I-event affinity ( figure 6 ) . [SEP]
[CLS] a30 and c30 arrested the growth at the nonagon shape and g20 arrested the growth at the hexagonal intermediate B-property . [SEP]
[CLS] the intermediates B-property that were trapped at an early stage had a smaller diameter as compared to the later intermediates B-property . [SEP]
[CLS] hence , the particle diameter followed a trend where particles with t30 had the largest diameter , while particles with a30 and c30 had the smallest diameter . [SEP]
[CLS] since the samples were subjected to the same amount of gold B-material precursor , the amount of precursor that was not consumed in the diameter increment is now available for growth in nanoparticle B-nanoparticle thickness . [SEP]
[CLS] hence , the second phase of growth progression is the growth in thickness , which depends upon the amount of precursor available for reduction . [SEP]
[CLS] the particle thickness obviously had an opposite trend to diameter . [SEP]
[CLS] particles synthesized in the presence of a30 had the highest thickness and particles with t30 had the lowest particle thickness . [SEP]
[CLS] the above experiments have identified three factors that dna operates through : 1 ) binding B-event of I-event dna I-event to the precursor , 2 ) dna B-event binding I-event affinity to the np B-nanoparticle surface and 3 ) density of dna on the seed ' s surface . [SEP]
[CLS] cyclic voltammetry studies revealed that dna B-event binding I-event to the precursors only influences the diffusion of the precursor towards au { 111 } surface , which is indicated by the change in cathodic current with change in dna sequence in the order , t ( which allows fastest precursor diffusion ) > g > c > a . this complies with the second factor , i . e . , the dna B-event binding I-event affinity trend to au where a > c > g > t . [SEP]
[CLS] although dependent on the binding affinity , the density of dna on the seed ' s surface by itself plays a significant role . [SEP]
[CLS] an experiment where the concentration of t30 was increased to consequently increase the strand density on the seed limited the lateral growth of the prism , i . e . decreased the diameter and increased the thickness . [SEP]
[CLS] increasing the t30 dna concentration to up to 10 times resulted in hexagonal particles , suggesting the kinetic trapping of hexagonal intermediate B-property owing to the increased dna density that compensates for the low affinity of the sequence . [SEP]
[CLS] the surface property of the nanoparticles B-nanoparticle with a30 is rough in nature as compared to a smooth round particle formed in the presence of c30 . [SEP]
[CLS] the roughness may be attributed to the mobility B-property of the base on the au surface , specifically au { 111 } . the calculated mobility B-property of the bases follows the order a < g = t < c . [SEP]
[CLS] the low mobility B-property of a on the au surface allows au deposition only in areas where the a30 strand is absent , thereby producing a rough surface . [SEP]
[CLS] on hybridizing t30 functionalized 5 nm aunp B-nanoparticle with a30 particles resulted in the localization of the 5 nm aunps B-nanoparticle in the cervices of the rough particle , consistent with the base mobility - based hypothesis . [SEP]
[CLS] a more complicated system involving dna - encoded growth of two different metal B-nanoparticle nanoparticles I-nanoparticle tells a mechanistic story that not only complements well with the monometallic system described above , but also provides new insights into how dna operates at a more intricate level . [SEP]
[CLS] the pd cubic seed which is used as a seed in this study contains the { 111 } and { 110 } sites , whose residing atoms B-material lack a fulfilled coordination number , which makes them relatively higher in energy . [SEP]
[CLS] the dna B-event binding I-event affinity and dna density on the seed influence two major factors on the incoming metal B-material atoms B-material , 1 ) the deposition and 2 ) the diffusion . [SEP]
[CLS] the relative rate of metal B-material atom I-material deposition and diffusion determines the morphology of the shell B-material formed and the dna of different sequences tunes both rates ( figure 7a ) . [SEP]
[CLS] the low binding affinity of the t10 does not substantially passivate the seed ' s surface , promoting the deposition of the incoming metal B-material on the high energy sites , as observed in the sem . [SEP]
[CLS] hence , the rate of deposition is much higher than the rate of metal B-material diffusion on the seeds surface , ultimately constricting the shell B-material formation to the corners and edges , forming a pd - au core - frame structure . [SEP]
[CLS] c10 on the other hand , does passivate the high energy sites due to higher binding affinity and allows the rates of deposition to be lower than the rate of diffusion , to form a complete cuboctahedron shell B-material . [SEP]
[CLS] g10 tends to form a secondary structure with the metal B-material precursors and thus the seed ' s surface is passivated with a rigid structure that inherently has low mobility B-property . [SEP]
[CLS] the rate of atom B-material deposition is higher than the rate of atom B-material diffusion , which initially promotes the formation of islands on the cubic core B-material . [SEP]
[CLS] the deposition therefore occurs in areas where the rigid secondary structure is absent . [SEP]
[CLS] eventually , after metal B-material atom B-material diffusion events , the formation of an undulated pd - au shell B-material takes place . [SEP]
[CLS] further increase in g10 concentration completely passivates the surface and does not allow any growth on the palladium B-material cube . [SEP]
[CLS] a10 has high binding affinity with both the seed and the precursor . [SEP]
[CLS] thus , the high energy sites on the seed are passivated and the metal B-material deposition and diffusion takes place uniformly to form a rhombi cuboctahedron . [SEP]
[CLS] the a10 sequence also binds to the precursor to form smaller unstable nanocrystallites that undergo inter - particle fusion and subsequently deposit onto the seed , a major step in an aggregative growth mechanism or ostwald ripening . [SEP]
[CLS] the high density of a10 on the seed and its binding to the precursor both equally contribute to this mechanism . [SEP]
[CLS] the uv - vis kinetic study , where the growth in presence of different dna sequences was monitored in - situ by the absorption profiles revealed that a10 displays a sigmoidal growth curve in comparison to the more common exponential growth curve exhibited by the other bases ( figure 7b ) . [SEP]
[CLS] the sigmoidal growth curve is characteristic of the aggregative growth mechanism . [SEP]
[CLS] further investigation of a similar system consisting of a concave palladium B-material seed instead of a cubic seed , revealed the key influence of the core B-material in a dna - mediated synthesis . [SEP]
[CLS] the existence of high - energy sites on the seed influences the initial deposition of gold B-material onto the seed surface in the presence of dna with low or medium binding affinity , such as t10 and g10 , c10 , respectively , resulting in an overall increased growth rate . [SEP]
[CLS] dna with higher binding affinity , such as a10 , still retains the ability to bind to the precursor and form smaller nanocrystallites , validated by the sigmoidal curve observed in the kinetic absorption studies . [SEP]
[CLS] in the absorbance vs . time kinetic profiles , the increase in absorbance takes place within ~ 5 min for the concave cube vs . ~ 10 min in the presence of the simple cube , which agrees with the increased growth rate . [SEP]
[CLS] in all of the cases , the rate of deposition is greater than the rate of diffusion owing to high surface energy of the protruding edges and corners of the concave seed . [SEP]
[CLS] the deposition along the < 111 > direction was almost always greater than au deposition along the < 100 > direction , implying that γ increased in the following order : { 100 } < { 110 } < { 111 } . [SEP]
[CLS] a major influence of the seed was observed in the timebased sem studies on the morphological evolution . [SEP]
[CLS] with a simple cubic core B-material , the influence of dna was evident from the start of growth , whereas , the initial growth in the concave cube core B-material was heavily dictated by the surface energy of the seed . [SEP]
[CLS] therefore , when the growth was arrested at an initial time phase or when less gold B-material precursor was used , the morphology that resulted in the presence of t10 , g10 and c10 were almost indistinguishable , consistent with their similar spr properties . [SEP]
[CLS] however , after the initial surface energy passivation by gold B-material deposition , the influence of the dna in the further deposition of gold B-material comes in . [SEP]
[CLS] the fundamental study of the mechanisms involved in dna - mediated synthesis of metal B-nanoparticle nanoparticles I-nanoparticle helps learn the deciding factors in what exemplifies fine - control of nanomaterial B-material morphology . [SEP]
[CLS] although the salient principles under which dna - mediated shape - control occurs are often interdependent , certain parameters such as seed facets , preexisting capping ligands , seed composition , can be decoupled and further studied . [SEP]
[CLS] some of the most distinct advantages of nanotechnology are its interesting surface , optical and chemical properties that sparked widespread interest for its applications in the fields of biology , electronics , medicine , batteries , solar cells B-material , and sensors . [SEP]
[CLS] when bulk materials are shrunk to the nanoscale , it takes on different optical and chemical properties . [SEP]
[CLS] over the past decade , there have been tremendous advancements to design and synthesize new nanoscale materials to engineering solutions to different problems . [SEP]
[CLS] hence , it is important to understand the properties of the novel dna - mediated nanoparticles B-nanoparticle with different morphologies and explore how these materials can be employed for the above applications . [SEP]
[CLS] surface engineering of nanomaterials B-material is the bridge between nanotechnology and biology . [SEP]
[CLS] for nanomaterials B-material to be used effectively in biology , there is a need for effective and efficient modification of biocompatible B-property ligands , allowing the nanoparticles B-nanoparticle to be soluble B-property and stable in the complex biological medium . [SEP]
[CLS] however , surface modification of nanomaterials B-material has generally been met with some level of difficulty and complexity [SEP]
[CLS] while it is commonly argued that successful surface modification of nanomaterials B-material allowed effective use of these nanomaterials B-material in biological applications , these synthetic routes have often been either complicated or inefficient in conjugation techniques . [SEP]
[CLS] the majority of nanoparticle B-nanoparticle synthesis uses the liquid - solid - solution ( lss ) phase transfer synthetic route to yield semiconducting , upconversion , conducting polymer B-material , organic optoelectronic semiconducting and magnetic B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] nanomaterials B-material synthesized via these routes often require post modification since the capping ligand is generally hydrophobic B-property in nature and not biologically feasible for applications . [SEP]
[CLS] gold B-material and silver B-nanoparticle nanoparticles I-nanoparticle on the other hand require post modifications after synthesis to load dna via a tedious and time consuming salt B-material ageing process or acid treatment [SEP]
[CLS] newer , faster freeze directed protocols have been reported but are successful only for au nanoparticles B-nanoparticle coated with citrate ligands . [SEP]
[CLS] the discovery of dna - mediated morphological growth of mono or bimetallic nanomaterials B-material offers a completely novel approach for an effective dna B-event modification I-event to render metallic nanostructures not only water B-material soluble B-property but more importantly with a bio - recognition capability via a one - step synthesis reaction . [SEP]
[CLS] the dna mediated morphological growth of nanoparticles B-nanoparticle resulted in the retention of bio - recognition properties of dna , while dna itself being used as a ligand to stabilize the nanoparticle B-nanoparticle . [SEP]
[CLS] it was observed that by incubating B-technique a30encoded nanoparticles B-nanoparticle ( i . e . , rough - round au particles from au nanoprism ) with 5 nm aunps B-nanoparticle functionalized complementary dna ( t30 ) , satellite nanostructures were formed . controls were performed with 5 nm gold B-nanoparticle nanoparticles I-nanoparticle functionalized non - complementary dna ( a30 ) resulted in no satellites nanoassemblies ( figure 8a ( i ) ) . interestingly , as reported by wu et al , even though only 10 bases of deoxynucleotides were used in the dna mediated growth of silver B-material nanostructures , satellite nano - assemblies were observed to form in the presence of complementary dna functionalized 5 nm gold B-nanoparticle nanoparticles I-nanoparticle ( figure 8a ( ii ) ) . [SEP]
[CLS] these results provide a strong evidence , that only a few bases within the oligonucleotides were used as functional ligand to stabilize the morphological growth of mono - metallic B-nanoparticle nanoparticles I-nanoparticle and majority of the dna is left exposed to the surrounding , allowing the nanostructure be functional for further dna hybridization . [SEP]
[CLS] thiolated dna functionalized on a nanoparticle B-nanoparticle surface can be replaced with shorter alkyl thiols B-material such as mercaptoethanol , on the other hand it has been demonstrated that for aunfs synthesized in the presence of dna , it is difficult to replace the surface dna using shorter thiols B-material , validating the robust nature of the functionalization . [SEP]
[CLS] hence , these intriguing findings allows the synthesis of nanoparticles B-nanoparticle with bio - recognition abilities without post synthesis modification . [SEP]
[CLS] metal B-nanoparticle nanoparticles I-nanoparticle exhibit a unique property known as surface plasmon resonance ( spr ) wherein the conduction electrons on the surface of the nanoparticle B-nanoparticle interact with incident light . [SEP]
[CLS] the spr depends heavily on the shape and the size of the nanoparticle B-nanoparticle and this specific property can be used for several applications such as photocatalysis , surfaceenhanced raman - based detection , and biomedical imaging . [SEP]
[CLS] dna mediated growth of mono - metallic B-nanoparticle nanoparticles I-nanoparticle has also allowed the effective tuning of surface plasmonic properties of nanoparticles B-nanoparticle . [SEP]
[CLS] one of the most intriguing results yielded from dna mediated morphological growth of nanomaterials B-material was the ability to rationally tune the absorption of au nanorods B-nanoparticle from the nir i window to the nir ii window . [SEP]
[CLS] it was observed that in the presence of a20 , the l - lspr peak was intensified and redshift to 856 nm . this is accompanied by the formation of dumbbell shape at the ends of the au nanorods B-nanoparticle ( figure 3c ) . [SEP]
[CLS] more interestingly , with the addition of phosphorothioate modified a20 , the l - lspr peak was found to be further turned to 1011 nm with four phosphorothioate to the dna ( figure 4 ) . [SEP]
[CLS] this unique tuning to the nir ii window was attributed to the fact that with the increase of phosphorothioate modifications , there was larger intraparticle gaps with flattened ends to induce a red shift by a significant amount . [SEP]
[CLS] one of the main challenges facing nanotechnology in biological applications has been to render nanoparticles B-nanoparticle not only soluble B-property but also functional to detect , sense or target specific analytes in the biological matrix . [SEP]
[CLS] as mentioned previously , most nanoparticle B-nanoparticle types ranging from semiconductor nanoparticles B-nanoparticle to lanthanide B-nanoparticle nanoparticles B-nanoparticle and gold B-nanoparticle nanoparticles I-nanoparticle are synthesized as hydrophobic B-property nanoparticles B-nanoparticle due to the presence of oleic acid as capping ligands . [SEP] B-nanoparticle
[CLS] while these syntheses yielded highly homogenous small nanoparticles B-nanoparticle , complex surface engineering has to be performed to render these nanoparticles B-nanoparticle soluble B-property before it can be used as delivery agents or nanomedicine within the cells B-material for various applications . [SEP]
[CLS] to overcome the above limitation , wang et al reported that using dna mediated morphological growth method , the synthesized aunfs consist of high densities of dna on its surface . [SEP]
[CLS] dark field microscopy B-technique of aunf in cho cells B-material showed good distribution of nanoparticles B-nanoparticle within the cell B-material ( figure 8b ) . [SEP]
[CLS] by utilizing the light scattering properties of aunf , there was strong evidence for the effective delivery of nanoparticles B-nanoparticle without the use of transfection agents . [SEP]
[CLS] additionally , the same study shows that the dna tethered to the dna - encoded aunfs is more stable when treated with mercaptoethanol as compared to nanoparticles B-nanoparticle that were conjugated with thiolated dna , probably because the dna are embedded in between the nanoparticles B-nanoparticle during the growth , which prevents the dna from dissociating from the nanoparticles B-nanoparticle . [SEP]
[CLS] this particular finding is crucial in that biological environments contain many small thiolated molecules that can completely replace conjugated dna from the nanoparticle B-nanoparticle surface but not the dna that is present on a dnaencoded nanoparticle B-nanoparticle . [SEP]
[CLS] dna ligand is the only ligand used in the synthesis of these dna mediated nanoparticles B-nanoparticle , it is compelling that dna functionalized nanoparticles B-nanoparticle resulted in the efficient cellular uptake of these shaped aunf . [SEP]
[CLS] this can be further supported by another independent work by mirkin et al , where high dna density on the surface of the nanoparticles B-nanoparticle resulted in greater adsorption to surface proteins B-material on the cell B-material to induce better cellular uptake . [SEP]
[CLS] since dna mediated nanoparticles B-nanoparticle result in a facile synthesis of nanoparticles B-nanoparticle of different shapes , there could also be different cellular uptake rates influenced by the shape of the nanoparticles B-nanoparticle . [SEP]
[CLS] yang and coworkers reported that the silver B-material nanoprisms derived from dna mediated synthesis resulted in particles having good cell B-property viability I-property as compared to that of silver B-nanoparticle nanoparticles I-nanoparticle , while at the same time the silver B-material nanoprisms exhibit better antibacterial properties . [SEP]
[CLS] due to the nature of dna mediated nanomaterials B-material , these particles have been demonstrated to be facile in synthesis to yield highly biocompatible B-property properties and at the same time offers interesting biological application such as the ones observed in silver B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] sers properties have also been demonstrated in these dna - mediated nanoparticles B-nanoparticle . [SEP]
[CLS] 4methylbenenethiol ( 4 - mbt ) was used by the lu group to demonstrate enhancement factors around 10 5 and 10 6 using dna - mediated silver B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] the highest enhancement was reported to be with c10 on silver B-nanoparticle nanoparticles I-nanoparticle with good reproducibility . [SEP]
[CLS] at the same time , truncated ag nanoparticles B-nanoparticle with t10 could be used as hotspots for sers enhancement . [SEP]
[CLS] in addition , sers was also demonstrated in dna mediated aunrs . [SEP]
[CLS] enhancement factors between 10 4 and 10 6 was observed for different shapes such as rods , dumbbells , cracked octahedron and octahedron shapes using 4 - mbt dye . [SEP]
[CLS] similar sers enhancement was also reported by yang and coworkers using crystal violet . [SEP]
[CLS] it was observed that the sers effects were stronger using agnpr as compared to that of silver B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] furthermore , the nir absorbing pd @ au core - shell nanoparticles B-nanoparticle were used as sers substrates for the same substrate with enhancement factors of up to ~ 10 7 when excited by higher wavelength lasers . [SEP]
[CLS] the enhancement factors depended on the morphology of the nanoparticles B-nanoparticle tracing back to the sequence of dna utilized in the synthesis ( figure 8c ) . [SEP]
[CLS] this review summarizes recent advancements in the field of dna - mediated growth of mono - and bi - metallic nanomaterials B-material with controlled morphologies . [SEP]
[CLS] through the discovery of dna codes , the use of dna with different sequences to tune the morphologies has been demonstrated while using gold B-material prisms , spheres , rods , silver B-material and palladium B-material nanocubes as seeds . [SEP]
[CLS] as a result , different shapes and surface structures of nanomaterials B-material were synthesized where dna was demonstrated not only as a stabilizing capping ligand to the nanomaterials B-material , but more importantly to systematically control the growth of nanomaterials B-material . [SEP]
[CLS] it has been found that the affinities of the nucleobases play a vital role in interacting and stabilizing different metal B-material surfaces that consequently changes the metal B-material deposition profiles in a sequence dependent matter . [SEP]
[CLS] the versatility that this approach introduces to metal B-nanoparticle nanoparticle I-nanoparticle synthesis is extraordinary . [SEP]
[CLS] changing the dna functionality by altering the sequences of dna bases introduces novel design rules in the generating new morphologies of nanoparticles B-nanoparticle with exclusive properties . [SEP]
[CLS] more interestingly , in a one - step reaction , these dna directed nanoparticles B-nanoparticle retained bio - recognition capabilities on the surface . [SEP]
[CLS] this is quite rare in gold B-material and silver B-material nanomaterials B-material without post modification with dna . [SEP]
[CLS] the rational growth of nanoparticles B-nanoparticle has also yielded in a systematic tuning of optical properties such as lspr from the visible range to the nir ii window . [SEP]
[CLS] several of the final morphologies exhibited excellent sers properties owing to the unique shapes obtained via dna - mediated synthesis . [SEP]
[CLS] these interesting improvements and modifications to the properties of nanomaterials B-material prompt further investigations into the role of dna to rationally tune these properties . [SEP]
[CLS] these facile routes to synthesize unique nanostructures have been proven to be reproducible and stable in aqueous media . [SEP]
[CLS] many questions still remain in the field of dna - mediated nanoparticle B-nanoparticle growth with different morphologies . [SEP]
[CLS] while dna interactions with metal B-material ions B-material and metal B-material intercalators have been known , there are only very few systematic studies on dna interactions with nanoparticle B-nanoparticle surface . [SEP]
[CLS] even in the current literatures of the dna interaction with bulk metal B-material surface , the results are often inconsistent between different studies . [SEP]
[CLS] to advance the field of using dna to mediate the growth of nanomaterials B-material , the detailed interaction of dna with metal B-material surfaces should be elucidated . [SEP]
[CLS] another area of interest would be to study the influence of different functional groups on the dna in controlling the growth of nanomaterials B-material . [SEP]
[CLS] the ease of base modification allows one to remove or add certain functional groups onto the nucleobases enabling one to decouple functional parameters that influence the final morphologies . [SEP]
[CLS] in addition , abasic analogues can establish the necessity of having purines and pyrimidines for the precise growth of nanoparticles B-nanoparticle . [SEP]
[CLS] these studies could further help in the rational design of capping ligands that can predictably influence the morphology of nanomaterials B-material and consequently their chemical and physical properties . [SEP]
[CLS] understanding the effects in these two areas will allow better control and design on mediating nanomaterial B-material growth and its use as not only a stabilizing ligand but more importantly , a modular ligand that is capable of controlling the nanomaterial B-material morphologies . [SEP]
[CLS] while the effect of different dna sequences on the morphologies of pd - au nanoparticles B-nanoparticle have been elucidated , it would be interesting to see if the finding from this study can be applied to studying of other bimetallic nanoparticles B-nanoparticle , such as au - ag nanoparticles B-nanoparticle , given the similar lattice constants of au and ag and the knowledge we have gained on how dna interacts with each of the metals B-material individually . [SEP]
[CLS] additionally , almost all of the current studies have been focused on noble metal nanoparticles B-nanoparticle , it is important to explore extension of the approach to study more complicated systems that involve other metals B-material and metal - oxide / semiconductor hybrid materials . [SEP]
[CLS] since wide band gap semiconductors have relatively small optical cross - sections in the visible spectrum , these materials interact weakly with visible light . [SEP]
[CLS] one way of enhancing this property is by integrating them with plasmonic metals B-material either as an oxide core - metal shell B-material or metal core - oxide shell B-material and the goal can be accomplished with dna encoded synthesis . [SEP]
[CLS] to provide deeper insights into the mechanism of dna - mediated nanoparticle B-nanoparticle formation , one can take advantage of in situ liquid cell B-material to image growth of nanomaterials B-material in real time under the transmission electron microscope ( tem ) [SEP]
[CLS] this technique has allowed the extensive studies on the defect formation , particle growth rate , oxidative etching , [ 163 , 164 ] and kirkendall effect . [SEP]
[CLS] [ 165 ] hence , to further advance the field , in situ tem techniques may be employed to study the growth of np B-nanoparticle in real time and to accurately observe the growth mechanism using both a wet cell B-material and flow cell method . [SEP]
[CLS] using this tool , the tem image and its facet changes at each stage of the nanoparticle B-nanoparticle growth can be accurately studied to fully understand what is happening at each stage of the nanoparticle B-nanoparticle growth . [SEP]
[CLS] these studies are important as current mechanisms were proposed by using mercaptoethanol to stop the nucleation of nanoparticles B-nanoparticle and there could be inaccuracies in determining the exact growth of the nanomaterials B-material . [SEP]
[CLS] to study the exact growth , in situ tem is current the best technique available to determine how the nanomaterial B-material nucleates and further grows into the final shape . [SEP]
[CLS] while dna mediated growth of gold B-material and silver B-nanoparticle nanoparticles I-nanoparticle has been reported , it is important to take extra precaution to avoid or minimize electron - beam induced reduction of metal B-material ions B-material in the solution that may contribute to some of the observations . [SEP]
[CLS] at the same time , advancements are being made to elucidate how shape and size of nanomaterials B-material influence the cellular uptake in cells B-material . [SEP]
[CLS] this is an important area of interest due to the emergence of nanomaterials B-material in sensing , imaging and drug delivery . [SEP]
[CLS] besides the conventional studies to investigate the cytotoxicity B-property of nanoparticles B-nanoparticle , another focus would be centered on understanding how shape influences the uptake of cells B-material to maximize delivery into the cells B-material as a delivery vehicle B-material . [SEP]
[CLS] these dna mediated mono - metallic nanostructures can be further functionalized in the most facile manner with functional aptamers for biomedical applications . [SEP]
[CLS] another area of interest is the fate of the nanoparticle B-nanoparticle shape after cellular internalization and the extent to which shape related properties such as spr can be utilized after the internalization event , which will be important for biomedical or bio - sensing applications . [SEP]
[CLS] finally , most investigations have focused on homo - oligomeric dna in order to elucidate effects of different nucleobases in controlling the morphology . [SEP]
[CLS] it is important to build upon the progress and explore deeper into the effect of different sequence combinations on the morphologies of nanomaterials B-material in order to be more analogs to biological genetic codes . [SEP]
[CLS] the holy grail is to develop clear accurate design principles that governs dna codes for abiological nanoparticle B-nanoparticle morphologies for all sequence combinations and across diverse ranges of metals B-material in single or complex hybrid nanosystems . [SEP]
[CLS] the realization of this goal would offer a myriad of new possibilities in the integration of these novel nanomaterials B-material with healthcare platforms and offering engineering solutions to current challenges in nanoscale fabrication . [SEP]
[CLS] schematic demonstrating a few amongst many functional modifications that can be made to the different components of dna , i . e . the phosphate backbone and the nucleobases . [SEP]
[CLS] schematic representing the different nanoparticle B-nanoparticle systems studied via dna mediated shape control approach . [SEP]
[CLS] systems using gold B-nanoparticle nanoparticles I-nanoparticle , specifically spherical aunp B-nanoparticle ( 1a ) , au nanoprism ( 1b ) and au nanorod B-nanoparticle ( 1c ) ; silver B-nanoparticle nanoparticles I-nanoparticle , specifically , ag nanocube ( 1d ) and spherical agnp ( 1e ) ; and bimetallic B-nanoparticle nanoparticles I-nanoparticle ( pd - au ) ( 1f ) . [SEP]
[CLS] a ) . [SEP]
[CLS] circular dichroism ( cd ) measurements demonstrating the disruption and formation of the i - motif and the g - quadruplex during the overgrowth of ag nanocubes in the presence of c10 and g10 respectively . [SEP]
[CLS] ( parts of the figure reproduced with permission . [SEP]
[CLS] copyright 2014 american chemical society ) [SEP]
[CLS] b ) . [SEP]
[CLS] showing different aunp B-nanoparticle morphologies grown from au prism seeds in the presence of different dna sequences ( the sizes ~ 200 nm ) . [SEP]
[CLS] ( reproduced with permission . [SEP]
[CLS] copyright 2012 wiley - vch ) [SEP]
[CLS] c ) . [SEP]
[CLS] overgrowth of aunr overgrowth using different dna sequence block combinations , monitored by uv / vis absorption and sem . [SEP]
[CLS] scale bars = 20 nm [SEP]
[CLS] ( reproduced with permission . [SEP]
[CLS] copyright 2015 wiley - vch ) ( reproduced with permission . [SEP]
[CLS] copyright 2015 wiley - vch ) a ) . [SEP]
[CLS] schematic representing the overgrowth of au nanorod B-nanoparticle seeds in the presence of dna . [SEP]
[CLS] the facets of the seed play an important role in the dna B-event binding I-event events . [SEP]
[CLS] ( reproduced with permission . [SEP]
[CLS] copyright 2015 wiley - vch ) [SEP]
[CLS] b ) . [SEP]
[CLS] the growth progression of the au nanoprism in the presence of t30 , starting from ( a ) nanoprism to ( b ) nonagon to ( c ) hexagon to a final ( d ) six - pointed star with corresponding model of the np B-nanoparticle and diffraction pattern ( insets ) . [SEP]
[CLS] blue and red lines on the models indicate the distance measured from the center to the corners or sides of the particles , respectively , plotted in the bottom right corner . [SEP]
[CLS] hr - tem image of the hexagonal . [SEP]
[CLS] scale bar is 2 nm . [SEP]
[CLS] red box shows the location where the higher - resolution image was observed . [SEP]
[CLS] ( reproduced with permission . [SEP]
[CLS] copyright 2015 american chemical society ) schematic representing the proposed mechanism of growth of au nanoprisms influenced by dna in two stages : shape control and thickness growth . [SEP]
[CLS] ( reproduced with permission . [SEP]
[CLS] copyright 2015 american chemical society ) [SEP]
[CLS] copyright 2016 american chemical society ) [SEP]
[CLS] copyright 2010 american chemical society ) [SEP]
[CLS] c ) . [SEP]
[CLS] the sers spectra of 4 - mbt modified on the surfaces of different pd @ au core - shell nanoparticles B-nanoparticle grown from a concave pd seed with various dna sequences . [SEP]
[CLS] ( figure reproduced with permission . [SEP]
[CLS] copyright 2018 springer nature ) . [SEP]
[CLS] left : plasmonic properties of the aunrs overgrown with a20 dna and a20 with 1 , 2 and 4 ps - modifications . [SEP]
[CLS] right : tem images of the as synthesized nanoparticles B-nanoparticle . [SEP]
[CLS] scale bars are 20 nm ( high magnification tem image ) and 50 nm ( low magnification tem image ) . ( reproduced with permission . [ 84 ] copyright 2015wiley - vch ) [SEP]
[CLS] a ) . [SEP]
[CLS] schematic representation of the proposed mechanism of growth of pd−au bimetallic nanostructures influenced by different sequences of dna . [SEP]
[CLS] the atomic models represent the cross - section of the 3 - d models along the black box in each individual case . [SEP]
[CLS] b ) . [SEP]
[CLS] top : kinetic uv−vis absorption spectra of nanoparticle B-nanoparticle growth in the presence of t10 . [SEP]
[CLS] bottom : the plot of absorbance vs time at the λ max values for each of the particles . [SEP]
[CLS] ( reproduced with permission . [SEP]
[CLS] copyright 2016 american chemical society ) [SEP]
[CLS] 8 . the post - synthetic properties of dna - encoded particles . [SEP]
[CLS] ( i ) . top : tem images of aunf synthesized with a30 assembled with 5 nm au nanoparticles B-nanoparticle functionalized with thiolated t30 ( left ) and with a thiolated non - complementary sequence ( right ) [SEP]
[CLS] bottom : aunps B-nanoparticle synthesized with t30 assembled with 5nm au nanoparticles B-nanoparticle functionalized with thiolated a30 ( left ) and with a thiolated non - complementary sequence ( right ) . [SEP]
[CLS] ( parts of the figure reproduced with permission . [ 80 ] copyright 2010 american chemical society ) . [SEP]
[CLS] ( ii ) . [SEP]
[CLS] au nanoparticle B-nanoparticle grown from nanoprism seed in the presence of a30 assembled with aunp B-nanoparticle functionalized with thiolated non - complementary sequence ( left ) and thiolated t30 ( right ) . [SEP]
[CLS] ( figure reproduced with permission . [ 104 ] copyright 2015 american chemical society ) . [SEP]
[CLS] b ) . [SEP]
[CLS] dark field image of the cho cells B-material treated with aunf particles ( left ) and without nanoparticle B-nanoparticle treatment ( right ) . [SEP]
[CLS] ( figure reproduced with permission . [ 80 ] copyright 2010 american chemical society ) . [SEP]
[CLS] c ) . [SEP]
[CLS] the sers spectra of 4 - mbt modified on the surfaces of different pd @ au core - shell nanoparticles B-nanoparticle grown from a concave pd seed with various dna sequences . [SEP]
[CLS] ( figure reproduced with permission . [ 135 ] copyright 2018 springer nature ) . [SEP]
[CLS] the facile construction of metal - dna complexes by using ' click ' reactions is reported here . [SEP]
[CLS] a series of 2 ' - propargyl - modified dna oligonucleotides were initially synthesized as structure scaffolds and were then modified through ' click ' reaction to incorporate a bipyridine ligand equipped with an azido group . [SEP]
[CLS] these metal chelating ligands can be placed in the dna context in site - specific fashion to provide versatile templates for binding various metal B-material ions B-material , which are exchangeable by using a simple edta washing - and - filtration step . [SEP]
[CLS] the constructed metal - dna complexes were found to be thermally stable . [SEP]
[CLS] their structures were explored by solving a crystal structure of a propargyl - modified dna duplex and installing the bipyridine ligands by molecular modeling and simulation . [SEP]
[CLS] these metal - dna complexes could have wide applications as novel organometallic catalysts B-property , artificial ribonucleases and potential metal B-material delivery systems . [SEP]
[CLS] dna , the fundamental biomolecule of life , has increasingly emerged over the past few decades as a key building block and structural framework creating a myriad of functional materials . [SEP]
[CLS] these dna based materials are very attractive in both fundamental research and practical applications . [SEP]
[CLS] for instance , owing to the unique base pairing specificity , predictability and versatile programmability , dna selfassembly has become an important strategy for the construction of nano - materials such as carbon B-nanoparticle nanotubes I-nanoparticle , nanobricks , metal B-nanoparticle nanoparticles I-nanoparticle and quantum B-nanoparticle dots I-nanoparticle , etc . [SEP]
[CLS] introductionthrough a bottomup method with precise design and control . [SEP]
[CLS] in addition , the electrostatic properties and electrical conductivity of dna as a highly charged polymer B-material makes it attractive nanowire B-nanoparticle to fabricate nanoscale electro devices . [SEP]
[CLS] more importantly , dna is biodegradable B-property and biocompatible B-property , and therefore can work as an excellent bio - functional framework for developing cell B-material imaging probes , biocatalysts , drug deliver systems , and therapeutics ( y . j . . [SEP]
[CLS] these functional dna molecules can be further used to construct small molecule - dna hybrids that will largely increase the scope and versatility of their functional applications . [SEP]
[CLS] it is well - known that single stranded dna / rna molecules , so called aptamers , can be selected through selex technology ( systematic evolution of ligands by exponential enrichment ) to bind almost every bimolecular target including proteins B-material , carbohydrates B-material , lipids B-material , nucleic B-material acids I-material and whole cells B-material with extraordinarily high binding specificity and affinity . [SEP]
[CLS] these dna aptamers can serve as good structure scaffolds for the site - specific incorporation of diverse functionalities into any desired positions of the binding targets . [SEP]
[CLS] in this sense , the hybrid complex of dna - small molecules represents a general strategy to diversify the dna building blocks and holds great promising to construct novel dna based functional materials . [SEP]
[CLS] however , although this dna hybrid strategy has gained increasing popularity in nanostructure constructions , its applications in other areas , such as biocatalysis and organic synthesis , are yet to be explored probably due to the lack of feasible synthetic methods and the detailed structure information about small molecule - dna interactions . [SEP]
[CLS] an interesting application that we are currently exploring involves constructing metallic dna molecules as novel artificial ribonucleases to cleave target pathogenic rna sequences , and as chiral catalysts B-property to facilitate stereoselective organic transformations . [SEP]
[CLS] these two functions can be achieved by the site - specific incorporation of metal B-material ions B-material into specific positions of dna scaffolds that serve as either rna targeting reagents or chiral environments . [SEP]
[CLS] several methods have been developed for the site - specific introduction of metal B-material ions B-material into nucleic B-material acids I-material mainly through nucleobasemetal chelating or ' click ' chemistry , a widely used bioorthogonal strategy to label macromolecules . [SEP]
[CLS] however , the construction of a general scaffold or template that can bind a variety of metal B-material ions B-material for different functional applications has not been fully explored . [SEP]
[CLS] in this paper , we demonstrated that the ' click ' conjugation could be used to build this type of general platform for metal B-material binding in a site - specific way . [SEP]
[CLS] we first synthesized a series of dna strands with previously reported 2 ' - propargyl modified nucleotide building blocks at different positions ( 1 , scheme 1 ) , which can be subsequently linked to a metal chelating bipyridine ligand with a preinstalled azido group ( 2 , scheme 1 ) to make dna - ligand - metal B-material complexes ( 3 , scheme 1 ) . [SEP]
[CLS] these site - specifically located bipyridines can work as versatile platforms to bind different metal B-material ions B-material that are easily exchangeable by using an edta - washing - and - filtration step ( 4 , scheme 1 ) . [SEP]
[CLS] in addition , the 3d structure information of this type of dna - metal B-material complex was also elucidated by x - ray crystallography and molecular simulation studies . [SEP]
[CLS] based on the synthetic feasibility of installing a reactive group into flexible locations of dna , we chose the 2 ' - propargyl modified ribonucleotides as ideal dna building blocks , which are well compatible with solid phase synthesis and post - synthetic treatments . [SEP]
[CLS] the minor - groove located propargyl groups have been demonstrated to produce modest perturbation of dna biophysical properties such as duplex stability ; and have been used to generate several fluorescent B-property dna probes . [SEP]
[CLS] for proof of principle , we synthesized a few single and double - stranded dna oligos containing each of the four 2 ' - propargyl ( 2 ' - prop ) - a , c , g and u in different sequence contexts . [SEP]
[CLS] the modified products were purified by reverse phase hplc and characterized by electrospray ionization B-property mass spectrometry ( esi - ms , table 1 ) . [SEP]
[CLS] the selection of the metal B-material binding ligands fell on a typical bipyridine structure , which has been previously applied to construct dna - based asymmetric catalysts B-property and functional nanomaterials B-material , as the starting material B-material to install the ' click ' active azido tags . [SEP]
[CLS] as shown in scheme 2 , the treatment of 5 , 5 ' dimethyl - bipyridine with n - bromosuccinimide ( nbs ) and azobisisobutyronitrile ( aibn ) in a heated ccl 4 solution for 17h produced a clean mixture of mono - and di - brominated bipyridine compounds with ~ 1 : 1 ratio , which can be separated by column chromatography B-technique . [SEP]
[CLS] the mono - brominated product 6 was subsequently transferred to the azido - bipyridine 2 through the substitution of bromine B-material by azide in a heated dmf solution with quantitative yield . [SEP]
[CLS] with these building blocks in hand , we carried out the ' click ' reaction using the standard cubr catalyst B-property in the presence of acetonitrile as cu + chelating reagent . [SEP]
[CLS] although only moderate synthesis yields could be achieved for this click conjugation , the products were easily separated from the substrates by our reverse phase hplc conditions ( see experimental section for conditions ) . [SEP]
[CLS] exemplified in fig . 1 , the incorporation of bipyridine ligand into dna oligonucleotides decreased their retention time by about 2 min in our chromatogram , which suggested that these bipyridine - dna complexes possessed a slightly higher overall polarity than the initial propargyl modified species . [SEP]
[CLS] the separated substrate could be reused for the next cycle of ' click ' reaction . [SEP]
[CLS] interestingly , the esi - ms data clearly showed the presence of copper B-material ion B-material in the complex ( table 2 , entry 1 ) after both reaction and purification , which can be explained by the strong binding of copper B-material ion B-material to the bipyridine ligand . [SEP]
[CLS] the copper B-material ion B-material was subsequently washed out by using 10 equivalents of edta through an ultrafiltration device with a 3k - cutoff membrane . [SEP]
[CLS] the concentrated solution was subsequently washed with water B-material to obtain a metal - free species that could serve as a general template for the binding of other metal B-material ions B-material including cu 2 + , ni 2 + , zn 2 + and cd 2 + . [SEP]
[CLS] the corresponding metal - dna complexes were shown to be very stable under the conditions employed for esi - ms analysis ( table 2 and fig . s1 - 7 ) . [SEP]
[CLS] in order to further test their chemical stability , the metal B-material - dna oligonucleotide complexes were heated in 10 mm phosphate buffer ( ph 7 ) under 60 °c for 24h and monitored by hplc for possible strand cleavage . [SEP]
[CLS] as exemplified in fig . s8 for dna6 - cu 2 + , the metallic dna oligonucleotides were thermally stable . [SEP]
[CLS] these data demonstrated that bipyridine - directed chelation represents a general and versatile strategy to achieve site - specific incorporation of metal B-material ions B-material into different sites of dna structure . [SEP]
[CLS] it has been shown that the propargyl modifications at the 2 ' - positions of dna strand could increase the duplex stability when the dna strand is hybridized with complementary rna , whereas it can slightly destabilize the duplex when hybridized with dna . [SEP]
[CLS] consistent with these observations , as exemplified by using dna 2 in table 1 , the 2 ' - propargyl modified dna duplex provided nearly the same t m as the unmodified native species ( black and blue curves in fig . 2 ) , whereas the overall thermal stability decreased by 6 . 6 °c for the duplex containing the additional bipyridine ligand ( red curve in fig . 2 ) , indicating certain extent of structural perturbation . [SEP]
[CLS] in order to explore the possible effects of these propargyl and bipyridine modifications on the dna duplex structure , we successfully crystallized and solved a high - resolution structure of the self - complementary octamer duplex containing two 2 ' - propargyl - a residues ( dna - 9 in table 1 ) [ 5 ' - g ( 2 ' - ome - du ) gt ( 2 ' - prop - a ) cac - 3 ' ] 2 . [SEP]
[CLS] in this construction , the 2 ′ - ome - du residue was used to facilitate crystallization and drive the dna sequence to fold into an a - form duplex without major structural perturbation , in analogy with the 2 ' - seme - du functionality . [SEP]
[CLS] this modified dna oligonucleotide could crystallize under several different conditions of nucleic B-material acid I-material mini - screening and natrix buffer kit within a few days . [SEP]
[CLS] the crystals with best diffraction were grown under buffer conditions containing 10 % 2methyl - 2 , 4 - pentanediol ( mpd ) , 40 mm sodium B-material cacodylate ( ph 7 . 0 ) , 12 mm spermine tetra - hcl , 40 mm licl , 80 mm strontium B-material chloride B-material and 20 mm magnesium B-material chloride . [SEP]
[CLS] the best crystal diffracted to 1 . 6 a and the data collection / structure refinement statistics are summarized in table 3 . [SEP]
[CLS] the overall duplex structure and local 2 ' - prop - a : u pairing pattern are shown in fig . 4 . [SEP]
[CLS] overall , there is no major structural perturbation caused by these two propargyl groups by comparing the modified dna duplex with its native counterpart ( fig . 4a , 4b ) , although the backbone of 2 ′ - propargyl - a residue is rotated for ~ 100 degree as observed in the base pairing pattern ( fig . 4c ) . [SEP]
[CLS] the 2 ′ - propargyl groups were located in the minor groove of the duplex and turned to the 3 ′ - direction of each strand . [SEP]
[CLS] this arrangement leaves plenty of space for further modifications of the propargyl group within the minor groove . [SEP]
[CLS] the next step aimed at the characterization of the complete bipyridine conjugates to elucidate how the metal B-material - chelating ligands aligned in the dna duplex . [SEP]
[CLS] unfortunately , all crystals obtained under many different conditions showed very weak and disordered diffraction patterns . [SEP]
[CLS] this outcome suggested that , although the ligand might not affect the overall duplex structure , it was likely to adversely affect the molecular packing . [SEP]
[CLS] as a possible alternative , we employed computational modeling to generate structures , in which two bipyridine ligands containing two copper B-material ions B-material were attached to the propargyl groups in the initial crystal structure . [SEP]
[CLS] the final model of the whole complex was energy minimized and then subjected to molecular dynamic simulations . [SEP]
[CLS] the comparison between the duplexes with and without the ligands revealed that the ligands did not significantly distruct the helical structure of the duplex during the course of the simulation ( see supplementary figure s9 ) . [SEP]
[CLS] however , it was interesting to note that the ligands adopted two distinct types of orientations as shown in fig . 5 . [SEP]
[CLS] the dominant configuration ( ~ 40 % ) adopted by the modified duplex involves one of the ligands reaching across the minor groove to form a saltbridge between the chelated copper B-material - ion B-material and the phosphate group of two nucleotides to the 3 ' side of the complementary strand ( fig . 5a & 5c ) . [SEP]
[CLS] this across - the - groove binding by one of the ligands restricts the ability of the other ligand to simultaneously form another saltbridge , and hence is accomadated in the minor groove with the copper B-material ion B-material facing outwards and solvated . [SEP]
[CLS] the second configuration ( ~ 30 % ) involves both ligands simultaneously bound to phosphate groups , which are two nucleotides to the 3 ' side of their respective strands ( fig . 5b & 5d ) . [SEP]
[CLS] while this configuration allows for simulataneous formation of two salt B-material - bridges , we speculate that the entropic penalty associated with this state makes it less favorable than the single salt - bridge state of the duplex , which also possesses a two - fold symmetry due to the symmetrical nature of the duplex . [SEP]
[CLS] even though we observed both the symmetrical states of the dominant configuration , we did not observe a transition between the two due to the large energy barrier B-property between the states . [SEP]
[CLS] we surmise that the multiple orientations adopted by the ligands might have resulted in the poor resolution of their positions in the crystal structure . [SEP]
[CLS] it is worthy to note that we performed the molecular dynamics simulations in 1m nacl solution . [SEP]
[CLS] at such concentrations of salt B-material , the copper B-material ion B-material is not expected to strongly coordinate with chloride B-material ions B-material , which is what we observed in our simulations . [SEP]
[CLS] instead , we found that phosphate - bound copper B-material ion B-material coordinates with three water B-material molecules , maintaining a six - membered co - ordination sphere ( see supplemental figure s10 ) . [SEP]
[CLS] in summary , we have described an efficient and facile method for constructing metallic dna oligonucleotides through a ' click ' conjugation between 2 ' - propargyl - modified dnas and a bipyridine metal B-material chelating ligand . [SEP]
[CLS] the bipyridine - modified dna can serve as ideal structural scaffolds to bind different metal B-material ions B-material , which can be easily exchangeable in our dna contexts . [SEP]
[CLS] these complexes are thermally stable and do not cause major structural perturbations to the dna duplex . [SEP]
[CLS] in order to obtain structural insights , we solved the crystal structure of a propargyl modified dna duplex and modeled two bipyridine ligands with two copper B-material ions B-material into the propargyl groups by molecular simulation . [SEP]
[CLS] we observed potential interactions between coordinated copper B-material and oxygen B-material atoms I-material in the dna backbone . [SEP]
[CLS] such dna - based metal chelating systems will be expected to have wide applications in the construction of artificial ribonucleases and chiral catalysis , as well as the delivery of metal B-material ions B-material . [SEP]
[CLS] the organic reagents and solvents were purchased from sigma - aldrich and used directly without further purification . [SEP]
[CLS] all solid reagents were dried under a high vacuum line prior to use . [SEP]
[CLS] air sensitive reactions were carried out under nitrogen B-material . [SEP]
[CLS] analytical tlc plates precoated with silica gel f 254 ( dynamic adsorbents B-property ) were used for monitoring reactions and visualized by uv light . [SEP]
[CLS] flash column chromatography B-technique was performed using silica gel ( 32 - 63 μm ) . [SEP]
[CLS] all 1 h , 13 c and 31 p nmr spectra were recorded on a brucker 400 spectrometer . [SEP]
[CLS] chemical shift values are in ppm . [SEP]
[CLS] 13 c nmr signals were determined by using apt technique . [SEP]
[CLS] high - resolution ms data were achieved by esi mass spectrometry at university at albany , suny . [SEP]
[CLS] to a stirred solution of 5 , 5 ' - dimethyl - 2 , 2 ' - bipyridine , 5 ( 1 . 03 g , 5 . 60 mmol ) and nbromosuccinimide ( 1 . 02g , 5 . 60 mmol ) in anhydrous ccl 4 ( 50 ml ) under argon B-material at ambient temperature was added catalytic aibn ( 0 . 092 g , 0 . 56 mmol ) . [SEP]
[CLS] the mixture was heated to reflux at 77 °c for 17h until the starting material B-material was completely consumed . [SEP]
[CLS] the mixture was filtered through silica gel when hot . [SEP]
[CLS] the solution was cooled and filtered again to remove most of the di - bromo product . [SEP]
[CLS] the filtrate was concentrated and purified by silica gel chromatography B-technique with 5 % meoh in ch 2 cl 2 ( contain 0 . 5 % net 3 ) to provide the product 0 . 59 g in 40 % isolate yield . [SEP]
[CLS] rf 0 . 8 , 5 % meoh in ch 2 cl 2 ( contain 0 . 5 % net 3 ) . [SEP]
[CLS] 1 h nmr ( 400 mhz , cdcl 3 ) : δ 8 . 68 ( s , 1h ) , 8 . 62 ( s , 1h ) , 8 . 37 ( d , 1h , j = 8 . 2 hz ) , 8 , 30 ( d , j = 8 . 2 hz , 1h ) , 7 . 85 ( d , j = 8 . 2 hz , 1h ) , 7 , 64 ( d , j = 8 . 2 hz , 1h ) , 4 . 55 ( s , 2h ) , 2 . 41 ( s , 3h ) . [SEP]
[CLS] 13 c nmr ( cdcl 3 , 100 mhz ) δ 156 . 14 ppm . [SEP]
[CLS] to a stirred solution of 5 - ( bromomethyl ) - 5 ' - methyl - 2 , 2 ' - bipyridine , 6 ( 1 . 31g , 5 . 0 mmol ) in meoh ( 25 ml ) was added sodium B-material azide 39 mmol ) and the mixture was heated to 70 °cfor overnight . [SEP]
[CLS] when the starting material B-material was completely consumed , the solvent was concentrated , diluted with ch 2 cl 2 ( 20 ml ) and the mixture was washed with brine , dried over sodium B-material sulfate . [SEP]
[CLS] the solvent was concentration and purified by silica gel chromatography B-technique using eluent 5 % meoh in ch 2 cl 2 ( with 1 % net 3 ) to give the product 1 . 06 g in 94 % yield . [SEP]
[CLS] rf 0 . 4 , 5 % meoh in ch 2 cl 2 ( with 1 % net 3 ) . [SEP]
[CLS] the 2 ' - propargyl modified phosphoramidites were purchased from chemgenes corporation . [SEP]
[CLS] all the dna oligonucleotides were synthesized at 1 . 0 - μmol scales by solid phase synthesis using a mermade mm8 synthesizer . [SEP]
[CLS] all the phosphoramidites were dissolved in acetonitrile to a concentration of 0 . 07 m . 0 . 02 m i 2 in thf / py / h 2 o solution was used as oxidizing reagent . [SEP]
[CLS] all the other reagents are standard solutions obtained from chemgenes or glen research . [SEP]
[CLS] synthesis was performed on the appropriate nucleoside immobilized via a succinate linker to control - pore glass ( cpg - 500 ) . [SEP]
[CLS] all oligonucleotides were prepared in dmtr - on form . [SEP]
[CLS] after synthesis , the oligos were cleaved from the solid support and fully deprotected with ama ( ammonium hydroxide B-material : methylamine = 1 : 1 ) at 65 °c for 30min . [SEP]
[CLS] the amines B-material were removed by speed - vac concentrator before hplc purification . [SEP]
[CLS] after the dmtr - on purification , the detritylation was carried out by the treatment of 3 % trichloroacetice acid for 3min , followed by the neutralization with triethylamine to ph 7 . 0 . [SEP]
[CLS] the dmtr - off oligonucleotides were purified again by hplc . [SEP]
[CLS] the oligonucleotides were purified by reverse phase hplc using a zorbax sb - c18 column at a flow rate of 6 ml / min . [SEP]
[CLS] buffer a was 20 mmtriethylammonium acetate , ph 7 . 1 ; buffer b contains 50 % acetonitrile in 20 mmtriethylammonium acetate , ph 7 . 1 . [SEP]
[CLS] a linear gradient from buffer a to 80 % buffer b in 25 min was used to elute the oligos . [SEP]
[CLS] the analysis was carried out by using the same type of analytical column with the same eluent gradient . [SEP]
[CLS] all the dna oligos were analyzed by esi - ms . [SEP]
[CLS] the propargyl modified dna oligonucleotides ( 0 . 38 mm , 200ul in h 2 o ) and azidobipyridine compound 2 ( 10 mm , 114 ul , h 2 o ) were placed in a 1 . 5 ml vial . [SEP]
[CLS] in a separate vial , 17 ul cubr solution ( 100 mm in dmso / tbuoh 3 : 1 ) and 34 ul ch 3 cn solution ( 100 mm in dmso / tbuoh 3 : 1 ) were mixed and added to the dna solution . [SEP]
[CLS] the mixture was shaken at room temperature for overnight before being evaporated to near dryness in a speed - vac under 65 °c . [SEP]
[CLS] sodium B-material acetate ( 0 . 3 m , 100 ul ) was then added and the suspension was stirred for 1 h before 1 ml of ethanol was added . [SEP]
[CLS] the vial was vortexed well and stored in a freezer ( −80 °c ) for 1h before centrifugation for 15 min at 13000 rpm . [SEP]
[CLS] the supernatant was carefully removed from the dna pellet . [SEP]
[CLS] 70 % cold ethanol ( −20 °c ) was used to wash the pellet for three times . [SEP]
[CLS] finally , the pellet was left drying on air and dissolved in water B-material for hplc purification . [SEP]
[CLS] samples were analysed by direct infusion esi on a thermofisher scientific ( waltham , ma ) velos ltq - orbitrapvelos mass spectrometer . [SEP]
[CLS] all analyses were performed in nanoflow mode by using quartz emitters produced in house with a sutter instruments co . ( novato , ca ) p2000 laser pipette puller . [SEP]
[CLS] up to 5μl samples were typically loaded onto each emitter by using a gel - loader pipette tip . [SEP]
[CLS] a stainless steel wire was inserted in the back - end of the emitter to supply an ionizing B-property voltage that ranged between 0 . 8 and 1 . 2 kv . source temperature and desolvation conditions were adjusted by closely monitoring the incidence of ammonium adducts and water B-material clusters . [SEP]
[CLS] the instrument was calibrated by using a mixture of 0 . 5 mg / ml of csi in 50 % methanol , which provided 1 - 3 ppm mass accuracy . [SEP]
[CLS] solutions of the duplex dnas ( 0 . 5 μm ) were prepared by dissolving the purified dnas in sodium B-material phosphate ( 10 mm , ph 6 . 5 ) buffer containing 100 mm nacl . [SEP]
[CLS] the solutions were heated to 85 °c for 3 min , then cooled down slowly to room temperature , and stored at 4°c for 2h before t m measurement . [SEP]
[CLS] thermal denaturation was performed in a cary 300 uv - visible spectrophotometer with a temperature controller . [SEP]
[CLS] the temperature reported is the block temperature . [SEP]
[CLS] each denaturizing curves were acquired at 260 nm by heating and cooling from 5 to 80°c for four times in a rate of 0 . 5 °c / min . [SEP]
[CLS] all the melting curves were repeated for at least four times . [SEP]
[CLS] dna samples ( 0 . 5 mm duplex ) were heated to 80 °c for 3 minutes , cooled slowly to room temperature , and placed at 4 °c overnight before crystallization . [SEP]
[CLS] nucleic B-material acid I-material mini screen and natrix kits ( hampton research ) were used to screen crystallization conditions at different temperatures using the hanging - drop vapour diffusion method . [SEP]
[CLS] perfluoropolyether was used as cryoprotectant for the crystal mounting . [SEP]
[CLS] data was collected under a liquid nitrogen B-material stream at −174°c . [SEP]
[CLS] the diffraction data was collected at beam lines als 8 . 2 . 2 in lawrence berkeley national laboratory . [SEP]
[CLS] data were collected at a wavelength of 1 . 0 a . crystals were exposed for 1 second per image with a 1 degree oscillation angle . [SEP]
[CLS] all data were processed using hkl2000 and denzo / scalepack . [SEP]
[CLS] the 2 ' - propargyl modified dna structure presented here was solved by molecular replacement with phaser using pdb structure 1z7i as the search model , followed by the refinement using refmac . [SEP]
[CLS] the usual refinement protocol includes positional refinement , restrained b - factor refinement , and bulk solvent correction . [SEP]
[CLS] the stereo - chemical topology and geometrical restraint parameters of dna / rna were applied . [SEP]
[CLS] after several cycles of refinement , a number of highly ordered waters B-material were added . [SEP]
[CLS] cross - validation with a 5 % test set was monitored during the refinement . [SEP]
[CLS] the σa - weighted maps of the ( 2m | fo | - d | fc | ) and the difference ( m | fo | - d | fc | ) density maps were computed and used to build the 2 ' propargyl group . [SEP]
[CLS] we performed extensive md simulations of the dna duplex with and without the modifications totalling over a microsecond of simulation data . [SEP]
[CLS] the crystal structure of dna duplex with two 2 ' - propargyl groups was used as the template to which the two bipyridine ligands with copper B-material ions B-material are added . [SEP]
[CLS] the ligands ( one per strand ) was constructed in an " open " type conformation , i . e ) pointing away from the duplex . [SEP]
[CLS] we attached copper B-material ions B-material to the ligands imparting a + 2 charge , followed by md simulations to predict the structural preferences of the ligand with respect to the rest of the duplex . [SEP]
[CLS] molecular dynamics simulations were performed using gromacs - 4 . 6 . 3 . [SEP]
[CLS] the simulation system included the dna duplex with / without ligands in a solution of 1m nacl solution in a 3d periodic box . [SEP]
[CLS] the box size was 5×5×5 nm 3 containing 85 na + ions B-material , 75 cl - ions and 3769 water B-material molecules . [SEP]
[CLS] the systems were subjected to energy minimization to prevent any overlap of atoms B-material , followed by a 10 ns equilibration run . [SEP]
[CLS] configurations were chosen at 2 ns intervals from the equilibration run , for five independent 100 ns production runs , totalling 0 . 5 microsecond of trajectory for analysis . [SEP]
[CLS] the md simulations incorporated leap - frog algorithm with a 2 fs time step to integrate the equations of motion . [SEP]
[CLS] the system was maintained at 300k using the velocity rescaling thermostat . [SEP]
[CLS] the long - ranged electrostatic interactions were calculated using particle mesh ewald ( pme ) algorithm with a real space cutoff of 1 . 2 nm . [SEP]
[CLS] lj interactions were also truncated at 1 . 2 nm . [SEP]
[CLS] tip3p model was used represent the water B-material molecules , and lincs algorithm was used to constrain the motion of hydrogen B-material atoms I-material bonded to heavy atoms . [SEP]
[CLS] co - ordinates of the dna molecule were stored every 10 ps for further analysis . [SEP]
[CLS] parmbsc1 forcefield , with modified backbone , glycosidic and sugar torsions was used for the dna simulations . [SEP]
[CLS] amber - type force - field parameters were generated for the propargyl group as follows . [SEP]
[CLS] for obtaining the partial charges on the atoms B-material , we used the online resp charge - fitting server , red . [SEP]
[CLS] the geometry of the modified nucleoside was energy minimized , and hartree - fock level theory and 6 - 31g * basis - sets were employed to arrive at a set of partial charges . [SEP]
[CLS] the parameters for the propargyl modified nucleoside and the bipyridine ligands were listed in the end of the supporting information . [SEP]
[CLS] uv - melting temperature studies of native , 2 ' - propargyl modified , and dipyridine ' clicked ' dna duplexes . [SEP]
[CLS] sequences : dna - 2 in table 1 , 5 construction of metal - dna complex by the site - specific incorporation of metal B-material ions B-material into dna through a ' click ' reaction between the propargyl modified dna1 and an azido modified bipyridine 2 as the metal B-material chelating ligand . [SEP]
[CLS] synthesis of azido - bipyridine ligand 2 starting from 5 , 5 ' - dimethyl - bipyridine compound 5 . [SEP]
[CLS] analytical hplc chromatograms obtained from 2 ' - propargyl - dna 1 [ 5 ' - ccaa ( 2 ' - prop - a ) ggttacaggtaag ] before ( a ) and after ( b ) ' click ' reaction with the bipyridine ligand . [SEP]
[CLS] panel ( c ) is the chromatogram obtained from the co - injected samples . [SEP]
[CLS] ' - ccaaaggttacaxgtaag - 3 ' , where x represents native dg ( black ) , 2 ' - prop - g ( blue ) and 2 ' - dipyridine - g ( red ) respectively , pairs with their native complementary strand 5 ' - cttacctgtaacctttgg - 3 ' [SEP]
[CLS] their t m values are 55 . 4 , 55 . 1 , and 48 . 8 °c respectively . [SEP]
[CLS] 4 . structural features of octamer dna duplex [ 5 ' - g ( 2 ' - ome - du ) gt ( 2 ' - prop - a ) cac - 3 ' ] 2 . [SEP]
[CLS] ( a ) and ( b ) 180° rotation views of duplex superimposed comparison between propargyl modified 8mer ( red ) and native 8mer ( cyan ) ( pdb id : 1dns ) , with the two 2 ' - propargyl groups stick into minor grooves . [SEP]
[CLS] the observed r . m . s . d is 1 . 38 a . [SEP]
[CLS] ( c ) base pair comparison of propargyl - a : t ( red ) and native a : t ( cyan ) . [SEP]
[CLS] the oxygen B-material and carbon B-material atoms I-material are showed as red and blue spheres respectively . [SEP]
[CLS] 5 . computational models of bipyridine modified octamer dna duplex [ 5 ' - g ( 2 ' - ome - u ) gt ( 2 ' - prop - a ) cac - 3 ' ] 2 after ' click ' reaction . [SEP]
[CLS] ( a , b ) surface view of the complex with the bipyridine rings extending cross and within the minor groove . [SEP]
[CLS] ( c , d ) stick view of the overall duplex structures with the two configurations of ligands and copper B-material ions B-material . [SEP]
[CLS] 2 ' - propargyl modified dna oligonucleotides . [SEP]
[CLS] molecular masses of dna oligonucleotide [ 5 ' - tcggg ( 2 ' - prop - u ) acccga - 3 ' ] after ' click ' reaction and edta - aided ions B-material exchange . [SEP]
[CLS] the analysis was performed by esi - ms as described in the experimental section . [SEP]
[CLS] x - ray data collection and structural refinement statistics of 2 ' - propargyl modified dna 8mer duplexgt ( 2 ' - prop - a ) cac - 3 ' ] 2 [SEP]
[CLS] the effects of spherical nucleic B-material acid I-material ( sna ) gold nanoparticle B-nanoparticle conjugates on the activation of macrophages in vitro and release of cytokines in vivo were explored . [SEP]
[CLS] herein , we show that gquadruplexes , the formation of which is enhanced on gold B-nanoparticle nanoparticle I-nanoparticle surfaces , elicit an increase in cytokine release from mouse and human macrophages and induce the upregulation of activation receptors as well as no 2 production in vitro . [SEP]
[CLS] moreover , these g - rich snas can induce cytokine release when injected intravenously , though there were no severe , long - term effects observed . [SEP]
[CLS] these results further reinforce the notion that nucleic B-material acid I-material sequence and structure play an important role in how snas interact in biological milieu and highlight a key design parameter . [SEP]
[CLS] spherical nucleic B-material acids I-material ( snas ) are an emerging class of nanomaterials B-material consisting of a nucleic B-material acid I-material shell B-material surrounding a nanoparticle B-nanoparticle core B-material . [SEP]
[CLS] due , in part , to the dense , oriented oligonucleotide structure , snas behave differently than their linear counterparts and exhibit enhanced interactions with serum and cell B-material surface proteins B-material , resulting in higher cellular association and uptake . [SEP]
[CLS] consequently , snas have been developed for many gene regulation applications , including psoriasis and glioblastoma , as well as immunomodulation B-property applications , and are in clinical trials for both . [SEP]
[CLS] previously , it has been shown that immediately after snas are introduced to serum , serum proteins B-material bind to the sna and form a protein B-material corona I-material . [SEP]
[CLS] furthermore , the identity , number , and types of proteins B-material that bind are dependent on the dna sequence . [SEP]
[CLS] specifically , the presence of g - quadruplexes on the sna alters protein B-material corona I-material formation and results in differences in cellular uptake and in vivo biodistribution . [SEP]
[CLS] evidence of enhanced uptake of guanine - rich ( g - rich ) snas by macrophages in vitro and macrophage - rich tissues , such as the liver and spleen , in vivo suggest that g - rich snas may also induce macrophage activation . [SEP]
[CLS] although snas do not inherently elicit an immune response in epithelial cells B-material in vitro , they may be designed to modulate the immune response using oligonucleotide motifs common to pathogens . [SEP]
[CLS] in this report , we explore for the first time how changes in nonimmunogenic nucleic B-material acid I-material sequences of snas can impact their interactions with macrophages in vitro . [SEP]
[CLS] understanding the role of the nucleic B-material acid I-material sequence on macrophage activation is crucial for the design of safe and efficacious snas . [SEP]
[CLS] herein we explore the effect of nucleic B-material acid I-material sequence on macrophage activation in vitro and in vivo through the use of two sna constructs : gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) fractionalized with either a g - rich sequence that forms g - quadruplexes or a poly - t sequence ( scheme 1 ) . [SEP]
[CLS] we find that g - rich snas in the presence of serum proteins B-material do indeed interact with macrophages differently than their poly - t counterparts and induce both cytokine and nitric oxide B-material ( no ) production as well as upregulate a cell B-material surface I-material receptor I-material linked with early macrophage activation in vitro . [SEP]
[CLS] furthermore , g - rich snas induce increases in cytokine production in mice hours post injection , but no severe , long - term immune response was observed . [SEP]
[CLS] these results highlight the role of the surface chemistry of nanoparticles B-nanoparticle , and nucleic B-material acid I-material sequence specifically , in modulating their interactions with cells B-material both in vitro and in vivo . [SEP]
[CLS] citrate stabilized aunps B-nanoparticle ( 13 nm ) were synthesized via the frens method 27 and their size was characterized using dynamic B-technique light I-technique scattering I-technique ( dls , malvern ) . [SEP]
[CLS] thiolated g - rich and poly - t dna were synthesized using an mm48 oligonucleotide synthesizer ( bioautomation ) using standard solid - phase synthesis reagents ( glen research ) . [SEP]
[CLS] dna was deprotected from its solid support and purified using high pressure liquid B-technique chromatography I-technique ( hplc ) on a c18 column ( varian ) . [SEP]
[CLS] dna ( 30 nmol , stock solution at 100 μm ) was added to 10 ml of 10 nm aunps B-nanoparticle and incubated B-technique for 2 h at room temperature with agitation . [SEP]
[CLS] nacl was added to the mix of dna and aunps B-nanoparticle gradually until the final concentrations reached 0 . 5 m ( g - rich snas ) and 1 m ( poly - t snas ) to ensure similar loading densities , as discussed in our previous work . [SEP]
[CLS] after salting B-event , snas were concentrated using centrifugation ( max speed = 16 873g ) , washed , and suspended in 1× pbs , ph 7 . 4 at a concentration of 100 nm ( by sna ) for all downstream in vitro and in vivo experiments . [SEP]
[CLS] the concentration of dna and aunps B-nanoparticle was measured using uv - visible spectrophotometry ( agilent ) at 260 and 524 nm , respectively . [SEP]
[CLS] once formulated , the sna concentration was determined using uv - vis spectrophotometry at 524 nm , with ε = 2 . 7 × 10 8 l / ( mol cm ) . [SEP]
[CLS] then , the number of dna strands per particle was determined by dissolving the aunp B-nanoparticle cores B-material using 40 mm kcn and measuring the dna concentration via an oligreen assay ( life technologies ) . [SEP]
[CLS] thp - 1 ( atcc tib - 202 ) , raw 264 . 7 ( atcc tib - 271 ) , and raw - blue ( invivogen ) cells B-material were cultured in a 5 % co 2 incubator B-technique following atcc ' s recommended instructions . [SEP]
[CLS] thp - 1 cells B-material were cultured in rpmi 1640 ( gibco ) containing 10 % fetal bovine serum ( fbs ) and 1 % penicillin / streptomycin supplemented with 0 . 05 mm 2 - mercaptoethanol . [SEP]
[CLS] raw 264 . 7 and raw - blue cells B-material were cultured in dmem ( gibco ) containing 10 % fbs and 1 % penicillin / streptomycin and subcultured by scraping . [SEP]
[CLS] these cell B-material lines were chosen to represent macrophages derived from human and mouse origin . [SEP]
[CLS] a total of 100 000 thp - 1 cells B-material were seeded per well in a 96 - well plate with rpmi 1640 containing 20 % fbs and 100 nm phorbol myristate acetate ( pma , invivogen ) to differentiate for 72 h . [SEP]
[CLS] cells B-material were then incubated B-technique with 1 or 5 nm sna for 4 h in optimem ( gibco ) with or without 10 % human serum ( sigma - aldrich ) in 96 - well plates ( 100 μl total volume ) . [SEP]
[CLS] the media was then replaced with rpmi 1640 containing 10 % fbs for 18 h . [SEP]
[CLS] cell B-material supernatants were collected and analyzed using the bd cytometric bead array - human inflammatory cytokine kit ( bd biosciences ) to determine il - 1β , il - 6 , il - 10 , and tnfα levels . [SEP]
[CLS] a total of 100 000 raw 264 . 7 cells B-material were seeded per well in a 96 - well plate with optimem ( 100 μl total volume ) containing 10 % human serum and incubated B-technique with snas ( 1 to 20 nm ) for 24 h . [SEP]
[CLS] cell B-material supernatants were collected and analyzed using a luminex magnetic B-property bead assay ( r & d biosciences ) for levels of il - 6 , mcp - 1 , rantes , and tnfα . [SEP]
[CLS] a total of 30 000 raw 264 . 7 cells B-material were seeded per well in a 96 - well plate . [SEP]
[CLS] after 24 h , cells B-material were incubated B-technique with 1 , 5 , 10 , or 20 nm snas in optimem with or without 10 % human serum for 1 or 4 h at a total well volume of 100 μl . the media was then removed and replaced with dmem containing 10 % fbs for 18 h . [SEP]
[CLS] cell B-material supernatants were collected and analyzed using a griess reagent system ( promega ) to quantify no 2 − release . [SEP]
[CLS] a total of 100 000 raw 264 . 7 cells B-material were seeded at a density of 10 6 cells B-material / ml in a 12 - well plate for 24 h in dmem supplemented with 10 % fbs before they were incubated B-technique with snas ( either 1 or 5 nm ) in optimem supplemented with 10 % human serum at a total volume of 1 ml . [SEP]
[CLS] murine splenocytes , seeded at a density of 10 6 cells B-material / ml in a 12 - well plate , were incubated B-technique with snas ( 1 or 5 nm ) in optimem supplemented with 10 % human serum . [SEP]
[CLS] after a 24 h incubation B-technique , cells B-material were washed three times with pbs and stained with antibodies B-material for cd69 , cd86 , and mhc - ii ( bd biosciences ) for 30 min at 4 °c and then fixed using 4 % paraformaldehyde at 4 °c for 30 min . [SEP]
[CLS] for murine splenocytes , f4 / 80 antibody B-material was also incubated B-technique alongside the other antibodies B-material to identify macrophages via flow B-technique cytometry I-technique . [SEP]
[CLS] samples were then analyzed on an lsrii flow cytometer ( bd biosciences ) . [SEP]
[CLS] the percent of upregulated cd69 , cd86 , and mhc - ii for each sample was plotted following dead cell B-material exclusion . [SEP]
[CLS] in vivo cytokine determination . [SEP]
[CLS] the in vivo experiments were conducted using a protocol approved by the institutional animal care and use committee at northwestern university . [SEP]
[CLS] c57bl / 6 mice ( 6 - 8 weeks old ) were injected with 200 μl of 100 nm g - rich sna , poly - t sna , or 1× pbs intravenously through the tail vein . [SEP]
[CLS] blood samples were collected from mice at 8 and 24 h post injection via a retro - orbital blood draw . [SEP]
[CLS] samples were spun down , and serum was used to conduct cytokine analysis using a cytometric bead array - mouse inflammatory cytokine kit ( bd biosciences ) . [SEP]
[CLS] livers and spleens were collected from mice that were transcardially perfused and sacrificed at 8 h , 24 h , and 28 days post injection . [SEP]
[CLS] tissues were immediately fixed using a 10 % neutral buffered formalin solution , paraffin - embedded and sectioned , and stained using hematoxylin and eosin ( h & e ) or terminal deoxynucleotidyl transferase dutp nick - end labeling ( tunel ) staining ( roche ) . [SEP]
[CLS] macrophage activation leads to secretion of nitric oxide B-material ( no ) , a whole host of cytokines ( i . e . , tnfα , il - 12 , il - 1 , il - 1β , il - 6 , il - 10 ) and chemokines ( i . e . , ip - 10 , mip - 1a , mcp - 1 ) , as well as upregulation of biological markers ( i . e . , mhc - ii , cd86 ) . [SEP]
[CLS] to determine the extent of macrophage activation via the secretion of proinflammatory cytokines and chemokines , human ( pma - differentiated thp - 1 ) and mouse ( raw 264 . 7 ) macrophages were treated with g - rich and poly - t snas and analyzed for cytokine secretion . [SEP]
[CLS] the results of a 4 h sna incubation B-technique followed by an 18 h media replenishment period ( with 10 % fbs ) in thp - 1 macrophages indicate that g - rich snas , more so than poly - t snas , cause cells B-material to generate the proinflammatory cytokines il - 1β , il - 6 , and tnfα in the presence of serum proteins B-material at higher treatment concentrations ( 5 nm ) ( figure 1a ) . [SEP]
[CLS] specifically , although baseline levels of tnfα and il - 1β secretion were detected after treatment with low concentrations of g - rich and poly - t snas ( 1 nm ) , the presence of serum proteins B-material in addition to both snas induced a minor amount of il - 6 secretion . [SEP]
[CLS] in addition , the levels of il - 1β and il - 6 secretion were higher for cells B-material treated with g - rich snas with and without the addition of serum proteins B-material as compared to their poly - t sna counterparts , whereas tnfα secretion was especially pronounced for cells B-material treated with g - rich snas in the presence of serum proteins B-material . [SEP]
[CLS] interestingly , levels of il - 10 , an anti B-property - I-property inflammatory I-property cytokine that can activate macrophages through the alternative , or type ii , pathway were indistinguishable between control and experimental samples ( figure s1 ) . [SEP]
[CLS] experimental samples were run alongside a bacterial lipopolysaccharide ( lps , 200 ng / ml ) positive control , which exhibited similar il - 1β and tnfα production but much higher il - 6 production ( figure 1a ) . [SEP]
[CLS] this indicates that while g - rich snas can induce cytokines , it is less severe than the induction achieved by lps . [SEP]
[CLS] in mouse macrophages , the trend is similar : g - rich snas coated with serum proteins B-material after a 24 h incubation B-technique induce proinflammatory cytokines and chemokines ( il - 6 , mcp - 1 , rantes , and tnfα ) at higher levels than that of poly - t snas ( figure 1b ) . [SEP]
[CLS] interestingly , upon exploring higher sna concentrations , a dose dependency arises . [SEP]
[CLS] meanwhile , cytokine production remains unchanged for both linear g - rich and poly - t dna ( figure s2 ) for cells B-material treated with and without serum , suggesting that the serum protein B-material interaction with the nucleic B-material acid I-material shell B-material on the surface of the sna is responsible for the observed results . [SEP]
[CLS] one explanation for the enhanced macrophage activation from serum - coated g - rich snas could be due to the fact that they have a higher affinity for and bind more opsonin proteins B-material ( such as complement c3b ) compared to poly - t snas , flagging themselves for macrophage uptake and eliciting activation . [SEP]
[CLS] it is of note that when compared to a similar dose of immunostimulatory dna ( cpg , 1826 ) at 1 μm by dna ( equivalent in dna amount to about 15 nm of sna ) , sna activation levels are significantly lower ( figure 1b ) . [SEP]
[CLS] although cytokine production in both human and mouse macrophages indicates a greater extent of macrophage activation upon treatment with g - rich snas in the presence of serum proteins B-material , a second measure of activation was also probed : degree of nitric oxide B-material ( no ) production . [SEP]
[CLS] raw 264 . 7 mouse macrophages were incubated B-technique for 1 or 4 h with g - rich and poly - t snas in the absence and presence of serum before the media ( containing 10 % fbs ) was replenished for 18 h and analyzed for nitrite ( no 2 − ) , the primary oxidation product of no under physiological conditions ( figure 2a ) . [SEP]
[CLS] a griess reagent assay ( to quantify nitrite concentration in the media ) indicated that after a 1 h incubation B-technique , cells B-material treated with proteincoated g - rich snas induced significantly more no secretion than g - rich snas given without human serum or poly - t snas at 1 or 5 nm , with or without the presence of human serum . [SEP]
[CLS] at 10 and 20 nm , however , both types of snas induced no generation , suggesting macrophage activation , an observation that is consistent with the increase in proinflammatory cytokines and chemokine production at higher sna concentrations ( figure 1 ) . [SEP]
[CLS] after a 4 h incubation B-technique , all cells B-material exhibit enhanced no production compared to cells B-material treated for 1 h . [SEP]
[CLS] interestingly , incubation B-technique with protein - coated g - rich snas for 4 h exhibits near - saturation levels at a concentration as low as 1 nm , a similar level that was observed at 20 nm after 1 h ( figure 2a ) . [SEP]
[CLS] although enhanced no production was seen across all constructs with increasing nanoparticle B-nanoparticle concentration and incubation B-technique time , protein - coated g - rich snas induced the highest levels of no production across all tested groups at each concentration , a conclusion that is consistent with the cytokine profile results ( figure 1 ) . [SEP]
[CLS] moreover , upon a deeper analysis into nf - κb , a transcription B-event factor for proinflammatory genes , expression levels in raw 264 . 7 mouse macrophages also reach the same conclusion : g - rich snas induce higher levels of macrophage activation than poly - t snas , or g - rich or poly - t dna ( figure s3 ) . [SEP]
[CLS] this is to be expected , as increased nf - κb expression can be triggered by tnfα and no production . [SEP]
[CLS] these results further reinforce the notion that changes in nucleic B-material acid I-material sequence can alter sna - protein B-material interactions and consequently affect sna - cell B-material interactions . [SEP]
[CLS] activated macrophages can also exhibit changes in the expression levels of cell B-material surface I-material receptors I-material . [SEP]
[CLS] to test this , the upregulation of cell B-material surface I-material receptors I-material cd69 , cd86 , and mhc - ii was measured in mouse macrophages ( raw 264 . 7 , figure 2b ) using flow B-technique cytometry I-technique . [SEP]
[CLS] interestingly , neither g - rich nor poly - t snas induce enhanced expression of cd86 or mhc - ii in mouse macrophages , typical markers of macrophage activation . [SEP]
[CLS] however , g - rich snas do exhibit enhanced cd69 activation , a marker of early activation , 34 at higher dosages ( 5 nm ) , with even higher levels than that induced by lps dosed at 200 ng / ml ( figure 2b ) . [SEP]
[CLS] although cd69 is not constitutively active in murine macrophages , it can be activated through tnfα , which is secreted in a dose dependent manner from raw 264 . 7 macrophages upon interaction with g - rich snas in the presence of human serum ( figure 1a ) [SEP]
[CLS] to further explore the impact of g - rich snas on cd69 activation , g - rich and poly - t snas and dna were added to mouse splenocytes ( figure 3 ) . [SEP]
[CLS] flow B-technique cytometry I-technique was used to look at the upregulation of cell B-material surface I-material receptors I-material of primary macrophages ( f4 / 80 + ) : cd40 , cd86 , and mhc - ii and cd69 , in which g - rich snas induced cd69 and cd40 upregulation while poly - t snas did not . [SEP]
[CLS] these results indicate that although the mouse macrophages show early stages of activation , they are not fully activated through canonical pathways because of the absence of commonly upregulated cell B-material surface I-material receptors I-material , further emphasizing that snas will not cause long - term , nonspecific side effects , which are commonly seen when delivering nucleic B-material acid I-material therapies by other means . [SEP]
[CLS] thus far , all the results have indicated that g - rich snas , likely because of the composition of the serum proteins B-material in the protein B-material corona I-material , can induce higher levels of macrophage activation than poly - t snas . [SEP]
[CLS] next , it was important to determine if a similar phenomenon occurs in vivo . [SEP]
[CLS] to test this , g - rich and poly - t snas were injected intravenously in mice at a dosage of approximately 20 pmol of sna ( 200 μl at 100 nm sna ) or approximately 1 . 5 mg of dna per kg of body weight . [SEP]
[CLS] cytokine analysis of the blood after 8 and 24 h shows that g - rich snas induced little inflammatory , mcp - 1 and il - 12p70 , or anti B-property - I-property inflammatory I-property , il - 10 , cytokine production over a pbs control injection , except for statistically significant higher levels of il - 6 ( figure 4 ) . [SEP]
[CLS] this level of il - 6 , however , is orders of magnitude lower than if mice were given true immune stimulatory agents , such as bacterial lps , or even snas designed to activate tlr9 . [SEP]
[CLS] meanwhile , the level of cytokine secretion from poly - t snas is not statistically significant compared to a pbs control . [SEP]
[CLS] these results suggest that although by 8 h , g - rich snas are interacting with serum proteins B-material and exhibit an increase in il - 6 production , by 24 h post injection , all cytokine levels have returned to the baseline , suggesting no long - term macrophage activation . [SEP]
[CLS] this correlates well with previous studies that have probed the longer - term effects of sna treatment in mice . [SEP]
[CLS] furthermore , neither g - rich nor poly - t snas exhibit gross histological changes in macrophage - rich tissues , such as the liver or spleen , as determined through hemotoxylin and eosin ( h & e ) staining ( figure 5 ) at 24 h post injection , again confirming that snas do not inherently elicit immune responses that lead to long - term , negative consequences in vivo . [SEP]
[CLS] this work probes the impact of sequence on an sna ' s interaction with macrophages in biological milieu . [SEP]
[CLS] these observations are important for several reasons . [SEP]
[CLS] the data suggests that changes in sequence , which are known to lead to differences in the sna ' s protein B-material corona I-material composition , can also induce acute macrophage activation . [SEP]
[CLS] furthermore , these changes in sequence also translate to an increase in cytokine release shortly after administration in vivo . [SEP]
[CLS] taken together , these results might hint at unwanted side effects ; however , although sequence can alter the sna ' s interactions with macrophages , there is no lasting observable cellular activation as determined through systemic cytokine release at later time points and macrophage - rich tissue histology . [SEP]
[CLS] finally , this work highlights the importance of nucleic B-material acid I-material sequence in designing more efficacious snas , because the formation of nucleic B-material acid I-material tertiary structures can change the short - term extent of macrophage activation in vitro and cytokine release in vivo . [SEP]
[CLS] for example , these results could be particularly advantageous for immune modulation purposes , that is , the adjuvancy of a tlr9 activating sna is likely to be enhanced by either incorporation of g - rich sequences on the surface of the sna ( in addition to the tlr9 activating dna ) or by enriching the tlr9 activating dna with additional guanine tails at the sna surface . [SEP]
[CLS] refer to web version on pubmed central for supplementary material B-material . [SEP]
[CLS] ( a ) cytokine production from human macrophages ( pma - differentiated thp - 1 ) after a 4 h treatment with g - rich or poly - t snas in the presence and absence of 10 % human serum ( hs ) , followed by complete media replenishment for 18 h , with 10 % fbs , as analyzed using a bd cytometric bead array . [SEP]
[CLS] ( b ) cytokine production from mouse ( raw 264 . 7 ) macrophages treated with g - rich or poly - t snas at varying concentrations for 24 h , in media containing 10 % human serum , as analyzed using a luminex magnetic B-property bead assay . [SEP]
[CLS] statistical analysis was conducted using tukey ' s multiple comparison test ( * for p < 0 . 05 , * * for p < 0 . 01 , and * * * * for p < 0 . 0001 ) . [SEP]
[CLS] h & e stained spleen and liver 24 h post injection . [SEP]
[CLS] scale bar represents 100 μm . [SEP]
[CLS] ( a ) griess reagent quantifying the levels of no 2 − secretion by raw 264 . 7 mouse macrophages incubated B-technique with g - rich or poly - t snas in the presence and absence of human serum ( hs ) after 1 or 4 h . [SEP]
[CLS] ( b ) flow B-technique cytometry I-technique analysis of cell B-material surface I-material receptor I-material ( cd69 , cd86 , and mhc - ii ) expression levels in raw 264 . 7 macrophages after a 24 h incubation B-technique with g - rich or poly - t snas . [SEP]
[CLS] tukey ' s multiple comparison test was used to determine statistical significance , where * * * denotes p < 0 . 001 , and * * * * denotes p < 0 . 0001 . [SEP]
[CLS] 3 . upregulation of cell B-material surface I-material receptors I-material ( cd40 , cd69 , cd86 , and mhc - ii ) in macrophages ( f4 / 80 + ) after primary splenocytes were treated with a 24 h incubation B-technique with g - rich or poly - t dna or snas ( at 1 and 5 nm ) in the presence of human serum , as determined using flow B-technique cytometry I-technique [SEP]
[CLS] * denotes p < 0 . 01 , and * * * * denotes p < 0 . 0001 for g - rich snas in relation to untreated , as determined using tukey ' s multiple comparison test . [SEP]
[CLS] 4 . cytokine release in vivo in response to intravenously injected g - rich or poly - t snas . [SEP]
[CLS] * denotes p < 0 . 05 , * * denotes 0 . 001 , and * * * * denotes p < 0 . 0001 for all groups compared to pbs control [SEP]
[CLS] 1 . gold B-nanoparticle nanoparticles I-nanoparticle are coated with thiolated g - rich and poly - t dna strands to form snas [SEP]
[CLS] this paper describes a label free technique for determining ligand loading on metal B-nanoparticle nanoparticles I-nanoparticle using a variant of secondary ion B-material mass spectrometry . [SEP]
[CLS] au 400 4 + clusters bombard dnafunctionalized anisotropic gold B-material nanostars and isotropic nanospheres B-nanoparticle with similar surface areas to determine ligand density . [SEP]
[CLS] for each projectile impact , co - localized molecules within the emission area of a single impact ( diameter of 10 - 15 nm ) were examined for each particle . [SEP]
[CLS] individual nanoparticle B-nanoparticle analysis allows for determination of the relationship between particle geometry and dna loading . [SEP]
[CLS] we found that branched particles exhibited increased ligand density versus nanospheres B-nanoparticle and determined that positive and neutral curvature could facilitate additional loading . [SEP]
[CLS] this methodology can be applied to optimize loading for any ligand - core interaction independent of nanoparticle B-nanoparticle core B-material , ligand , or attachment chemistry . [SEP]
[CLS] gold B-nanoparticle nanoparticles I-nanoparticle have emerged as a useful probe for biological systems because of their functionality , easy characterization , and biological compatibility . [SEP]
[CLS] attaching molecules like dna to the surface of nanoparticles B-nanoparticle stabilizes the particles in culture media and provides functionality . [SEP]
[CLS] additionally , the nanoscopic environment created by the shape and curvature of the nanoparticle B-nanoparticle changes the chemical properties of the tethered molecules . [SEP]
[CLS] the curvature of nanoparticles B-nanoparticle can alter the distribution , concentration , or pk a of surface ligands , which can affect the structure of absorbed proteins B-material and the therapeutic efficacy of each nanoparticle B-nanoparticle . [SEP]
[CLS] specifically , how nanoparticles B-nanoparticle interact with cellular systems depends on the surface area : volume ratio of the core B-material . [SEP]
[CLS] therefore , studying the fundamental relationships between single nanoparticles B-nanoparticle and ligands could reveal information about multivalent effects and energy - charge transfer . [SEP]
[CLS] concurrent characterization of the nanoparticle B-nanoparticle core B-material and attached ligands is essential for maximizing the efficacy of the designed nanoconstruct . [SEP]
[CLS] common methods for nanoparticle B-technique characterization I-technique are based on electron B-technique microscopy I-technique due to its high spatial resolution . [SEP]
[CLS] however , electron B-technique microscopy I-technique has proven to be challenging as a method for determining the distribution of ligands on the surface of a metal B-nanoparticle nanoparticle I-nanoparticle due to the large contrast difference between the high - z elements of the core B-material and the low - z elements of the ligands . [SEP]
[CLS] consequently , current quantification of the ligand distribution on metal B-nanoparticle nanoparticle I-nanoparticle surfaces is limited to ensemble methods : indirect measurements ( e . g . , unbound ligand sers detection ) , labeled ligands ( e . g . , fluorescently B-property tagged ligands or dye molecules ) , or elemental analysis ( e . g . , au and s via inductively coupled plasma mass spectrometry ) . [SEP]
[CLS] these measurements do not provide an accurate analysis of ligands on individual nanoconstructs and do not evaluate how the ligands interact with the environment . [SEP]
[CLS] studies using x - ray photoelectron spectroscopy B-technique ( xps ) have shown that ligand coverage can be evaluated on flat surfaces and symmetrical nanoparticles B-nanoparticle . [SEP]
[CLS] xps measurements can reveal the interaction between metal B-material atoms B-material or metal B-nanoparticle nanoparticles I-nanoparticle with dna , identifying the interaction sites for neutral and charged metal B-material atoms B-material with dna molecules . [SEP]
[CLS] while singleparticle measurements of fluorophore - labeled ligands have been achieved , highthroughput analysis with such methods is limited . [SEP]
[CLS] to gain statistically relevant data and determine how curvature influences collective and multivalent ligand properties , measurements need to be taken on a large number of intact , individual particles . [SEP]
[CLS] time of flight secondary ion B-material mass spectrometry ( tof - sims ) is well suited for analyzing nanoscale surfaces due to high lateral ( ca . 100 - 400 nm ) and depth resolution ( ca . 5 - 10 nm ) . [SEP]
[CLS] we used a variant of tof - sims , event - by - event detection mode , that uses individual projectiles to analyze single nanoparticles B-nanoparticle . [SEP]
[CLS] the method has two innovative features : ( 1 ) the mode of bombardment and recording of the secondary ions B-material and ( 2 ) impacting the surface with relatively massive 2 nm projectiles ( au 400 4 + ) that produces abundant secondary ion B-material emissions from the sample . [SEP]
[CLS] this one - of - a - kind tool is suited for the quantification of the ligand distribution on an individual metal B-nanoparticle nanoparticle I-nanoparticle independent of morphology and method of ligand attachment ( e . g . , covalent or electrostatic bonds ) . [SEP]
[CLS] by stochastically sampling with a few million projectile impacts , statistical tools can be used to evaluate correlations among co - emitted species . [SEP]
[CLS] grouping projectile impacts on like objects and evaluating the number and type of co - emitted ligand species allow the ligand loading to be determined . [SEP]
[CLS] in this work , we directly quantified label free ligands on single nanoparticles B-nanoparticle with similar surface areas but different morphologies . [SEP]
[CLS] specifically , au nanostars ( auns ) were sorted on the basis of the size and shape and 50 nm nanospheres B-nanoparticle . [SEP]
[CLS] tof - sims in event - by - event detection mode was used to quantify the number and position of bound ligands in positive , negative , or neutral nanoconstruct surfaces . [SEP]
[CLS] we found that the ligand loading of a nanosphere B-nanoparticle ( positive curvature only ) was lower than that of auns with varying amounts of negative , neutral , and positive curvature . [SEP]
[CLS] within the sorted auns fractions , we determined that particles with a greater amount of negative curvature had loading efficiencies lower than those of auns with larger amounts of positive and neutral curvature . [SEP]
[CLS] secondary ion B-material mass spectroscopy B-technique . [SEP]
[CLS] 1 shows a simplified setup of the experimental approach ; a detailed instrumental scheme has been previously reported . [SEP]
[CLS] the instrumental setup was designed to bombard a sample with one projectile at a time and record the secondary ions B-material from each impact separately . [SEP]
[CLS] these ions B-material are concurrently mass analyzed by tof and recored before the subsequent projectile impact , enabling chemical characterization at the nanoscale . [SEP]
[CLS] sims analysis was performed using custom - built instrumentation equipped with a gold B-material liquid metal B-material ion B-material source ( lmis ) , which produces the gold B-material projectiles used to bombard the sample . [SEP]
[CLS] in brief , au 400 4 + projectiles were produced by a lmis and selected with a wien filter , both of which are installed on the 120 kv platform to increase the bombardment energy . [SEP]
[CLS] after exiting the high - voltage platform , the beam of au 400 4 + was pulsed at a rate of 1000 projectiles per second ( 0 . 1 projectile per pulse ) , ensuring each projectile was separated in time and space . [SEP]
[CLS] the sample was biased to −10 kv , resulting in a total projectile kinetic energy of 520 kev for au 400 4 + . [SEP]
[CLS] a weak magnetic B-property field was used to separate the emitted electrons from the negative secondary ions B-material to direct the electrons to a microchannel platebased detector . [SEP]
[CLS] the detected electrons act as the start of the tof measurement . [SEP]
[CLS] the secondary ions B-material continued through the magnetic B-property field and were mass analyzed with a reflectron tof mass spectrometer ( mass resolution , full width at half - maximum , at m / z 48 = 1600 ) . [SEP]
[CLS] the detector was microchannel plate - based with a circularly symmetric anode segmented into eight parts , which allows for up to eight isobaric ions B-material to be detected from a single impact . [SEP]
[CLS] for each projectile impact , the start and stop signals were collected by a multistop time - to - digital converter ( tdcv4 , institute of nuclear physics , orsay , france ) and then stored in a personal computer as an individual mass spectrum . [SEP]
[CLS] the gold B-material nanoconstruct samples were stochastically analyzed with 2 - 4 × 10 6 au 400 4 + projectiles on an area with a radius of ca . 125 μm , corresponding to 2 - 4 × 10 6 individual mass spectra for each sample . [SEP]
[CLS] in the sims analysis , ca . 0 . 3 % of the surface was analyzed stochastically ; thus , each projectile impacted an unperturbed portion of the sample ( super static regime ) . [SEP]
[CLS] nanoparticle B-nanoparticle synthesis and information . [SEP]
[CLS] auns were synthesized using gold ( iii ) chloride B-material trihydrate , haucl 4 ( sigma - aldrich , st . louis , mo ) , in good ' s buffer 4 - ( 2 - hydroxyethyl ) - 1 - piperazineethanesulfonic acid , hepes buffer ( sigma - aldrich ) , both a shape and reducing B-property agent I-property , to produce anisotropic gold B-nanoparticle nanoparticles I-nanoparticle with branches varying in number and size . [SEP]
[CLS] the 1 m stock hepes solution was made by dissolving the buffer salt B-material in millipore water B-material ( 18 . 2 mω cm ) using a mediumsized stir bar to ensure thorough mixing . [SEP]
[CLS] the ph of the hepes solution was measured using a thermo scientific ph meter and adjusted using concentrated solutions of naoh . [SEP]
[CLS] for fine ph adjustments , hcl was added dropwise . [SEP]
[CLS] auns were synthesized by adding 0 . 2 mm ( final concentration ) haucl 4 to 100 mm hepes buffer . [SEP]
[CLS] each solution was vortexed in a 50 ml falcon tube for 1 min before the addition of haucl 4 and for 1 min afterward . [SEP]
[CLS] after vortexing , the growth solution was left undisturbed at room temperature for 24 h . [SEP]
[CLS] au nanospheres B-nanoparticle of varying diameters ( 5 , 10 , 20 , 30 , 40 , 60 , 80 , 100 , and 150 nm ) were purchased from sigma - aldrich . [SEP]
[CLS] dgc was used to separate auns on the basis of size and shape . [SEP]
[CLS] the sorted auns were analyzed separately to understand how differences in shape and curvature ( positive , negative , and neutral ) affected dna loading . [SEP]
[CLS] sucrose density gradients were formed using a gradient maker ( biocomp instruments , fredericton , nb ) with 9 ml starting solutions of 50 % and 60 % ( w / v ) sucrose in water B-material . [SEP]
[CLS] on the basis of previous methods , we created a linear gradient through a custom mixing program alternated five times between the following two steps : ( 1 ) time of 5 s , angle of 76° , and speed of 30 rpm and ( 2 ) time of 15 s , angle of 76° , and speed of 0 rpm . [SEP]
[CLS] for dgc , 500 μl of a concentrated solution of bare auns ( 35 - 40 nm ) was layered on top of the density gradient in an ultra - clear sw28 centrifuge tube ( beckman coulter , pasadena , ca ) and then centrifuged at 4400g for 3 h using a thermo fisher scientific sorvall legend xt 120v benchtop centrifuge ( thermo fisher scientific , waltham , ma ) . [SEP]
[CLS] the samples were fractionated at intervals of 4 mm from the meniscus . [SEP]
[CLS] each of the 10 fractions , f1 - f10 , was dialyzed in thermo fisher 20k slide - a - lyzer dialysis cassettes for 24 h to remove sucrose from the solution ( thermo fisher scientific ) . [SEP]
[CLS] the as - synthesized and sorted solutions were characterized by ultraviolet - visible spectroscopy B-technique to measure the bulk optical properties and by transmission B-technique electron I-technique microscopy I-technique ( tem ) to visualize individual auns . [SEP]
[CLS] particles with a greater number of branches , longer branches , and a larger overall size tended to sediment to the bottom of the gradient , while particles closer in shape to a small sphere moved more slowly through the gradient . [SEP]
[CLS] these characteristic differences between nanospheres B-nanoparticle and heterogeneous auns allowed for sorting based on structural features . [SEP]
[CLS] three milliliters of a auns solution was placed in a 1 cm plastic brookhaven cuvette , and the absorbance spectra were measured from 400 to 1400 nm using a cary 5000 ultravioletvisible - near - infrared spectrophotometer ( agilent technologies , santa clara , ca ) . [SEP]
[CLS] characterization of the size and shape of the auns was undertaken using tem . [SEP]
[CLS] the tem grids , carbon B-material type b , 300 mesh copper B-material grids ( ted pella , redding , ca ) , were treated with 0 . 1 % ( w / v ) poly - l - lysine B-material ( sigma - aldrich ) for 5 min prior to particle attachment . [SEP]
[CLS] the particles were deposited by pipetting 40 μl of a 10 - fold concentrated au nanoparticle B-nanoparticle solution onto prepared tem grids . [SEP]
[CLS] the samples were left to rest on the treated grids for 30 - 60 s , after which the solution was wicked away with filter paper . [SEP]
[CLS] a jeol 1230 tem instrument ( jeol ltd . , tokyo , japan ) was used to image the particles with an 80 kv accelerating potential . [SEP]
[CLS] representative images were collected from different areas of the grid . [SEP]
[CLS] structural features , such as circularity and feret diameter , were characterized using the analyze particles plugin on imagej for ≥500 particles per sample . [SEP]
[CLS] a circularity threshold of 0 . 8 was used to define spherical particles . [SEP]
[CLS] the feret diameter , which corresponds to the largest tip - to - tip distance on a particle , was also measured . [SEP]
[CLS] the branch number was manually counted from at least 10 different zoomed - out images for ≥400 particles in each fraction . [SEP]
[CLS] the branch length and the number of branches were measured manually from the tip to the base of the branch . [SEP]
[CLS] we created a standard curve ( figure s1 ) of surface area using nine au nanospheres B-nanoparticle ( 5 , 10 , 20 , 30 , 40 , 60 , 80 , 100 , and 150 nm ) that were functionalized with a small molecule , thiolated gd chelates . [SEP]
[CLS] because the gd chelates present little steric hindrance to bind to the surface of gold B-nanoparticle nanoparticles I-nanoparticle , the number of gd chelates on the surface of gold B-material nanospheres B-nanoparticle of known diameter can be compared to the number of auns , estimating the surface area of the auns . [SEP]
[CLS] the nanosphere B-nanoparticle solutions and sorted auns fractions were vortexed with a 100fold excess of gd chelate for 12 - 24 h with 0 . 01 % tween . [SEP]
[CLS] the gd / nanoparticle B-nanoparticle solutions were purified through three rounds of centrifugation ( 9600 rcf , 10 min ) and resuspension in milli - q water B-material . [SEP]
[CLS] au and gd quantification was performed by acid digestion of samples followed by inductively coupled plasma mass spectrometry [ icp - ms analysis on a thermo icap qc icp - ms instrument ( thermo fisher scientific ) ] . [SEP]
[CLS] icp - ms samples were digested in a 1 : 1 nitric acid / hydrocholoric acid ( hno 3 , > 69 % ; traceselect hcl , 37 % ) mixture . [SEP]
[CLS] milli - q water B-material and a multielement internal standard containing bi , ho , in , li , sc , tb , and y ( inorganic ventures , christiansburg , va ) were added to produce a solution of 2 % ( v / v ) hno 3 , 2 % ( v / v ) hcl , and 5 . 0 ng / ml internal standard up to a final volume of either 3 or 10 ml . [SEP]
[CLS] serial dilutions of gd and au standards ( inorganic ventures ) were prepared in the same matrix as the samples . [SEP]
[CLS] the gd / auns were then interpolated on the au nanoparticle B-nanoparticle standard to determine the surface area per particle . [SEP]
[CLS] synthesis of dna - functionalized nanoparticles B-nanoparticle . [SEP]
[CLS] we functionalized even - numbered auns fractions ( f2 , f4 , f6 , f8 , and f10 ) and 50 nm nanospheres B-nanoparticle with single - stranded 24mer poly - t dna by deprotection of the disulfide and salt B-material aging . [SEP]
[CLS] the 24mer poly - t was chosen to limit the different types of secondary ions B-material for this proof of concept experiment . [SEP]
[CLS] functionalized nanoparticles B-nanoparticle were purified by three rounds of centrifugation ( 10000 rpm , 10 min ) and resuspension in milli - q water B-material with 0 . 01 % tween . [SEP]
[CLS] the samples were stored at 4 °c after synthesis . [SEP]
[CLS] dna - functionalized nanoparticles B-nanoparticle were prepared for sims analysis using the marangoniflow - assisted method on cleaned 1 cm × 1 cm silicon B-material wafers . [SEP]
[CLS] this method ensured sufficient isolation between individual particles to allow for measurements of individual particles ( figures s2 - s4 ) . [SEP]
[CLS] separation and characterization of sorted hepes auns . [SEP]
[CLS] the auns synthesis conditions produced heterogeneous particles with zero to eight branches with varying curvatures ( figure 1 and figure s5 ) and an average surface area per particle similar to that of a 50 nm nanosphere B-nanoparticle . [SEP]
[CLS] rate - zonal dgc , as described in our previous work , 39 created enriched populations of auns ; each fraction showed greater particle homogeneity than the unsorted solution . [SEP]
[CLS] additionally , the fractions exhibited different surface areas per volume of particles , which demonstrated the change in curvature between the fractions ( figure s6 ) . [SEP]
[CLS] an increasing fraction number corresponds to an increase in the number of branches , branch length , and particle volume . [SEP]
[CLS] we analyzed the auns and 50 nm nanospheres B-nanoparticle using sims with individual projectiles . [SEP]
[CLS] from these individual mass spectra , characteristic ions B-material were identified and grouped together on the basis of the type of species : carbon B-material ( purple ) , the si support ( orange ) , auns ( black ) , dna ( pink ) , and salt B-material ( green ) ( figure 2 and figure s7 ) . [SEP]
[CLS] these mass spectra are the summation of those of ions B-material collected from all impacts on the surface . [SEP]
[CLS] from the si support , two silicon B-material oxide I-material clusters were observed : sio ] . [SEP]
[CLS] all regions of the sample were identified , and the results showed that every dna - related species was co - localized with the auns . [SEP]
[CLS] to evaluate the co - localization of species on the sample , i . e . , ligand loading , we measured ions B-material that were simultaneously emitted and correlated the rate of coemission . [SEP]
[CLS] to calculate the rate of coemission , a characteristic ion B-material was identified ( coincidence ion B-material ) and all mass spectra that contain this ion B-material were summed . [SEP]
[CLS] coemission mass spectra are presented in figure s8a - d for each region on the sample surface : nanoconstructs , salt B-material crystals , and silicon B-material support . [SEP]
[CLS] in the coemission spectra , the intensity of a second ion B-material of interest ( evaluated ion B-material ) was measured . [SEP]
[CLS] the correlation coefficient between these two ions B-material was determined ( eq 1 ) and tested against a random correlation with a 99 % confidence interval ( see the supporting information and figure s9 ) . [SEP]
[CLS] where a and b are the evaluated and coincidence ions B-material , respectively , i a is the intensity of a , i b is the intensity of b , and i a , b is the intensity of the coemission of a with b . [SEP]
[CLS] 3 displays a representative two - dimensional heat map of low to medium branched auns ( fraction 4 ) , where this correlation test was performed pairwise on all characteristic ions B-material . [SEP]
[CLS] to demonstrate that all dna coemissions were captured , every region of the sample was identified and displayed , including the silicon B-material wafer and salt B-material crystals ( figures s10 - s14 ) . [SEP]
[CLS] silicon B-material oxide I-material clusters ( si + others ) co - localized with only si species . [SEP]
[CLS] this si colocalization demonstrated nanoconstructs were isolated from each other on the wafer between open regions . [SEP]
[CLS] additionally , we found that nacl clusters were positively correlated with one another due to salt crystal formation ( salt B-material + salt ) ; however , they exhibited negative correlation with all other species , which demonstrated no interactions with the dna or auns measurements . [SEP]
[CLS] auns are represented by the small au clusters ( au n , where n = 1 - 9 ) in the bottom left corner of the heat map . [SEP]
[CLS] auns and dna - related ions B-material were positively correlated both to one another and to themselves but not to salt B-material or si - related species . [SEP]
[CLS] because a positive correlation means the secondary ions B-material were co - emitted in the same impacts , the dna and auns were within 10 - 15 nm of one another , demonstrating co - localization . [SEP]
[CLS] in sims , the observed intensity of an ion B-material is a product of the number of ejected molecules , the chance that a molecule or fragment will become charged ( ionization B-property probability ) , the transmission of the mass spectrometer , and the detection efficiency ; thus , the intensity does not directly reflect the species concentration . [SEP]
[CLS] comparing the intensity of an ion B-material between different samples proves to be challenging , because changes in the chemical environment may affect ionization B-property probability and skew the results . [SEP]
[CLS] thus , to compare between samples , multivariate analysis is often used . [SEP]
[CLS] here we used a correlation coefficient to compare between samples . [SEP]
[CLS] a key advantage of the correlation coefficient as shown in eq 1 ( correlation coefficient ) is that the ionization B-property probability of each ion B-material appears in both the numerator and denominator and does not affect the calculation . [SEP]
[CLS] the same is true for surface coverage ( eq 2 ) . therefore , the dna surface coverage of each auns fraction and the nanospheres B-nanoparticle can be compared . [SEP]
[CLS] we evaluated the surface coverage of each sample component ( e . g . silicon B-material wafer , salt B-material crystals , and auns ) by identifying ions B-material that originate from the same types of impacts , i . e . , those with a positive correlation . [SEP]
[CLS] we then determined the fraction of impacts ( i . e . , surface coverage ) containing the two species together using the following equation : [SEP]
[CLS] where i a is the intensity of a , i b is the intensity of b , i a , b is the intensity of the coemission of a with b , and n 0 is the number of measurements . [SEP]
[CLS] the fraction of impacts occurring on the auns was calculated for all samples by selecting measurements in which two au species were co - emitted ( figure s15 ) . [SEP]
[CLS] the number of impacts containing dna was in good agreement with the number containing both dna and au , which verifies that the nanoconstructs were still intact upon sampling . [SEP]
[CLS] however , only 65 % of the impacts with dna also contained the coemission of two au species , which was needed to calculate the number of times a auns was bombarded . [SEP]
[CLS] this difference could be due to two different effects . [SEP]
[CLS] ( 1 ) auns coated with a single - stranded 24mer poly - t dna may result in decreased emissions from underlying regions . [SEP]
[CLS] where ligands were bound to the nanoparticle B-nanoparticle , the au 400 4 + projectile interacted with the ligand before striking the auns surface , which resulted in decreased au emission . [SEP]
[CLS] ( 2 ) the emission of au clusters was affected by the asymmetric shape of the auns . [SEP]
[CLS] the core B-material of a particle has a greater number of au atoms B-material than the branches . [SEP]
[CLS] the result was that impacts occurring on the core B-material emit more gold B-material than those occurring on the branches . [SEP]
[CLS] therefore , to compensate for these two effects , ligand coating B-material and anisotropic shape , and compare the dna distribution on different auns samples , we measured the relative coincidence yield and normalized to the 50 nm nanospheres B-nanoparticle . [SEP]
[CLS] to determine the loading distribution , we selected a gold B-material cluster ( au 7 − ) that was detected in all nanoconstruct samples and then measured the dna ions B-material that were co - emitted . [SEP]
[CLS] particles with more ligand loading resulted in increased coemission of dna - related ions B-material with au 7 − . [SEP]
[CLS] this method takes into account only impacts occurring on the nanoconstructs while avoiding those containing the silicon B-material support or salt B-material . [SEP]
[CLS] using eq 3 , we evaluated the coincidental yield , cy , of dna ions B-material when a gold B-material cluster was detected , to determine dna loading per particle : [SEP]
[CLS] where i a , b is the intensity of the coemission of a with b , n b is the number of impacts in which b was detected , and cy a , b is the coincidental yield of a in impacts in which b was detected . [SEP]
[CLS] the dna loading of each auns fraction ( cy ) was compared to that of the 50 nm nanospheres B-nanoparticle to determine shape or curvature effects by dividing cy auns by cy 50 nm spheres . [SEP]
[CLS] 4 shows the cy auns / cy 50 nm spheres ratio for thymine nucleotide ( c 5 h 6 n 2 o 2 po 4 c 5 h 7 o − ) co - detected with au 7 − . [SEP]
[CLS] a similar trend was found for all dnarelated species . [SEP]
[CLS] the dashed line at 1 . 0 represents the loading of a 50 nm nanosphere B-nanoparticle . [SEP]
[CLS] the results showed that the cy was higher in all auns fractions than in the spheres . [SEP]
[CLS] f4 auns , with approximately three branches per particle , displayed the highest relative cy , which indicates the highest density of dna loading . [SEP]
[CLS] the shape of auns in f4 increased the amount of neutral curvature compared to that of a 50 nm sphere but limited the amount of negative curvature , which optimized dna functionalization on the particles . [SEP]
[CLS] fraction 2 ( f2 ) , a mixture of single - branch and small spherical particles , had the lowest loading . [SEP]
[CLS] in fractions f6 - f10 , the number of branches increased and formed more regions of negative curvature where the branch meets the core B-material . [SEP]
[CLS] we hypothesize that an increase in negative curvature causes a decrease in dna loading , due to steric hindrance , and therefore attribute the increased loading per particle to the positive and neutral curvature on the auns structure . [SEP]
[CLS] the results presented here show that particle size and curvature influence ligand loading , specifically the abundance of positive and negative curvature . [SEP]
[CLS] to optimize ligand loading , particle geometries with an abundance of positive curvature should be considered . [SEP]
[CLS] the size and length of the target ligand would also need to be considered when determining the size and curvature of the nanoconstruct . [SEP]
[CLS] this study , carried out on size - selected auns , demonstrates the ability of tof - sims with individual projectiles to determine shape - dictated dna loading . [SEP]
[CLS] we found that the amount of functionalized dna on nanoparticles B-nanoparticle depended on their size and curvature . [SEP]
[CLS] auns with branches increased the amount of loaded dna versus that of 50 nm au nanospheres B-nanoparticle . [SEP]
[CLS] however , a greater branch number sterically hindered additional attachment , especially in regions of negative curvature where the branch meets the core B-material . [SEP]
[CLS] the methodology presented can be universally applied to probe any ligand - nanoparticle B-nanoparticle interaction . [SEP]
[CLS] previous experiments have shown that the particle size and shape alter the intensity and relative abundance of metal B-material clusters emitted from metal B-material particles . [SEP]
[CLS] these findings suggest the method may be further developed to evaluate ligand loading with the concurrent identification of an individual particle ' s size and shape . [SEP]
[CLS] a each projectile was separated in time and space and upon impacting the sample causes emission from a volume that is 10 - 15 nm in diameter . [SEP]
[CLS] for each projectile impact , the coemitted ions B-material are mass analyzed by tof and collected in a mass spectrum . [SEP]
[CLS] this scheme is not to scale [SEP]
[CLS] color scale of the correlation coefficient , with red corresponding to positive correlation and blue to negative correlation . [SEP]
[CLS] ions B-material that do not have a significant correlation were not plotted . [SEP]
[CLS] the evaluated ions B-material are listed on the y - axis , and the coincidental mass spectrum is listed on the x - axis . [SEP]
[CLS] 1 . ( a ) photographs of a centrifuge tube before ( top ) and after ( bottom ) centrifugation of a concentrated auns solution in a sucrose linear density gradient . [SEP]
[CLS] ( b ) branch number distribution within each fraction based on manual branch counting of ≥400 particles per fraction . [SEP]
[CLS] ( c ) tem images ( scale bar of 100 nm ) of unsorted auns ( top ) and different fractions after dgc ( bottom ) . [SEP]
[CLS] 2 . ( a ) m / z 0 - 160 and ( b ) m / z 180 - 310 . [SEP]
[CLS] notable peaks are identified and color - coded : purple for carbon B-material clusters , pink fore dna - related , orange for silicon B-material - related , green for salt B-material , and black for au clusters . [SEP]
[CLS] 4 . cy ratio for c 5 h 6 n 2 o 2 po 4 c 5 h 7 o − compared to that of the 50 nm spheres . [SEP]
[CLS] f4 demonstrated 6 - fold enhancement of loading compared with that of the spheres as shown through the cy ratio . [SEP]
[CLS] 3 h − and si 2 o 5 h − [SEP]
[CLS] salt B-material clusters formed due to preparation under aqueous conditions , na x cl y − . [SEP]
[CLS] three types of ions B-material related to the [SEP]
[CLS] the synthesis and evaluation of spherical nucleic B-material acids I-material ( snas ) incorporating two physically and chemically distinct classes of oligonucleotides ( odns ) at programmed ratios are described . [SEP]
[CLS] these snas are single entity agents that enter the same target cell B-material at defined stoichiometries , and as such allow one to control important cell B-material signaling and regulatory processes . [SEP]
[CLS] to study the effect of sequence multiplicity within such structures , we synthesized snas consisting of a mixture of class a cpg and class b cpg , immunostimulatory odns that activate two different toll - like receptor B-material 9 signaling pathways , each in a sequence - specific fashion . [SEP]
[CLS] these dual - cpg snas exhibit high cellular uptake and codelivery of the two odns , relative to mixtures of the linear odn counterparts , and remain highly associated inside the cell B-material over time . [SEP]
[CLS] furthermore , the dual - cpg snas augment dendritic cell B-material maturation , compared to the same amounts of oligonucleotides delivered in linear or sna form but not conjugated to one another . [SEP]
[CLS] consequently , these structures constitute a platform for designing oligonucleotide - based combination therapeutics with highly tailorable activities . [SEP]
[CLS] odns has shown striking synergy , resulting in in vivo mouse survival after tumor B-material challenge . [SEP]
[CLS] 20 while this synergistic effect has been observed in in vivo tumor B-material models , immune activation as a function of the ratio of cpg - a and cpg - b has not been elucidated . [SEP]
[CLS] the sna platform is ideal for carrying out such studies due to its modularity and programmability . [SEP]
[CLS] delivering multiple entities on the same particle to immune cells B-material is beneficial over administration of simple mixtures of the individual components , because the nanoparticle B-nanoparticle scaffold allows for delivery of programmed ratios of each component to the same target cell B-material . [SEP]
[CLS] in this study , we incorporated two physically ( e . g . , structural features ) and chemically ( e . g . , backbone chemistry ) distinct oligonucleotide sequences , cpg - a ( odn 1585 ) and cpg - b ( odn 1826 ) , into lsnas ( scheme 1 ) to study the intracellular trafficking of the snas and their downstream activation of dcs . [SEP]
[CLS] to generate snas , 3 ′ - cholesterol - terminated oligonucleotides and 50 nm 1 , 2 - dioleoyl - snglycero - 3 - phosphocholine ( dopc ) liposomes B-nanoparticle were mixed in a predetermined ratio to achieve the desired cpg odn loading on the sna scaffold . [SEP]
[CLS] first , single - component snas , comprised of either all cpg - a or all cpg - b dna , were synthesized and characterized to understand the impact of sequence and secondary dna structure on the dna loading of snas . [SEP]
[CLS] in particular , cpg - b snas ( figure s2 ) have been well - characterized and studied as immunotherapeutics with a maximal loading of 75 cpg - b odn per 50 nm liposome B-nanoparticle . [SEP]
[CLS] conversely , cpg - a odn has been proposed to have nanoparticle - like properties because it contains a poly ( g ) sequence that forms g - quadruplexes that can potentially stabilize the palindrome sequence . [SEP]
[CLS] thus , cpg - a is rarely incorporated into nanomaterials B-material and has never been used as a component of snas . [SEP]
[CLS] however , these higherorder structures of cpg - a are salt - and temperature - dependent [SEP]
[CLS] results and discussionthus , we hypothesized that cpg - a aggregation could be circumvented by incorporating cpg - a into snas during the sna assembly process ( see supporting information ) . therefore , we first studied the oligonucleotide content of cpg - a in snas and determined through agarose B-technique gel I-technique electrophoresis I-technique that up to 150 cholesterol - terminated cpg - as could be inserted into 50 nm dopc liposomes B-nanoparticle ( figure 1a ) . [SEP]
[CLS] this observation was confirmed by the size increase , as a function of oligonucleotide equivalents , as determined by dynamic B-technique light I-technique scattering I-technique ( dls , figure 1b ) . [SEP]
[CLS] cryogenic transmission B-technique electron I-technique microscopy I-technique ( cryotem ) analysis revealed that unfunctionalized liposomes B-nanoparticle pack tightly together , presumably due to the absence of negatively charged surface ligands and therefore minimized electrostatic repulsion between particles ( figure 1c ) . [SEP]
[CLS] however , when the liposomes B-nanoparticle were transformed into snas through their external modification with cpg - a , there was a marked increase in interparticle spacing ( figure 1d ) , a trend that was also observed with cpg - b snas ( figure s2 ) . [SEP]
[CLS] for example , cpg - a sna interparticle spacing increased with increased oligonucleotide loading from 21 . 0 ± 3 . 5 nm ( 50 strands / sna , figure s3 ) to 27 . 7 ± 3 . 7 nm ( 150 strands / sna , figure 1d ) . [SEP]
[CLS] these results not only show that higher oligonucleotide loading density leads to higher particle surface coverage , but also creates a more radially oriented odn shell B-material within the sna structure . [SEP]
[CLS] when the dna / liposome B-nanoparticle ratio exceeded 150 : 1 , an upward shift in mobility B-property by gel B-technique electrophoresis I-technique was observed ( figure 1a ) , indicating a significant size increase in the population , presumably due to aggregation of the snas caused by sequence selfcomplementarity ( see discussion in supporting information ) . [SEP]
[CLS] to avoid aggregation - specific effects on cellular uptake and downstream immune activation , snas incorporating 150 strands of cpg - a odn per particle were used in further studies . [SEP]
[CLS] snas incorporating both classes of cpg odn on the same nanoparticle B-nanoparticle scaffold were next synthesized by incorporating cpg - a and cpg - b into unfunctionalized dopc liposomes B-nanoparticle at defined ratios ( table s2 ) , based on the determined saturation limits of each singlecomponent sna . [SEP]
[CLS] agarose B-technique gel I-technique electrophoresis I-technique reveals that both cpg classes are incorporated into snas ( figure s8 ) . [SEP]
[CLS] to confirm the successful dual functionalization of snas with both cpg odn , forster resonance energy transfer ( fret ) , the energy transfer process from an excited fluorophore to an acceptor fluorophore ( such as from fluorescein to cy3 ) when the donor and acceptor are within the forster radius , was employed . [SEP]
[CLS] dual - cpg snas functionalized with 50 % fluorescein - labeled cpg - a and 50 % cy3 - labeled cpg - b showed a significantly higher fret efficiency as compared to a 50 : 50 mixture of two distinct sets of snas , each containing one fluorophore - labeled class of cpg odn ( figure 2a ) . [SEP]
[CLS] such significantly higher fret efficiency indicates that the two cpg odn classes are in closer proximity when functionalized on the same sna compared to when they are functionalized on separate snas , reflecting successful dual sequence functionalization on the same sna . [SEP]
[CLS] to evaluate the uptake and immunogenicity B-property of dual - cpg snas , mouse bone marrowderived dendritic cells B-material ( dcs ) , a tlr9 - presenting cell B-material line widely utilized for in vitro immune stimulation studies using cpg odns , were used as a model system . [SEP]
[CLS] dcs incubated B-technique with snas showed significantly higher uptake as compared to cells B-material treated with linear odns ( figure 2b , c ) after 30 min . [SEP]
[CLS] dual - cpg snas showed similar uptake to snas functionalized with a single class of cpg odn ( figures 2b , c , s8 ) on a per particle basis . [SEP]
[CLS] as expected , the fluorescence B-property of cells B-material treated with dual - cpg snas incorporating fluoresceinlabeled cpg - a and cy5 - labeled cpg - b at a 50 : 50 mol ratio was roughly half the intensity of cells B-material treated with single - component snas , as each cpg component encompassed half of the total cpg odn incorporated into the dual - cpg sna scaffold . [SEP]
[CLS] the pearson correlation coefficient ( ρ ) is a measure of the linear correlation between two variables , where values greater than 0 . 8 indicate a very strong positive correlation . [SEP]
[CLS] in cells B-material incubated B-technique with 50 : 50 dual - cpg snas , the value of ρ when analyzing both cpg - a and cpg - b uptake indicates that there is a very strong positive correlation between the uptake of the two sequences ( ρ = 0 . 849 ± 0 . 005 ) . [SEP]
[CLS] in addition , the population of doubly positive cells B-material ( cpg - a + / cpg - b + ) was roughly 5 - fold higher in cells B-material treated with dual - cpg snas as compared to cells B-material treated with a 50 : 50 mixture of the two linear cpg odns not on snas ( figures 2e , f and s9 ) . [SEP]
[CLS] together , these results indicate that dual - cpg snas lead to higher codelivery of cpg - a and cpg - b to cells B-material . [SEP]
[CLS] interestingly , confocal microscopy B-technique revealed that even after extended periods ( e . g . , 48 h ) , the fluorescence B-property from the two labeled cpg components of 50 : 50 dual - cpg snas still strongly overlapped as quantified by high manders overlap coefficients , indicating that these materials remain in close proximity during cellular B-event processing I-event , while fluorescence B-property from cells B-material treated with a 50 : 50 mixture of both linear labeled cpg odns showed little overlap ( figures 2g , h and s10 ) . [SEP]
[CLS] although cpg odns activate tlr9 and lead to the secretion of cytokines and interferons , different classes of cpg odns induce different immune responses due in part to different intracellular trafficking of the oligonucleotides . [SEP]
[CLS] after entering cells B-material , cpg - a binds to tlr9 preferentially in the early endosome , which activates the interferon regulatory factor 7 ( irf7 ) pathway and leads to the secretion of type i ifn ( e . g . , ifn - α ) . [SEP]
[CLS] in contrast , cpg - b activates tlr9 primarily in the late endosome , where the nuclear factor kb ( nf - κb ) pathway is predominately activated and secretes proinflammatory cytokines . [SEP]
[CLS] the detailed mechanisms of different activation pathways are still not completely understood , but it is proposed that both the sequence of the oligonucleotides and the ph of the intracellular organelles may contribute . [SEP]
[CLS] both factors likely impact the morphology of the cpg - tlr9 complex and consequently the extra - organelle portion of tlr9 responsible for downstream activation . [SEP]
[CLS] thus , we generated a series of dual - cpg snas with different ratios of cpg - a to cpg - b loading , ranging from 0 % cpg - b ( 100 % cpg - a ) to 100 % cpg - b ( 0 % cpg - a ) to study the effects of codelivery on downstream activation , with oligonucleotide loading confirmed by agarose B-technique gel I-technique electrophoresis I-technique ( figure s8 ) . [SEP]
[CLS] after incubating B-technique dcs with 1 μm snas ( by total cpg concentration ) for 24 h , we found that high cpg - b loading promoted cytokine secretion ( figures 3a , b and s11 ) , indicative of activation of the nf - κb pathway , while high cpg - a loading led to high ifn - α production , indicative of activation of the irf7 pathway ( figure 3c ) . [SEP]
[CLS] further , cells B-material treated with single - component cpg - a snas or cpg - b snas stimulated higher cytokine and interferon production as compared to their linear counterparts . [SEP]
[CLS] interestingly , although cytokine secretion increased steadily with increasing cpg - b loading in snas , the linear odn mixture reached a maximum level of cytokine secretion at 30 % cpg - b with no enhanced cytokine production observed at higher ratios of cpg - b to cpg - a ( figure 3a , b ) . [SEP]
[CLS] similar behavior was observed for ifn - α production by linear odn mixtures as a function of cpg - a ratios ( figure 3c ) . [SEP]
[CLS] we hypothesized that the differences in immune activation by snas as compared to linear odns was a result of the difference in intracellular trafficking of the two constructs . [SEP]
[CLS] in the case of linear cpg odns , each component traveled independently to its preferred intracellular pathway for downstream activation , since the two components were not structurally associated . [SEP]
[CLS] however , when cpg - a and cpg - b were coupled onto the same entity in dual - cpg snas , they remained highly associated over time ( figure 2g ) . [SEP]
[CLS] thus , cpg - a and cpg - b were trafficked to the same location , leading to only a portion of each cpg type delivered to its preferred intracellular organelle for tlr9 activation at any given time . [SEP]
[CLS] to confirm this hypothesis , we studied the intracellular trafficking of different snas using confocal microscopy B-technique ( figures s12 - s14 ) , as well as the localization of cpg in intracellular organelles ( figures 3d , e , s15 ) . [SEP]
[CLS] we found that the trafficking of cpg - a and cpg - b odns was primarily dictated by the sna scaffold with sequence identity having a minimal effect on intracellular trafficking when the odns were on an sna . [SEP]
[CLS] conversely , the trafficking of mixtures of linear odns was dictated by each odn sequence identity . [SEP]
[CLS] cpg snas , regardless of sequence , were trafficked to early endosomes , late endosomes , and lysosomes at approximately the same rates and on the same time scales ( figure 3d ) . [SEP]
[CLS] in general , snas colocalized with the early endosome after uptake , followed by a rapid decrease with a concomitant increase of colocalization with the late endosome . [SEP]
[CLS] there was only an ~ 0 . 5 h difference between cpg - a and cpg - b snas that colocalized with the early endosome , and all snas showed colocalization with the late endosome with a peak at ~ 2 - 3 h . [SEP]
[CLS] colocalization of snas in lysosomes steadily increased , reaching a plateau at 4 h which was maintained up to 24 h . on the contrary , mixtures of linear cpg - a and cpg - b odns were trafficked very differently ( figure 3e ) . [SEP]
[CLS] after uptake , linear cpg - a odn maintained high colocalization with the early endosome , where cpg - a / tlr9 binding is preferred , with a slight decrease in colocalization over time . [SEP]
[CLS] in contrast , linear cpg - b odn showed high colocalization with the late endosome at early time points , indicating very rapid traveling to late endosomes , where cpg - b / tlr9 binding is favored . [SEP]
[CLS] these results may guide the design of snas for preferentially delivering various oligonucleotide sequences to specific intracellular organelles following this particular spatiotemporal path , which is otherwise impossible to control for the corresponding linear odn counterparts . [SEP]
[CLS] we further hypothesized that maximum dc activation could be achieved by codelivering an intermediate B-property ratio of cpg - a and cpg - b on the same sna . [SEP]
[CLS] costimulation markers are antigen nonspecific signaling proteins B-material that are necessary for antigen - presenting cells B-material ( e . g . , dcs ) to activate t cells B-material . [SEP]
[CLS] in addition to cytokine , chemokine , and interferon productions , tlr9 activation also leads to the upregulation of cd80 and cd86 ( also known as b7 - 1 and b7 - 2 , respectively ) , two proteins B-material that can bind to cd28 on t cells B-material for critical costimulation and t cell activation . [SEP]
[CLS] after incubating B-technique dcs with dual - cpg snas for 24 h , snas at different cpg - a / b ratios demonstrated almost consistently superior performance to incubation B-technique as compared to linear cpg odns at the same ratios ( figures 4 , s16 ) . [SEP]
[CLS] expression of cd80 and cd86 peaked at 70 mol % cpg - a and 30 mol % cpg - b ratio on snas , and the percentage of the cd80 + / cd86 + double - positive population was also the highest at this cpg ratio . [SEP]
[CLS] these results suggest that dual - cpg snas with controlled oligonucleotide ratios can achieve maximal dc maturation , potentially because of the intracellular trafficking pathway taken by snas , allowing cells B-material to take advantage of activation by both cpg classes . [SEP]
[CLS] after snas enter cells B-material , a relatively higher amount of cpg - a than cpg - b was needed to effectively bind tlr9 in the early endosome and induce significant downstream activation , because snas only remain in the early endosome for a brief time ( figure 3d ) . [SEP]
[CLS] conversely , a relatively lower amount of cpg - b was needed because the snas remain colocalized with the late endosome for extended periods , where cpg - b is processed . [SEP]
[CLS] the sna architecture allows for the delivery of diverse odn classes at programmable ratios and with high uptake and codelivery to the same target cell B-material . [SEP]
[CLS] by generating a series of snas incorporating cpg - a and cpg - b on the same nanoparticle B-nanoparticle scaffold at different ratios , we determined that the sna architecture provides high intracellular association of these two distinct odn classes . [SEP]
[CLS] furthermore , codelivery of both cpg classes on the dual - cpg sna scaffold leads to synergistic dc costimulatory molecule activation , where cytokine and ifn secretion is proportional to the fraction of each of their activating cpg classes ( cpg - b and cpg - a , respectively ) . [SEP]
[CLS] although the two sequences used in this report are both tlr9 ligands , the high association and intracellular trafficking behaviors are governed by the sna scaffold and are independent of sequence , as these sequences are trafficked to the same location despite being both physically and chemically diverse . [SEP]
[CLS] therefore , these findings should be generalizable to the design and synthesis of snas containing any combination of multisequence snas for desired applications . [SEP]
[CLS] 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ( dopc ) was purchased from avanti polar lipids B-material as a solution in chloroform . [SEP]
[CLS] tlr9 ligands cpg odn 1585 ( class a ) , 1826 ( class b ) , and their corresponding gpc control sequences were synthesized on an abi 3900 dna oligonucleotide synthesizer with all reagents purchased from glen research ( see supporting information ) . [SEP]
[CLS] bone marrow - derived dcs were collected from c57bl / 6 wildtype mice and processed following the literature protocol , and the cells B-material were cultured in rpmi 1640 medium ( gibco ) supplemented with 10 % fetal bovine serum ( fbs , atlanta biologicals ) , 100 u / ml penicillin - streptomycin ( pen - strep , gibco ) , and 20 ng / ml recombinant mouse granulocytemacrophage colony stimulating factor ( gm - csf , ebioscience ) . [SEP]
[CLS] jawsii , an immortal murine bone marrow - derived dc cell B-material line , was purchased from atcc ( crl - 11904 ) and cultured in alpha minimum essential medium ( gibco ) supplemented with 1 mm sodium B-material pyruvate ( gibco ) , 5 ng / ml gm - csf , 20 % fbs , and 100 u / ml pen - strep . [SEP]
[CLS] in a typical synthesis , 4 ml of a chloroform solution of dopc lipid B-material ( 25 mg / ml ) was transferred into a glass vial , and the lipid B-material was dried under nitrogen B-material gas for 30 min and stored in vacuo overnight to completely remove organic solvent and generate a thin lipid B-material film . [SEP]
[CLS] the film was then rehydrated in 10 ml of pbs . [SEP]
[CLS] the resulting suspension was vortexed for 30 s and sonicated for 30 min . [SEP]
[CLS] following five freeze - thaw cycles , the suspension underwent serial extrusions in 200 , 100 , 80 , and 50 nm track - etch membranes to obtain uniform - size liposomes B-nanoparticle . [SEP]
[CLS] liposomes B-nanoparticle were concentrated using 50k mwco microkros tangential flow filtration ( spectrum laboratories ) , and the final concentration was measured using the phosphatidylcholine assay kit ( sigma ) . [SEP]
[CLS] to functionalize oligonucleotides onto the surface of liposomes B-nanoparticle , 100 μm 3 ′ - cholesterol - functionalized oligonucleotides were mixed with a predetermined amount of liposomes B-nanoparticle , calculated following table s2 . [SEP]
[CLS] samples were held overnight at 37 °c under shaking at 500 rpm . [SEP]
[CLS] to assess cpg - a incorporation into snas , agarose B-technique gel I-technique electrophoresis I-technique was performed using 1 % agarose ( sigma ) with sybr safe dna gel stain in 0 . 5× tris / borate / edta ( tbe ) ( thermofisher scientific ) under 120 v for 1 h . [SEP]
[CLS] to assess dual - cpg loading , fluorophorelabeled oligonucleotides ( fluorescein - cpg - a and cy5 - cpg - b ) were used , and agarose B-technique gel I-technique electrophoresis I-technique was performed using 0 . 5 % agarose in 1× tbe without further staining . [SEP]
[CLS] dynamic B-technique light I-technique scattering I-technique ( dls ) and zeta B-property potential I-property measurements were performed using a malvern zetasizer nano with ~ 10 nm samples by particle . [SEP]
[CLS] cryotem was performed with a hitachi ht7700 tem with a gatan cryo - transfer holder , and imaging was performed under a 120 kv accelerating voltage . [SEP]
[CLS] cryotem samples were prepared by loading 4 μl of sample ( 100 μm cpg sna stock solution or 1 . 33 μm liposome B-nanoparticle solution ) onto 300 - mesh copper B-material tem grids with lacey carbon B-material films ( electron B-technique microscopy I-technique sciences ) using a fei vitrobot mark iv with its chamber equilibrated at 4 °c and 100 % humidity . [SEP]
[CLS] the samples were blotted for 5 s and plunged into liquid ethane before they were transferred and stored in liquid nitrogen B-material . [SEP]
[CLS] fret measurements were performed using a synergy plate reader ( biotek ) with 10 nm samples by oligonucleotides . [SEP]
[CLS] fret efficiency ( e ) was calculated as [SEP]
[CLS] where f da and f d are the donor fluorescence B-property intensities with and without an acceptor , respectively . [SEP]
[CLS] cells B-material ( 2 × 10 4 jawsii ) in 0 . 5 ml phenol red - free cell B-material culture medium were plated on gelatin - coated 15 mm coverslips in 24 - well plates overnight and incubated B-technique with 5 μm sna by cpg oligonucleotide concentration . [SEP]
[CLS] fluorescein - labeled cpg - a odn and / or cy5labeled cpg - b odn at high concentration were used to ensure a detectable signal after a prolonged time . [SEP]
[CLS] to track the same amount of snas trafficking over time , all cells B-material were treated for 30 min . [SEP]
[CLS] for the 30 min time points in the intracellular trafficking studies , cells B-material were incubated B-technique with media containing either snas or linear cpg odns for 30 min , then washed , removed from media , fixed , and stained . [SEP]
[CLS] for all other time points , cells B-material were incubated B-technique in sna or cpg - containing media for 30 min , washed twice , and incubated B-technique with fresh media for the additional amount of time prior to fixation and staining . [SEP]
[CLS] at the end of the treatment , coverslips were incubated B-technique in 4 % paraformaldehyde in pbs ( diluted from a 32 % aqueous solution from electron B-technique microscopy I-technique sciences ) for 15 min for fixation and 0 . 2 % triton - x in pbs ( diluted from triton - x from sigma ) for 10 min for permeation . [SEP]
[CLS] for the cpg association study ( figure 2 ) , cells B-material were incubated B-technique in image - it fx signal enhancer ( thermofisher scientific ) for 30 min and then in alexa fluor 568 phalloidin for 30 min . [SEP]
[CLS] phalloidin labels the actin of the cytoskeleton , which here serves as the confirmation of dc structure ( the dendrite growth over time may also serve as confirmation of dc maturation , though it is difficult to quantify ) . [SEP]
[CLS] for the intracellular trafficking study , coverslips were incubated B-technique in 2 % bovine serum albumin ( bsa ) and 0 . 2 % triton - x in pbs for 1 h for blocking . [SEP]
[CLS] rabbit anti - eea1 ( abclonal ) , rabbit anti - rab9 ( abclonal ) , rabbit anti - lamp1 ( abcam ) , and rabbit anti - lc3 ( sigma - aldrich ) primary antibodies B-material diluted 1 : 1000 in 1 % bsa , 0 . 1 % triton - x in pbs were used for early endosome , late endosome , lysosome , and autophagosome labeling , respectively . [SEP]
[CLS] alexa fluor 568 - labeled goat antirabbit igg h & l secondary antibody B-material ( abcam ) diluted 1 : 1000 in 1 % bsa , 0 . 1 % triton - x in pbs [SEP]
[CLS] was used to fluorescently B-property tag the intracellular organelle markers . [SEP]
[CLS] all treatment and staining steps used 0 . 4 ml volumes , and coverslips were washed twice using 1 ml of pbs or thrice using pbs - triton - x ( for antibody B-material washes ) after each step . [SEP]
[CLS] after staining , coverslips were mounted onto glass slides using prolong glass with nucblue nuclear stain ( thermofisher scientific ) and cured overnight . [SEP]
[CLS] confocal microscopy B-technique was performed using leica tcs sp8 lscm with hybrid detectors ( hyd ) and hyvolution . [SEP]
[CLS] data processing and colocalization calculations were performed using the leica application suite x ( for hyvolution ) and an in - house matlab script . [SEP]
[CLS] a total of 25 cells B-material were included for the analysis of each sample . [SEP]
[CLS] images of individual detection channels were exported as grayscale maps and denoised using deep neural network . [SEP]
[CLS] z - stack layers of whole cells B-material were arranged into a three - dimensional array as pseudo - 3d reconstruction of the cell B-material image for each channel . [SEP]
[CLS] colocalization analysis provides a quantitative means to evaluate the qualitative confocal image data . [SEP]
[CLS] manders overlap and colocalization coefficients were used in this study to analyze the colocalization of color pairs from the confocal images . [SEP]
[CLS] for analyzing the association of the two cpg odns , the manders overlap coefficient , a parameter describing the degree of overlap between two components in an image , was calculated . [SEP]
[CLS] the manders overlap coefficient , a derivative of the pearson ' s correlation coefficient ( r ) , is defined as [SEP]
[CLS] where a i and b i are the gray scale values of the individual voxels i of the color components a and b , which in this case , are the fluorescein and cy5 signals , respectively . [SEP]
[CLS] for analysis of colocalization of cpg odn in the organelles , the manders colocalization coefficient ( m ab ) is used which is defined by [SEP]
[CLS] so that m ab measures the fluorescence B-property fraction of a colocalized with b , which in this case is the odn signal ( fluorescein and / or cy5 ) colocalized in the organelle ( alexa fluor 568 ) . [SEP]
[CLS] this coefficient is useful since it does not consider the pixels of the second signal ( organelles ) that do not overlap with the first signal ( odn ) . [SEP]
[CLS] both manders overlap and colocalization coefficients are signal intensity independent , which make them advantageous in this study to compare markers that may have different signal intensities caused by different uptake amount ( sna vs linear oligonucleotides ) , cpg signal decrease over time , and difference in organelle antibody B-material labeling . [SEP]
[CLS] all antibodies B-material and staining buffers were purchased from bd biosciences , and all antibodies B-material were mixed and diluted to 1 : 200 in staining buffer . [SEP]
[CLS] for the uptake study , 3 × 10 5 cells B-material were treated with fluorescein - labeled cpg - a and / or cy5 - labeled cpg - b oligonucleotides with a total oligonucleotide concentration of 1 μm in 1 . 2 ml microtubes . [SEP]
[CLS] after a 30 min incubation B-technique at 37 °c , cells B-material were washed three times with 1 ml of pbs and stained with 100 μl of antibody B-material solution containing rat antimouse cd11b and hamster antimouse cd11c . [SEP]
[CLS] pearson correlation calculations ( ρ ) were performed using fluorescein ( g ) and cy5 ( r ) signals of each cell B-material into the formula [SEP]
[CLS] where μ g and σ g are the mean and standard deviation of g , respectively , and μ r and σ r are the mean and standard deviation of r , respectively . [SEP]
[CLS] for the dendritic cell B-material activation study , 3 × 10 5 cells B-material were incubated B-technique with 200 μ l of 1 μ m cpg ( linear or sna ) containing media for 24 h in 1 . 2 ml microtubes . [SEP]
[CLS] the cells B-material were then washed three times with 1 ml of pbs and stained with 100 μ l of antibody B-material solution containing rat antimouse cd11b , hamster antimouse cd11c , hamster antimouse cd80 , and rat antimouse cd86 for 15 min at 4 °c . [SEP]
[CLS] the cells B-material were then fixed with 3 . 7 % paraformaldehyde in pbs for 20 min at room temperature . [SEP]
[CLS] flow B-technique cytometry I-technique was completed with a bd lsrfortessa flow cytometer system . [SEP]
[CLS] a total of 30 , 000 events were collected for each sample , and dcs gated with cd11c + population . [SEP]
[CLS] color compensation was performed using the abc total antibody B-material compensation bead kit ( thermofisher scientific ) . [SEP]
[CLS] data analysis was performed using flowjo and matlab . [SEP]
[CLS] cytokine and ifn measurements . [SEP]
[CLS] cytokine and ifn release were measured using luminex 200 . [SEP]
[CLS] assays were performed using mouse magnetic B-property luminex assay kits purchased from r & d systems for cytokines and thermofisher scientific for ifn - α . [SEP]
[CLS] cells B-material ( 2 × 10 5 ) were treated with 200 μ l of media containing 100 nm cpg ( linear or sna ) in a round - bottom 96 - well plate for 24 h ; after centrifugation for 5 min at 1000 rpm , the cell B-material supernatants were collected and transferred to the luminex assay plate without further dilution . [SEP]
[CLS] sample processing and measurements were performed following the manufacturer ' s specific protocols . [SEP]
[CLS] data analysis was performed using milliplex analyst software . [SEP]
[CLS] characterization of cpg - a snas . [SEP]
[CLS] ( a ) agarose B-technique gel I-technique electrophoresis I-technique of liposomes B-nanoparticle functionalized with different amounts of cholesterol - labeled cpg - a odn ; the symbol ∞ indicates free cholesterol - labeled cpg - a odns . [SEP]
[CLS] ( b ) hydrodynamic diameter of cpg - a snas as measured by dls . [SEP]
[CLS] cryotem images of ( c ) bare liposomes B-nanoparticle and ( d ) snas functionalized with 150 cpg - a odns . [SEP]
[CLS] scale bars : 100 nm . [SEP]
[CLS] insets : schematics of the corresponding constructs . [SEP]
[CLS] characterization of dual - cpg snas ( 50 : 50 mol ratio cpg - a to cpg - b ) . [SEP]
[CLS] ( a ) fret efficiency comparison between fluorescein - cpg - a sna / cy3 - cpg - b sna mixture and dual - cpg sna . [SEP]
[CLS] fluorescence B-property signals of ( b ) fluorescein - cpg - a and ( c ) cy5 - cpg - b after 30 min treatments in single - component or dual - cpg form . [SEP]
[CLS] ( d ) pearson correlation coefficient ( ρ ) between cpg - a and cpg - b uptake in dual - cpg sna and linear cpg mixture forms . [SEP]
[CLS] representative flow B-technique cytometry I-technique data of ( e ) dual - cpg sna and ( f ) linear cpg mixture uptake . [SEP]
[CLS] ( g ) manders overlap coefficients between cpg - a and cpg - b in dual - cpg sna and linear cpg mixture forms over time after cellular uptake . [SEP]
[CLS] ( h ) representative confocal microscopy B-technique images of dual - cpg sna 30 min , 4 h , and 24 h after initial uptake . [SEP]
[CLS] color assignments : fluorescein - cpg - a ( green ) , cy5 - cpg - b ( red ) , nucleus ( blue ) , actin ( gray ) . [SEP]
[CLS] scale bars : 5 μm . [SEP]
[CLS] 3 . cytokine and ifn production following intracellular localization . [SEP]
[CLS] production of ( a ) tnf - α , ( b ) il - 1α , and ( c ) ifn - α ( # , signal below the lower limit of detection for untreated samples ) after 24 h of treatment . [SEP]
[CLS] ( * * , p < 0 . 01 ; * , p < 0 . 05 ) . [SEP]
[CLS] manders colocalization coefficients of ( d ) cpg - snas and ( e ) cpg odn in intracellular markers over time . [SEP]
[CLS] dual - cpg data shown in ( d , e ) incorporated a 1 : 1 ratio of cpg - a to cpg - b . [SEP]
[CLS] 4 . dc costimulation marker activation . [SEP]
[CLS] ( a ) representative flow B-technique cytometry I-technique data for cd80 and cd86 expression after 24 h treatment of cpg snas or linear cpg odns . [SEP]
[CLS] mean fluorescence B-property intensity ( mfi ) of ( b ) cd80 and ( c ) cd86 expression . [SEP]
[CLS] ( d ) percentage of cd80 + / cd86 + double - positive cell B-material population after treatment . [SEP]
[CLS] ( * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 ) [SEP]
[CLS] spherical nucleic B-material acids I-material ( snas ) are a class of nanomaterials B-material with a structure defined by a radial distribution of densely packed , short dna or rna sequences around a nanoparticle B-nanoparticle core B-material . [SEP]
[CLS] this jason a . wertheim [SEP]
[CLS] limited largely due to challenges with effective delivery . [SEP]
[CLS] delivery of unmodified , linear oligonucleotides results in rapid clearance , nuclease - mediated degradation , and poor internalization by cells B-material . [SEP]
[CLS] spherical nucleic B-material acids I-material ( snas ) are a class of nucleic B-material acids I-material composed of a dense shell B-material of radially oriented oligonucleotides surrounding a nanoparticle B-nanoparticle core B-material . [SEP]
[CLS] this architecture allows snas to overcome many of the limitations associated with delivery of linear oligonucleotides . [SEP]
[CLS] sna architectures rapidly enter over 50 different cell B-material types , resist nuclease degradation , and transcytose across different biological barriers B-property , including the skin , 8 blood - brain barrier B-property , and blood - tumor B-material barrier B-property . [SEP]
[CLS] there are a number of factors , such as nanoparticle B-nanoparticle size , shape , and surface charge , that affect the bioavailability B-property of systemically administered nanomedicines . [SEP]
[CLS] this includes their interaction with serum proteins B-material , mechanism of cellular entry , and clearance from the body . [SEP]
[CLS] previously , we determined that gold B-material - based snas ( au snas ) primarily distribute to the liver and spleen with minor changes due to varying the presented dna sequence or backfilling the sna surface with peg . [SEP]
[CLS] this follows the pattern of many nanoparticles B-nanoparticle , which are cleared by the cells B-material of the mononuclear phagocyte system ( mps ) located primarily in organs such as the liver , spleen , and bone marrow . [SEP]
[CLS] because the au snas are not extensively used clinically , we sought to explore the more modular and clinically relevant liposomal B-nanoparticle sna ( lsna ) in order to exploit structural changes to direct dna biodistribution . [SEP]
[CLS] the highly modular lsna architecture enables modification of both the nanoparticle B-nanoparticle core B-material and surface chemistry . [SEP]
[CLS] with lsna architectures , the affinity of the dna shell B-material to the liposome B-nanoparticle template can be modified to control overall nanostructure stability and the release rate of oligonucleotides from the liposome B-nanoparticle core B-material . [SEP]
[CLS] for example , increasing the hydrophobicity B-property of the 3 ′ tail of the dna sequence by changing it from a cholesterol group to a c16 diacyl lipid B-material anchor ( dppe ) increases the affinity of the dna shell B-material for the liposome B-nanoparticle template . [SEP]
[CLS] in serum - containing media , this modification increases the half - life of dna attachment to the lsna ' s lipid B-material bilayer I-material by greater than this increased stability leads to greater cellular uptake and potency with respect to innate immune receptor B-material stimulation . [SEP]
[CLS] these observations highlight how lsna stability may dictate interactions with immune cell B-material populations in vivo as well as the tissues and cell B-material populations to which the lsnas distribute . [SEP]
[CLS] to determine these in vivo structure - function relationships , we synthesized lsnas with either cholesterol - or lipid B-material - anchored dna and measured the immune response , tissue distribution , and cellular level distribution of each lsna construct in immunecompetent mice . [SEP]
[CLS] the results highlight the advantages of lsna architectures over linear dna and the importance of lsna stability in tuning the delivery of systemically administered oligonucleotides to target difficult - to - reach tissues . [SEP]
[CLS] the biodistribution profiles of intravenously injected lsnas were studied in healthy , wildtype c57bl / 6 mice using a cyanine 5 ( cy5 ) fluorophore - labeled , phosphorothioate backbone single - stranded dna ( ssdna ) sequence . [SEP]
[CLS] liposomes B-nanoparticle onto which the dna was functionalized were labeled with 10 % tetramethylrhodamine ( tamra ) - pc ( tables s1 , s2 ) . [SEP]
[CLS] the dna used to synthesize cholesterol - tail lsnas ( chol - lsnas ) was terminated with cholesterol , while the dna used to synthesize dppe - tail lsnas ( dppe - lsnas ) was terminated with a dibenzocyclooctyne - modified thymidine nucleobase ( dbco - dt ) ( figure 1a ) . [SEP]
[CLS] the sequence used was odn 2138 , a sequence designed as a gpc nonimmunogenic control to the cpg - containing tlr9 agonist odn 1826 . [SEP]
[CLS] lsnas were synthesized by first forming a 50 nm diameter small B-material unilamellar I-material vesicle I-material ( suv ) template composed of 100 % 1 , 2 - dioleyl - sn - glycero - 3 - phosphocholine ( dopc ) . [SEP]
[CLS] for dppe lsnas , dopc suvs were modified with 5 % ( mol / mol ) azide - capped 1 , 2 - dipalmitoyl - sn - glycero - 3 - phosphoethanolamine ( dppe - azide ) ( figure 1b ) . [SEP]
[CLS] following suv formation , dna sequences and liposome B-nanoparticle templates were mixed and shaken overnight at room temperature in 20 mm hepes - buffered saline . [SEP]
[CLS] this facilitated cholesterol - terminated dna insertion into the suv bilayer , forming chol - lsnas , as well as the copper - free click reaction of dbco - terminated dna with dppe - azide lipids B-material on the suv surface , forming dppe - lsnas . [SEP]
[CLS] the liposome B-nanoparticle size and spherical architecture were confirmed using dynamic B-technique light I-technique scattering I-technique ( dls ) and transmission B-technique electron I-technique microscopy I-technique ( tem ) ( figures s1 , s2 ) . [SEP]
[CLS] using uvvis spectroscopy B-technique to measure dna concentration and inductively coupled plasma optical emission spectrometry ( icp - oes ) to quantify phosphorus B-material content , we determined the number of dna strands per lsna . [SEP]
[CLS] the mixing ratio of cholesterol - tailed dna to lipids B-material that results in the maximum number of dna strands per liposome B-nanoparticle was determined to be approximately 15 . 4 μm dna to 1 mm lipids B-material ( figure s3 ) . [SEP]
[CLS] the dna loading into each respective formulation is comparable at this reaction stoichiometry , as the average number of strands per liposome B-nanoparticle was 123 ± 28 for the chol - lsna and 96 ± 18 for the dppe - lsna . [SEP]
[CLS] as a control , we used a mixture ( mix ) of linear dna with no hydrophobic B-property tail ( tables s1 , s2 ) with the same number of liposomes B-nanoparticle , such that the mix contains the same ssdna sequence , but cannot form lsnas . [SEP]
[CLS] lsnas elicit a reduced cytokine response compared to equivalent linear dna sequences . [SEP]
[CLS] systemically administered linear oligonucleotides often lead to off - target effects , such as nonspecific cytokine production and stimulation of inflammatory pathways . [SEP]
[CLS] to quantify the difference between linear dna and lsna structures in this context , we measured the production of various cytokines in the serum after intravenous administration of lsnas into c57 / bl6 mice . [SEP]
[CLS] at 30 min post - injection , linear dna increased production of the pro - inflammatory I-property cytokine mcp - 1 by 2 . 46 - fold over chol - lsnas and 1 . 80 - fold over dppe - lsnas . [SEP]
[CLS] linear dna also induced ifnγ production when it was not detected in lsna - treated mice ( figure 2a ) . [SEP]
[CLS] this suggested a more severe acute inflammatory response to linear dna than for the equivalent dose of lsnas . [SEP]
[CLS] il - 6 and tnf production were also increased in linear dna treated mice compared to lsna formulations , but changes were not statistically significant . [SEP]
[CLS] enhanced production of anti B-property - I-property inflammatory I-property cytokine il - 10 was also observed in response to linear dna compared to untreated mice ( figure 2a ) . [SEP]
[CLS] by 24 h , most cytokines produced in response to linear dna and lsnas returned to basal levels , with il - 6 and tnf not detected . [SEP]
[CLS] the only exception was mcp - 1 , which showed elevated levels due to linear dna , 2 . 39 - fold enhancement over chol - lsnas and 2 . 71 - fold over dppe - lsnas ( figure 2b ) . [SEP]
[CLS] these changes in cytokine levels suggest that the lsna architectures studied herein are inherently less inflammatory than linear dna in vivo , an observation we previously described in vitro . [SEP]
[CLS] because the inflammatory cytokines mcp - 1 and ifnγ are known to recruit and activate cells B-material of the mps , the difference in production of these cytokines in response to each form of dna ( linear or lsna ) is likely linked to differences in trafficking and sequestration of each of these architectures after injection . [SEP]
[CLS] organ level distribution of lsnas is structure dependent . [SEP]
[CLS] to evaluate the tissue level distribution of administered dna , mice received a peripheral intravenous ( iv ) injection of either lsna structure or a control mixture ( mix ) containing the same amount of cy5 - dna - and tamra - pc - labeled liposomes B-nanoparticle ( table s2 ) . [SEP]
[CLS] after 30 min or 24 h of circulation , organs were recovered and analyzed . [SEP]
[CLS] using the spectral unmixing function on the ivis instrument , fluorescence B-property of the 10 % tamra - pc liposomes B-nanoparticle was separated from that of the cy5 - tagged dna . [SEP]
[CLS] when comparing all organs at 30 min , the greatest fluorescence B-property from both dna and the liposome B-nanoparticle cores B-material of lsnas came from the liver , kidneys , and spleen ( figures 3a , s4 - s6 ) . [SEP]
[CLS] however , when each organ was imaged separately , more significant differences were apparent . [SEP]
[CLS] for most organs examined , except the small intestine and pancreas , dna derived from either the chol - lsna or dppe - lsna had greater tissue accumulation relative to linear dna . [SEP]
[CLS] in the liver and serum , twice as much dna fluorescence B-property was observed with lsna - treated mice than linear dna . [SEP]
[CLS] the lsna core B-material fluorescence B-property was at a comparable ratio , with both lsnas exhibiting 1 . 5 - to 2 - fold enhancement in the liver and greater than 50 - fold enhancement in the serum . [SEP]
[CLS] there was a skewing toward chol - lsna trafficking to the lungs and lymph nodes , where we observed 5 - fold and 3 - fold increased cy5 - dna fluorescence B-property compared to linear dna ( figures 3b , s5 ) . [SEP]
[CLS] compared to dppe - lsnas , dna from chol - lsnas accumulated in the lungs by greater than 2 - fold . [SEP]
[CLS] the liposome B-nanoparticle core B-material fluorescence B-property was also 2 - fold greater with chol - lsnas , which suggested that the cholesterol - dna may not be released from the liposome B-nanoparticle core B-material before lung accumulation ( figure 3b ) . [SEP]
[CLS] in contrast , dna from dppe - lsnas trafficked in greater amounts to the heart , brain , and kidneys ( figures 3b , s5 , s7 ) . [SEP]
[CLS] most notably , dppe - lsnas exhibited nearly 2 - fold enhanced cy5 - dna fluorescence B-property in the kidneys compared to the mix and chol - lsnas , but showed no tamra fluorescence B-property enhancement ( figures 3b , s6 ) . [SEP]
[CLS] this suggests that the enhanced kidney delivery may be due to dna dissociation from dppe - lsnas prior to kidney accumulation . [SEP]
[CLS] similarly , at 24 h , the liver and kidneys exhibited the overall highest dna accumulation ( figures 3c , s9 ) . [SEP]
[CLS] both lsnas exhibited higher liposome B-nanoparticle core B-material fluorescence B-property than the mix in the serum but had little cy5 - dna fluorescence B-property at this time point . [SEP]
[CLS] the highest serum dna signal was from chol - lsnas ( figure 3d ) . [SEP]
[CLS] in the liver , both lsnas continued to show higher cy5 fluorescence B-property compared to linear dna ( figure 3d ) . [SEP]
[CLS] cy5 - dna fluorescence B-property from chol - lsnas remained higher than linear and dppe - lsnas in the lungs and lymph nodes ( figures 3d , s9 , s11 ) and also had the highest accumulation in the brain and bone marrow . [SEP]
[CLS] chol - lsna - treated mice exhibited the highest tamra liposome B-nanoparticle fluorescence B-property in the liver and lungs at this time point ( figures 3d , s8 , s10 ) . [SEP]
[CLS] dppe - lsnas continued to deliver the most dna to the kidneys , with nearly 3 - fold cy5 fluorescence B-property relative to chol - lsnas ( figures 3d , s9 ) . [SEP]
[CLS] akin to the 30 min time point , there was little tamra - pc fluorescence B-property in the kidneys from dppe - lsnas , suggesting cy5 - dna release before accumulation . [SEP]
[CLS] linear dna accumulation , which was higher in the pancreas and small intestine at 30 min ( figure s7 ) , was not significantly different from either lsna in these organs at 24 h ( figure s11 ) . [SEP]
[CLS] to further probe tissue - level distribution and the colocalization of both labeled lsna components , we imaged cryosectioned tissues from mice injected with both dual fluorophore - labeled lsnas and the mix control . [SEP]
[CLS] in agreement with the ivis data , livers from animals treated with either lsna had greater levels of cy5 fluorescence B-property at both time points ( figure 4a , e ) . [SEP]
[CLS] although lsnas accumulated in the liver in greater total amounts , the location of linear dna and both lsnas appeared similar within our organ sections ( figures 4a , e and s12 ) , suggesting that dna from lsnas and linear dna may be sequestered by similar cell B-material types . [SEP]
[CLS] this was confirmed by flow B-technique cytometry I-technique ( figure 5a ) . [SEP]
[CLS] for the lungs , the highest fluorescence B-property was observed in the case of the chol - lsna , both at 30 min ( figure 4b ) and 24 h ( figure 4f ) , also consistent with ivis imaging ( figure 3b , d ) . [SEP]
[CLS] while ivis imaging of whole organs cannot distinguish the location of both fluorophore - labeled components within tissues , cryosections indicated that the liposome B-nanoparticle and dna of chol - lsnas were colocalized within lung tissue ( figure s13 ) . [SEP]
[CLS] this confirmed that the cholesterol - tail dna is not released from chol - lsnas before lung accumulation . [SEP]
[CLS] the spleen showed a high level of linear dna accumulation in what appears to be a blood vessel at 30 min ( figure 4c ) . [SEP]
[CLS] in contrast , the spleens from lsna - treated animals exhibited more evenly distributed fluorescence B-property throughout the organ ( figures 4c , s14 ) . [SEP]
[CLS] at 24 h , this evenly distributed fluorescence B-property signal remained in the spleens from animals treated with dppe - lsnas and was not observed in spleens from animals treated with linear dna ( figure 4g ) . [SEP]
[CLS] the tubules of the kidney showed very high cy5 signals for both linear dna - and dppe - lsna - treated animals compared to animals treated with chol - lsnas , 30 min ( figure 4d ) and 24 h ( figure 4h ) post - injection , consistent with the ivis data . [SEP]
[CLS] while it is not surprising that linear dna is observed in the tubules , accumulation of dna from dppe - lsnas in these structures was surprising . [SEP]
[CLS] the 50 nm diameter of the dppe - lsnas is large compared to glomerular capillary pores , the largest of which have radii of approximately 80 a . [SEP]
[CLS] thus , dppe - lsnas cannot be filtered if the lsna is intact . [SEP]
[CLS] we hypothesize that dna derived from dppe - lsnas dissociates from the liposome B-nanoparticle template in the glomerulus , leaving lipid - tail dna alone to pass through the glomerular fenestrations . [SEP]
[CLS] to confirm that the dna was released , we checked for colocalization of the liposomes B-nanoparticle and dna of dppe - lsnas in the cryosections . [SEP]
[CLS] we observed high cy5 - dna signal in the kidneys , but little tamra fluorescence B-property above the untreated background , suggesting that the dna must have been released from dppe - lsnas prior to accumulation within the tubules ( figure s15 ) . [SEP]
[CLS] this is a finding specific to lsnas , as significant accumulation of other snas in the kidneys has not been previously observed . [SEP]
[CLS] we reported previously that au snas exhibit the greatest accumulation in the liver and spleen with very little accumulation in the kidneys . [SEP]
[CLS] in contrast , lsnas exhibited the greatest dna accumulation in four different organs : the liver , lungs , spleen , and kidneys . [SEP]
[CLS] this suggests that the more dynamic nature of lsnas compared to au snas may play a role in their in vivo bioavailability B-property . [SEP]
[CLS] we also observed dna trafficking from lsnas in the small intestines , lymph nodes , and pancreas , suggesting that there may be other possible tissue targets for future lsna therapeutic development ( figures s7 , s11 ) . [SEP]
[CLS] after examining macroscopic differences in dna distribution between each architecture using ivis and fluorescence B-technique microscopy I-technique , we determined structure - distribution relationships on the cell B-material population level . [SEP]
[CLS] we developed two flow B-technique cytometry I-technique panels , one for staining immune cells B-material and another for nonimmune cells B-material , which would capture the majority of cell B-material types present in each organ . [SEP]
[CLS] within the immune cell B-material panel , single cell B-material suspensions derived from each organ were stained for a general immune cell B-material marker ( cd45 ) , t cells B-material ( cd3 ) , b cells ( cd19 ) , neutrophils ( cd11b ) , dendritic cells B-material ( cd11c ) , and macrophages ( cd68 ) . [SEP]
[CLS] the nonimmune cell B-material panel included the general immune cell B-material marker ( cd45 ) , to exclude those cells B-material that stained positively , as well as markers for epithelial cells B-material ( epcam ) , endothelial cells B-material ( cd31 ) , fibroblasts ( cd140a ) , and blood - derived stem cells B-material ( cd34 ) . [SEP]
[CLS] the general gating strategy used is depicted in figure s16 . [SEP]
[CLS] at the 30 min time point ( after injection of labeled linear dna or lsna ) , there was very little difference between groups in the total number of cells B-material from the liver ( figure 5a ) , spleen ( figure 5c ) , and kidneys ( figure 5d ) that tested positive for the cy5 - dna . [SEP]
[CLS] however , an investigation of specific cell B-material types within the spleen and kidneys revealed differences in the total amount of dna being delivered to cells B-material by each respective construct . [SEP]
[CLS] in the spleen , there was a trend for higher accumulation of dna from the dppe - lsna in the nonimmune cells B-material , particularly in epithelial cells B-material ( figure 5c ) , and higher accumulation of dna from chol - lsna in cd11b + immune cells B-material . [SEP]
[CLS] the kidney showed higher linear dna accumulation in nonimmune cells B-material , but no difference between groups in immune cells B-material . [SEP]
[CLS] this was an expected outcome because the count of total immune cells B-material ( cd45 + cells B-material ) in the kidney was very low ( < 1 % of cell B-material counts ) . [SEP]
[CLS] the most significant difference at 30 min for total cy5 - positive cells B-material was observed in the lungs ( figure 5b ) , where chol - lsnas showed the highest dna accumulation , which is consistent with ivis and fluorescence B-technique imaging I-technique results . [SEP]
[CLS] in particular , dna from chol - lsnas was preferentially trafficked to the nonimmune cells B-material of the lungs , and there was no significant difference between chol - and dppe - lsnas in immune cells B-material , although the total dna fluorescence B-property from both lsnas in immune cells B-material was higher than linear dna . [SEP]
[CLS] at the later time point , the trend for total cy5 accumulation in each organ remained the same , with the liver , spleen , and kidney showing no significant difference between groups and the lungs showing higher chol - lsna delivery . [SEP]
[CLS] however , there were differences in the total dna in specific cell B-material types . [SEP]
[CLS] in the liver at 24 h ( figure 5e ) , both chol - and dppe - lsnas exhibited higher fluorescence B-property in nonimmune cells B-material , which suggested that lsna architecture is responsible for enhanced dna delivery to the liver . [SEP]
[CLS] noticeably , in the immune cells B-material within the liver , chol - lsnas showed higher delivery to t cells B-material , but all three treatments were less distinguishable in b cells B-material , neutrophils , dendritic cells , and macrophages . [SEP]
[CLS] in the spleen ( figure 5g ) [SEP]
[CLS] , the same trend was observed in nonimmune cells B-material as at 30 min . [SEP]
[CLS] in contrast , immune cells B-material sequestered dna from dppe - lsnas in greater amounts in cd11b + cells B-material than dna from chol - lsnas . [SEP]
[CLS] a slight preference of chol - lsna dna trafficking to cd11c + and cd68 + cells B-material at 24 h was also observed . [SEP]
[CLS] in the kidney ( figure 5h ) , there was a dramatic reduction in the trafficking or accumulation of dna from chol - lsnas in all nonimmune and cd19 + immune cells B-material . [SEP]
[CLS] whereas linear dna showed the highest accumulation at 30 min , dppe - lsnas exhibited the highest fluorescence B-property intensities from most cell B-material types at 24 h . [SEP]
[CLS] specifically , fibroblasts , epithelial cells B-material , and t cells B-material showed very high dppe - lsna accumulation . [SEP]
[CLS] finally , in the lungs ( figure 5f ) , chol - lsnas exhibited higher cy5 fluorescence B-property intensity than linear dna in all cell B-material types , with a preference for the trafficking of dna from chol - lsnas to immune cells B-material . [SEP]
[CLS] in summary , the results from this study suggest that lsnas are not immediately cleared from circulation and that they may be used to direct nucleic B-material acids I-material to cells B-material and organs outside of those rich in cells B-material of the mononuclear phagocyte system . [SEP]
[CLS] this is an important finding , as the mps is a major hurdle in the delivery of nanoparticle - based therapeutics , 46 - 48 but understanding how structure dictates where lsnas ( or the nucleic B-material acids I-material that comprise snas ) accumulate upon intravenous administration allows for the rational design of targeted lsna therapeutics . [SEP]
[CLS] this insight broadens the scope of the clinical indications that could benefit from lsna therapies . [SEP]
[CLS] we show that the architecture of lsnas offers a delivery advantage over linear dna , as the dna derived from lsnas is observed in greater quantity in most tissues and circulation after 30 min and 24 h post - injection . [SEP]
[CLS] distribution differences between linear dna and lsnas are likely due to a decreased inflammatory cytokine response and a different clearance mechanism . [SEP]
[CLS] the sna architecture ' s ability to enhance nucleic B-material acid I-material transport across barriers B-property within the body and uptake into many cell B-material types may also drive these distribution differences . [SEP]
[CLS] in addition , we have shown that the affinity of dna to its liposome B-nanoparticle template affects the distribution of lsnas in vivo . [SEP]
[CLS] this is a particularly important design consideration for therapeutic lsna development . [SEP]
[CLS] chol - lsnas show high dna trafficking to the lungs , which could lead to therapeutic development for indications such as chronic obstructive pulmonary disease , pulmonary fibrosis , or lung cancer . [SEP]
[CLS] dppe - lsnas show high dna accumulation in the kidneys at the time points examined , which could be beneficial for treating glomerular diseases . [SEP]
[CLS] these lsnas also exhibit high accumulation in the spleen , indicating potential as cancer vaccines . [SEP]
[CLS] the dense dna shell B-material on lsnas also changes the tissue - level distribution of the liposome B-nanoparticle core B-material . [SEP]
[CLS] the chol - lsna architecture enhanced the delivery of the liposome B-nanoparticle core B-material to the liver and lungs , while the dppe - lsna architecture significantly increased the delivery of liposomes B-nanoparticle to the brain . [SEP]
[CLS] as the liposome B-nanoparticle components of lsnas can be loaded with other drug cargoes , lsnas have the potential to co - deliver drugs and nucleic B-material acids I-material to several major organs . [SEP]
[CLS] we envision that the readily tailorable distribution we describe in this article will inform further applications of this technology , especially in targets where structure dictates significant delivery enhancement . [SEP]
[CLS] dna oligonucleotides were synthesized using automated solid support phosphoramidite synthesis ( model : mm12 , bioautomation , inc . ) . [SEP]
[CLS] the sequence used , odn 2138 , has been previously shown to be nonimmunogenic in linear and sna form . [SEP]
[CLS] the free strand nontargeting ( odn 2138 ) sequence is 5 ′ - tccatgagcttcctgagctt - cy5 - ( spacer18 B-material ) - ( spacer18 ) - 3 ′ . [SEP]
[CLS] on the nanoparticle B-nanoparticle , the nontargeting ( odn 2138 ) sequence is 5 ′ - tccatgagcttcctgagctt - cy5 - ( spacer18 B-material ) - ( spacer18 ) - cholesterol - 3 ′ . [SEP]
[CLS] the dbcomodified nontargeting ( odn 2138 ) sequence is 5 ′ tccatgagcttcctgagctt - cy5 - ( spacer18 B-material ) - ( spacer18 ) dbcodt - 3 ′ . [SEP]
[CLS] all oligonucleotides were synthesized with a phosphorothioate ( ps ) backbone . [SEP]
[CLS] sequences were purified by high - pressure liquid B-technique chromatography I-technique ( hplc , agilent technologies ) and characterized using matrix - assisted laser desorption ionization - time - of - flight ( maldi - tof , bruker autoflex iii ) . [SEP]
[CLS] 1 , 2 - dioleoylsn - glycero - 3 - phosphocholine ( dopc ) , 1 , 2 - dipalmitoyl - sn - glycero - 3 - phosphoethanolamine - n - ( 6 - azidohexanoyl ) ammonium salt B-material ( azido - cap pe ) , and 1palmitoyl - 2 - ( dipyrrometheneboron difluoride ) undecanoyl - sn - glycero - 3 - phosphocholine ( topfluor tamra - pc ) , all purchased from avanti polar lipids B-material , inc . , were dissolved in chloroform and prepared into a lipid B-material film . [SEP]
[CLS] the solvent was evaporated under nitrogen B-material , and trace chloroform was removed under vacuum for several hours . [SEP]
[CLS] following this , the lipid B-material film was rehydrated in a buffer containing 20 mm hepes and 150 mm nacl ( ph 7 . 4 ) and freezethaw cycled several times . [SEP]
[CLS] the solution was then extruded through polycarbonate membranes of increasingly smaller pore size ( 100 nm , 80 nm , 50 nm ) until the resulting small B-material unilamellar I-material vesicles I-material were monodisperse with a hydrodynamic radius of ~ 50 nm as ascertained by dls ( malvern instruments ) . [SEP]
[CLS] the concentration of lipids B-material was determined via elemental analysis using icp - oes ( thermo fisher scientific ) . [SEP]
[CLS] dna loading to each nanoparticle B-nanoparticle was determined by measuring the dna absorbance at 260 nm of lsnas dissociated in 0 . 1 % sodium B-material dodecyl sulfate using a uv - vis spectrophotometer and measuring total phosphorus B-material concentration using icp - oes . [SEP]
[CLS] based on analysis of maximum cholesterol dna loading on 50 nm liposomes B-nanoparticle shown in figure s2 , 1 . 3 mm of total lipids B-material was mixed with 20 μm cholesterol - or dbco - terminated dna for 3 - 4 h at 37 °c under constant agitation . [SEP]
[CLS] the hydrodynamic radius and polydispersity were measured by dls . [SEP]
[CLS] lsna samples were negative stained with 2 % ( w / v ) uranyl acetate . [SEP]
[CLS] lsnas were drop cast on tem grids containing a carbon B-material film on 300 copper B-material mesh ( ted pella , inc . ) . [SEP]
[CLS] after 30 s , the liquid was wicked away using filter paper and the sample was rinsed twice with 20 mm hepes containing 150 mm nacl to remove particles not adhered to the grid . [SEP]
[CLS] subsequently , uranyl acetate stain solution was dropped onto the grid and removed four times using filter paper , and the grid was air - dried . [SEP]
[CLS] a jeol 1230 tem ( jeol , ltd . ) was used for imaging . [SEP]
[CLS] male mice ( c57bl / 6 ) in the age range of 8 - 12 weeks were obtained from the jackson laboratory and maintained in conventional housing . [SEP]
[CLS] all animals used were handled according to methods and procedures approved by the institutional animal care and use committee at northwestern university . [SEP]
[CLS] briefly , mice were given a single bolus injection of 50 μm linear dna or lsnas via peripheral intravenous injection . [SEP]
[CLS] at predetermined periods of time , mice were anesthetized using a 1 : 1 mixture of ketamine / xylazine , and blood was collected via cardiac puncture . [SEP]
[CLS] organs were cleared of blood by transcardial perfusion with 1× phosphate - buffered saline ( pbs ) . [SEP]
[CLS] once blood was removed via cardiac puncture , it was allowed to clot on ice . [SEP]
[CLS] samples were centrifuged at a minimum of 400g for 5 min . [SEP]
[CLS] the supernatant was isolated , immediately frozen , and stored until analysis . [SEP]
[CLS] the amounts of il - 6 , il - 10 , il - 12p70 , mcp - 1 , tnf , and ifnγ were measured using a flow - cytometry - based multiplexing assay ( cba mouse inflammation kit , bd biosciences ) on a facsymphony flow cytometer ( becton dickinson ) , and data were visualized using flowjo ( version 10 . 5 . 3 , flowjo llc ) . [SEP]
[CLS] organs for imaging were harvested , fixed in 10 % neutral buffered formalin overnight , and then stored in 1× pbs until imaging using an in vivo imaging system ( ivis , perkinelmer ) . [SEP]
[CLS] an excitation wavelength of 535 nm and an emission wavelength of 580 nm were used to visualize tamra - labeled lipids B-material , and an excitation wavelength of 640 nm and an emission wavelength of 680 nm were used to quantify the relative fluorescence B-property of the cy5 - labeled dna . [SEP]
[CLS] one - way anova was used to calculate significance between groups . [SEP]
[CLS] following ivis imaging , the same organs were placed in 15 - 30 % sucrose at 4 °c until the organs sunk to the bottom of the vial . [SEP]
[CLS] tissues were then embedded in a glycol / resin mixture ( tissue - tek o . c . t . ) and snap frozen using liquid nitrogen B-material . [SEP]
[CLS] tissues were cryosectioned to 5 μm slices and placed on glass slides . [SEP]
[CLS] the slides were stained with fluorescein - phalloidin and mounted with an antifade mountant containing 4 ′ , 6 - diamidino - 2 - phenylindole ( dapi ) ( thermo fisher scientific ) and imaged using an inverted microscope ( zeiss axio 7 inverted microscope with axiocam 506 mono ) . [SEP]
[CLS] organs for flow B-technique cytometry I-technique were harvested , minced , and incubated B-technique in an enzymatic digestion mixture ( collagenase with dnasei , with or without elastase ) for 30 min at 37 °c . [SEP]
[CLS] once digested , organs were sieved through a 70 μm cell B-material strainer and centrifuged . [SEP]
[CLS] red blood cell B-material lysis was performed as necessary ( gibco ) . [SEP]
[CLS] single cells B-material were washed with 1× pbs / 2 % bovine serum albumin and stored on ice . [SEP]
[CLS] cells B-material were stained for immune ( cd45 , cd3 , cd19 , cd11b , cd11c , cd68 ) and nonimmune ( epcam , cd31 , cd140a , cd34 ) markers ( becton dickinson , biolegend ) as well as with a fixable live / dead stain ( thermo fisher scientific ) . [SEP]
[CLS] cells B-material were fixed in neutral buffered formalin after staining . [SEP]
[CLS] analysis of dna association was done using a facsymphony flow cytometer ( becton dickinson ) , and data were visualized using flowjo ( version 10 . 5 . 3 , flowjo llc ) . [SEP]
[CLS] one - way anova was used to calculate significance between treatment groups . [SEP]
[CLS] all results are expressed as the mean ± se or mean ± sd and number of biological replicates ( n ) as noted in the figure captions . [SEP]
[CLS] outliers were removed using the rout method with a false discovery rate ( q ) of 1 % . [SEP]
[CLS] ivis data of whole organ fluorescence B-property of the cy5 - dna and tamra liposomes B-nanoparticle are normalized to the untreated organs ; hence all bar graphs are reported in fold fluorescence B-property enhancement over untreated organs . [SEP]
[CLS] one - way analysis of variance ( anova ) was performed and tukey ' s post hoc test was used for multiple comparisons when the result was significant ( p < 0 . 05 ) . [SEP]
[CLS] in analyzing the flow B-technique cytometry I-technique data ( figure 5 ) , significance tests were applied to the % cy5 positive cells B-material in each [SEP]
[CLS] oligonucleotide components used to synthesize each lsna . [SEP]
[CLS] ( a ) structures of 3 ′ dna tails that anchor dna to each liposome B-nanoparticle template . [SEP]
[CLS] ( b ) each dna sequence is reacted with either 50 nm liposomes B-nanoparticle composed of ( top ) 100 % dopc or ( bottom ) 95 % dopc / 5 % dppe - azide ( mol / mol ) to form each respective lsna . [SEP]
[CLS] effect of linear oligonucleotides and lsnas on cytokine production . [SEP]
[CLS] cytokines were measured following intravenous administration of linear dna , chol - lsnas , or dppe - lsnas at ( a ) 30 min and ( b ) 24 h post - injection in c57 / bl6 mice ( nd = not detectable ; statistical significance was calculated by one - way anova with tukey ' s post hoc test ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 , error bars represent standard error , n = 3 ) . [SEP]
[CLS] whole organ analysis of dna trafficking . [SEP]
[CLS] following iv injection of lsnas , organs were harvested at ( a , b ) 30 min and ( c , d ) 24 h and imaged ex vivo . [SEP]
[CLS] tamra - pc ( liposome B-nanoparticle ) and cy5 ( dna ) fluorescence B-property were separated using the spectral unmixing function of the ivis instrument . [SEP]
[CLS] relative tissue level distribution normalized to untreated mice was assessed by imaging all organs simultaneously ( a , c ) . [SEP]
[CLS] individual organs were imaged , and the relative fluorescence B-property was calculated at ( b ) 30 min and ( d ) 24 h ( n = 3 - 5 ; statistical significance was calculated by one - way anova with tukey ' s post hoc test ; * p < 0 . 05 , * * p < 0 . 01 , error bars represent standard error ) . [SEP]
[CLS] distribution of cy5 - labeled dna within tissues . [SEP]
[CLS] the distribution of cy5 - labeled linear dna , chol - lsnas , and dppe - lsnas in the liver , lungs , spleen , and kidneys at ( a - d ) 30 min and ( e - h ) 24 h post - injection ( blue = dapi ( nuclear stain ) , green = phalloidin ( actin filament stain ) , red = cy5 - dna , taken at 40× magnification , scale bar = 20 μm ) . [SEP]
[CLS] analysis of cellular distribution using flow B-technique cytometry I-technique . [SEP]
[CLS] flow B-technique cytometry I-technique was used to assess the total accumulation of cy5 - dna in immune and nonimmune cells B-material from linear dna and lsnas in the liver , lungs , spleen , and kidneys at ( a - d ) 30 min and ( e - h ) 24 h ( n = 3 postinjection ; statistical significance comparing percent cy5 + cells B-material was calculated by one - way anova with tukey ' s post hoc test ; * p < 0 . 05 , error bars represent standard deviation ) . [SEP]
[CLS] rapid , multiplex , and quantitative detection of sequencespecific or mutated genes associated with human diseases has played a central role in modern clinical treatments of molecular diagnostics and genomics research . [SEP]
[CLS] over the past decades , several advances of novel technologies and methods have been achieved . [SEP]
[CLS] there are however everincreasing requirements for improving analytical capabilities , in particular for signal multiplexing and precise quantification [SEP]
[CLS] nowadays , among various optical approaches , the fluorescence - based method has been mostly applied . [SEP]
[CLS] as it suffers from the drawbacks of spectral overlap and multiwavelength excitation , which fundamentally limit the number of available fluorophores , simultaneous detection of more than ten dna targets in a single analysis would be hardly performed even using quantum B-nanoparticle dots I-nanoparticle . [SEP]
[CLS] recently , color - coding microparticle technologies have been developed to solve this problem by making use of an immense number of available encoding beads . [SEP]
[CLS] one challenge is the accurate and precise quantification because of their polydispersity and nonbicompatibility , as they have micrometer sizes . [SEP]
[CLS] furthermore , the complex fabrication process may hamper their broad usability . [SEP]
[CLS] therefore , there has been interest to explore a nonoptical labeling and detecting method , which is able to simplify labeling process and provide high - level multiplexing as well as precise determination . [SEP]
[CLS] an elemental labeling strategy for bioassays B-technique has been regarded as an emerging method in which large biomolecules are labeled with elemental tags and subsequently detected by elemental mass spectrometry , such as inductively coupled plasma mass spectrometry ( icp - ms ) . [SEP]
[CLS] compared with other methods , it contains two inherent advantages : 1 ) owing to the benefits of a large number of elements or isotopes B-material ( up to 100 ) potentially used as elemental tags , as well as excellent mass resolution and multi - element detectors of icp - ms , high - level multiplexed analysis can be successfully obtained without the limitation of spectral overlap ; 2 ) icp - ms has allowed isotope B-material ratio measurement with good accuracy and precision , thus in combination with isotope B-material dilution analysis ( ida ) , absolute - quantitative measurement can be carried out as the complementary use of molecular mass spectrometry . [SEP]
[CLS] in the last decade , elemental labeling bioassays B-technique have been successfully applied in the fields of enzyme - linked immunosorbent assay ( elisa ) , quantitative protomics , and single - cell B-material biology . [SEP]
[CLS] unfortunately , the value of elemental labeling in nucleic B-material acid I-material analysis has not been fully understood , and there are only a few relevant studies in the previous publications . in our opinion , the reason might be that there has been no highly specific and efficient approaches for labeling nucleic B-material acid I-material with elemental tags . [SEP]
[CLS] another aspect might be that the current methods of absolute quantification commonly require to couple icp - ms with time - and labor - intensive separation procedures ; for example , chromatography B-technique or gel B-technique electrophoresis I-technique . [SEP]
[CLS] thus , great efforts should be taken to overcome these shortages of elemental labeling bioassays B-technique and extend its applications to quantitative nucleic B-material acid I-material analysis . [SEP]
[CLS] herein , we report for the first time multiplex nucleic B-material acid I-material assays based on the elemental labeling strategy , which take advantages of dna hybridization reactions for specific recognition , rare - earth elements for multiplex labeling , magnetic B-property microparticles for fast separation , and icp - ms for ultrasensitive detection . [SEP]
[CLS] importantly , both absolute and relative quantification could be performed for multiplex analysis of dna targets . [SEP]
[CLS] as a proof - of - concept study of highlevel multiplexing , 15 dna targets associated with clinical diseases were simultaneously detected by elemental labeling tags of y , la , ce , pr , nd , sm , eu , gd , tb , dy , ho , er , tm , yb , and lu . [SEP]
[CLS] the relative quantification was carried out by using internal calibration curves . [SEP]
[CLS] additionally , the absolute quantification with chromatography - free hybridization isotope B-material dilution analysis ( hida ) was developed . [SEP]
[CLS] taking advantage of labeling the artificially 161 dy - enriched and 168 er - enriched isotopes B-material with dilution probes , two dna targets were analyzed absolutely and simultaneously . [SEP]
[CLS] as shown in scheme 1 a , the procedure of labeling sequence - specific oligonucleotides with elemental tags involves two steps : 1 ) oligonucleotides , 3 ' end - functionalized with thiol B-material ( a sh ) groups , were specifically derivatized with malemide groups of 1 , 4 , 7 , 10 - tetraazacyclododecane - 1 , 4 , 7tris - aceticacid - 10 - maleimidoethylacetamide ( mma - dota ) , a compound commonly employed for chemical labeling of proteins B-material or peptides B-material ; 2 ) rare - earth elements ( rees ) were chelated with high kinetic and thermodynamic stability ( for reaction conditions ( ph , mole ratio of mma - dota to dna , time , and temperature ) , see the supporting information ) . [SEP]
[CLS] as there was only one a sh group in the dna , the stoichiometry between dna and ree ions B-material in rees - labeled dna complexes was a 1 : 1 mole ratio . [SEP]
[CLS] the distinguishing feature using rees as elemental tags is that they have similarly chemical properties , a low background in biological samples , and high the labeling procedures were monitored by matrixassisted laser desorption / ionization B-property time - of - flight mass spectrometry ( maldi - tof - ms ) . [SEP]
[CLS] the products of each step were purified by high - performance liquid B-technique chromatography I-technique ( hplc ) . [SEP]
[CLS] finally , the specificity of elemental labeling products was validated by icp - ms measurement . [SEP]
[CLS] for instance , hepatitis a virus ( hav ) target probe ( the sequence is given in the supporting information , table s2 ) was labeled with the element la . [SEP]
[CLS] 1 a showed the maldi - tof - ms results . [SEP]
[CLS] the determined molecular weight ( dw ) of hav probe was 7878 . 2 , corresponding to the theoretical molecular weight ( tw ) of 7873 . 2 ; the dw of the first - step product was 8406 . 9 , corresponding to dna - dota complex tw of 8401 . 7 ; the dw of the second - step product was 8541 . 4 , corresponding to dna - dota - la complex tw of 8537 . 6 . [SEP]
[CLS] the mass difference of 4 - 5 between the measured and theoretical molecular weight was due to the mass measurement errors of the maldi - tof - ms employed . [SEP]
[CLS] finally , after purification by hplc ( figure 1 b ) , the product of dna - dota - la complex was confirmed by icp - ms profile spectra ( m / z 135 to 180 ) in figure 1 c ( hav - la ) . [SEP]
[CLS] it was also confirmed that the other 13 sequence - specific dna probes were successfully labeled with 13 different rees , respectively . [SEP]
[CLS] the results of maldi - tof - ms are provided in the supporting information , figure s2 . [SEP]
[CLS] by using the same protocedure , 15 rees in total , including y , and two stable isotopes B-material ( 161 dy - enrichment and 168 er - enrichment ) , as well as indium B-material ( in ) , were successfully labeled with the sequence - specific dna probes , respectively . [SEP]
[CLS] the relative quantification of multiplexed dna assays was first investigated , and the principle is illustrated in scheme 1 b . [SEP]
[CLS] in the experiments , 15 dna targets with 25 - 30 bases related to clinical diseases ( cancer , heredopathia , and virus ) and a control target for internal standard were used as the model systems . [SEP]
[CLS] the sequences of these dna targets and their designed capture and report probes are given in the supporting information , table s2 . [SEP]
[CLS] the suspension of 2 . 8 mm magnetic B-property microparticles ( mmps ) was functionalized with 16 thiolated capture probes , which were complementary to one half region of the targets of interest . [SEP]
[CLS] then they were incubated B-technique with the mixture of dna targets and 15 ree - labeled report probes ( hav - la , hbv - ce , hcv - pr , hiv - nd , hpv - sm , ev - eu , tp - gd , vv - tb , ba - dy , ft - ho , sras - er , bc - tm , ad - yb , scd - lu , pc - y ) as well as inlabeled report probes ( is - in ) , which were complementary to the other half region of the targets . [SEP]
[CLS] the sandwich hybridization reactions led to the formation of mmp - target - element complexes . [SEP]
[CLS] subsequently , by using magnetic B-property separation , the excess rees - labeled report probes were removed . [SEP]
[CLS] finally , by increasing the temperature to 95 8c , ree - labeled report probes were allowed to be released from the mmp - target - element complexes to the supernatant and were measured by icp - ms ( for experimental details , see the supporting information ) . [SEP]
[CLS] in this assay , the identification of each dna target was performed by the recognition of m / z spectrum of its own labeled rees , and the quantification was dependent on the ion B-material sensitivity quantitatively measured by icp - ms . [SEP]
[CLS] the majority of rees had more than one stable isotope B-material , some of which have mass spectral overlap ( as shown in figure 1 c ) . [SEP]
[CLS] however , they had at least one overlap - free isotope B-material for the detection ( the isotopes B-material and their abundance of each ree are listed in the supporting information , table s3 ) . [SEP]
[CLS] thus , 15 rees could be simultaneously detected in a single analysis . [SEP]
[CLS] in this approach , the isotopes B-material of 89 y , 139 la , 140 ce , 141 pr , 146 nd , 147 sm , 153 eu , 158 gd , 159 tb , 163 dy , ho , 166 er , 169 tm , 172 yb , and 175 lu were utilized for the simultaneous detection of 15 dna targets without mass spectral overlap . [SEP]
[CLS] consequently , the amounts of dna targets were referring to the signals of these isotopes B-material in icp - ms . [SEP]
[CLS] 2 a illustrates the analytical results of 15 dna targets with four levels of concentrations ( 5 , 10 , 15 , and 20 pmol ) . [SEP]
[CLS] in the spectra , each unique isotope B-material identified one dna target and the channel counts of isotopes B-material increased according to the amount of dna targets . [SEP]
[CLS] however , different intensities had been obtained among different dna targets with the same concentration . [SEP]
[CLS] the reason was that the measured isotopes B-material had the different abundance . [SEP]
[CLS] in the quantification procedure by calibration curves , an internal standard ( is ) target with constant concentration ( 10 pmol ) , the probe of which was labeled with the element in , was analyzed along with the samples . [SEP]
[CLS] in each assay , the is target was added to the sample solutions and operated under the same condition as other targets . [SEP]
[CLS] thus , it could account for sample losses and variation from sample preparation and separation steps , and correct the biased measurement of icp - ms . [SEP]
[CLS] as a result , the calibration curves of relative intensities of rees ( corresponding to the signal ratio between rees and in ) versus the concentrations of the dna targets had been obtained with good linearity . [SEP]
[CLS] for example , figure 2 b illustrates the calibration curves of 175 lu / 115 in versus the scd target . [SEP]
[CLS] the achieved detection limits of the dna targets in this method were as low as 0 . 5 - 2 pmol . [SEP]
[CLS] the linear range was from 0 . 5 to 20 pmol and could be extended by increasing the dosage of the mmps . [SEP]
[CLS] moreover , as there are nearly 40 isotopes B-material of rees , the multiplexing capability of the proposed method could be further improved . [SEP]
[CLS] as mentioned above , the relative - quantification approach was a comparative analysis and quantified the analytical data of dna samples by calibration curves by using known amount standard nucleic B-material acids I-material , which were perfectly matched with the sequences of the targets . [SEP]
[CLS] however , an absolutequantification bioassay B-technique , requiring no standard references relying on the targets , could directly provide the precise amount of targets . [SEP]
[CLS] in recent studies , there has been no examples of sequence - specific and multiplex dna analysis in this type of absolute - quantification method . [SEP]
[CLS] to address this issue , we propose a novel method for the absolute dna quantification by coupling isotope B-material dilution analysis with dna hybridization reactions . [SEP]
[CLS] the principle is shown in scheme 2 . [SEP]
[CLS] it can be seen that dilution probes ( dps ) , which were labeled with artificially enriched isotopes B-material , could directly hybridize with the capture probes ( cps ) immobilized on the mmps regardless of dna targets . [SEP]
[CLS] in contrast , report probes ( rps ) , which were labeled with natural rees , are coupled with cps through the hybridization of dna targets . [SEP]
[CLS] for a constant amount of dps and excess amount of rps , the dna targets determined the ratio of rps / dps ( r rps / dps ) in the hybridized complexes of cps . [SEP]
[CLS] r rps / dps could be indicated by the ratio of m 2 / m 1 ( r m 2 / m 1 ) . [SEP]
[CLS] therefore , by quantifying the amount of dps , as well as measuring r m 2 / m 1 in the hybridized cps , rps , and dps , the amount of dna targets could be calculated according to isotope B-material dilution functions ( see the supporting information ) . [SEP]
[CLS] to demonstrate the feasibility of the hida method for multiplex dna assay , a two - strand system was designed as shown in figure 3 , where two sequence - specific cps were used for simultaneous hybridization with ba and sars targets , respectively . [SEP]
[CLS] the natural dy and er were labeled with their rps , while 161 dy - enriched and 168 er - enriched isotopes B-material were labeled with their dps . [SEP]
[CLS] the mass numbers of 161 , 163 , scheme 2 . [SEP]
[CLS] the hybridization isotope B-material dilution analysis ( hida ) strategy . [SEP]
[CLS] the hida system was composed of four parts : capture probes , report probes , dilution probes , and dna targets . [SEP]
[CLS] the capture probes had two regions : the former region directly hybridized with dilution probes , and the latter region from sandwich hybridization with dna targets and report probes . [SEP]
[CLS] ln was a natural rare - earth element , while * ln was an artificially enriched isotope B-material . [SEP]
[CLS] they had similar chemical and physical properties , but the isotope B-material abundance at the mass numbers of m 1 and m 2 . [SEP]
[CLS] isotope B-material ratio measurement was carried out by icp - ms to analyze the change in the isotope B-material ratio of m 2 / m 1 resulting from the hybridization of dna targets . [SEP]
[CLS] . 166 , and 168 were monitored by icp - ms for isotope B-material ratio measurement , and there was no mass spectral overlap of them in the elements dy and er . [SEP]
[CLS] in the experiments , 100 ml of mmps ( 1 - 2 10 9 beads ml a1 ) was first mixed with 500 pmol of ba and sras target cps , allowed to react for 4 h with gentle shaking at room temperature , then washed with coupling buffer to remove unreactive cps . [SEP]
[CLS] after the preparation of capture - probe - functionalized mmps , 200 pmol of excess ba and sras target rps and a certain amount of the samples were added . [SEP]
[CLS] subsequently , 159 . 89 ae 0 . 35 ng of 161 dylabeled dps and 171 . 89 ae 0 . 28 ng of 168 er - labeled dps , quantitatively measured by icp - ms previously , were spiked to the mixtures . [SEP]
[CLS] thereafter , dna hybridization , sample separation , and icp - ms measurement were operated in a similar fashion to the relative quantification . [SEP]
[CLS] 4 indicated the results of icp - ms profile spectra of three samples containing different concentrations of ba and sars targets ( amount increasing from 1 to 3 ) . [SEP]
[CLS] as a result , the signals of mass number 163 and 166 increased more remarkable than 161 and 168 , with the increase of dna targets . [SEP]
[CLS] original isotope B-material ratio of 161 / 163 ( r 161 / 163 ) in natural dy and 161 dy - enriched isotope B-material were 0 . 76 and 106 . 37 , respectively ; the original isotope B-material ratio of 168 / 166 ( r 168 / 166 ) in natural er and 168 er - enriched isotope B-material were 0 . 80 and 157 . 68 , respectively . [SEP]
[CLS] compared with them , in the hida assays of the sample 1 , 2 , and 3 , r 161 / 163 changed from 8 . 21 ae 0 . 05 , 4 . 18 ae 0 . 04 , to 2 . 78 ae 0 . 05 ; and r 168 / 166 changed from 11 . 29 ae 0 . 07 , 5 . 42 ae 0 . 04 , to 3 . 20 ae 0 . 05 . [SEP]
[CLS] therefore , the dna targets coupled rps with cps and resulted in the change of r rps / dps . [SEP]
[CLS] 1 shows the results of absolute quantification of ba and sars targets in the sample 1 , 2 , and 3 , according to the measured isotope B-material ratios , the amount of dps spiked , and the isotope B-material dilution equations ( supporting information , equations s1 and s2 ) . [SEP]
[CLS] to validate the accuracy of the hida method , the uv absorption B-technique spectroscopy I-technique was applied to quantify the amount of ba and sars targets in the samples by measuring optical density in 260 nm . [SEP]
[CLS] these results could be considered as reference values , and the results obtained by the hida method were in good agreement with them . [SEP]
[CLS] the recovery was 93 . 7 - 106 . 5 % , which indicated that the proposed approach was an accurate method . [SEP]
[CLS] furthermore , the multiplexing ability of this method could be extended by simultaneously detecting more isotopes B-material of rees . [SEP]
[CLS] however , as one dna target required two isotopes B-material of rees for the absolute analysis , its multiplexing capability was reduced compared with the relative approach mentioned above . [SEP]
[CLS] furthermore , compared with species - specific or species - unspecific isotope B-material dilution methods in proteomics , hida for dna analysis , taking advantage of sequence - specific hybridization reactions , led to absolute quantification without using chromatographic or electrophoretic separation B-technique techniques I-technique . [SEP]
[CLS] in conclusion , we have demonstrated a novel elemental labeling method for the absolute and relative quantification of multiplex dna assays . [SEP]
[CLS] this is the first report that thiolfunctionalized dna was covalently coupled with rees by mma - dota , and 15 dna targets could simultaneously be detected in a single analysis . [SEP]
[CLS] furthermore , the isotope B-material dilution analysis based on hydrolization reactions was successfully achieved for absolute dna quantification . [SEP]
[CLS] the method features high - level multiplexing and precise quantification for dna analysis . [SEP]
[CLS] it is noteworthy that because the elemental tags labeled with dna probes are not limited to rees , this method could be further extended in its multiplexing by the use of other elements or isotopes B-material , such as noble and transition metals B-material . [SEP]
[CLS] furthermore , in contrast to other molecular detection techniques , the signal of icp - ms is independent of the species and sample matrix , which would provide accurate determination in the real samples . [SEP]
[CLS] although this work is a proof - ofconcept study , it could be anticipated that this method would boost the development of genetic analysis research and has potential applications in molecular diagnostics of personalized medicine . [SEP]
[CLS] our ongoing work will investigate the feasibility of elemental labeling strategy in the field of absolutequantitative pcr assay and high - throughput multiplex dna sequencing . [SEP]
[CLS] reference results [ ng ] determined results [ ng ] reference results [ ng ] [ a ] determined by the hida method ( n = 5 ) . [SEP]
[CLS] [ b ] determined by uv absorption spectrometry at 260 nm ( n = 5 ) . [SEP]
[CLS] * ] g . han , s . zhang , z . xing , prof . x . zhang beijing key laboratory for microanalytical methods and instrumentation , department of chemistry tsinghua university beijing 100084 ( china ) [SEP]
[CLS] e - mail : xrzhang @ mail . tsinghua . edu . cn sensitivity in icp - ms . [SEP]
[CLS] importantly , dota - ree chelates as labeling reagents are water B-property - I-property soluble B-property and biocompatible B-property . [SEP]
[CLS] multiplex dna assays based on the elemental labeling strategy . [SEP]
[CLS] scheme 1 . a ) labeling dna with elemental tags . [SEP]
[CLS] the oligonucleotide with an sh group was conjugated with mma - dota , and then rareearth elements , stable isotopes B-material , and indium B-material were chelated in the macrocycle dota . [SEP]
[CLS] b ) multiplex dna assay procedures . [SEP]
[CLS] first , dna targets were added to the suspension array of capture - probe functionalized magnetic B-property microparticles ( mmps ) , and mixed with elemental labeling dna probes . [SEP]
[CLS] subsequently , the sandwich conjugates of mmp - dna - element complexes were synthesized by hybridization reactions . [SEP]
[CLS] then , by magnetic B-property separation and temperature increase above the melting temperature ( t m ) , the elemental labeling dna probes were released to the supernatant , which was quantitatively measured by icp - ms . [SEP]
[CLS] characterization of rare - earth - labeled dna probes . [SEP]
[CLS] figure 1 . a ) the maldi - tof - ms results of la - labeled hav probes . [SEP]
[CLS] the determined mass of 7878 . 2 was from thiol - functionalized dna , 8406 . 9 from the product of dna - dota complexes , and 8541 . 4 from the product of dna - dota - la complexes . [SEP]
[CLS] b ) purity test of dna - dota - la complexes by hplc . [SEP]
[CLS] the retention time was 13 . 55 min . [SEP]
[CLS] c ) icp - ms profile spectra of 14 dna probes labeled with different rees from la to lu . [SEP]
[CLS] in the determination of each purified product , the icp - ms scan region was from m / z 135 to 180 , with a dwell time of 100 ms . [SEP]
[CLS] figure 2 . a ) icp - ms profile spectra of 15 dna targets with four levels of concentrations . [SEP]
[CLS] in consideration of some mass overlaps , isotopes B-material of rees were chosen for the simultaneous determination , including 89 y , 139 la , 140 ce , 141 pr , 146 nd , 147 sm , 153 eu , 158 gd , 159 tb , 163 dy , 165 ho , 166 er , 169 tm , 172 yb , and 175 lu . 115 in was monitored as an internal standard in the experiments . [SEP]
[CLS] additionally , 15 clinically relevant genetic targets , respectively related to hav , hbv , hcv , hiv , hpv , ev , tp , vv , b . anthracis , f . tularensis , sars , coronavirus , breast cancer , prostate cancer , alzheimer ' s disease , as well as sickle cell B-material disease , were simultaneously analyzed with four concentrations of 5 , 10 , 15 , and 20 pmol . [SEP]
[CLS] the internal standard target was 10 pmol . [SEP]
[CLS] b ) the internal calibration curves of relative intensities of 175 lu / 115 in versus the concentrations of the scd targets ( containing blank sample ) . [SEP]
[CLS] for other calibration curves , see the supporting information , figure s3 . [SEP]
[CLS] received : august 26 , 2012 published online : december 13 , 2012 . [SEP]
[CLS] keywords : absolute quantification • dna assay • elemental labeling • mass spectrometry • rare earth elements [SEP]
[CLS] dna sequences and labeled elements of hida for ba ( top ) and sars ( bottom ) targets . [SEP]
[CLS] the ba report probe 3 ' end was labeled with natural dy ( 161 , abundance of 18 . 91 % ; 163 , abundance of 24 . 90 % ) ; the ba dilution probe 3 ' end was labeled with 161 dy - enriched isotope B-material ( 161 , abundance of 95 . 73 % ; 163 , abundance of 0 . 90 % ) . [SEP]
[CLS] the sras report probe 3 ' end was labeled with natural er ( 166 , abundance of 33 . 61 % ; 168 , abundance of 26 . 78 % ) ; the sras dilution probe 3 ' end was labeled with 168 er - enriched isotope B-material ( 166 , abundance of 0 . 62 % ; 168 , abundance of 97 . 76 % ) . [SEP]
[CLS] each capture probe had 47 bases in which 10 - base poly a was used as the spacer B-material in both 5 ' end and middle of the oligonucleotide . [SEP]
[CLS] icp - ms profile spectra of three samples ( 1 , 2 , 3 ) . [SEP]
[CLS] scan regions were m / z 160 . 5 - 161 . 5 , 162 . 5 - 163 . 5 , 165 . 5 - 166 . 5 , and 167 . 5 - 168 . 5 , with a dwell time of 50 ms . [SEP]
[CLS] the results of isotope B-material ratios of five determinations averaged were : i 161 / i 163 = 8 . 21 ae 0 . 05 ( 1 ) , 4 . 18 ae 0 . 04 ( 2 ) , 2 . 78 ae 0 . 05 ( 3 ) and i 168 / i 166 = 11 . 29 ae 0 . 07 ( 1 ) , 5 . 42 ae 0 . 04 ( 2 ) , 3 . 20 ae 0 . 05 ( 3 ) . [SEP]
[CLS] absolute quantification analysis of two dna targets . [SEP]
[CLS] going beyond the limits of optical biosensing motivates exploration of signal amplification strategies that convert a single molecular recognition event into a response equivalent to hundreds of fluorescent B-property dyes . [SEP]
[CLS] in this respect , forster resonance energy transfer ( fret ) with bright fluorescent B-nanoparticle nanoparticles I-nanoparticle ( nps B-nanoparticle ) is an attractive direction , but it is limited by poor efficiency of nps B-nanoparticle as fret donors , because their size is typically much larger than the forster radius ( ~ 5 nm ) . [SEP]
[CLS] here , we established fret - based nanoparticle B-nanoparticle probes that overcome this fundamental limitation by exploiting a phenomenon of giant light harvesting with thousands of strongly coupled dyes in a polymer B-material matrix . [SEP]
[CLS] these nanoprobes B-nanoparticle are based on 40 - nm dye - loaded poly ( methyl methacrylate - co - methacrylic acid ) ( pmma - ma ) nps B-nanoparticle , so - called light - harvesting nanoantennas , which are functionalized at their surface with oligonucleotides . [SEP]
[CLS] to achieve this functionalization , we developed an original methodology : pmma - ma was modified with azide / carboxylate B-material bi - functional B-material group I-material that enabled assembly of small polymeric nps B-nanoparticle and their further cu - free click coupling with oligonucleotides . [SEP]
[CLS] the obtained functionalized nanoantenna behaves as giant energy donor , where hybridization of target nucleic B-material acid I-material ( encoding survivin cancer marker ) with ~ 23 grafted oligonucleotides / cy5 - acceptors switches on / off fret from ~ 3200 rhodamine - donors of the nanoantenna , leading to 75 - fold signal amplification . [SEP]
[CLS] in solution and on surfaces at single - particle level , the nanoprobe B-nanoparticle provides sequence - specific two - color ratiometric response to nucleic B-material acids I-material with limit of detection reaching 0 . 25 pm . [SEP]
[CLS] it displays unprecedented brightness for a fret biosensor : it outperforms analogous fret - based molecular probe by > 2000 - fold and qdot - 605 by ~ 100 - fold . [SEP]
[CLS] the developed concept of amplified sensing will increase orders of magnitude sensitivity of fluorescent B-property probes for biomolecular targets . [SEP]
[CLS] the problem of limited brightness of fluorescent B-property dyes 1 in biomolecular sensing stimulated development of amplification mechanisms that convert a single molecular recognition event into a response equivalent to hundreds of fluorescent B-property dyes . [SEP]
[CLS] beyond strategies that multiply ( chemically / enzymatically ) the number of molecular species , 3 concepts of direct amplification of dye emission have been developed . [SEP]
[CLS] first one is based on conjugated polymers B-material , where a large number of aromatic unites strongly coupled by pi - conjugation can efficiently transfer the excitation energy to a single energy acceptor . [SEP]
[CLS] second one uses plasmonic nanostructures ( nanoantennas ) , where fluorescence B-property of single molecules located in the " hot spots " can be amplified . [SEP]
[CLS] fluorescent B-nanoparticle nanoparticles I-nanoparticle ( nps B-nanoparticle ) , 6 such as quantum B-nanoparticle dots I-nanoparticle ( qdots ) , 7 dye - doped silica , 8 aggregation - induced emission , 9 conjugated polymer B-material 10 and dye - loaded polymer B-material nps B-nanoparticle , being much brighter than organic dyes , are promising scaffolds for fabrication of biosensors with signal amplification . [SEP]
[CLS] to convert fluorescent B-property nps B-nanoparticle into biosensors ( nanoprobes B-nanoparticle ) , forster resonance energy transfer ( fret ) is generally used , where the particle serves as energy donor , while energy acceptors with target - recognition units are grafted to its surface . [SEP]
[CLS] 12 however , to achieve direct signal amplification in this case , efficient fret from the whole particle to a single acceptor should be achieved , which is a fundamental challenge because the particle size is generally much larger than the forster radius ( ~ 5 nm ) . [SEP]
[CLS] for instance , in case of qdots , which are probably the most popular nps B-nanoparticle for fabrication of biosensors , 12b efficient fret can be obtained only when significant amounts of fret acceptors ( 10 - 50 ) are attached to their surface through a thinnest possible organic B-material shell I-material . [SEP]
[CLS] dye - loaded polymeric nps B-nanoparticle are particularly attractive alternative to qdots , because of their superior brightness , biocompatible B-property / biodegradable B-property matrix and low cytotoxicity B-property . [SEP]
[CLS] very recently , following a concept used by nature in chlorophyll , we designed dye - loaded polymeric nps B-nanoparticle operating as giant light - harvesting nanoantenna . [SEP]
[CLS] its > 10 , 000 rhodamine dyes , assembled together by bulky counterions within in 60 - nm poly ( methyl methacrylate - co - methacrylic acid ( pmma - ma ) - based nanoparticle B-nanoparticle , transferred efficiently the energy to few fret acceptors inside the particle , leading to amplification of their emission > 1000fold . [SEP]
[CLS] it surpassed plasmonics - based amplification values , and enabled first observation of single molecules in sunlight conditions . [SEP]
[CLS] however , in order to transform them into probes for biomolecules , surface chemistry of these unique nanomaterials B-material remains to be developed . [SEP]
[CLS] ubiquitous nature of nucleic B-material acids I-material ( nas ) makes them highly important targets for detection in biological research and medical diagnostics . [SEP]
[CLS] their ultra - low concentrations in biological samples , below the detection limit of fluorescent B-property molecular probes , require molecular multiplication techniques , notably polymerase chain reaction ( pcr ) , a multi - step process that uses complex mixture of reagents , sophisticated equipment and well - trained staff . [SEP]
[CLS] chemists developed variety of fluorescent B-property probes that can access much lower concentrations of nas , up to few pm , but they also use molecular multiplication , based on enzymes 3b or hybridization chain reactions . [SEP]
[CLS] to achieve direct one - step amplification , without molecular multiplication , cationic B-material conjugated polymers B-material were initially proposed , 4b because they can report on sequence specific hybridization improving detection limits down to 10 pm range . [SEP]
[CLS] however , their limitations are non - specific interaction with other na duplexes and proteins B-material , and strong dependence on the ionic strength . [SEP]
[CLS] noble metal nanoparticles I-nanoparticle offered new opportunities in na detection . [SEP]
[CLS] very recently , tinnefeld and co - workers pioneered the amplified detection of nas based on plasmonics nanoantenna positioned precisely in space with help of dna origami . [SEP]
[CLS] the amplification achieved was 7 . 3 on average , although higher values were observed for some nps B-nanoparticle . [SEP]
[CLS] in this respect , our organic nanoantennas are expected to offer new possibilities . [SEP]
[CLS] we hypothesize that giant light - harvesting in the organic nanoantenna can be used to establish a concept of amplified fret - based sensing , where a few nucleic B-material acid I-material hybridization events at the nanoantenna surface can trigger fluorescence B-property response of thousands of dyes . [SEP]
[CLS] to this end , a methodology of chemical functionalization of the pmma - ma - based nanoantenna with oligonucleotides has to be developed first . [SEP]
[CLS] moreover , to achieve efficient fret , the size of the nanoantenna particle should be optimal ( 40 - 50 nm ) and the distance from its surface to the fret acceptor should be minimized . [SEP]
[CLS] however , small pmma - ma - based nps B-nanoparticle bearing nucleic B-material acids I-material have not been reported to date . [SEP]
[CLS] the problem is inefficiency of the direct coupling of hydrophobic B-property polymers B-material ( including pmma - ma derivatives ) with oligonucleotides because of difficulty to solubilize together highly apolar and polar species , 26 so that solid phase synthesis is required . [SEP]
[CLS] on the other hand , the examples of direct modification of polymeric nps B-nanoparticle by oligonucleotides are limited to block - copolymers containing relatively large hydrophilic B-property units with peg linkers , which is incompatible with our fret strategy . [SEP]
[CLS] based on our recent work , showing that a single charge per polymer B-material is sufficient to assemble small nps B-nanoparticle by nanoprecipitation , we expect that addition of a reactive group next to this charged group will enable preparation of functionalizable nanoantennas . [SEP]
[CLS] in the present work , we developed a robust approach to functionalize small ( < 50 nm ) polymeric nps B-nanoparticle with oligonucleotides and thus provided the first platform based on dye - loaded organic nanoantennas for amplified sensing of biomolecules . [SEP]
[CLS] due to light - harvesting principle , we achieved efficient fret from ~ 3200 donor dyes inside nanoantenna to ~ 23 acceptors hybridized at the particle surface , leading to average signal amplification of 75 . [SEP]
[CLS] being ~ 100 - fold brighter than qdot - 605 , the nanoprobes B-nanoparticle produce donor / acceptor emission ratio response to just 3 - 4 na hybridizations per particle , which makes them the brightest nanoprobes B-nanoparticle reported to date . [SEP]
[CLS] the nanoprobe B-nanoparticle was designed to detect a fragment of nucleic acid encoding survivin protein B-material , an important anti - apoptotic cancer marker . [SEP]
[CLS] the obtained nanoprobe B-nanoparticle operates in solution and on surfaces delivering limit of detection for the na target of 5 and 0 . 25 pm , respectively , which is 1000 - 10 , 000 fold lower than that achieved using molecular probes . [SEP]
[CLS] given the universality of fret - based detection concept , our functionalizable organic nanoantennas open the route to a broad range of ultrabright probes for amplified detection of biomolecules . [SEP]
[CLS] at the surface of nanoparticle B-nanoparticle . [SEP]
[CLS] target na displaces short oligonucleotide with fret acceptor ( tcs - cy5 ) from the np B-nanoparticle surface , resulting in the fret loss , so that the emission of the probe switches from red ( acceptor ) to green ( donor ) . [SEP]
[CLS] chemical structures of the rhodamine derivative r18 and its bulky counterion f5 - tpb are also shown . [SEP]
[CLS] design of nanoprobes B-nanoparticle for nucleic B-material acids I-material . [SEP]
[CLS] in our design , the light - harvesting nanoantenna ( fret donor ) is based on poly ( methyl methacrylate - co - methacrylic acid ( pmma - ma , 1 . 6 % methacrylic acid ) nps B-nanoparticle loaded with ion pair of hydrophobic B-property rhodamine ( r18 ) with bulky hydrophobic B-property counterion tetrakis ( pentafluorophenyl ) borate ( f5 - tpb ) . [SEP]
[CLS] the latter serves as spacer B-material between dyes to minimize their aggregation - caused quenching inside nps B-nanoparticle 16a and ensures ultrafast excitation energy migration required for efficient light harvesting ( figure 1 ) . [SEP]
[CLS] this nanoantenna is modified at the surface with a capture oligonucleotide , complementary to a nucleic B-material acid I-material fragment encoding cancer marker survivin ( 20mer , surc ) . [SEP]
[CLS] surc is hybridized with a short target - competitive sequence bearing fret acceptor cy5 ( 12mer , tcs - cy5 ) , so that the acceptor is localized close to the nanoantenna surface . [SEP]
[CLS] cy5 dye was selected because its absorption spectrum has a good spectral overlap with the emission spectrum of r18 / f5 - tpb - loaded nps B-nanoparticle with a forster radius of 6 . 1 nm . [SEP]
[CLS] combination of these two oligonucleotides was originally proposed by mirkin and co - workers in nanoflares , 20e , 31 which ensured quenching of dyes located close to gold B-material nps B-nanoparticle . [SEP]
[CLS] in our case , this design is used to induce fret form nanoantenna to cy5 acceptor . [SEP]
[CLS] here , thousands of rhodamine dyes inside nanoantenna pump the energy to a few cy5 acceptors at the surface , resulting in strong acceptor ( red ) emission amplified by the light harvesting ( figure 1 ) . [SEP]
[CLS] then , the target nucleic B-material acid I-material displaces tcs - cy5 acceptors and blocks fret , so that nanoprobe B-nanoparticle should switch to green emission of nanoantenna . [SEP]
[CLS] importantly , a few hybridization events are expected to switch emission of thousands of dyes , provide this basis for signal amplification . [SEP]
[CLS] to obtain such a nanoprobe B-nanoparticle , the primary challenge is to functionalize our polymeric nanoantenna with controlled number of oligonucleotides . [SEP]
[CLS] dna - modified polymeric B-nanoparticle nanoparticles I-nanoparticle [SEP] B-nanoparticle
[CLS] in the first approach , we modified pmma - ma polymer B-material with an azide group ( pmma - n3 ) , which was further reacted with oligonucleotide bearing dibenzocyclooctyne ( dbco ) unit through a cu - free click cycloaddition . [SEP]
[CLS] the latter was reported to provide efficient coupling without dna damage . [SEP]
[CLS] however , this direct coupling failed because we could not find an appropriate solvent to solubilize both polar oligonucleotide and apolar polymer B-material . [SEP]
[CLS] moreover , in contrast to pmma - ma , nanoprecipitation of pmma - n3 gave only very large aggregates ( figure s1 ) , because charged carboxylate B-material group I-material , essential for obtaining small nps B-nanoparticle , was absent in pmma - n3 . [SEP]
[CLS] therefore , we designed a polymer B-material bearing both charged carboxylate B-material and reactive azide groups in close proximity . [SEP]
[CLS] in addition to formation of small nps B-nanoparticle , this additional charged group can favor the exposure of reactive azide group at the nps B-nanoparticle surface ( figure 2 ) . [SEP]
[CLS] for this purpose , we developed a three - functional linker containing : ( i ) carboxyl B-material as a charged group ; ( ii ) azide for coupling with the oligonucleotide and ( iii ) amino group for conjugation with the polymer B-material ( figure 2 ) . [SEP]
[CLS] as a building block , we chose the bi - protected aspartic B-material acid I-material , commonly used for solid - phase peptide B-material synthesis , fmoc - asp ( otbu ) - oh . [SEP]
[CLS] its carboxylic function was modified with 3 - chloropropanamine , and then the chlorine B-material was substituted with an azide , which resulted also in fmoc removal giving target linker asp ( otbu ) - n3 ( figure 2 ) . [SEP]
[CLS] the latter was coupled with pmma - ma through an amide B-material bond . [SEP]
[CLS] nmr spectra confirmed good yield of the polymer B-material modification . [SEP]
[CLS] finally , tert - butyl group was removed giving a final polymer B-material ( pmma - aspn3 ) for preparation of functionalized nanoantennas ( figure 2 ) . [SEP]
[CLS] nanoprecipitation of acetonitrile solution of pmma - aspn3 polymer B-material with fluorescent B-property dye ( r18 / f5 - tpb ) into aqueous buffer ( ph 7 . 4 ) gave small nps B-nanoparticle ( 35 nm , figure 3a ) , highlighting the crucial role of the additional charged carboxylate B-material group I-material . [SEP]
[CLS] moreover , with modified nanoprecipitation protocol , concentration of pmma - aspn3 nps B-nanoparticle was further improved ~ 10 - fold ( figure s2 ) , while keeping small particle size ( 42 nm , figure 3a ) . [SEP]
[CLS] despite high 30 wt % loading of r18 / f5 - tpb ( e . g . ~ 3200 dyes per 40 nm particle ) [SEP]
[CLS] , the fluorescence B-property quantum yield of nps B-nanoparticle was very high ( 47 % , table s1 ) , which renders our nanoantenna particles exceptionally bright . [SEP]
[CLS] for grafting oligonucleotides , the obtained concentrated dye - loaded nps B-nanoparticle were reacted with dbcobearing oligonucleotide surc - dbco for 18h at different temperatures , followed by annealing with shorter oligonucleotide tcs - cy5 bearing fret acceptor . [SEP]
[CLS] the purification of the dna - conjugated nps B-nanoparticle from non - reacted oligonucleotides was achieved by successive filtrations through 100 kda filters , as verified by absorption B-technique spectroscopy I-technique ( figure s3 ) . [SEP]
[CLS] fluorescence B-property spectra suggested that at 4 and 20°c the reaction did not occur , while at 40°c we observed a clear fret signal from nps B-nanoparticle to hybridized tcs - cy5 , indicating successful conjugation reaction ( figure s4 ) . [SEP]
[CLS] moreover , grafting reaction at 40°c was confirmed by absorption B-technique spectroscopy I-technique , where the signal of the hybridized tcs - cy5 was observed in the purified samples , in contrast to negative control based on pmma - ma nps B-nanoparticle without azide group mixed with surc - dbco ( figure 3b ) . [SEP]
[CLS] the heating at 40°c is probably necessary in order to increase the probability of collision between dbco group grafted to oligonucleotide and azide group at the particle surface , in line with relatively slow dbco / azide reaction kinetics reported recently for other type of polymeric nps B-nanoparticle . [SEP]
[CLS] 28c however , in phosphate buffer containing 12 mm mg 2 + ions B-material , required for formation of stable duplexes , dna - nps B-nanoparticle conjugates ( np B-nanoparticle - surc ) showed relatively large size ( figure 3a ) , probably because of partial aggregation of the obtained nps B-nanoparticle . [SEP]
[CLS] stability of dna - nps B-nanoparticle conjugates can be improved by increasing number of oligonucleotides per particle , as it was recently shown for nps B-nanoparticle built of ring - opening metathesis block copolymers . [SEP]
[CLS] however , to achieve the highest optical amplification through the light - harvesting mechanism , minimal ratio of acceptors to donors should be used , i . e . minimal number of fret acceptors should be grafted to the particle surface . [SEP]
[CLS] to address these both issues , our nps B-nanoparticle were reacted with a mixture of coding ( surc - dbco ) and non - coding ( t20 - dbco ) oligonucleotides . [SEP]
[CLS] according to absorption B-technique spectroscopy I-technique , addition of t20 - dbco ( 20 µm ) to surc - dbco ( 3 µm ) did not inhibit the grafting of surc to nps B-nanoparticle surface ( figure 3b ) , probably because azide groups of nps B-nanoparticle were in excess ( 26 µm ) . [SEP]
[CLS] fluorescence B-property spectra of these nps B-nanoparticle ( np B-nanoparticle - surc - t20 / cy5 ) showed strong emission of the fret acceptor tcs - cy5 around 670 nm ( figure 3c ) . [SEP]
[CLS] moreover , the emission maximum of cy5 shifted to the red and its anisotropy value increased compared to free tcs - cy5 ( figure s5 ) , confirming the tcs - cy5 / surc hybridization . [SEP]
[CLS] these results suggested successful grafting of surc - dbco to nps B-nanoparticle in the presence of t20 - dbco . [SEP]
[CLS] remarkably , np B-nanoparticle - surc - t20 / cy5 particles remained small in phosphate buffer with mg 2 + ions B-material and their size did not change even after 2 month incubation B-technique in this medium ( figure 3a ) . [SEP]
[CLS] moreover , their emission spectrum with characteristic fret signal remained practically invariant for this 2 month period ( figure s6 ) . [SEP]
[CLS] therefore , grafting excess of non - coding dna ( t20 ) is essential for stability of our polymeric nps B-nanoparticle . [SEP]
[CLS] in addition , transmission B-technique electron I-technique microscopy I-technique ( tem ) confirmed that conjugation of nps B-nanoparticle with oligonucleotides did not modify their spherical shape and monodispersity , while their size increased only by ca 5 nm ( figure 3d - g ) . [SEP]
[CLS] np B-nanoparticle - aspn3 after reaction with surc followed by hybridization with tcs - cy5 ( np B-nanoparticle and np B-nanoparticle - surc / cy5 , respectively ) , and np B-nanoparticle - aspn3 after reaction with surc and t20 followed by hybridization with tcs - cy5 ( np B-nanoparticle - surc - t20 / cy5 ) . [SEP]
[CLS] ( c ) fluorescence B-property spectra of np B-nanoparticle - surc - t20 ( 43 pm ) without and with tcs - cy5 ( 1 nm ) and comparison with spectra of corresponding molecular probe cy3 - surc / tcs - cy5 ( 1 nm ) at the same instrumental conditions . [SEP]
[CLS] for better comparison the latter spectrum was also multiplied 100fold , showing that it is > 100 fold less bright than the np B-nanoparticle - probe . [SEP]
[CLS] tem images of np B-nanoparticle - aspn3 ( d ) and np B-nanoparticle - surc - t20 ( f ) and corresponding size distribution statistics ( e , g ) . [SEP]
[CLS] scale bar 50 nm . [SEP]
[CLS] ( h ) normalized fluorescence B-property spectra of np B-nanoparticle - surc - t20 at different number of hybridized tcs - cy5 per particle . [SEP]
[CLS] 20 mm phosphate buffer ( ph 7 . 4 ) with 30 mm of nacl and 12 mm of mgcl 2 was systematically used . [SEP]
[CLS] ( c , h ) excitation wavelength was 530 nm . [SEP]
[CLS] signal amplification by nanoantenna . [SEP]
[CLS] our further studies were focused on the most promising nps B-nanoparticle bearing both surc and t20 . [SEP]
[CLS] their hybridized form with fret acceptor tcs - cy5 ( np B-nanoparticle - surc - t20 / cy5 ) was called " np B-nanoparticle - probe " . [SEP]
[CLS] the synthesis of these nanoprobes B-nanoparticle was repeated four times showing good reproducibility of their size and spectroscopic properties ( table s2 ) . [SEP]
[CLS] based on the absorption data and the particle size from tem ( 4112 nm ) we estimated that 23±3 ( s . e . m . n = 4 ) fret acceptors ( tcs - cy5 ) were grafted per nanoantenna particle containing 3200±400 donor dyes . [SEP]
[CLS] in this respect , two outstanding features of np B-nanoparticle - probe should be mentioned : ( i ) high fluorescence B-property quantum yield ( 46 % , table s1 ) and ( ii ) 60±6 % fret efficiency from 3200donor dyes inside nanoantenna of 20 - nm radius to 23 acceptors at the surface , i . e . through distances far beyond the forster radius ( 6 . 1 nm ) . [SEP]
[CLS] this is a highly efficient light - harvesting phenomenon with minimal energy losses , where giant ensemble of donors " pumps " the energy to few acceptors , which implies strong amplification of the acceptor emission ( antenna effect ) . [SEP]
[CLS] the antenna effect , measured as an intensity ratio of the donor to acceptor in the excitation spectra ( figure s7 ) , was 58 ±1 with high reproducibility for all four preparations ( table s2 ) . [SEP]
[CLS] this result means that excitation via nanoantenna amplifies acceptor ( tcs - cy5 ) emission 58 - fold . [SEP]
[CLS] to show the power of our nanoantenna amplification approach , we compared the np B-nanoparticle - system with a molecular fret probe ( cy3 - surc / tcs - cy5 ) , where surc conjugate with cy3 dye was hybridized with tcs - cy5 . [SEP]
[CLS] the cy3 dye was chosen because it exhibited similar absorption and emission properties to rhodamine dye used in our nps B-nanoparticle ( table s1 ) . [SEP]
[CLS] remarkably , for the same fret acceptor concentration ( 1 nm of tcs - cy5 ) , the signal from our np B-nanoparticle - probe was > 100 - fold higher compared to the molecular probe ( figure 3c ) , in line with the antenna effect values . [SEP]
[CLS] as the np B-nanoparticle - probe concentration in this case was 43 pm , the single np B-nanoparticle - probe was > 2 , 000 - fold brighter than the single molecular probe . [SEP]
[CLS] finally , we verified the minimal number of tcs - cy5 that np B-nanoparticle - probe can distinguish by its fret signal . [SEP]
[CLS] to this end , tcs - cy5 was hybridized with surc - modified nps B-nanoparticle at the different ratios ( figure s8 ) . [SEP]
[CLS] the increase in number of fret acceptors per np B-nanoparticle improved the fret signal ( figure 3h ) . [SEP]
[CLS] remarkably , hybridization of 3 - 5 tcs - cy5 per particle produced clearly detectable change in the dual emission of the nanoprobe B-nanoparticle , indicating that just a few molecular recognition events can control emission of ~ 3200 donor dyes inside nanoantenna . [SEP]
[CLS] this unique sensitivity of our nanoantenna - based probes to few hybridization events together with strong signal amplification phenomenon are of key importance for detection of the target nucleic B-material acids I-material . [SEP]
[CLS] in the presence of the na target encoding survivin np B-nanoparticle - probe totally lost fret signal at 670 nm , in contrast to the control sample without the target ( figure 4a ) . [SEP]
[CLS] importantly , the response is ratiometric , displaying > 5 - fold change in the a / d intensity ratio . [SEP]
[CLS] this result shows that the target sequence displaced fret acceptor tcs - cy5 at nps B-nanoparticle surface , thus stopping fret . [SEP]
[CLS] moreover , the response of np B-nanoparticle - probe decreased drastically already for a single mismatch in the target sequence , which was located in the middle of the region corresponding to the target - competitive sequence . [SEP]
[CLS] second and third mismatches further deteriorated the response to the oligonucleotide ( figure 4a , figure s9 ) , showing very good sequence - specificity of the np B-nanoparticle - probe . [SEP]
[CLS] we further verified the importance of the mg 2 + ions B-material in the response of np B-nanoparticle - probe . [SEP]
[CLS] remarkably , below 12 mm of mg 2 + , np B-nanoparticle - probe showed a decrease in the fret signal after 3h incubation B-technique without the target ( figure s10a ) , confirming that 12 mm concentration of mg 2 + ensures stability of the duplex between the capture sequence and tcs - cy5 . [SEP]
[CLS] importantly , the response of np B-nanoparticle - probe to the target also decreased at lower mg 2 + concentrations ( figure s10 ) , indicating that mg 2 + could also assist the displacement of tcs - cy5 by the target sequence . [SEP]
[CLS] therefore , 12 mm concentration of mg 2 + was systematically used in all further experiments . [SEP]
[CLS] then , the np B-nanoparticle - probe diluted to tcs - cy5 concentration of 10 pm ( i . e . ~ 0 . 4 pm np B-nanoparticle - probe ) was tested in growing concentrations of na target . [SEP]
[CLS] the relative intensity of the fret acceptor , expressed as semi - empirical parameter of fret efficiency , a / ( a + d ) , gradually decreased in the concentration range of the na target from 20 to 200 pm ( figure 4b , c ) . [SEP]
[CLS] the estimated limit of detection ( lod ) was 5 pm , which is remarkably low for a standard fluorometer . [SEP]
[CLS] then , we checked whether our nanoprobe B-nanoparticle is operational in different biological media . [SEP]
[CLS] in all studied media , namely phosphate buffered saline ( pbs ) , pbs with bovine serum albumin ( bsa ) , opti - mem ( cell B-material culture medium without serum ) , opti - mem with fetal bovine serum ( fbs ) , and opti - mem with human serum , the probe showed robust ratiometric response to the na target : decrease in the relative intensity of the acceptor emission ( figure 4d ) . [SEP]
[CLS] moreover , the probe preserved the response in the excess of the denatured calf thymus dna , which served as model of random na sequence . [SEP]
[CLS] thus , our np B-nanoparticle - probe is compatible with highly complex media containing variety of biomolecules , including proteins B-material and nas . [SEP]
[CLS] targets containing 1 , 2 and 3 mismatches were tested at the same conditions . [SEP]
[CLS] fluorescence B-property spectra ( b ) and values of fret response a / ( a + d ) ( c ) of np B-nanoparticle - probe after incubation B-technique with target at different concentrations . [SEP]
[CLS] a and d are the pick intensities of the acceptor ( at 665 nm ) and the donor ( at 580 nm ) , respectively . [SEP]
[CLS] concentration of the np B-nanoparticle - probe corresponds to 10 pm of tcs - cy5 . [SEP]
[CLS] 20 mm phosphate buffer ( ph 7 . 4 ) with 30 mm of nacl and 12 mm of mgcl 2 was systematically used . [SEP]
[CLS] ( d ) fret response of the np B-nanoparticle - probe ( 100 pm of tcs - cy5 ) to the na target ( 500 pm ) in different biological media ( concentration of mg 2 + was adjusted to 12 mm ) : control ( 20 mm phosphate buffer with 30 mm of nacl ) , pbs , 10 µg / ml in pbs ) , bsa ( 0 . 5 mg / ml in pbs ) , optimem , fbs ( inactivated , 10 % in optimem ) , human serum ( inactivated , 10 % in optimem ) and dna from calf thymus ( ct - dna , heat denatured . [SEP]
[CLS] ( c , d ) [SEP]
[CLS] error bars are standard deviation of the mean ( n = 3 ) . [SEP]
[CLS] excitation wavelength was 530 nm . [SEP]
[CLS] evaluation of np B-nanoparticle - probe at the single - particle level . [SEP]
[CLS] the ultimate test for the performance of the nanoprobe B-nanoparticle is to verify whether it can operate at the level of single particle . [SEP]
[CLS] to this end we modified the glass surface with a20 sequence , which is complementary to that of non - coding t20 sequence of our np B-nanoparticle - probe ( figure 5a ) . [SEP]
[CLS] after incubation B-technique with the np B-nanoparticle - probe fluorescent B-property particles were clearly seen displaying remarkable homogeneity of their fluorescence B-property intensity ( figure 5b , c ) . [SEP]
[CLS] importantly , the overall ( donor + acceptor ) intensity recorded per np B-nanoparticle - probe was similar to that for control np B-nanoparticle - t20 ( without acceptor ) , being ~ 100 - fold higher compared to qddot - 605 excited at optimal ( 470 nm ) wavelength ( figure 5b , c , table s3 ) . [SEP]
[CLS] this unprecedented brightness for a fret biosensor is explained by high overall fluorescence B-property quantum yield of np B-nanoparticle - probe ( 46 % ) and ~ 3200 rhodamine dyes inside nanoantenna with total molar extinction coefficient of ~ 3 . 210 8 m - 1 cm - 1 at 550 nm ( vs . 1 . 110 6 m - 1 cm - 1 for qdot - 605 at 488 nm ) that serve as giant ensemble of energy donors . [SEP]
[CLS] to evaluate fret signal at the single particle level , the images of nps B-nanoparticle were recorded simultaneously at the green ( donor ) and red ( acceptor ) channels . [SEP]
[CLS] it can be seen that np B-nanoparticle - probe showed similar intensities at these two channels and appeared mostly yellow in the overlay ( figure 5d ) , whereas control np B-nanoparticle - t20 showed signal only in the donor channel ( figure 6a ) . [SEP]
[CLS] these results provide clear evidence that , after immobilization at the surface , our np B-nanoparticle - probe preserved strong fret , as in the spectroscopy B-technique measurements ( figure 3c ) . [SEP]
[CLS] then , to evaluate antenna effect at the single - particle level , we compared emission of the acceptor excited via nanoantenna ( at 550 nm ) vs that directly excited at 640 nm . [SEP]
[CLS] remarkably , 50 - fold higher excitation power density ( irradiance ) at 640 nm was required to achieve emission intensity comparable to that excited at 550 nm ( figure 5d ) . [SEP]
[CLS] quantitative image analysis revealed 7530 fold amplification of acceptor emission ( figure 5e ) , in line with the spectroscopic data in solution . [SEP]
[CLS] we should note that , this is the first report where this high amplification is reported at the single particle level for a biosensor . [SEP]
[CLS] previous report that used qdot as a fret donor for na detection at the single particle level did not exploit light - harvesting concept because a large number of acceptors per particle ( ~ 50 ) was required to achieve efficient fret . [SEP]
[CLS] 14b indeed , the ratio of extinction coefficients of qdot - 605 ( 1 . 110 6 m - 1 cm - 1 at 488 nm ) to 50 acceptors ( 2 . 510 5 50 = 1 . 2510 7 m - 1 cm - 1 ) is 0 . 09 , suggesting that excitation through qdot - 605 is less efficient than direct excitation of the acceptors . [SEP]
[CLS] in case of our nanoantenna , the ratio of extinction coefficients of nanoantenna and 23 acceptors at the used excitation wavelengths ( 550 and 640 nm ) is 3 . 210 8 / 4 . 510 6 = 71 , in agreement with the amplification values measured at the single - particle level . [SEP]
[CLS] the amplification at the single - particle level within a biosensor was reported very recently for a plasmonic system using dna origami , where the average amplification was 7 . 3 . [SEP]
[CLS] 5 . single - particle imaging of immobilized nanoprobes B-nanoparticle . [SEP]
[CLS] ( a ) scheme of nanoprobes B-nanoparticle immobilization on glass surface modified with bsa - biotin , neutravidin , and a20 - biotin . [SEP]
[CLS] the interaction with the surface occurs due to hybridization of a20 - biotin with t20 of np B-nanoparticle . [SEP]
[CLS] ( b ) wide - field fluorescence B-technique microscopy I-technique of the immobilized nps B-nanoparticle : quantum B-nanoparticle dots I-nanoparticle ( qdot - 605 ) excited at 470 nm , control np B-nanoparticle - t20 and nanoprobe B-nanoparticle ( sum of donor and acceptor channels is shown ) . [SEP]
[CLS] to obtain comparable signals excitation power density for qdot - 605 was 25 - fold higher than that for np B-nanoparticle - t20 and nanoprobe B-nanoparticle : 14 vs 0 . 56 w cm - 2 , respectively . [SEP]
[CLS] ( c ) histogram of single - particle intensity distribution for these three types of nps B-nanoparticle . [SEP]
[CLS] at least 1000 nps B-nanoparticle were analyzed in each case . [SEP]
[CLS] ( d ) wide - field microscopy B-technique images of the nanoprobe B-nanoparticle at the acceptor ( direct excitation at 640 nm ) , antenna - amplified acceptor ( fret - acceptor ) , donor ( fretdonor ) and merged fret - acceptor and donor channels ( at the same intensity scale ) . [SEP]
[CLS] the excitation of nanoprobe B-nanoparticle ( fret - donor and fret - acceptor ) was at 550 nm with excitation power density 0 . 4 w cm - 2 , while direct excitation of acceptor was done at 640 nm using 50 - fold higher power density ( 20 w cm - 2 ) . [SEP]
[CLS] signals from fret - donor and fret - acceptor were recorded at < 640 and > 640 nm , respectively . [SEP]
[CLS] integration time was systematically 200 ms . [SEP]
[CLS] ( e ) amplification of fret - acceptor emission ( antenna effect ) by the nanoprobe B-nanoparticle at the single - particle level presented as a distribution histogram . [SEP]
[CLS] at least 1500 nps B-nanoparticle were analyzed . [SEP]
[CLS] all channels are represented at the same intensity scale . [SEP]
[CLS] the excitation wavelength was at 550 nm with 0 . 4 w cm - 2 power density . [SEP]
[CLS] signals from the donor and the acceptor were recorded at < 640 and > 640 nm , respectively . [SEP]
[CLS] a and d are the integrated densities of the signal from individual particles recorded at the acceptor and the donor channels , respectively . [SEP]
[CLS] finally , we tested the response of our nanoprobe B-nanoparticle to the target at the single - particle level ( figure 6 ) . [SEP]
[CLS] after incubation B-technique of immobilized np B-nanoparticle - probe with the target the emission in the red channel strongly decreased , in line with our observations by fluorescence B-technique spectroscopy I-technique . [SEP]
[CLS] the semi - quantitative fret efficiency parameter , a / ( a + d ) , decreased from 0 . 48 down to 0 . 20 ( figure 6b ) , reaching values close to that for the control np B-nanoparticle - t20 without fret acceptor ( 0 . 12 ) . [SEP]
[CLS] immobilized np B-nanoparticle - probe was able to detect na target in the concentration range of 1 - 1000 pm ( figure 7a and s11 ) with a remarkably low lod of 0 . 25 pm ( down to 12 . 510 - 18 moles in 50 µl ) . [SEP]
[CLS] at low target concentration , the response of np B-nanoparticle - probe was significantly improved for longer incubation B-technique times ( figure 7a ) , showing that sensitivity of our system was only limited by the kinetics of hybridization with the target na . [SEP]
[CLS] the latter is known to be on the time scale of hours for pm na concentrations . [SEP]
[CLS] robust detection of 10 pm na target at the singleparticle level was achieved in the presence of bsa , bovine and human serum ( figure 7b , c and s12 ) , showing potential of our surface - immobilized np B-nanoparticle - probe for development of future diagnostics assays . [SEP]
[CLS] finally , we should stress that ~ 23 hybridization events at the surface of the single np B-nanoparticle - probe ( corresponding to the number of tcs - cy5 per np B-nanoparticle ) resulted in the color switching of a particle exhibiting the brightness of > 3000 rhodamine dyes or ~ 100 qdot - 605 . [SEP]
[CLS] this outstanding performance of the developed np B-nanoparticle - probe has two important consequences . [SEP]
[CLS] first , the observed color switching implies that , at the single particle level , the np B-nanoparticle - probe could readily detect just a few copies of the nucleic B-material acid I-material target . [SEP]
[CLS] indeed , according to spectroscopy B-technique data , hybridization of 3 - 5 nucleotides can provide detectable change in the fret signal ( figure 3h ) . [SEP]
[CLS] second , due to the signal amplification produced by light harvesting with > 3000 dyes , the target nucleic B-material acids I-material could be detected at very low excitation power density ( 0 . 4 w cm 2 ) of led in an epi - fluorescence B-property mode , which is > 100 fold - lower than required in the single - molecule detection measurements . [SEP]
[CLS] the use of low power significantly decreases the background noise and makes possible detection of a few copies of nucleic B-material acids I-material using relatively weak and inexpensive light sources and a simple imaging setup . [SEP]
[CLS] here , we introduce a concept of amplified fluorescence B-property sensing based on giant light - harvesting ensemble of organic dyes in a polymeric B-nanoparticle nanoparticle I-nanoparticle ( nanoantenna ) . [SEP]
[CLS] this nanoantenna serves as super - efficient energy donor in a fret - based nucleic B-material acid I-material detection assay , where a few hybridization events at the surface of the nanoparticle B-nanoparticle switch on / off energy transfer from thousands of dyes . [SEP]
[CLS] as chemical modification of small polymeric B-nanoparticle nanoparticles I-nanoparticle with oligonucleotides remains a challenge , we developed an original strategy for functionalization of the polymeric nanoantennas . [SEP]
[CLS] bi - functional moiety bearing charged carboxylate B-material and azide was grafted to a biocompatible B-property hydrophobic B-property polymer B-material ( pmma - ma ) , which ensured ( i ) preparation of small nanoparticles B-nanoparticle ( ~ 40 nm ) by nanoprecipitation and ( ii ) effective exposure of the azide group for coupling with oligonucleotides by cu - free click reaction . [SEP]
[CLS] using this strategy , we functionalized the nanoantenna particles with simultaneously two oligonucleotides . [SEP]
[CLS] the first one is the molecular recognition unit that after hybridization with the target nucleic B-material acid I-material ( encoding a fragment of cancer marker survivin ) removes the energy acceptor ( cy5 dye ) from the nanoantenna surface , triggering the loss of fret . [SEP]
[CLS] the second one is a non - coding oligonucleotide , which is found to be essential for nanoparticle B-nanoparticle stability in biological media . [SEP]
[CLS] the obtained nanoprobes B-nanoparticle display efficient fret ( > 60 % ) from ~ 3200 rhodamine - based donor dyes of nanoantenna to only 23 cy5 - acceptors . [SEP]
[CLS] this efficient light - harvesting phenomenon results in the amplification of the acceptor emission ( antenna effect ) reaching 75 . [SEP]
[CLS] the nanoprobe B-nanoparticle detects target nucleic B-material acid I-material by changing the ratio of its donor / acceptor intensity ratio and can distinguish down to 3 - 5 hybridizations at its surface . [SEP]
[CLS] our nanoantenna based probe is ~ 100 - fold brighter than quantum B-nanoparticle dots I-nanoparticle ( qdot - 605 ) and > 2000 - fold brighter than analogous fret - based molecular probe . [SEP]
[CLS] to our knowledge , it is the first nanoparticle - based probe for biomolecules that combines this exceptional brightness and such high signal amplification . [SEP]
[CLS] the nanoprobe B-nanoparticle operates in solution and on surfaces at the single - particle level delivering limit of detection of 5 and 0 . 25 pm , respectively , the latter being limited only by the hybridization kinetics . [SEP]
[CLS] we expect that outstanding brightness and amplification characteristics of nanoantenna probes will greatly simplify detection of nucleic B-material acids I-material with low - cost portable fluorescence B-property detection instruments , which is of highest importance for detection biomolecular markers of diseases in point of care diagnostics . [SEP]
[CLS] moreover , our functionalized organic nanoantenna , owing to the exceptional capacity to donate the energy from thousands of donors to few acceptors at its surface , can increase by orders of magnitude brightness of practically any fret based detection assay . [SEP]
[CLS] oligonucleotides [SEP]
[CLS] lyophilized single strand dna sequences were purchased from iba , dissolved in milli - q water B-material , aliquoted and stored at - 20 °c for further experiments . [SEP]
[CLS] the oligonucleotide sequences used in this study are shown below . [SEP]
[CLS] surc - dbco , 5 ' - ccc agc ctt cca gct cct tga - ( dbco ) - 3 ' ; t20 - dbco , 5 ' - ttt ttt ttt ttt ttt ttt tt - ( dbco ) - 3 ' ; tcs - cy5 , 5 ' - ( cy5 ) - tca agg agc tgg - 3 ' ; target , 5 ' - caa gga gct gga agg ctg gg - 3 ' ; single mismatch , 5 ' - caa gca gct gga agg ctg gg - 3 ' ; two mismatches , 5 ' - caa gca gct gga agc ctg gg - 3 ' ; three mismatches , 5 ' - caa gca gct cga agc ctg gg - 3 ' ; a20 - biotin , 5 ' - ( bio ) - aaa aaa aaa aaa aaa aaa aa - 3 ' [SEP]
[CLS] nanoparticle B-nanoparticle preparation . [SEP]
[CLS] protocol for diluted nps B-nanoparticle : 50 μl of the pmma - aspn3 polymer B-material solution in acetonitrile ( 1 mg ml - 1 containing r18 / f5 - tpb at 30 wt % relative to the polymer B-material ) were added quickly using a micropipette to 450 μl of 20 mm phosphate buffer , ph 7 . 4 at 21 °c under shaking ( thermomixer comfort , eppendorf , 1100 rpm ) . [SEP]
[CLS] the particle solution was then quickly diluted 5 - fold with the same buffer . [SEP]
[CLS] for preparation of nps B-nanoparticle functionalized with dna the protocol was modified to increase nps B-nanoparticle concentration : 100 μl of the pmma - aspn3 polymer B-material solution in acetonitrile ( 2 mg ml - 1 with 30 wt % r18 / f5 - tpb relative to the polymer B-material ) were then added quickly using a micropipette to 900 μl the same buffer under shaking with thermomixer ( 1 , 100 rpm ) . [SEP]
[CLS] then , the residues of acetonitrile were evaporated . [SEP]
[CLS] np B-nanoparticle - probe synthesis . [SEP]
[CLS] aliquots of dna ( 3 μm surc - dbco , with or without 20 μm t20 - dbco ) were added to 300 μl of nanoparticles B-nanoparticle ( concentration of azide groups were ~ 26 µm ) . [SEP]
[CLS] the reaction was mixed and kept overnight at 40 °c in thermomixer without shaking protected from light . [SEP]
[CLS] then the reaction was cooled down to room temperature . [SEP]
[CLS] for annealing with tcs - cy5 , the aliquot of tcs - cy5 in ratio 1 : 1 with surc - dbco was added and mixture was heated to 70 °c in water B-material bath for 3 min . [SEP]
[CLS] to complete hybridization the reaction was cooled down to room temperature and kept in the dark for 2 h . then [SEP]
[CLS] the mixture was diluted with 20 mm phosphate buffer containing 12 mm mgcl 2 and 30 mm nacl to 4 ml and purified by centrifugation using centrifuge filters ( amicon , 0 . 5ml , 100k ) on 1000 g at 20 °c for 2 min . [SEP]
[CLS] the procedure of centrifugation was repeated 5 times to remove the non - reacted oligonucleotides . [SEP]
[CLS] the obtained np B-nanoparticle - probes in volume of 1 ml were kept in the dark at 4 °c . [SEP]
[CLS] detection of survivin nucleic B-material acid I-material target . [SEP]
[CLS] for detection in solution the np B-nanoparticle - probe was diluted in 20 mm phosphate buffer containing 30 mm nacl and 12 mm mgcl 2 to a desired concentration ( corresponding to tcs - cy5 ) and aliquot of target oligonucleotide was added . [SEP]
[CLS] before measurements the mixture was incubated B-technique in the dark at 4 °c for 20h , unless specified . [SEP]
[CLS] for detection of target na on surfaces , the aliquot of the na target was added to the labtek chamber with immobilized np B-nanoparticle - probes . [SEP]
[CLS] the mixture was kept in the dark at the room temperature for 1 and 4 h incubation B-technique or at 4 °c for 20h incubation B-technique . [SEP]
[CLS] for na detection in different biological media , the pbs and optimem were adjusted to 12 mm mgcl 2 concentrations and further used for sample preparations . [SEP]
[CLS] bsa was diluted to 0 . 5 mg / ml concentration in pbs . [SEP]
[CLS] the deoxyribonucleic B-material acid I-material sodium B-material salt B-material from calf thymus ( ct - dna ) was dissolved to concentration of 2 mg / ml in water B-material and kept overnight at 4 °c for complete dissolution . [SEP]
[CLS] then , it was heated for 3 min at 90 °c followed by rapid transfer into ice bath . [SEP]
[CLS] for measurements it was diluted to concentration of 10 µg / ml in pbs . [SEP]
[CLS] foetal bovine serum ( fbs ) and human serum were heat - inactivated by exposing them for 30 min to 56 °c before they were used . [SEP]
[CLS] after that the fbs and human serum were diluted to 10 % concentration in optimem . [SEP]
[CLS] for measurements in solution the np B-nanoparticle - probe was diluted in the corresponding media and the target oligonucleotide was added . [SEP]
[CLS] before the measurements were made , the mixtures of np B-nanoparticle - probe with and without target oligonucleotide were incubated B-technique in the dark at 4 °c for a given time . [SEP]
[CLS] for the measurements on the surface , 300 µl of the media was mixed with aliquot of the target oligonucleotide and then the mixture was added to the labtek chamber with immobilized np B-nanoparticle - probes . [SEP]
[CLS] prepared mixtures were incubated B-technique for 20 hours at 4 °c before measurements . [SEP]
[CLS] 1 . concept of polymeric nanoantenna for amplified detection of nucleic B-material acids I-material . [SEP]
[CLS] the dye - loaded nanoparticle B-nanoparticle is schematically presented , bearing non - coding ( in gray ) and capture ( dark red ) oligonucleotides . [SEP]
[CLS] yellow arrows schematically show the light - harvesting process toward fret acceptor [SEP]
[CLS] figure 2 . of synthesis of nanoprobes B-nanoparticle for nucleic B-material acids I-material . [SEP]
[CLS] it includes synthesis of linker molecule ( asp ( otbu ) - n3 ) , its conjugation to pmma - ma and deportation affording target polymer B-material pmma - aspn3 , nanoprecipitation of pmma - aspn3 to obtain nps B-nanoparticle bearing azide groups , dna grafting to the surface of nps B-nanoparticle and hybridization with cy5 - tcs . [SEP]
[CLS] characterization of dna conjugates with nanoantenna particles . [SEP]
[CLS] ( a ) size by dls of nps B-nanoparticle bearing the motif aspn3 ( np B-nanoparticle - aspn3 ) , nps B-nanoparticle bearing aspn3 prepared at high concentration ( np B-nanoparticle - asp - n3 - conc ) , nps B-nanoparticle bearing aspn3 and target - specific oligonucleotide surc ( np B-nanoparticle - surc ) , nps B-nanoparticle bearing asp - n3 , surc and non - specific oligonucleotide t20 ( np B-nanoparticle - surc - t20 ) , and the latter after 2 months storage ( np B-nanoparticle - surc - t20 , 2 months ) . [SEP]
[CLS] ( b ) absorption spectra of control pmma - ma nps B-nanoparticle ( without azide groups ) and [SEP]
[CLS] response of nanoprobe B-nanoparticle to the nucleic B-material acid I-material target . [SEP]
[CLS] ( a ) fluorescence B-property spectra of np B-nanoparticle - probe ( 100 pm of tcs - cy5 ) after incubation B-technique for 20h at 4°c without and with survivin na target ( 1 nm ) . [SEP]
[CLS] targets [SEP]
[CLS] response of surface - immobilized np B-nanoparticle - probe to the na target at the single - particle level . [SEP]
[CLS] ( a ) wide - field fret images ( donor channel , acceptor channels and overlay ) and ( b ) corresponding histograms of relative fret efficiency , a / ( a + d ) , of the np B-nanoparticle - probe ( first row ) , nanoprobe B-nanoparticle after incubation B-technique with 100 pm survivin na target for 20h at 4c ( second row ) and control np B-nanoparticle - t20 ( third row ) . all channels are represented at the same intensity scale . [SEP]
[CLS] the excitation wavelength was at 550 nm with 0 . 4 w cm - 2 power density . [SEP]
[CLS] signals from the donor and the acceptor were recorded at < 640 and > 640 nm , respectively . [SEP]
[CLS] a and d are the integrated densities of the signal from individual particles recorded at the acceptor and the donor channels , respectively . [SEP]
[CLS] single - particle evaluation of np B-nanoparticle - probe ratiometric response in different conditions . [SEP]
[CLS] ( a ) ratiometric response of the immobilized np B-nanoparticle - probe to different concentrations of survivin na target after three different incubation B-technique times : 1 and 4 h at room temperature , and 20 h at 4 c . [SEP]
[CLS] ( b ) reponses of np B-nanoparticle - probe 10 pm na target after 20 h incubation B-technique at 4 c in different biological media : bsa , fbs and human serum ( hs ) in pbs . [SEP]
[CLS] ( a , b ) the error bars are s . e . m . [SEP]
[CLS] at least 1000 particles were analyzed for each condition . [SEP]
[CLS] ( c ) example of overlaid images of donor and acceptor channels for np B-nanoparticle - probe incubated B-technique with 10 pm na target after 20 h incubation B-technique at 4 c . the excitation wavelength was 550 nm with power density of 0 . 4 w cm - 2 ; integration time was 200 ms . [SEP]
[CLS] scale bar , 5 µm . [SEP]
[CLS] oligonucleotide - based agents have the potential to treat or cure almost any disease , and are one of the key therapeutic drug classes of the future . [SEP]
[CLS] bioconjugated oligonucleotides , a subset of this class , are emerging from basic research and being successfully translated to the clinic . [SEP]
[CLS] in this review , we first briefly describe two approaches for inhibiting specific genes using oligonucleotides - antisense dna ( aso ) and rna interference ( rnai ) followed by a discussion on delivery to cells B-material . [SEP]
[CLS] we then summarize and analyze recent developments in bioconjugated oligonucleotides including those possessing galnac , cell B-material penetrating peptides B-material , αtocopherol , aptamers , antibodies B-material , cholesterol , squalene , fatty acids , or nucleolipids . [SEP]
[CLS] these novel conjugates provide a means to enhance tissue targeting , cell B-material internalization , endosomal escape , target binding specificity , resistance to nucleases , and more . [SEP]
[CLS] we next describe those bioconjugated oligonucleotides approved for patient use or in clinical trials . [SEP]
[CLS] finally , we summarize the state of the field , describe current limitations , and discuss future prospects . [SEP]
[CLS] bioconjugation chemistry is at the centerpiece of this therapeutic oligonucleotide revolution , and significant opportunities exist for development of new modification chemistries , for mechanistic studies at the chemical - biology interface , and for translating such agents to the clinic . [SEP]
[CLS] the molecular biology central dogma describes the transfer of biological information stored in genes to proteins B-material using biomacromolecules . [SEP]
[CLS] the biomacromolecule deoxyribonucleic B-material acid I-material ( dna ) is found in all nucleated eukaryotic cells B-material and it stores information via a specific sequence present in dna . [SEP]
[CLS] the first - step in this unidirectional process of genes to proteins B-material is transcription B-event whereby ribonucleic B-material acid I-material ( rna ) is generated from dna ( i . e . , transcription B-event ) . [SEP]
[CLS] rna is then spliced into the nucleus to retain only the coding part of the rna , namely the exons . [SEP]
[CLS] the resulting messenger rna ( mrna ) is transferred to the cytoplasm , where the ribosome reads the information and produces a protein B-material ( i . e . , translation ) . [SEP]
[CLS] these biomacromolecules play key roles in all aspects of life from life cycle , pathogenesis , to healing and , thus , opportunities exist to control a biological outcome by intervening in transcription B-event or translation . [SEP]
[CLS] in 1967 , synthetic nucleoside derivatives were reported with the intent for specific base pairing with target sequences . [SEP]
[CLS] in 1978 , summerton developed methods for inactivating sequences through crosslinking , and zamecnik and stephenson reported that short synthetic dna fragments ( called oligodeoxynucleotides , odn ) from the rous sarcoma virus , possessing sequence binding complementary to rna molecules , inhibited viral replication . [SEP]
[CLS] this seminal discovery documented replication prevention of a viral rna strain using a spe - cific odn - today known as antisense treatment [SEP]
[CLS] several studies have also elucidated many of the molecular mechanisms underlying different human and animal pathologies . [SEP]
[CLS] thus , altering the expression of pathological genes , whether genetic or modified by mutation , is of keen interest and technologies are being developed for such purposes . [SEP]
[CLS] antisense dna ( aso ) and rna interference ( rnai ) are two very promising technologies for inhibiting specific genes . [SEP]
[CLS] the former utilizes a dna fragment complementary to the target mrna sequence . [SEP]
[CLS] the latter approach , first described by r . jorgensen and extensively studied by andrew z . fire and craig mello , uses small interfering rnas ( sirnas ) to target the mrna . [SEP]
[CLS] antisense oligonucleotides induce different inhibition mechanisms , which can occur in the cytoplasm and in the nucleus ( figure 1 ) . [SEP]
[CLS] after transcription B-event of the dna into premessenger rna , several post - transcriptional modifications are performed , including splicing , capping at the 5 ' end , or the polyadenylation of the 3 ' end . [SEP]
[CLS] the mrna is then translocated into the cytoplasm by exportins , where it is then translated into proteins B-material by ribosomes . [SEP]
[CLS] a synthetic oligonucleotide elicits its activity in two cellular compartments , the cytoplasm or the nucleus . [SEP]
[CLS] after hybridization of the oligonucleotide on mrna , two inhibitory mechanisms are possible : i ) mrna degradation or ii ) translational repression . [SEP]
[CLS] mrna is degraded upon hybridization with an oligonucleotide following rnase h recognition . [SEP]
[CLS] antisense dna can also inhibit the translation step via a steric hin - drance mechanism at the ribosome binding site or by prohibiting the ribosomal motion along the mrna . [SEP]
[CLS] several mechanisms of inhibition are also possible in the nucleus , including the inhibition of post - transcriptional modifications , such as capping at the 5 ' end , or by binding to polyadenylation sites at the 3 ' utr ends . [SEP]
[CLS] additionally , antisense may also inhibit or promote the inclusion of exons to alter the splicing of pre - mrna ( figure 1 ) . [SEP]
[CLS] in principle , protein B-material regulation is accomplished by the use of either agonist or antagonist strategies , with the caveat that the target rna sequence is known . [SEP]
[CLS] because they are exogenous molecules , synthetic oligonucleotides are substrates for endo - and exo - nucleases , and thus exhibit short half - lives both in vitro and in vivo . [SEP]
[CLS] for example , after intravenous ( iv ) injection in monkeys , phosphodiester oligonucleotide analogues are quickly degraded with a half - life of only 5 minutes . [SEP]
[CLS] in order to overcome this low stability , oligonucleotides are chemically modified . [SEP]
[CLS] sites of chemical modification include the base , ribose , and the phosphate linkage B-property . [SEP]
[CLS] the types of modifications vary from classical to non - classical bioisosteres . [SEP]
[CLS] additionally , one chemical modifications does not always address the same half - life limitation ( s ) , and often two or more modifications are combined to increase oligonucleotide stability . [SEP]
[CLS] during the last few decades , many different modification chemistries have been developed ( figure 2 ) . [SEP]
[CLS] one of the first chemical modifications reported is the replacement of the phosphodiester by a phosphorothioate linkage B-property ( pto ) to minimize oligonucleotide degradation . [SEP]
[CLS] interestingly , this modification does not interfere with the recruitment of rnase h and target rna cleavage . [SEP]
[CLS] structures of the different chemical modifications and the knockdown mechanisms are shown in figure 2 . [SEP]
[CLS] to improve the binding affinity and nuclease resistance , chemical modifications including the 2 ' - o - methyl ( 2 ' - ome ) , 2 ′ - o - methoxyethyl ( 2 ′ - moe ) , and / or 2 ' - fluoro ( 2 ' - f ) modifications of rna are inserted into the structure . [SEP]
[CLS] to enhance the binding affinity , locked nucleic B-material acid I-material ( lna ) modifications are used where the conformational freedom of the ribose is restricted . [SEP]
[CLS] more recently , tricyclo - dna ( tcdna ) are reported as another constrained nucleotide featuring a three - ring scaffold . [SEP]
[CLS] importantly , these types of chemical modifications efficiently address the degradation issues . [SEP]
[CLS] however , they do not improve the cellular uptake or targeting of the oligonucleotide to a specific site at the cellular , tissue , or organ level . [SEP]
[CLS] indeed , these hydrophilic B-property polyanions do not easily cross a physiologic barrier B-property ( e . g . , skin ) or a cell B-material membrane . [SEP]
[CLS] therapies must adhere to a wide range of stringent specifications in order to advance into clinical trials and succeed . [SEP]
[CLS] an ideal oligonucleotide therapy is safe to administer , affordable to produce , exhibits a 2 year shelf - life , possesses an extended half - life in blood and serum ( > 12 hrs ) , and inhibits intended targets effectively with low nonspecific or off - target interactions . [SEP]
[CLS] that said , differing disease indications will require fine tuning of the design requirements for optimal performance . [SEP]
[CLS] as discussed below , a wide range of modifications to oligonucleotides improve one or many of these parameters , and the use of bioconjugation to oligonucleotides is primarily focused on improving the delivery of oligonucleotides to the cytosol or nucleus . [SEP]
[CLS] bioconjugates address a number of different oligonucleotide delivery challenges , such as improving biodistribution to a specific region or cell B-material type ( e . g . , antibodies B-material ) , promoting endosomal escape ( e . g . , cpp ) , increasing receptor - mediated transport ( e . g . , galnac ) , and / or increasing lipophilicity B-property ( e . g . , cholesterol ) . [SEP]
[CLS] in this review , we discuss recent advances in the delivery of oligonucleotides . [SEP]
[CLS] we briefly describe self - assembly approaches based on electrostatic , hydrophobic B-property , or h - bonding interactions to give , for example , lipoplexes , and refer the reader to several comprehensive reviews on this topic . [SEP]
[CLS] we focus on modified bioconjugated oligonucleotides for delivery , including galnac , cell B-material penetrating peptides B-material , α - tocopherol , aptamers , antibodies B-material , cholesterol , squalene , fatty acids , nucleolipids , and bis - conjugates . [SEP]
[CLS] we also describe relevant linking chemistries between the oligonucleotide and the bioconjugate with respect to molecular design . . [SEP]
[CLS] additionally , the therapeutic applications of such bioconjugated oligonucleotides and outcomes from clinical trials are highlighted . [SEP]
[CLS] finally , we summarize the state of the field and discuss future prospects . [SEP]
[CLS] today , significant opportunities exist for development of new modification chemistries , for mechanistic studies at the chemical - biology interface , and for engineering systems for efficient delivery to the target site . [SEP]
[CLS] cellular uptake is one of the key steps in order for oligonucleotides to elicit their biological activity , as the target mrnas and the cellular machinery are located in the cytoplasm and in the nucleus . [SEP]
[CLS] several transfection agents have been developed and commercialized for the intracellular delivery of oligonucleotides such as oligofectamine 2000 ( invitrogen ) , jetpei ( polyplus transfection ) or k2 ( biontex ) . [SEP]
[CLS] many of these transfecting products are composed of cationic B-material lipids B-material , polyethylenimine , or deae - dextran , which self - organize and self - assemble in aqueous medium via electrostatic interactions with the negatively charged oligonucleotides . [SEP]
[CLS] this strategy compacts the oligonucleotide into aggregates for subsequent endocytosis B-event by the cell B-material . [SEP]
[CLS] once inside the cell B-material , the oligonucleotides are released into the cytoplasm . [SEP]
[CLS] however , toxicity B-property remains a disadvantage of these agents , as most of transfecting agents are cationic B-material , but alternatives are being investigated . [SEP]
[CLS] additionally , many of these complexes are not stable in the presence of serum , limiting or preventing their in vivo use . [SEP]
[CLS] following intravenous administration , the resultant oligonucleotide / transfecting agent complexes accumulate primarily in the liver , due to their size . [SEP]
[CLS] consequently , significant research efforts are dedicated to the delivery challenges in order to improve the transfection of nucleic B-material acids I-material . [SEP]
[CLS] for example , poly ( 2 - dime - thylaminoethyl ) methacrylate , when mixed with oligonucleotides , forms micelles B-material of controlled sizes based on the n / p ratio . [SEP]
[CLS] upon adding an albumin coating B-material to the surface , the cytotoxic B-property effect of the polymer B-material is minimized and cancerous cells B-material are preferentially transfected relative to healthy cells B-material . [SEP]
[CLS] another strategy employs modified viruses , which internalize nucleic B-material acids I-material in cells B-material . [SEP]
[CLS] however , this strategy can afford off - target toxicity B-property since the oligonucleotides are not delivered in their synthetic form , but rather integrated into the viral genome . [SEP]
[CLS] lipid B-nanoparticle nanoparticles I-nanoparticle ( lnp ) are also being investigated as the non - viral transfecting cargo . [SEP]
[CLS] however , the transfection B-property efficiency I-property is generally lower than that observed for viral transfection systems . [SEP]
[CLS] recently , lnps loaded with sirna targeting polo - like kinase 1 ( plk1 ) protein B-material , present in the triple negative breast cancer cell B-material line ( mda - mb - 231 ) , have been modified with antibodies B-material to target tumors B-material . [SEP]
[CLS] biodistribution studies of labeled sirna - lnps demonstrated that antibody modified lnp ( antibody B-material against heparin - binding egf - like growth factor , αhb - egf ) effectively delivered sirna to tumor B-material tissue in mice . [SEP]
[CLS] interestingly , the plk1 protein B-material expression was inhibited after intravenous injection of the lnps and tumor B-material growth was decreased . [SEP]
[CLS] these results indicate that antibody modified lnps loaded with sirna are a promising therapeutic approach for breast cancer . [SEP]
[CLS] another recent article investigated the bioconjugation of antibodies B-material to lnp for targeting endothelial cells B-material lining the vascular lumen . [SEP]
[CLS] these new particles were non toxic both in vitro and in vivo as well as preferentially accumulated in the lungs . [SEP]
[CLS] in contrast , the same nanoparticles B-nanoparticle without conjugated antibodies B-material quickly accumulated into the liver , indicating that targeting moieties ( antibodies B-material / lnp bioconjugate ) can avoid the hepatic uptake with endogenous serum protein B-material like apo - e . [SEP]
[CLS] alternatively , bioinspired molecules such as nucleolipids ( nl ) are being used to construct lnps . [SEP]
[CLS] nls self - assemble to form unique supramolecular structures , and the lnps based nls loaded with nucleic B-material acids I-material successfully transfect plasmid dna , sirna , and antisense oligonucleotides to a number of different cell B-material lines : human breast adenocarcinoma mcf - 7 cells B-material , human liver ( hepg2 ) , mouse fibroblast ( nih 3t3 ) , chinese hamster ovarian ( cho ) cells B-material , and human prostate cancer ( pc - 3 ) cells B-material . [SEP]
[CLS] furthermore , these lnps can be further modified to be stimuli - responsive , such as responding to changes in ph to enhance the delivery of nucleic B-material acids I-material . [SEP]
[CLS] the above examples are representative and by no means comprehensive , as there are many formulations described for nucleic B-material acid I-material vectorization ( figure 3 ) , and the reader is referred to several comprehensive reviews on the subject . [SEP]
[CLS] the re - targeting of nucleic B-material acids I-material using viral vectors was investigated by reynolds et al . in the 2000 ' s . [SEP]
[CLS] viral vectors are attractive candidates for in vivo gene delivery because the infection efficacy is higher compared to other non - vial approaches . [SEP]
[CLS] for example an adenovirus vector was prepared containing both a fab fragment of an anti - ad5 knob antibody B-material and the anti - ace monoclonal B-material antibody I-material mab 9b9 . [SEP]
[CLS] this bi - specific conjugate exhibited enhanced pulmonary distribution by a synergic effect ( transductional and transcriptional B-event ) . [SEP]
[CLS] the major drawback of using this vector is sequestration by kupffer cells B-material into liver tissue . [SEP]
[CLS] the conjugation of specific molecules to oligonucleotides is a promising therapeutic approach for nucleic acid based drugs . [SEP]
[CLS] consequently , bioconjugates are of increasing presence in the pharmaceutical development pipeline [SEP]
[CLS] the major advantages of working with a bioconjugate include : 1 ) a new chemical entity ; 2 ) of defined composition ; and , 3 ) synthesized using chemical methods as opposed to bioprocesses . [SEP]
[CLS] these attributes , unlike formulations with polymers B-material or other transfecting reagents that give heterogeneous mixtures requiring extensive multi - pronged characterization analyses , will facilitate translation to the clinic . [SEP]
[CLS] additionally , these covalently conjugated molecules play one or more roles in recognition , targeting of cells B-material or tissues , cellular internalization , and pharmacokinetics . [SEP]
[CLS] the review of covalently conjugated oligonucleotides necessitates discussion on appropriate and effective linkers and linking chemistries , as interference strategies can be stymied by functionalization ( s ) [SEP]
[CLS] both cleavable and stable linkers are successfully used . [SEP]
[CLS] bio - orthogonal " click " chemistry approaches such as akyne - azide , and thio - maleimide are two common approaches to form stable linkages B-property with highly specific reactions . [SEP]
[CLS] cleavable bonds such as reducible disulfide B-property linkages I-property , esters ( cleavable through hydrolysis or esterases ) , phosphodiesters ( cleavable through nucleases ) and peptides B-material ( cleavable through proteases ) are also being utilized and examples are discussed below . [SEP]
[CLS] additionally , alternatives such as end functionalization ( 5 ' or 3 ' ) , and other cleavable strategies will be discussed in the context of different conjugated moieties . [SEP]
[CLS] peg modification [SEP]
[CLS] poly ( ethylene glycol ) ( peg ) was first conjugated to oligonucleotides more than two decades ago by different academic groups , including bonora , and burcovich in order to improve stability , avoid rapid degradation , and enhance cellular uptake . [SEP]
[CLS] in 2003 , ji hoon jeong et al . described peg attached to an antisense oligonucleotide sequence ( c - myb ) to afford a surrounding corona , which would prohibit interactions with plasma proteins B-material and increase aqueous solubility B-property . [SEP]
[CLS] intracellular uptake is achieved due to the formation of a micellar system resulting from the combination of peg conju - gates with fusogenic cationic B-material peptides B-material . [SEP]
[CLS] these formulations exhibit higher antiproliferative activity against smooth muscle cells B-material compared to the unconjugated c - myb antisense . [SEP]
[CLS] galnac modification [SEP]
[CLS] the galnac ( n - acetylgalactosamine ) modification , introduced by nair et al . , lead to hydrophilic B-property glycoconjugates ( figure 4 ) , increasing the cellular internalization in the liver due to binding to the asialoglyco - protein receptor B-material ( asgpr ) . [SEP]
[CLS] these lectin type c receptors are highly expressed on the plasma membrane of hepatocytes and both galactose and galnac are the preferred ligands for these receptors . [SEP]
[CLS] after binding to asgpr , the complex is quickly internalized to form early endosomes via a clathrin dependent mechanism . [SEP]
[CLS] as a result , asgpr targeting conjugates are readily delivered to the liver through intraveneous injection . [SEP]
[CLS] once inside the cell B-material , the oligonucleotides must escape from the endosome intact to elicit their bioactivity . [SEP]
[CLS] at neutral ph , the dissociation constant between these modified oligonucleotides and their receptor B-material is very low ( kd in the nanomolar 4 ) . [SEP]
[CLS] rnai - mediated degradation of the targeted mrna mechanism ( 5 ) . [SEP]
[CLS] inhibition of protein B-material translation ( 6 ) . [SEP]
[CLS] asgpr receptors are recycled in the plasma membrane ( 7 ) . range ) , indicating a strong association with asgpr receptors . [SEP]
[CLS] once inside the endosome , the acidic ph allows for dissociation of the ligand - receptor B-material complex , resulting in the return of asgpr to the membrane ( figure 5 ) and subsequent release of the ligand . [SEP]
[CLS] the galnac modification facilitates cell B-material internalization . [SEP]
[CLS] finally , the galnac modified oligonucleotides escape the endosome . [SEP]
[CLS] to prepare these bioconjugates , the galnac modification is introduced into the oligonucleotide either during dna synthesis as a non - nucleoside monomer B-material or at the end of the oligonucleotide synthesis as a " triantennular " form ( figure 4 ) . [SEP]
[CLS] the synthesis of such oligonucleotide conjugates is compatible with standard automated solid phase synthesis , where the galnac modification is attached either at the 3 ' , 5 ' extremities or within the oligonucleotide sequence . [SEP]
[CLS] the non - nucleoside monomer B-material is attached through a phosphodiester linkage B-property during oligonucleotide synthesis as a synthetic phophoramidite . [SEP]
[CLS] all of the different chemical modifications ( 2 ' - ome , 2 ' - f , pto ) , which can be inserted within the same sequence , are compatible with the gal - nac conjugation approach . [SEP]
[CLS] manoharan et al reported that these modifications improve the in vivo stability of sirna−galnac conjugates . [SEP]
[CLS] interestingly , after subcutaneous injection , the sirna−galnac conjugates distribute to the liver with subsequent gene expression decrease of the targeted mrna , thus enabling this approach for treating a wide range of liver diseases . [SEP]
[CLS] the pharmacokinetics of galnac - sirna conjugates were evaluated in monkeys and it was found that phosphorothioate linkages B-property in the 5 ' - region of sirna−galnac strands minimally affect plasma pharmacokinetics but result in a greater liver exposure . [SEP]
[CLS] this provides a prolonged duration of gene silencing due to improved metabolic stability . [SEP]
[CLS] the technology was also developed for asos targeting apolipoprotein with a successful improvement ( 10 - fold ) of inhibition effect in vivo . [SEP]
[CLS] in this study , the active aso is liberated only after internalization into cells B-material and not in the plasma , demonstrating that aso - galnac conjugates are a functional prodrug . [SEP]
[CLS] many gal - nac conjugated oligonucleotides are currently in preclinical and clinical trials . [SEP]
[CLS] for example , fitusiran 77 , 78 is a sirna developed by alnylam for the treatment of hemophilias . [SEP]
[CLS] the collaboration between ionis and akcea yielded aso ionis - apo ( a ) lrx for the treatment of hyperlipoproteinemia and cardiovascular diseases . [SEP]
[CLS] additionally , two anti - mirnas are being developed by regulus to treat viral diseases , such as hepatitis c ( rg - 101 ) , and a third is being developed in collaboration with astrazeneca for the treatment of type 2 diabetes ( rg - 125 ) . [SEP]
[CLS] peptide B-material sequences : cell B-material penetrating peptide B-material ( cpp ) and rgd . [SEP]
[CLS] cpps are short peptidic sequences , generally not exceeding thirty residues , which possess the ability to cross a cellular membrane and facilitate endosomal escape ( figure 6 ) . [SEP]
[CLS] cpp are classified into three categories : 1 ) protein - derived peptides B-material ; 2 ) chimeric peptides B-material formed by the fusion of two natural sequences ; and , 3 ) synthetic peptides B-material based on structure / function studies . [SEP]
[CLS] these short peptides B-material are ligands for specific receptors that facilitate cell B-material internalization by endocytosis B-event and destabilization of endosomes compartments . [SEP]
[CLS] for example , lonn et al . speculated that the ptd / cpp - eed domains enhance cellular delivery by insertion of a hydrophobic B-property patch into the lipid B-material bilayer I-material at a critical distance ( 18 bonds ) from the delivery domain . [SEP]
[CLS] this resulting concentration in the endosome results in a strong localized membrane destabilization leading to enhanced escape into the cytoplasm . [SEP]
[CLS] all cpps do not exhibit the same penetration mechanism , membrane leakage rates , or toxicity B-property . [SEP]
[CLS] in 2007 , a cpp ( ( rxr ) 4 ( x = 6 - aminohexanoic acid ) ) conjugated to a pmo oligomer was investigated . [SEP]
[CLS] the cpp - pmo conjugates did not show toxicity B-property below a dose of 15 mg / kg after intravenous injection ( bolus ) . [SEP]
[CLS] however at a dose of 30 mg / kg , animal body weight decreased while at a dose of 150 mg / kg , the animals were lethargic with elevated levels of creatinine . [SEP]
[CLS] blood biochemisty analysis of albumin , electrolytes , and bilirubin showed constant levels at all doses . [SEP]
[CLS] a major design issue for these conjugates is the complexation between the anionic oligonucleotides and the cationic B-material cpps . [SEP]
[CLS] in a previous review , steven dowdy focused on the potential use of cpp to deliver sirna , and the reader is referred to this manuscript . [SEP]
[CLS] the endocytosis - mediated uptake of such peptides B-material depends on three important steps : cell B-material association , internalization , and finally endosomal escape . [SEP]
[CLS] dowdy et al . also discuss the array of different cargos that have been delivered by cationic B-material ptds / cpps as well as cellular B-event processes I-event and biological responses that have been modulated . [SEP]
[CLS] cpps share many common features responsible for efficient activity including positive amino B-material acids I-material ( arginine B-material and lysine B-material ) and hydrophobic B-property residues ( tryptophan B-material and phenylalanine B-material ) . [SEP]
[CLS] alternatively , non - ionic oligonucleotides ( pmo , pna , phosphoryl guanidine ) are good candidates to bypass this charge - charge complexation requirement . [SEP]
[CLS] such systems are used successfully for exon skipping . [SEP]
[CLS] conventional formulations that carry oligonucleotides are composed of cpp via physical mixing , but the conjugation of these molecules to the oligonucleotide by a covalent bond provides additional benefits . [SEP]
[CLS] the chemical insertion of multiple peptides B-material on the same oligonucleotide improves the sensitivity of the conjugate for its receptor B-material . [SEP]
[CLS] in the case of sirna conjugated to cyclic rgd , the internalization mechanism is achieved by caveola - dependent endocytosis B-event . [SEP]
[CLS] in another study , the bombesin peptide B-material , coupled to the 5 ' end of a splice - shifting oligonucleotide ( sso ) , corrects splicing of an aberrant intron . [SEP]
[CLS] this fourteen amino B-material acid I-material peptide B-material , which is a neurotransmitter acting on the g protein receptors , facilitates oligonucleotide delivery to the nucleus of transfected prostate cancer pc3 cells B-material . [SEP]
[CLS] ye et al . demonstrated that a 3 ' cpp covalently conjugated to sirna increased their intra cellular delivery ( figure 6 ) . [SEP]
[CLS] the report investigated cpps with different conjugation chemistries with a peg space , one cleavable disulfide B-property linkage I-property , and another through a thiol - maleimide coupling . [SEP]
[CLS] the incorporation of a stable versus cleavable linkage B-property led to differences in knockdown and efficacy . [SEP]
[CLS] the linear tripeptide rgd and its cyclic - rgd analogs are well known peptides B-material for cell B-material targeting . [SEP]
[CLS] these peptide B-material ligands bind αvβ3 integrins which are overexpressed in several cancers , including melanomas , ovarian cancer , and breast cancer as well as in metastasis B-event [SEP]
[CLS] grafting such ligands to therapeutic oligonucleotides provides a means for targeting and efficient delivery . [SEP]
[CLS] however , a drawback of using the rgd as a targeting moiety is that the αvβ3 integrin is expressed on many cell B-material types as well as platelets , and , as such off - target effects and toxicities B-property are of concern . [SEP]
[CLS] although this class of peptide B-material - modified bioconjugates is being actively investigated for the delivery of nucleic B-material acid I-material in vitro , positive results still remain to be confirmed in vivo . [SEP]
[CLS] additionally , biodistribution and resistance to proteolytic degradation are not reported . [SEP]
[CLS] α - tocopherol . [SEP]
[CLS] α - tocopherol , commonly known as vitamin e , is used for the treatment of hypercholesterolemia . [SEP]
[CLS] this liposoluble B-property moiety is often coupled to oligonucleotides to facilitate cell B-material membrane interactions . [SEP]
[CLS] for example , yutaro asami et al . coupled tocopherol to a sirna at the 5 ' end of the 29 - nucleotide antisense strand for reducing apolipoprotein b ( apob ) levels . [SEP]
[CLS] improved inhibitory activity is observed relative to the parent cholesterol derivative . [SEP]
[CLS] in fact , only 2 mg / kg is needed to significantly reduce apob mrna in mouse liver after intravenous injection . [SEP]
[CLS] in similar conditions , the amount of cholesterol conjugate required to observe the same activity was 50 to 100 mg / kg . [SEP]
[CLS] additionally , α - tocopherol , coupled to an aso , increases endogenous gene inhibition in a mouse liver compared to an unconjugated aso . [SEP]
[CLS] simply conjugating tocopherol to the oligonucleotide structure did not induce an inhibitory effect , and a spacer B-material of several ( 4 - 7 ) unlocked nucleic B-material acids I-material ( una ) was required between the oligonucleotide 5 ' extremity and tocopherol to maintain biological activity . [SEP]
[CLS] in this study , the oligonucleotides within the aso selected were gapmers , oligonucleotides containing a mixture of chemically modified nucleotides ( lna , 2 ' - o - moe , for example ) at each extremities and a central sequence of dna ( the " gap " ) , allowing the rnase h cleavage after forming the mrna / dna complementary heteroduplex . [SEP]
[CLS] the una residues , on the spacer B-material , are cleaved in the cell B-material affording the aso using the rnase h mechanism for inhibition of mrna . [SEP]
[CLS] the inhibitory efficacy increased by 3 . 5 fold compared to the intravenous injection of non - conjugated oligonucleotides . [SEP]
[CLS] importantly , tocopherol improved the pharmacokinetics of aso affording accumulation in the liver ( 9 - after injection at a dose of 3 mg / kg for the conjugated and unconjugated aso , respectively ) , unlike the unconjugated aso , which distributed primarily to the kidneys ( 1 - 2 μg / g and 12 - 16 μg / g were found in the kidneys 6 h after injection at a dose of 3 mg / kg for conjugated and unconjugated aso , respectively ) . [SEP]
[CLS] aptamers [SEP]
[CLS] aptamers are oligonucleotide sequences ( dna or rna ) possessing a three - dimensional structure for biological recognition . [SEP]
[CLS] using a method called selex ( systematic evolution of ligands by exponential enrichment ) specific oligonucleotides are identified that bind a target with high affinity ( kd in the range of nanomolar to picomolar ) . [SEP]
[CLS] sullenger et al . used this approach to select an aptamer capable of targeting the prostate - specific membrane antigen ( psma ) receptor B-material at the surface of prostate cells B-material . [SEP]
[CLS] the designed rna molecule contained both the aptamer and sirna moieties . [SEP]
[CLS] this chimeric aptamer - sirna features a dsrna ( sirna ) motif that is a substrate for the intracellular dicer enzyme , an endoribonuclease . [SEP]
[CLS] as a proof of concept , lncap and pc - 3 prostate cells B-material were treated with the chimeric aptamer - sirna containing a therapeutic sirna sequence that targets genes overexpressed in human tumors B-material ( plk1 and bcl2 ) . [SEP]
[CLS] successful knockdown resulted in a decrease in cell B-material proliferation and an increase in apoptosis B-event . [SEP]
[CLS] as a control , an aptamer chimera bearing two point mutations exhibited an absence of specificity for its target , indicating the specific binding between the aptamer and its receptor B-material . [SEP]
[CLS] in another study , a sirna targeting the tat / rev viral genes was conjugated via a short polynucleotide B-material link to an aptamer that recognizes the glycoprotein gp120 of the hiv viral envelope . [SEP]
[CLS] this conjugate suppressed hiv infection in human cell B-material lines and in humanized mouse models . [SEP]
[CLS] aptamer - sirna chimeras are of significant interest and the reader is referred to a recent review summarizing the state - of - the - art . [SEP]
[CLS] the additional advantages of such chimeras include lower production costs , low batch - to - batch variation , longer shelf - life and little - to - no immunogenicity B-property and toxicity B-property compared to antibody B-material conjugates ( discussed below ) . [SEP]
[CLS] given that the three - dimensional structure is required for recognition and that aptamers are degraded by nucleases , significant research efforts are directed at modifying nucleotides to increase the stability without losing target recognition . [SEP]
[CLS] there are several locations and types of modifications being investigated including : the terminals of nucleic B-material acids I-material , the phosphodiester linkage B-property , the sugar ring and modifications on the bases , as well as 3 ' end capping with inverted thymidine and peg conjugation . [SEP]
[CLS] antibodies B-material . [SEP]
[CLS] monoclonal antibodies B-material are known for their recognition properties of biological targets . [SEP]
[CLS] in 2005 lieberman et al . reported the use of a monoclonal B-material antibody B-material conjugate for the delivery of sirna in order to inhibit the protein B-material production of hiv . [SEP]
[CLS] the antibody B-material was conjugated to the aso oligonucleotide via a click reaction involving an azidemodified antibody B-material and a cyclooctyne aso . [SEP]
[CLS] this strategy was further developed by satake et al . to conjugate the anti - cd22 antibody B-material to the mxd3 oligonucleotide . [SEP]
[CLS] this oligonucleotide targets a dimeric protein B-material ( mxd3 ) associated with myc , a transcription B-event factor responsible for the survival of b cell precursor cells B-material in acute lymphoblastic leukemia . [SEP]
[CLS] the conjugate induces inactivation of the mxd3 protein B-material responsible for apoptosis B-event in vitro in leukemic cells B-material as well as affords cytotoxicity B-property in normal b cells B-material but not in hematopoietic cells , including stem cells . [SEP]
[CLS] the in vivo results are promising as only a dose of 0 . 2 mg / kg twice a week for 3 weeks is required to double survival ( median 42 . 5 vs . 20 . 5 days ) . [SEP]
[CLS] based on these encouraging results , antibody - aso conjugates are a new therapeutic option to decrease the doses and to favor the accumulation of asos at designated locations . [SEP]
[CLS] however , the selection of antibody B-material or antibody B-material fragment for targeting a specific cell B-material type is challenging . [SEP]
[CLS] for example , genentech published an article describing the investigation of antibodies B-material conjugated to aso by a maleimide B-property linkage I-property , for targeting prostate carcinoma cells B-material after systemic administration . [SEP]
[CLS] this challenge is further complicated by the fact that the route of antigen internalization also affects silencing . [SEP]
[CLS] although antibody B-material conjugates reach the intended target site , the subsequent steps of cellular internalization and endosomal escape are also required for efficacy . [SEP]
[CLS] cholesterol . [SEP]
[CLS] lipid B-material moieties , and particularly cholesterol derivatives , are often conjugated to oligonucleotides for delivery purposes ( figure 7 ) . [SEP]
[CLS] oligonucleotides modified with cholesterol are recognized by high and low - density lipoproteins ( hdl and ldl ) in vivo and internalized via cholesterol binding B-event receptors I-event . [SEP]
[CLS] after intravenous or intraperitoneal injection , these oligonucleotide conjugates escape renal clearance , thus greatly impacting their pharmacokinetics by extending the time that the nucleic B-material acids I-material remain in the plasma . [SEP]
[CLS] a number of studies have demonstrated the advantage of coupling a cholesterol moiety to the oligonucleotide in order to reduce its renal clearance . [SEP]
[CLS] also , cholesterol conjugation increases cellular uptake of oligonucleotides . [SEP]
[CLS] godeau et al . reported improved in vitro cellular internalization of an oligonucleotide functionalized with cholesterol via a " click chemistry " approach . [SEP]
[CLS] additionally , these conjugates inhibited viral translation of hepatitis via an ires blocking mechanism . [SEP]
[CLS] additional studies in the kb - 8 - 5 cell B-material line showed that the cholesterol moiety allows endosomal escape without the use of any additional transfecting reagents . [SEP]
[CLS] after 4 hours , the sirna conjugates accumulate in the intercellular space . [SEP]
[CLS] after 24 hours , the distribution became homogeneous with an accumulation of the oligonucleotides in the cytoplasm of the cells B-material . [SEP]
[CLS] from a design perspective , the spacer B-material between the oligonucleotide and cholesterol is crucial to maintain the biological activity . [SEP]
[CLS] directly grafting the cholesterol to the 5 ' oligonucleotide end reduced the inhibitory activity of the nucleic B-material acids I-material ( sirna or aso ) . [SEP]
[CLS] in another study , apob was targeted to the liver after a daily intravenous injection of sirna - cholesterol conjugates at a dose of 50 mg / kg . [SEP]
[CLS] additionally , the huntingtin protein B-material was inhibited after a single intrastriatal injection of a cholesterol - coupled sirna . [SEP]
[CLS] these two examples demonstrate that sirna - cholesterol conjugation improves the delivery into different cell B-material types other than liver cells B-material . [SEP]
[CLS] a photo - responsive cholesterol - sirna conjugate ( on both sense or antisense strands ) is recently reported containing a photo - cleavable linkage B-property ( figure 7 ) [SEP]
[CLS] the orthonitrobenzyl group photo - cleaves in response to 365 nm uv light to yield the native 5 ' end . [SEP]
[CLS] three different cholesterol derivatives were prepared to inhibit 3 different genes ( 2 exogenous , luciferase and gfp ; and an endogenous , eg5 ) in hepatocarcinoma hepg2 cells B-material . [SEP]
[CLS] in the absence of light , this conjugation allows the internalization of the amphiphilic B-property cholesterol - sirna without inhibiting the target . [SEP]
[CLS] in the presence of a light stimulus , the link between the cholesterol and the sirna is cleaved , and the inhibitory activity of sirna is introduced [SEP]
[CLS] cholesterol is conjugated to oligonucleotides at different locations including the 5 ' or 3 ' end , as well as within the sequence . [SEP]
[CLS] a recent study reported six constructs conjugated with cholesterol covalently bound either at the ends or after thymidines within the sequence . [SEP]
[CLS] the oligonucleotide selected was a gapmer with 2 and 3 lnas at both the 5 ' and 3 ' ends , respectively . [SEP]
[CLS] higher in vitro activities are obtained when the cholesterol is inserted either at the 5 ' end or within the sequence . [SEP]
[CLS] in vivo , the cholesterol conjugates accumulate in the liver independent of the lipid B-material conjugation location ( 3 ' , 5 ' - ends or within the sequence ) . [SEP]
[CLS] furthermore , a regular phosphodiester linkage B-property exhibited superior in vivo performance in terms of biological activity ( by a factor of 5 ) compared to the analog phosphorothioate as a link between the lipid B-material and the aso . [SEP]
[CLS] nakajima et al . suggests that the unmodified phosphate is cleaved in the liver freeing the non - conjugated aso . [SEP]
[CLS] in 2007 , moschos et al . covalently attached the cholesterol moiety to the oligonucleotide sequence via a disulfide linker that is cleaved under intracellular reductive conditions . [SEP]
[CLS] in this study , the authors suggest that conjugation to cholesterol extends but does not increase sirnamediated mrna knockdown in the lung ( map kinase mrna in mouse lung ) . [SEP]
[CLS] squalene . [SEP]
[CLS] squalene is a triterpene molecule and biosynthetic precursor of cholesterol found in plants and animals . [SEP]
[CLS] squalene is often conjugated to the 3 ' - end of the sense strand of sirna via a thiol - maleimide coupling . [SEP]
[CLS] due to the resulting amphiphilic B-property character of the squalene - oligonucloetide conjugate , these conjugates spontaneously formed spherical assemblies of 165 ± 10 nm diameter with a zeta B-property potential I-property of − 26 mv in aqueous media . [SEP]
[CLS] these nanoparticles B-nanoparticle show enhanced oligonucleotide stability in the presence of serum and higher catalytic potency in vitro and in vivo against the targeted ret / ptc1 mrna compared to the unmodified sirna . [SEP]
[CLS] up to 70 % inhibition of the tumor B-material growth is observed after 19 days following intravenous injections of 2 . 5 mg / kg modified sirna at day 0 , 2 , 4 , 7 and 10 . [SEP]
[CLS] fatty acids . [SEP]
[CLS] currently in clinical trial ( phase 2 ) for the treatment of myelofibrosis , grn163l is a telomerase targeting , antisense oligonucleotide of thio - phosphoramidate with a covalently linked palmitic acid at the 5 ' - end ( figure 8 ) . [SEP]
[CLS] the palmitic acid moity is conjugated though a pto linkage B-property to the backbone of the aso . [SEP]
[CLS] telomerase activity is up - regulated in numerous cancers and , thus , is a viable target for treatment . [SEP]
[CLS] telomere shortening and lower cell B-property viability I-property are observed after inhibition of telomerase activity in cancer cell B-material lines . [SEP]
[CLS] the ic50 values ranged from 50 to 200 nm for telomerase expression in 10 different pancreatic cell B-material lines in the presence of grn163 . [SEP]
[CLS] telomerase reactivation and elongation are also observed when the antisense is no longer present , indicating that sequences targeting the rna template region of telomerase are effective inhibitors of telomerase . [SEP]
[CLS] grn163l is a potential adjuvant for cancer treatment as it halts the progression of the tumor B-material by restoring a normal , correct phenotype for the cancerous cells B-material . [SEP]
[CLS] the in vitro inhibition of telomerase activity by aso is improved with the palmitoyle modification . [SEP]
[CLS] an inhibition factor ranging from 1 . 4 to 39 is observed depending on the cell B-material type ( cervix , glioblastoma , hepatocarcinoma , lungs , melanoma , myeloma , ovary and prostate ) . [SEP]
[CLS] in vivo efficacy ( up to 56 % inhibition of telomerase activity after 24h compared to the uncoupled antisense or pbs ) is also observed in a murine xenograft model following intravenous injection ( 50 mg / kg ) in the absence of any additional transfecting reagent . [SEP]
[CLS] additionally , investigations reveal a synergistic effect when grn163l is coadministered with trastuzumab , a recombinant monoclonal B-material antibody I-material targeting her2 + receptors in breast cancers . [SEP]
[CLS] the composition of the lipid B-material ( oleic vs palmitic acid in the lipid B-material oligonucleotide conjugate ) was also evaluated , but with no noticeable difference in efficacy . [SEP]
[CLS] ketal nucleolipid conjugate . [SEP]
[CLS] nucleolipids , first reported by our groups , are also being investigated . [SEP]
[CLS] for example , the ketal uridine phosphoramidite with two alkyl chains at the 2 ' and the 3 ' positions , anchors oligonucleotides to liposomal B-nanoparticle membranes I-material . [SEP]
[CLS] these lipid - oligonucleotides are amphiphilic B-property with micellar aggregation properties that are used as a functional reservoir for hydrophobic B-property drugs . [SEP]
[CLS] the drug payload of the micelles B-material is released in the presence of the oligonucleotides complementary to the lipid B-material - dna conjugate . [SEP]
[CLS] similarly modified asos are investigated for the treatment of prostate cancer . [SEP]
[CLS] an antisense strategy based on targeting the translational controlled tumor B-material protein B-material ( tctp ) is described for the treatment of castration resistant prostate cancer ( crpc ) , a disease currently with a very low survival rate ( median survival reported between 9 and 30 months ) . [SEP]
[CLS] tctp is minimally expressed in healthy prostatic tissue , moderately in prostatic cancer cells B-material , and overexpressed in crpc cells B-material . [SEP]
[CLS] therefore , designing an inhibitory sequence is key for this promising strategy for the treatment of crpc . [SEP]
[CLS] an aso sequence of 20 nucleotides with pto backbone was selected to impede tctp expression . [SEP]
[CLS] normally , this aso sequence requires the assistance of a vectorization agent such as oligofectamine to be efficient in vitro . [SEP]
[CLS] the conjugation of the aso with the ketal nucleolipid enables the inhibition of tctp in the absence of transfecting agent in vitro and in vivo . [SEP]
[CLS] bis - conjugates . [SEP]
[CLS] multi - functionalized , bis - conjugated oligonucleotides are also being investigated . [SEP]
[CLS] tajik - ahmadabad et al . [SEP]
[CLS] designed an amphiphilic B-property cpp - modified aso ( in the pmo series ) capable of self - assembly . [SEP]
[CLS] the aso is functionalized by coupling a 3 - maleimidopropanoic acid moiety to the free secondary B-material amine I-material group I-material at the 3 ' end . [SEP]
[CLS] this maleimido - pmo was purified by hplc method . [SEP]
[CLS] the fatty - acid modified peptide B-material was prepared by coupling myristic anhydride at the n - terminus of the resin - bound apoe . [SEP]
[CLS] after deprotection and resin cleavage , the apoe peptide B-material with c - terminal cysteine B-material was conjugated to the aso by thiol - maleimide click reaction . [SEP]
[CLS] the spontaneous self - assembly of this aso occurs when myristic acid is covalently attached to the n - terminus of the peptide B-material . [SEP]
[CLS] the bis - conjugated aso inhibits exon 7 splicing of the pre - mrna smn2 gene , which is responsible for the pathology of spinal muscular atrophy . [SEP]
[CLS] these amphiphilic B-property species exhibit a 4 - fold increase in potency relative to the cpp - aso mono - conjugate . [SEP]
[CLS] a similar approach is described by wada et al . , who used cholesterol and galnac as modifiers at the 5 ' - end of an aso targeting apob ( figure 9 ) . [SEP]
[CLS] the conjugation was prepared using phosphoramidite chemistry , amenable to solid support synthesis of oligonucleotides . [SEP]
[CLS] the monovalent galnac phosphoramidite used was described earlier by yamamoto et al . [SEP]
[CLS] such a dual conjugation strategy reduces the renal biodistribution of the oligonucleotide . [SEP]
[CLS] in that case , the cholesterolconjugated aso also possessed a 3 - nucleotide - long linker with phosphodiester linkages B-property susceptible to endonuclease hydrolysis in the targeted tissues or cells B-material . [SEP]
[CLS] accumulation of the galnacmodified aso is low in the kidney just like the double 5 ' - modified cholesterol - galnac aso . [SEP]
[CLS] renal accumulation is five times lower when the aso is modified at both extremities one with the lipid B-material and the other with galnac . [SEP]
[CLS] as expected , the gal - nac modified asos preferentially accumulate in the liver . [SEP]
[CLS] thus , the cholesterol - galnac dual strategy represents an effective strategy for reducing the nephrotoxic potential of asos , while maintaining the gene silencing activity in the liver . [SEP]
[CLS] spherical nucleic B-material acids I-material [SEP]
[CLS] spherical nucleic B-material acids I-material ( snas ) are a class of nucleic B-material acid I-material conjugates differentiated from other conjugates here through their three dimensional nanostructure . [SEP]
[CLS] first developed by chad mirkin , this structure is composed of oriented single or double stranded nucleic B-material acids I-material in a dense spherical array ( figure 10 ) . [SEP]
[CLS] first generation snas possessed a gold B-nanoparticle nanoparticle I-nanoparticle core B-material with au - s linkages B-property to the nucleic B-material acids I-material , and since then have been expanded to contain other cores B-material for specific applications [SEP]
[CLS] sna structures are amenable to further modification to contain targeting B-material ligands I-material such as antibodies B-material , and immune agents such as antigens . [SEP]
[CLS] snas can also be formed with rna , modified backbones [SEP]
[CLS] , and sugar modifications [SEP]
[CLS] snas exhibit increased nuclease resistance due to their structure and are readily endocytosed into cells B-material without additional transfection making them an attractive bioconjugate class to enhance delivery [SEP]
[CLS] liposomal B-nanoparticle snas 138 are formulated from 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine and a tocopherol - oligonucleotide conjugate which contains a phosphodiester linkage B-property and a peg spacer B-material ( discussed in its own section above ) . [SEP]
[CLS] tocopherol - oligonucleotide conjugates form micellar structures , but liposomal B-nanoparticle snas provide additional benefit [SEP]
[CLS] . liposomal B-nanoparticle sna formulations are being evaluated as a novel therapy for immune modulation for example , snas are effective for both immune tlr agonism and inhibition through either target sequence dependent binding B-event to I-event tlr I-event receptors I-event or the receptor for mrna translation [SEP]
[CLS] receptor B-material agonism strategies focus on agonism through tlrs , and the activity of the sna conjugates is dependent on linker chemistry . [SEP]
[CLS] sna and tocopherol oligonucleotide conjugates show effective immune stimulation and tu - mor growth reduction in vivo , and sna formulations demonstrate the highest efficacy . [SEP]
[CLS] in a recent publication , a melanoma - specific peptide B-material antigen was conjugated via three different linkers to a tlr9 agonizing oligonucleotide adjuvant . [SEP]
[CLS] the complimentary oligonucleotide was conjugated to an antigen . [SEP]
[CLS] three different antigen - oligonucleotide linkages B-property were evaluated : one stable ( n - ( β - maleimidopropyloxy ) succinimide ester ) , one cleavable ( succinimidyl 3 - ( 2 - pyridyldithio ) propionate ) , and a traceless linkage B-property ( 4 - nitrophenyl 2 - ( 2 - pyridyldithio ) ethyl carbonate B-material ) . [SEP]
[CLS] see figure 10 for structural comparison . [SEP]
[CLS] the traceless linkage B-property undergoes an internal cyclization in addition to a disulfide cleavage to yield the antigen ' s native nterminus . [SEP]
[CLS] the choice of linker conjugation significantly influenced activity . [SEP]
[CLS] the sna formulations possessing the cleavable , traceless delivery linkage B-property yielded the greatest t cell activation and proliferation . [SEP]
[CLS] importantly , these findings highlight the critical role the linker plays . [SEP]
[CLS] moreover , they also demonstrate the potential for oligonucleotide conjugates to expand to new applications and a few sna formulations are progressing through preclinical into early clinical development ( e . g . , nct03086278 , table 1 and discussion below ) . [SEP]
[CLS] as exemplified in the preceding sections , numerous oligonucleotides have been investigated for a range of diseases . [SEP]
[CLS] the majority of these bioconjugated oligonucleotides are in preclinical studies ( i . e . , in vitro and small animal in vivo efficacy studies ) . [SEP]
[CLS] importantly , several formulations are regulatory approved and used in the clinic ( table 1 ) . [SEP]
[CLS] the data accumulated in table 1 are from company web sites and the clinicaltrials . gov web resource . [SEP]
[CLS] the first regulatory approved oligonucleotide ( in 1998 by the fda and in 1999 by the ema ( european medicines agency ) ) was for the treatment of cytomegalovirus retina , a viral infection of the eye . [SEP]
[CLS] if untreated , vision loss can occur in immune compromised patients . [SEP]
[CLS] specifically , fomivirsen ( vitravene ® ) was developed by ionis pharmaceutical ( isis at the time ) in collaboration with novartis ophthalmics . [SEP]
[CLS] this biomacromolecule , composed of 21 pto nucleotides , is complementary to one viral mrna . [SEP]
[CLS] the posology consists of a weekly intravitreal injection of 165 µg of a fomivirsen solution at 6 . 6 mg / ml . [SEP]
[CLS] the market authorization was withdrawn on july 30th 2002 at the request of the manufacturer for business reasons . [SEP]
[CLS] vitravene ® is still authorized in switzerland and can be administered in the european union upon specific request . [SEP]
[CLS] importantly , this success laid the foundation for a number of oligonucleotides being ushered into commercial development and the reader is referred to several reviews on this topic [SEP]
[CLS] as discussed in the introduction of this review , the design of oligonucleotides for use in the clinic requires specific design considerations and optimizations other than improved delivery . [SEP]
[CLS] for enhanced in vivo efficacy , oligonucleotides containing chemical modifications such as 2 ' - o - moe , 2 ' ome , lna and pmo are used to protect against degradation and increase target binding affinity . [SEP]
[CLS] to date , the most common oligonucleotide modifications are pto dna and 2 ' - o - moe . [SEP]
[CLS] additionally , 2 ' ome and lna are used , but most of these oligonucleotides are still in phase 1 or 2 clinical trials . [SEP]
[CLS] as of 2010 , all of the oligonucleotides approved by the fda and in phase 3 clinical trials contain pto linkages B-property . [SEP]
[CLS] the selection of oligonucleotide modifications defines the mechanism ( s ) of inhibition ( figure 2 ) . [SEP]
[CLS] the majority of oligonucleotides in phase 1 or 2 trial participate in an rnase h based inhibition mechanism , but are of more diverse chemical composition and functionalization . [SEP]
[CLS] that said , the inhibition mechanisms available include intron - exon splicing , rnase h activation , or blockage of translation through steric hindrance . [SEP]
[CLS] the following subsections are organized according to targeted disease with a discussion of the bioconjugated oligonucleotides used as well as their mechanism of action . [SEP]
[CLS] bone marrow disorders . [SEP]
[CLS] ( myelofibrosis and myelodysplastic disorders ) . [SEP]
[CLS] myeleofibrosis is caused by the replacement of bone marrow with scar tissue . [SEP]
[CLS] the resulting scarring leads to a reduction of red blood cell B-material formation . [SEP]
[CLS] myelodysplastic syndrome is a type of cancer in the bone marrow . [SEP]
[CLS] risk factors are genetic or caused by or arise during some cancer treatments . [SEP]
[CLS] imetelstat is a palmitoyl lipid B-material oligonucleotide conjugate in clinic trials to treat these diseases . [SEP]
[CLS] this 13 nucleotide - long aso targets telomerase activity , using palmitate as the targeting B-material ligand I-material , with thio - phospharamidite linkages B-property including the linkage B-property to the palmitate group ( figure 11 ) . [SEP]
[CLS] the drug has completed multiple phase ii clinical trials for metastatic breast cancer and multiple myeloma cancers patients . [SEP]
[CLS] the dose is limited by thrombocytopenia . [SEP]
[CLS] imetelstat ( grn163l ) received a fast track status in october 2017 from the fda for certain patients with a myelodysplastic syndrome . [SEP]
[CLS] hepatitis b . hepatitis b is a common liver infection . [SEP]
[CLS] the genome of the hepatitis b virus ( hbv ) is an attractive target because there are multiple sites on the hbv genome that can be targeted simultaneously . [SEP]
[CLS] there are several conjugates in the pipeline for the treatment of this disease . [SEP]
[CLS] for example , arc - 520 is an equimolar combination of two cholesterol conjugated sirnas , sihbv74 and sihbv77 , targeting an 18 nucle - otide long sequence in the hbv genome . [SEP]
[CLS] in addition to the sir - nas , a galnac conjugated peptide B-material is included as an excipient B-property to aid in delivery and endosomal escape . [SEP]
[CLS] a phase trial i was completed in 2018 , but the results have not been publicized as of september 2018 . [SEP]
[CLS] other clinical trials with arc - 520 have been withdrawn / terminated , likely due to the difficulty in knocking down expression due to hbv dna integrated into the host genome . [SEP]
[CLS] arrowhead pharmaceuticals has a combined phase i / ii clinical trial currently recruiting for aro - hbv . [SEP]
[CLS] alnylam terminated a phase i clinical trial using aln - hbv01 , a 23 nucleotide long , galnac conjugated , sirna . [SEP]
[CLS] this galnac conjugated sirna incorporated numerous modifications for solubility B-property and stability , including 2 ' - f , 2 - ome , with ps linkages B-property only near the sirna termini . [SEP]
[CLS] the discontinuation is part of a new agreement with vir biotechnology to begin development on aln - hbv02 , also a galnac conjugate , citing reduced off target effects due to the incorporation of glycol nucleic B-material acids I-material ( gnas ) in the backbone . [SEP]
[CLS] hemophilia [SEP]
[CLS] hemophilia is a blood clotting disorder . [SEP]
[CLS] fitusiran ( aln - at3sc ) is a galnac conjugate targeting antithrombin currently entering phase iii clinical trials . [SEP]
[CLS] in a phase i study , the mean antithrombin ( at ) knockdown was reported to be 59 % . [SEP]
[CLS] the study included 4 healthy volunteers ( a ) and 12 patients with severe hemophilia ( b ) . [SEP]
[CLS] in one hemophilia patient , a maximal knockdown of 86 % was reported along with 114 days bleed free . [SEP]
[CLS] this conjugate was administrated via subcutaneous injection . [SEP]
[CLS] first phase results showed the reduction of levels of the antithrombin protein B-material and the restoration of the balance for the production of clotting factors in adult a and hemophilia b patients , after a single monthly injection . [SEP]
[CLS] the second phase of the trial was suspended because one hemophilia a patient died due to a cerebral edema . [SEP]
[CLS] the suspension of the clinical trial was lifted after the trial ' s protocol was amended to better mitigate risks . [SEP]
[CLS] alnylam and sanofi are scheduled to conduct a phase 3 clinical trial in 2018 / 2019 with the atlas program , which will include patients with hemophilia a and b with or without inhibitors , and patients previously receiving on - demand or prophylactic therapy . [SEP]
[CLS] psoriasis [SEP]
[CLS] psoriasis is a chronic skin disease where skin cells B-material undergo a rapid cell B-material cycle . [SEP]
[CLS] snas are being developed to treat dermatologic indications such as mild to moderate psoriasis . [SEP]
[CLS] recently completed results from phase i studies for il17ra and tnfα protein B-material targets are promising ( purdue pharma ) . [SEP]
[CLS] the snas are absorbed through the skin and deliver sirnas into the epidermis providing effective knockdown of targets within keratinocytes with minimal immune response . [SEP]
[CLS] topical administration offers a local treatment option , which minimizes systemic off - target effects . [SEP]
[CLS] cancer [SEP]
[CLS] cancer is the unwanted abnormal growth and invasion of cells B-material in the body . [SEP]
[CLS] of the various treatment classessurgery , radiation , chemotherapy , and immunotherapyimmunotherapy is a particularly exciting one as it provides a means to use the immune system to fight the disease . [SEP]
[CLS] for example , ac - tivation of toll - like receptor B-material 9 ( tlr9 ) initiates a cascade of innate and adaptive immune responses including increased interferon gene expression . [SEP]
[CLS] increased interferon gene expression correlates with improved responses to programmed death 1 ( pd - 1 ) inhibition . [SEP]
[CLS] thus , patients who did not respond or stopped responding to anti - pd - 1 therapy are potential candidates for combined tlr9 agonists and anti - pd - 1 therapy . [SEP]
[CLS] exicure has successfully completed a phase i trial of tlr9 agonizing snas administered subcutaneously in healthy volunteers ( nct03086278 ) . [SEP]
[CLS] a phase ib / ii study is planned to study intratumorally delivered snas both alone and in combination with intravenously administered pembrolizumab , an antibody B-material targeting the pd - 1 receptor B-material . [SEP]
[CLS] the target cohort is in patients that have not yet responded to anti - pd - 1 or anti - pd - l1 therapies ( nct03684785 ) . [SEP]
[CLS] age - related macular degeneration . [SEP]
[CLS] age - related macular degeneration ( armd ) is the gradual worsening of vision and eye health over time due to retina damage . [SEP]
[CLS] the blurring or loss of vision associated with armd is characterized by the proliferation of blood vessels behind the retina . [SEP]
[CLS] in " wet " macular degeneration , neovascularization can worsen symptoms and is activated by vascular endothelial growth factor ( vegf ) signaling . [SEP]
[CLS] macugen ® ( pegaptanib ) is a 27 - nucleobase aptamer targeting vegf165 binding , and possesses a 3 ' - 3 ' deoxythymidine extremity and a 5 ' end with two branched polyethylene glycol chains of 20 kda . [SEP]
[CLS] the 2 ' positions are modified for nuclease resistance purposes by 2 ' ome for bases purines and fluorinated pyrimidines ( figure 12 ) . [SEP]
[CLS] importantly , the aptamer binds the heparin site of vegf with a picomolar affinity . [SEP]
[CLS] the fda approved this aptamer for the treatment of " wet " or age - related macular degeneration . [SEP]
[CLS] specifically , macugen ® , injected intravitreally , locally reduces the proliferation / expansion of blood vessels and a lack of adverse events is still observed at a dose ten times higher than clinically used . [SEP]
[CLS] the product is available in the form of pre filled syringes of 90 µl solution containing 0 . 3 mg of active substance . [SEP]
[CLS] several other drugs have been developed for the treatment of armd . [SEP]
[CLS] in the eu , lucentis ® ( ranibizumab , novartis ) , a monoclonal B-material antibody I-material fragment that targets vegf , and eylea ® ( aflibercept , bayer ) a recombinant fusion protein B-material that binds to vegf - a , vegf - b , and placental growth factor ( pigf ) with higher affinity than their native receptors . [SEP]
[CLS] avastin ® ( bevacizumab , roche ) is a humanized recombinant monoclonal B-material antibody I-material that binds vegf and competitively inhibits the binding between vegf and flt - 1 and kdr . [SEP]
[CLS] it adds to the list of macugen ® competitors in the united states . [SEP]
[CLS] the other nucleic B-material acid I-material treatment for armd is a modified sirna called bevasiranib . [SEP]
[CLS] it is a duplex of a 21 - nt long rna that targets vegf . [SEP]
[CLS] bevasiranib has not been approved but shows efficacy when combined with lucentis ® in clinical trials . [SEP]
[CLS] the effects of bevasiranib appear only six weeks after the beginning of the treatment , but the combination of lucentis ® / bevasiranib provides better visual acuity than lucentis ® alone . [SEP]
[CLS] hepatic porphyrias [SEP]
[CLS] these conditions are a combination of rare diseases that all overlap in that they all feature the accumulation of porphyrins in the blood . [SEP]
[CLS] the build - up of these proteins B-material causes nervous or dermatological symptoms . [SEP]
[CLS] currently in an ongoing phase iii clinical trial , givosiran ( aln - as1 ) , is a galnac conjugate sirna targeting aminovulinate synthase 1 ( alas1 ) present in the liver . [SEP]
[CLS] in a phase i trial with 23 patients , 11 patients reported mild or moderate adverse events ( ae ) with 1 reporting an unrelated , severe ae of abdominal pain and another reported unrelated ae of bursitis . [SEP]
[CLS] common mild / moderate aes of abdominal pain , diarrhea , nasopharyngitis , and hypoesthesia , pruritus , and rash were noted , but were not serious enough to stop treatment . [SEP]
[CLS] the product was injected by single or multiple subcutaneous injections and compared to an injection of sterile normal saline solution . [SEP]
[CLS] the purpose of the next phase 1 trial was to evaluate the safety and tolerability of givosiran ( aln - as1 ) in acute intermittent porphyria ( aip ) patients as well as to characterize pharmacokinetics ( pk ) and pharmacodynamics ( pd ) of aln - as1 in aip patients . [SEP]
[CLS] the safety and efficacy of givosiran was evaluated in 17 patients with acute aip . [SEP]
[CLS] the patients were initially monitored for three months and those who experienced one porphyria attack were eligible to receive once monthly injections of 2 . 5 or 5 . 0 mg / kg givosiran or placebo for six months . [SEP]
[CLS] durable inhibition of alas1 was observed after givosiran treatment . [SEP]
[CLS] the lower tested dose of 2 . 5 mg / kg reduced the annualized attack rate by 83 % and afforded a reduction of 88 % of hemin compared to the placebo . [SEP]
[CLS] a larger dose did not produce any amelioration . [SEP]
[CLS] in 2018 , data showed that at 22 months of therapy , patients were still experiencing a robust beneficial effect from givosiran . [SEP]
[CLS] famyloid polyneuropathy . [SEP]
[CLS] familial amyloid polyneuropathy ( fap ) is a genetic autosomal dominant disorder resulting from a mutation of the ttr ( transthyretin ) gene , leading to ttr protein B-material aggregation , which negatively affects the nervous system . [SEP]
[CLS] oligonucleotide therapeutic agents designed for the treatment of fap , also known as transthyretin - related hereditary amyloidosis ( attr ) , target the mrna sequence of transthyretin ( ttr ) for degradation . [SEP]
[CLS] three formulations are in clinical trials to decrease expression of ttr : inotersen ( developed by ionis and gsk ) , onpattro ( patisiran developed by alnylam ) , and revusiran ( developed in collaboration between alnylam and sanofi genzyme ) . [SEP]
[CLS] these therapeutics ( 2 sirnas - one being a galnac conjugate - and an aso ) are in phase 3 clinical trials . [SEP]
[CLS] one study compared the inhibitory activity of inotersen ( aso ) and patisiran ( sirna ) . [SEP]
[CLS] both treatments led to a 80 % decrease in ttr expression . [SEP]
[CLS] inotersen ( ionis - ttrrx ) , is a 20 - nucleotide long gapmer pto aso with 5 2 ' - o - moe modifications at both extremities . [SEP]
[CLS] the aso targets pre - mrna that is degraded following rnase h activity . [SEP]
[CLS] inotersen sodium B-material is subcutaneously injected weekly at a dose of 300 mg ( 284 mg of inotersen ) . [SEP]
[CLS] of note , three cases ( out of the 172 patients tested over 64 weeks ) reported thrombocytopenia . [SEP]
[CLS] inotersen has received marketing authorization approval from the european commission ( ec ) for the treatment of stage 1 or stage 2 polyneuropathy in adult patients with hereditary transthyretin amyloidosis ( hattr ) . [SEP]
[CLS] inotersen is commercialized under the name of [UNK] . [SEP]
[CLS] patisiran ( aln - ttr02 , [UNK] ) is an advanced sirna under development and is administered intravenously as lipidic B-nanoparticle nanoparticles I-nanoparticle ( lnp ) at noticeably low doses of 0 . 3 mg / kg every three weeks over an 18 - month period . [SEP]
[CLS] the most common adverse events observed in patients were peripheral edema and infusion related reactions . [SEP]
[CLS] in august 2018 , patisiran received u . s . food and drug ( fda ) approval for the treatment of the polyneuropathy of hereditary transthyretin - mediated amyloidosis in adults . [SEP]
[CLS] regulatory filings in other markets , including japan , are planned . [SEP]
[CLS] the european commission also granted marketing authorization for [UNK] on august 30th , 2018 . [SEP]
[CLS] revusiran [SEP]
[CLS] a galnac sirna conjugate administered subcutaneously , was previously undergoing phase 3 clinical trials . [SEP]
[CLS] this molecule has two pto linkages B-property , 22 2 ' - ome nucleotides , and another 22 modified residues with 2 ' f modifications . [SEP]
[CLS] however , development was stopped due to numerous aes leading to deaths in phase 3 clinical studies . [SEP]
[CLS] causes of theses side effects have not been published but the high weekly dose of 500 mg was suspected . [SEP]
[CLS] there have been numerous developments over the past 50 years that have led to the clinical efficacy of oligonucleotides for treating diseases . [SEP]
[CLS] the timeline featuring significant accomplishments is presented in figure 12 . asos were the first oligonucleotides to reach clinical trials and to receive regulatory approval . notably , this conjugated ( pegylated ) aptamer has been in use for more than 14 years . [SEP]
[CLS] the development of sirna therapeutics advanced significantly in the early 2000s and their first use in humans occurred in 2005 ( bevasiranib ) . [SEP]
[CLS] historic timeline of developmentssirna therapeutics that functioned as splicing modulators followed next in development and commercialization . [SEP]
[CLS] antagomirs ( mirna inhibitors ) were developed in 2009 and were followed by mirna mimetics in 2013 . [SEP]
[CLS] these two former classes of oligonucleotides utilize mirnas to modulate the activity of a given gene . [SEP]
[CLS] for example , miravirsen sequesters microrna - 122 and is used for the treatment of chronic hepatitis c , whereas mimics of microarn - 34a exhibit potentiated antitumor B-event activity I-event . [SEP]
[CLS] over 30 oncogenes are known to be inhibited by this microrna . [SEP]
[CLS] several bioconjugated oligonucleotides are regulatory approved and used in the clinic . [SEP]
[CLS] these bioconjugates possess a carbohydrate B-material ( patisiran ) , a polymer B-material ( pegaptanib ) , or a lipid B-material ( imetelstat - grn163l , currently on fast track status ) covalently linked to the oligonucleotides . [SEP]
[CLS] a number of additional ones are in preclinical development as well as clinical studies ( see table 1 . ) . [SEP]
[CLS] the primary function of these added conjugates is to enhance targeting , facilitate vectorization , reduce oligonucleotide susceptibility to nuclease activity , eliminate or reduce need for toxic B-property transfection agents , and generally improve pharmacological activity and efficacy . [SEP]
[CLS] increasing the therapeutic window and reducing the likelihood for adverse events is a top priority , and the use of conjugated oligonucleotides is an effective approach . [SEP]
[CLS] as discussed above , these conjugates are lipophilic B-property or a targeting moiety . [SEP]
[CLS] most of the therapies currently in late - stage development target the liver and liver associated diseases . [SEP]
[CLS] this is largely due to the success of the galnac modification , and the fact that the liver is an efficient filter for the blood . [SEP]
[CLS] other highly efficient targeting moieties need to be developed to target other organs or tissueslung , kidney , heart , bone marrow , brain , muscle , etc . [SEP]
[CLS] poor delivery to target sites necessitates higher doses , decreasing safety with greater chances of aes . [SEP]
[CLS] one strategy to reduce the severity of aes is to modulate or knockdown the effect after administration . [SEP]
[CLS] for example , a single subcutaneous 3 mg / kg galnac - sirna dose can be effectively reversed with a 0 . 1 mg / kg galnac oligonucleotide conjugate targeting the seed region of the incorporated sirna strand . [SEP]
[CLS] the result demonstrates both effective knockdown and return of ttr protein B-material levels in preclinical in vivo studies . [SEP]
[CLS] the evolution of safe antidotes for oligonucleotide therapies , such as the reversir strategy reported by zlatev et al . , poses benefits for safety and adoption into the clinic . [SEP]
[CLS] the possibility to finely control rnai pharmacology is a desired feature for highly efficient nucleic based therapeutic treatments . [SEP]
[CLS] at the molecular level , advances in oligonucleotide chemistry are providing new methods and site - specific reactions to modify oligonucleotides at the base , sugar , phosphate , or end groups . [SEP]
[CLS] additionally , natural bases can be substituted with synthetic analogs . [SEP]
[CLS] these developments afford a large diversity of new and unique biomacromolecules . [SEP]
[CLS] still , significant opportunities exist in this area to improve on reaction efficiency , to expand on the compositional diversity of modifications , and to realize structure - property relationships for nanoscale , self - assembling , and supramolecular systems . [SEP]
[CLS] these improvements will allow for more modular , diverse , and effective formulations . [SEP]
[CLS] similarly , molecular biology studies are providing new therapeutic targets with an improved understanding of pathways and resulting pathologies . [SEP]
[CLS] as such , the development of combination therapies is forthcoming . [SEP]
[CLS] the possible combinations will target either one or multiple mrnas or combine a bioconjugated oligonucleotide with a small molecule or protein B-material in order to improve efficacy and safety profiles . [SEP]
[CLS] as discussed in this review , modifications such as galnac , cell B-material penetrating peptides B-material , α - tocopherol , aptamers , antibodies B-material , cholesterol , squalene , fatty acids , and nucleolipids are new promising therapeutic candidates in the field . [SEP]
[CLS] despite the clinical progress to date , the design of modified oligonucleotides encompassing both the optimal delivery and efficient biological activity remains an important challenge . [SEP]
[CLS] diseases such as coronary heart disease , lower respiratory infections , cancer , diabetes , and alzheimer ' s are in need of new therapies , and , as oligonucleotides can target and bind nearly any expressed mrna , the possibility to develop oligonucleotide therapeutics exist . [SEP]
[CLS] key to these future successes will be understanding the pharmacokinetic and biodistribution profiles of these new therapeutics . [SEP]
[CLS] despite the promising bioconjugated oligonucleotides reported so far , oligonucleotide delivery represents a major hurdle still to be addressed . [SEP]
[CLS] as mentioned previously , most of the oligonucleotide conjugates used in clinic bio - accumulate in the liver . [SEP]
[CLS] furthermore , a clear relationship between the composition and structure of the chemical modification and its resulting effect on pharmacokinetic / biodistribution must be both quantified and established with appropriate positive and negative controls in place . [SEP]
[CLS] for example , membrane interacting bioconjugates often interact with many targets and are known for their promiscuity . [SEP]
[CLS] such a molecular rationale would allow scientists to design the chemical structure of the oligonucleotides depending on the specific disease , target tissue / organ , cell B-material type etc . [SEP]
[CLS] in summary , the use of oligonucleotides in the clinic is in its infancy and the potential for significant advances in patient care exist . [SEP]
[CLS] given the clinical success of monoclonical antibodies B-material ( mab ) as a biologic , we speculate that oligonucleotides will have a greater impact due to the specificity inherent to the target sequence , the ability to target nearly any protein B-material , and their stability both pre - administration and in vivo . [SEP]
[CLS] the full realization and maturation of the bioconjugated oligonucleotide therapeutic field will require timee . g . , 19 mab drugs were on the market only after 20 years of developmentbut this should not curtail enthusiasm . [SEP]
[CLS] in fact , findings from these mab studies as well as from polymer conjugated enzymes and small molecules used in the clinic will catalyze development and subsequent clinical applicability . [SEP]
[CLS] bioconjugation chemistry is at the centerpiece on this therapeutic oligonucleotide revolution . we encourage all to investigate this area where conceptualizing , designing , and engineering at the molecular level new functionalized oligonucleotides is key to success in the clinic . [SEP]
[CLS] 1 : mechanisms of antisense oligonucleotides ( adapted from chan j . h . p . et al . 2006 12 ) normal expression of the gene and protein B-material in the absence of aso ( 1 ) . [SEP]
[CLS] after internalization , aso hybridize to mrna via complementary sequence pairing . [SEP]
[CLS] the aso / mrna heteroduplex induces activation of rnase h and degradation of the mrna ( 2 ) or ribosome blockage by steric hindrance ( 3 ) . [SEP]
[CLS] both mechanisms result in inhibition of target protein B-material production . [SEP]
[CLS] alternatively , penetration of aso into the nucleus may lead to regulation of mrna via inhibition of capping formation at the 5 ' end ( 4 ) , inhibition of splicing ( 5 ) or the activation of rnase h ( 6 ) . figure 2 : chemical modifications of synthetic oligonucleotides ( adapted from khvorova a . and watts j . k . 2017 21 ) chemical modifications are incorporated at the phosphate , ribose or at the 5 ' or 3 ' ends of the oligonucleotides . [SEP]
[CLS] the type and the position of the modification induce three main mechanisms ( steric blockers in green , activation of rnase h in blue and rna interference ( rnai ) in orange ) . [SEP]
[CLS] in the case of 2 ' modification ( 2 ' - ome , 2 ' - o - moe , 2 ' nh2 , 2 ' - f - rna and 2 ' - f - ana ) , rnase h and rnai mechanism are possible with a gapmer design . [SEP]
[CLS] pto : phosphorothioate ; cna : dioxaphosphorinane - constrained nucleic B-material acid I-material ; pna : peptide B-material nucleic B-material acid I-material ; pmo : phosphorodiamidate morpholino oligonucleotide ; e - vp : ( e ) - vinylphosphonate ; ddc : dideoxycytosine ; 2 ' - ome : 2 ' o - methyl ; 2 ' - o - moe : 2 ' - o - methoxy ethyl ; 2 ' - f rna ; 2 ' - f ana : arabinonucleic acid ; lna : locked nucleic B-material acid I-material ; ena : 2 ' - o , 4 ' - c - ethylene - bridged nucleic B-material acid I-material ; cet : s - constrained ethyl ; tcdna : tricyclodna ; fhna : fluoro hexitol nucleic B-material acid I-material ; una : unlocked nucleic B-material acid I-material [SEP]
[CLS] 1 : mechanisms of antisense oligonucleotides ( adapted from chan j . h . p . et al . 2006 12 ) normal expression of the gene and protein B-material in the absence of aso ( 1 ) . [SEP]
[CLS] after internalization , aso hybridize to mrna via complementary sequence pairing . [SEP]
[CLS] the aso / mrna heteroduplex induces activation of rnase h and degradation of the mrna ( 2 ) or ribosome blockage by steric hindrance ( 3 ) . [SEP]
[CLS] both mechanisms result in inhibition of target protein B-material production . [SEP]
[CLS] alternatively , penetration of aso into the nucleus may lead to regulation of mrna via inhibition of capping formation at the 5 ' end ( 4 ) , inhibition of splicing ( 5 ) or the activation of rnase h ( 6 ) . figure 2 : chemical modifications of synthetic oligonucleotides ( adapted from khvorova a . and watts j . k . 2017 21 ) chemical modifications are incorporated at the phosphate , ribose or at the 5 ' or 3 ' ends of the oligonucleotides . [SEP]
[CLS] the type and the position of the modification induce three main mechanisms ( steric blockers in green , activation of rnase h in blue and rna interference ( rnai ) in orange ) . [SEP]
[CLS] in the case of 2 ' modification ( 2 ' - ome , 2 ' - o - moe , 2 ' nh2 , 2 ' - f - rna and 2 ' - f - ana ) , rnase h and rnai mechanism are possible with a gapmer design . [SEP]
[CLS] pto : phosphorothioate ; cna : dioxaphosphorinane - constrained nucleic B-material acid I-material ; pna : peptide B-material nucleic B-material acid I-material ; pmo : phosphorodiamidate morpholino oligonucleotide ; e - vp : ( e ) - vinylphosphonate ; ddc : dideoxycytosine ; 2 ' - ome : 2 ' o - methyl ; 2 ' - o - moe : 2 ' - o - methoxy ethyl ; 2 ' - f rna ; 2 ' - f ana : arabinonucleic acid ; lna : locked nucleic B-material acid I-material ; ena : 2 ' - o , 4 ' - c - ethylene - bridged nucleic B-material acid I-material ; cet : s - constrained ethyl ; tcdna : tricyclodna ; fhna : fluoro hexitol nucleic B-material acid I-material ; una : unlocked nucleic B-material acid I-material [SEP]
[CLS] 3 : strategies for the delivery of aso and / or sirna . [SEP]
[CLS] adapted from jarver et al . 2012 57 a : formation of a complex between a biomolecule or a polymer B-material and the oligonucleotide via electrostatic interactions and / or the hydrophobic B-property effect , b : formation of liposomal B-nanoparticle supramolecular structure with targeting or transfecting agent ( peg , peptide B-material , cpp , protein B-material , lipid B-material glycoconjugate , etc . ) , c : covalent conjugation with the oligonucleotide by a stable or cleavable bond . [SEP]
[CLS] 4 : molecular structures of galnac based conjugates , adapted from matsuda et al . 2015 72 diagram showing an oligonucleotide coupled to galnac ( left ) and molecular structure ( right ) . [SEP]
[CLS] the triantennular galnac is represented in the upper part , the galnac incorporated in sequence in the lower part . [SEP]
[CLS] 5 : cellular internalisation of galnac oligonucleotide conjugates , adapted from alnylam . [SEP]
[CLS] the galnac moiety is a ligand of the asgpr receptors . [SEP]
[CLS] the formation of the complex induces a clathrindependent endocytosis B-event mechanism . [SEP]
[CLS] acidification dissociates the sirna - releasing substrate / receptor B-material complex ( 3 ) into the cytoplasm ( 4 ) . [SEP]
[CLS] rnai - mediated degradation of the targeted mrna mechanism ( 5 ) . [SEP]
[CLS] inhibition of protein B-material translation ( 6 ) . [SEP]
[CLS] asgpr receptors are recycled in the plasma membrane ( 7 ) . [SEP]
[CLS] 6 : cpp grafting strategy on sirna . [SEP]
[CLS] a : noncleavable oligonucleotide cpp conjugate . [SEP]
[CLS] b : representation of the cleavable conjugate by reduction of the disulfide bridge : ( 1 ) bioconjugate enters into the cell B-material thanks to cpp . [SEP]
[CLS] ( 2 ) cleavage of the disulfide bridge allows detachment of the sirna from the cpp moiety . [SEP]
[CLS] ( 3 ) cpp enters into the nucleus . [SEP]
[CLS] ( 4 ) sirna elicits its inhibitory activity by an rnai mechanism . [SEP]
[CLS] adapted with permission from ye , j . , liu , e . , gong , j . , wang , j . , huang , y . , he , h . , and yang , v . c . ( 2017 ) high - yield synthesis of monomeric lmwp ( cpp ) - sirna covalent conjugate for effective cytosolic delivery of sirna . [SEP]
[CLS] theranostics 7 , 2495−2508 . 83 copyright 2017 , ivyspring . [SEP]
[CLS] 7 : molecular structure of a photocleavable cholesterol - conjugated oligonucleotide , adapted from yang j . et al . 2018 111 [SEP]
[CLS] 8 : molecular structure of grn163l . [SEP]
[CLS] the lipid B-material oligonucleotide grn163l , 5 ' - palm - tagggttagacaa - nh2 - 3 ' , with a thio - phosphoramidate inter - nucleosidic linkage B-property ( top ) and its structural formula ( down ) from the chemspider website . [SEP]
[CLS] 9 : cholesterol and galnac coupled aso , adapted from wada f . et al . 2018 129 teg : triethylene glycol ; galnac : nacetylgalactosamine ; aso : antisense oligonucleotide [SEP]
[CLS] 10 : spherical nucleic B-material acids I-material . [SEP]
[CLS] a . snas present nucleic B-material acids I-material in an outward facing spherical array . [SEP]
[CLS] b . oligonucleotidetocopherol conjugates form snas with liposomal B-nanoparticle cores B-material . [SEP]
[CLS] c . structure of linkage B-property and tocopherol moiety . [SEP]
[CLS] d . tlr agonizing oligonucleotides conjugated to tocopherol can be combined with antigens through a complimentary oligonucleotide and chemical conjugation through a linker . [SEP]
[CLS] e . different conjugate linkage B-property chemistries explored [SEP]
[CLS] 11 : structure of the thio - phospharamidite based aso with a telomerase activity . [SEP]
[CLS] the targeting moiety ( palmitate ) has been inserted at the 3 ' extremity . [SEP]
[CLS] 12 : secondary structure of pegaptanib , active substance of macugen ® adapted from ng e . w . m . et al . 2006 152 [SEP]
[CLS] 13 : timeline for key advances in therapeutic oligonucleotides . [SEP]
[CLS] adapted from lundin k . e . et al . 2015 159 the various substitutions and the advances of chemical modifications are shown in yellow , main discoveries and technological achievements are noted in blue and clinical studies are highlighted in green [SEP]
[CLS] bioconjugate oligonucleotides in the clinic . [SEP]
[CLS] in much the same way that the use of silicon B-material did in the 1970s , leading to the modern information technology industry , the development of advanced functional nanomaterials B-material will fuel many of the emerging industries that will address energy , healthcare , and environmental challenges as well as those in other areas . [SEP]
[CLS] therefore , a current challenge in the field of nanotechnology and materials science is the development of strategies to control the structuring and fabrication of functional materials and devices with potentially atomic scale precision on a large length scale , i . e . up to micro and even centimetre scales . [SEP]
[CLS] these new advanced functional materials aim at improving performances and developing new functionalities . [SEP]
[CLS] traditional miniaturization approaches , classically referred to as " top - down " approaches , are derived from the microelectronics industry . [SEP]
[CLS] these methods are primarily dedicated to semiconductor materials and are currently based on powerful and efficient techniques for structuring matter that are essentially operated by standard and industrial machines . [SEP]
[CLS] these topdown technologies face a number of issues , including cost and the need for machinery modifications and manufacturing lines to promote miniaturization and mass production . [SEP]
[CLS] importantly , these technologies also face the intrinsic physical limitations inherent to the miniaturization of classical materials , which necessitates fundamental modifications to the overall device architecture . [SEP]
[CLS] for instance , the concept of planar metal B-material oxide I-material semiconductor ( mos ) transistor was proposed and developed since the mid 1960s and is one reference of the road mapping in microelectronics . [SEP]
[CLS] self - assembly and , at large scales , " bottom - up " technologies are game changers in the way that materials can be envisaged . [SEP]
[CLS] along with the era of nanotechnology launched in the us in the 1990s , inspiration from the living world , where the directed assembly of molecules allows the production of organisms , was a disruptive technological alternative . [SEP]
[CLS] if we compare the dimensions of the devices reachable by the " top - down " technologies to those of the living world ( see figure 1 ) , two technological paths emerge that may lead to a re - foundation of the classical concepts of technologies in favour of systems that are highly heterogeneous in chemical and structural nature . [SEP]
[CLS] to date , numerous bottom - up strategies have been investigated . [SEP]
[CLS] these approaches fully rely on self - assembly , which consists of using the basic physical and chemical attractive / repulsive interactions ( van der waals , electrostatic , hydrogen B-material or covalent bonding ) as well as the basic processes of molecular recognition in biology . [SEP]
[CLS] the goal of these strategies is to interface and link various nano B-nanoparticle - I-nanoparticle objects I-nanoparticle , such as nanoparticles B-nanoparticle , nanotubes B-nanoparticle , biological molecules and other organic objects , without external intervention . [SEP]
[CLS] among these self - assembly strategies , a particularly interesting and versatile technique is to take advantage of the exquisite properties of deoxyribonucleic B-material acid I-material ( dna ) , i . e . the permutations and complementarity of nucleotide sequences , to programme and direct the assembly of nano B-nanoparticle - I-nanoparticle objects I-nanoparticle in potentially extremely complex architectures , which is not feasible by any other state - of - the - art technique . [SEP]
[CLS] this idea was first proposed in 1982 in the seminal work by nadrian seeman , who proposed the construction of nanometric " tiles " from elementary dna bricks and to build regular networks of tiles for the formation of larger 2d or 3d structures . [SEP]
[CLS] this initial revolutionary concept to deviate from the original biological function of dna and use it as a versatile and programmable technological brick was rapidly extended in many different directions . [SEP]
[CLS] more complex hybrid structures were produced by combining dna with other materials , inorganic or not , while also taking full advantage of the recognition capability of folded dna , so - called aptamers , and finally , of modified dna strands ( chemical terminations , for peptide B-material nucleic B-material acid I-material ( pna ) , etc . ) to extend its ability to marry other materials and fabrication techniques . [SEP]
[CLS] these studies led to a technologically wide and active field of study called dna nanotechnology in which dna is considered to be an engineering material B-material that can be used as an architectural material B-material , functional building blocks and devices for applications non strictly dedicated to biology ; in other words , a field in which dna is no more utilized as the carrier of the genetic information in the cell B-material . [SEP]
[CLS] the use of dna out of its biological context is now widely accepted by the scientific community , which has been accompanied by the massive industrial production of artificial dna . [SEP]
[CLS] this concept opens unparalleled perspectives for the nanoengineering of a multitude of " new advanced materials " for various applications such as energy , environment , health . . . in 2017 , there were 400 published articles ( source : web of science ) on dna nanotechnology in high impact journals . [SEP]
[CLS] section 2 will give a brief overview on the dna biomolecule and its primary features and properties , which will be followed by a description of the founding studies on dna nanotechnologies in section 3 . [SEP]
[CLS] then , sections 4 and 5 will focus on nanoparticle dnadirected assembly , accompanied with state - of - the - art techniques and a case study by the authors on al / cuo assembly . [SEP]
[CLS] dna nanotechnologies can be split into two fields : [SEP]
[CLS] - dna nanostructures ( section 3 ) : as proposed by nadrian seeman , relatively small dna strands are assembled to constitute elementary " bricks " , which can be further used to fabricate larger structures that essentially comprise dna and have one , two or even three dimensions . [SEP]
[CLS] the folding of much longer strands , such as the genome of the m13 virus , with the help of dna staples ( short dna strands having partitioned complementarity with the longer dna strand ) is another way of generating structures any shape one can imagine . [SEP]
[CLS] notably , small - sized dna strands can also fold into specific conformations , making it possible to have very specific interactions of the lock and key type , as proteins B-material typically do in biology . [SEP]
[CLS] these dna strands are called aptamers . [SEP]
[CLS] - dna as technological tool to drive nanoparticle B-nanoparticle assembly . [SEP]
[CLS] dna can be used as a " cement " to guide in the self - assembly of various organic and non - organic nano B-nanoparticle - I-nanoparticle objects I-nanoparticle . [SEP]
[CLS] this pathway , directly inspired by the work of nadrian c . seeman , was first demonstrated in 1996 by chad a . mirkin , who implemented the directed assembly of gold B-nanoparticle nanoparticles I-nanoparticle by dna strands . [SEP]
[CLS] this research field has become very active , accounting for approximately 10 % of all published articles on dna nanotechnology over the past five years ( source : web of science ) , whereas it represented less than 1 % in the 2000 ' s . [SEP]
[CLS] a wide variety of different building - block shapes as well as different strategies for assembling them have been reported thus far , as summarized in section 5 . [SEP]
[CLS] to date , nanoparticle B-nanoparticle and colloidal particle assemblies using synthetic dna have allowed for the production of mostly plasmonic , photonic or phononic metamaterials . [SEP]
[CLS] interestingly , another new application of dna nanotechnologies is the synthesis of highly ordered metal B-material / oxide I-material nanoparticle B-nanoparticle structures for energy - generating materials . [SEP]
[CLS] the properties of these materials , also called nanothermites , are highly dependent on the size of nanoparticles B-nanoparticle and their distribution in the 3d structure . [SEP]
[CLS] in this context , the use of dna strands to direct selfassembly shows great potential for the synthesis of nanothermites with outstanding energetic performances . [SEP]
[CLS] section 5 focusses on the nano - engineering of dna to self - assemble al / cuo nanothermite to illustrate the potential of dna nanotechnologies in obtaining high performance nanothermite for practical applications . [SEP]
[CLS] the nitrogenous bases are complementary , where adenine ( a ) and guanine ( g ) can specifically interact with thymine ( t ) and cytosine ( c ) , respectively , due to hydrogen B-material bonds ( two for the first " couple " and three for the second ) . [SEP]
[CLS] hybridization is the pairing of two complementary single - stranded dna sequences , which results in the formation of a double helix . [SEP]
[CLS] the energy gain resulting from the association of two single strands of dna is , in the first approximation , the sum of the association energies of each pair of base pairs , with 2 and 3 kbt ( where kbt is the thermal stirring energy ) for a - t and g - c , respectively . [SEP]
[CLS] note that hybridization is a complex process characterized by the interplay of mutual attraction forces , including hydrogen B-material bonds , solvent , and counter ions B-material to screen the negatively charged backbone . [SEP]
[CLS] the four primary characteristics that make dna an excellent technological brick for nanoconstruction are : [SEP]
[CLS] - reversible watson - crick pairing , which is based on the complex interplay between the solvent , dna backbone and hydrogen B-material bonds , including inter - base hydrogen B-material bonds . [SEP]
[CLS] external stimuli , such as the temperature , ph or ionic strength of the solution can denature the dna double helix , where it is separated into its single strand components . [SEP]
[CLS] in addition , dna can be repaired by dna B-event modification I-event enzymes , particularly ligases . [SEP]
[CLS] this specificity is especially used for the nanoconstruction of complex dna patterns . [SEP]
[CLS] - its size is ideally suited to nanoconstruction . [SEP]
[CLS] the diameter of the double helix is approximately 2 nm , the distance between bases is 0 . 34 nm , and the helical pitch is composed of 10 . 5 base pairs , which corresponds to approximately 3 . 5 nm . [SEP]
[CLS] - the stability and flexibility of an assembly can be adjusted . [SEP]
[CLS] the persistence length of double stranded dna is ~ 50 nm ( or 150 base pairs ) . [SEP]
[CLS] below this length , the strand can be considered as a rigid rod , whereas it becomes very flexible beyond this length . [SEP]
[CLS] there is also a difference in flexibility between double - and single - stranded dna , with the latter being much more flexible , with a persistence length of only 1 nm , allowing for the formation of true loops or curved structures . [SEP]
[CLS] - finally , 50 years of molecular biology research on dna has produced a consistent palette of tools for dna synthesis , manipulation , and modification . [SEP]
[CLS] many companies specifically offer synthetic dna with a great variety of chemical modifications . [SEP]
[CLS] it is now possible to order or create , in the laboratory , single or double dna strands consisting of the desired nitrogenous base sequence with a wide variety of chemical or biological functions that can be further integrated at the strand termini or at a specific locations within the strand . [SEP]
[CLS] in addition to the remarkable intrinsic properties of dna , all of these features lay the foundation for important expectations regarding dna nanotechnologies to control the structuring and fabrication of functional materials and devices . [SEP]
[CLS] the following examples shall illustrate the implementation of these technologies . [SEP]
[CLS] as mentioned earlier , the development of dna nanotechnologies began with the seminal work by nadrian seeman in the 1980s . [SEP]
[CLS] he envisaged associating several double strands of dna with the help of the hybridization of so - called " sticky - ends " to build more complex dna networks . [SEP]
[CLS] " sticky - ends " refers to dna termini that remain single stranded , offering hybridization possibilities with other basic dna elements with complementary single - stranded termini . [SEP]
[CLS] in doing so , he faced a number of issues due to dna helices being highly flexible , and the resulting assemblies did not exhibit the desired structural stability . [SEP]
[CLS] to address this problem , the seeman team came up with the idea of building complex structures of branched dna , strands nicknamed " tiles " , the simplest configuration of which was called dx ( for double crossover , figure 3 ) . [SEP]
[CLS] these structures are highly rigid owing to their double points of attachment and four points of external sticky - end attachments . [SEP]
[CLS] they were the first building blocks used for the construction of more complex networks based on dna , and the first two - dimensional networks were created by his team in 1998 . [SEP]
[CLS] 17 several studies followed based on these dx structures ( figure 3 ) , giving rise to more complex structures , such as tx - type tiles ( triple - crossover ) , and type px ( paranaemiccrossover ) . [SEP]
[CLS] in the mid 2000s , another key step in the emergence of dna nanotechnology was the development of dna origami by paul rothemund . [SEP]
[CLS] this breakthrough concept significantly increased the complexity , versatility , and size of dna structures . [SEP]
[CLS] the term " origami " refers to the japanese art of making various unique 3d forms with a paper sheet through folding techniques . [SEP]
[CLS] in dna origami , the paper sheet is a long dna strand ( obtained from dna extraction of the m13 virus ) , which can be considered to be a scaffold that is folded into the desired shape by means of hundreds of small oligonucleotides [ see figure 4 [SEP]
[CLS] a key advantage of this technology is that it is not necessary to precisely control the stoichiometry and purification of oligonucleotides , because the use of a long scaffold makes the assembly highly selective . [SEP]
[CLS] the minimum of energy is reached when all the staples are associated , regardless of the number of staples by unit volume . [SEP]
[CLS] dna origami achieved immediate success thanks to the overall reliability in reproducing complex 2d and 3d shapes and because of its conceptual and experimental simplicity . [SEP]
[CLS] progress in this technique has resulted in the formation and control of uniform structures with unprecedented complexity . [SEP]
[CLS] importantly , this method is associated with an automated computer design strategy . [SEP]
[CLS] indeed , all dna folding and associated staples must have their base sequences designed depending on each other and must be preprogramed with the help of computer - aided design procedures . [SEP]
[CLS] therefore , the staples are determined before the manufacturing of origami and be ordered from dna synthesis companies . [SEP]
[CLS] in addition , dna staples can be obtained with chemical or biological modifications , the subtle positioning of which within the origami structure can yield a chemically or biologically functional structure . [SEP]
[CLS] similarly , by including thiol B-material termini at specific locations of the origami , zhang after numerous studies and publications on two - dimensional dna origami , the concept has been extended to three - dimensional dna origami , which appeared for the first time in 2009 . [SEP]
[CLS] the gothelf and kjems team at the center for dna nanotechnology ( cdna ) at the university of aarhus in denmark demonstrated the connection of several 2d origami planar structures at their edges . [SEP]
[CLS] in their report , they described in detail the formation and characterization of a dna box with a controllable lid [ figure 4 ( d ) ] . [SEP]
[CLS] two sides of the box were hinged to one side and held closed along the opposite edge by a pair of " staple " dna strands named " locks " . [SEP]
[CLS] each lock can be opened by adding a dna strand complementary to the strands constituting the locks to the solution , releasing the lid of the dna box . [SEP]
[CLS] in summary , since the initial vision of seeman in the early 1980s , fundamental steps in the programming and engineering of dna nanostructures have succeeded one another , and the invention of the dna origami technique has allowed for the considerable development of these technologies , making possible to create complex structures with any shape and precise properties . [SEP]
[CLS] however , it is important to note that production yields tends to fall as soon as the complexity and density of structures increases . [SEP]
[CLS] furthermore , the lack of detailed information on the folding process remains an obstacle to the development of dna nanotechnologies . [SEP]
[CLS] thus , extensive studies on the fundamental thermodynamic versus kinetic aspects of the folding processes should be pursued . [SEP]
[CLS] an interest in using dna nanotechnology to programme the assembly of nanoparticles B-nanoparticle and colloids into larger hierarchical structures emerged in the 1990s ( alivisatos et al . , and mirkin et al . ) [SEP]
[CLS] using the controlled chemistry of sulfur B-material on gold B-material surfaces , both groups had the idea of grafting single - ended thiol - terminated dna strands ( - sh ) onto gold B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] the mirkin team made use of thiol - modified oligonucleotides ( - sh ) at one end of the strand for chemical grafting onto the surface of 13 - nm gold B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] two separate solutions of gold B-material nps B-nanoparticle modified with non - complementary strands were made and mixed in equal amounts . [SEP]
[CLS] a single - stranded dna sequence that was complementary to both strands was then added in excess to the above mixture . [SEP]
[CLS] this additional dna strand acted as a " linker " and induced the assembly of the nanoparticles B-nanoparticle into aggregates of several microns by hybridization of the linker strands with both strands grafted onto the nanoparticles B-nanoparticle . [SEP]
[CLS] the colour of the nanoparticle B-nanoparticle solution gradually changes from pink ( dispersed particles ) to blue ( aggregated particles ) . [SEP]
[CLS] an interesting aspect of this type of assembly is its reversibility . [SEP]
[CLS] indeed , at ~ 55 °c , the so - called melting temperature , dehybridization of the strands takes place , and the redispersion of the nanoparticles B-nanoparticle causes the solution to return to its initial pink colour ( see figure 5 ) . [SEP]
[CLS] alivisatos chose to work on particles that were 10 - fold smaller , onto which a single oligonucleotide sequence was covalently attached using alternating strand with distinct sequences . [SEP]
[CLS] then , hybridization was performed at different defined positions of a long single strand that resulted in the nanoscalecontrolled positioning of the nps B-nanoparticle along the one - dimensional dna strand . [SEP]
[CLS] undoubtedly , the controlled interplay of complementary and non - complementary dna strands makes dna nanotechnology one of the most powerful bottom - up approaches for building hierarchical architectures of nanoobjects ( noble metals B-material , semiconductors , oxides B-material , and polymers B-material ) with an almost infinite variety of high - performance programmable dna / nanoparticle B-nanoparticle hybrid materials . [SEP]
[CLS] since the seminal work by alavisatos and mirkin on gold B-nanoparticle nanoparticles I-nanoparticle , many dna / nanoparticle B-nanoparticle assembly processes have been reported , mostly by varying the dna length and processing conditions and to generate materials for applications in biodiagnostics , therapeutic agents , plasmon - enhanced spectroscopy B-technique , magneto - optical sensors , and catalysis and energetic materials , which will be presented in detail in section 5 . [SEP]
[CLS] the most successful and widespread technique for gold B-material surface functionalization technique used to graft dna strands uses thiol B-material chemistry , taking advantage of the well - known and controlled reaction between thiol B-material and metal B-material surfaces ( such as gold B-material , see figure 6 ) . [SEP]
[CLS] an alternative method using antigen / antibody B-material interactions has been reported to immobilize dna strands on surfaces , notably oxide B-material surfaces , extending the possible applications of the organized and controlled heterogeneous structures of nanoparticles B-nanoparticle . [SEP]
[CLS] the biotin / streptavidin interaction is recognized as one of the strongest non B-property - I-property covalent I-property interactions I-property in nature , having a complex dissociation constant of 4 10 - 14 mol . l - 1 and a formation enthalpy of 30 kbt . [SEP]
[CLS] this strong interaction is results from a perfectly adjusted protein B-material pocket for biotin such that its binding is insensitive to ph , salinity and temperature . [SEP]
[CLS] streptavidin is a protein B-material consisting of four tetrameric fragments , each of which can accommodate four biotin species , which are immobilized onto streptavidin and form hydrogen B-material , electrostatic and hydrophobic B-property bonds with the aromatic amine B-material groups I-material of streptavidin . [SEP]
[CLS] the use of this interaction has attracted a great deal of interest because of its versatility , and it can be applied to any type of surface , even gold B-material , [SEP]
[CLS] provided that the surface is sufficiently reactive to ensure that the protein B-material is immobilized [SEP]
[CLS] for example , cobbe et al . proposed an aggregation of gold B-nanoparticle nanoparticles I-nanoparticle previously functionalized with a biotin group using streptavidin and singlestranded dna . [SEP]
[CLS] the two strategies used in this study are schematically shown in figure 7 a and b respectively . [SEP]
[CLS] the self - assembly of heterogeneous nanoparticles B-nanoparticle by protein - dna interactions and their differences from gold B-material were also assayed by oleg gang and colleagues . [SEP]
[CLS] finally , we should mention the use of other chemical functions , such as carboxylic B-material acid I-material ( - cooh ) , which is more appropriate for metal B-material oxide I-material surfaces , or other oxides B-material , such as silicon B-material dioxide I-material ( see figure 6 ) . [SEP]
[CLS] while the literature commonly reports on organic coatings B-material to inhibit further oxidation of metallic B-nanoparticle nanoparticles I-nanoparticle , to the best of our knowledge , no mention of these possible chemical species has been addressed with respect to dna . [SEP]
[CLS] in practice , the overall optimization process for controlling dna - directed assembly in the liquid phase is a highly complex task . [SEP]
[CLS] over the last several decades , substantial progress has been achieved in understanding and controlling the wet chemical technical parameters for the patterning of matter at the nanoscale , especially the effect of salt B-material concentration on both the grafting density and hybridization of dna strands or the heterogeneity of dna grafted on anisotropic nanoparticles B-nanoparticle as demonstrated very recently . [SEP]
[CLS] highly ordered face centre cubic ( fcc ) and body centre cubic ( bcc ) structures of 5 - or 10 - nm gold B-nanoparticle nanoparticles I-nanoparticle have been described with different dna strand lengths and a programmable interparticle distance ( as illustrated in figure 8 ) and have more recently been reported with the crystallization of nanoparticles B-nanoparticle into more complex structures ( vb23 , 24 ) . [SEP]
[CLS] it is now known that several key parameters have a large effect on the assembly kinetics and architecture of the final structures : [SEP]
[CLS] - the ionic concentration in solution , such as the salt B-material concentration ( nacl , mgcl2 , . . . ) . [SEP]
[CLS] park et al . have shown that the salt B-material concentration tends to decrease the inter - particle distance by decreasing the repulsive interactions between nanoparticles B-nanoparticle . [SEP]
[CLS] - the spacer B-material nature of the oligonucleotide also influences the inter - particle distance . [SEP]
[CLS] indeed , a " slump " of an oligonucleotide on the nanoparticle B-nanoparticle was observed with a spacer B-material composed of repeated adenine bases , reducing the inter - particle distance by 2 . 5 - fold because of a greater affinity of this base with gold B-material compared to that observed with other bases . [SEP]
[CLS] - 41 - the dna strand length impacts crystallinity , where an excessively long strand lowers crystallinity because nanoparticles B-nanoparticle are less constrained , whereas a short link with respect to the size of the nanoparticle B-nanoparticle does not allow crystallization because of the size polydispersity of nanoparticles B-nanoparticle . [SEP]
[CLS] experimental phase diagrams showing the crystallinity of gold B-nanoparticle nanoparticles I-nanoparticle as a function of these parameters have been established . [SEP]
[CLS] owing to the flexibility of their structure , dna strands are also reprogrammable , allowing a crystal structure to be directly altered in solution by selecting the appropriate oligonucleotide sequence or by introducing dna interlayers , modifying the stiffness and temperature sensitivity of the dna bond . [SEP]
[CLS] for example , gang et al . recently determined gold B-nanoparticle nanoparticle I-nanoparticle phase diagrams based on stoichiometry ( i . e . the relative proportion of two populations of gold B-nanoparticle nanoparticles I-nanoparticle with complementary dna strands ) , the size of nanoparticles B-nanoparticle , and the length of dna linker , both experimentally and theoretically , using coarse grain models . [SEP]
[CLS] these phase diagrams are shown in figure 9 below , where gold B-nanoparticle nanoparticles I-nanoparticle can adopt the crystal phase of cscl , alb2 or cr3si depending on the size of the nanoparticles B-nanoparticle or the linker . [SEP]
[CLS] overall , there is good agreement between experimental points and theoretical results . [SEP]
[CLS] - the temperature of the solvent also influences crystallinity . [SEP]
[CLS] by aggregating at a temperature close to the melting temperature of dna , a transition can take place at which nanoparticles B-nanoparticle are allowed to rearrange into well - defined crystalline structures and on a larger scale . [SEP]
[CLS] 10 shows the principles of this rearrangement and gives the ingredients , thermal treatment and spacer B-material length required for obtaining crystals . [SEP]
[CLS] thus , the quality of the crystallization is strongly dependent on the temperature conditions . [SEP]
[CLS] cooling the colloidal solution at a sufficiently slow rate , which can take from two to three days , was recently shown to lead to the formation of micrometric rhombic dodecahedron " super crystals " . [SEP]
[CLS] in contrast , performing self - assembly at a constant temperature below the melting temperature results in the polycrystallization of the nanoparticles B-nanoparticle , which are then less organized despite having undergone the annealing process . [SEP]
[CLS] importantly , the formation of these dodecahedra is independent of the size of nanoparticles B-nanoparticle , showing that this structure is thermodynamically the most stable that is achievable for such systems . [SEP]
[CLS] finally , postcrystallization modifications have also made it possible to increase the temperature ranges or solvents in which these structures can be used by adding organic complexes or encapsulating dna in silica . [SEP]
[CLS] notably , it is also possible to control the number of functionalized strands on the surface of gold B-nanoparticle nanoparticles I-nanoparticle with one , two or more strands by " sorting " a colloidal solution of nanoparticles B-nanoparticle by electrophoresis B-technique . [SEP]
[CLS] this technique allows for the hybridization of two or three nanoparticles B-nanoparticle alone ( dimers or trimers ) that have optical properties dependent on the inter - particle binding , which is governed by the nature of the dna B-event binding I-event and can be dynamically altered in solution by ionic interactions and the chemical nature of the substrate . [SEP]
[CLS] thus , the chemical nature of the surface of the gold B-nanoparticle nanoparticles I-nanoparticle depends on the ligands used to stabilize the colloids . [SEP]
[CLS] lermusiaux et al . investigated the optical properties of dimers immobilized on the surface of gold B-nanoparticle nanoparticles I-nanoparticle as a function of the ionic concentration and the hydrophobicity B-property of the surfaces by varying the nature of the ligand . [SEP]
[CLS] the results presented in figure 11 show that the use of an amphiphilic B-property ligand ( i . e . having both a hydrophobic B-property and hydrophilic B-property group ) makes it possible to improve stability in terms of decreasing the unwanted aggregation of nanoparticles B-nanoparticle or in the conformation between two assembled nanoparticles B-nanoparticle . [SEP]
[CLS] the vast majority of work has focused on the self - assembly of gold B-material or silver B-nanoparticle nanoparticles I-nanoparticle , owing to their good colloidal stability , controlled synthesis and subsequent controlled thiol B-material chemistry . [SEP]
[CLS] however , extending their potential applications requires manipulating other materials . [SEP]
[CLS] gang et al . have proposed a general strategy of nanoparticle B-nanoparticle functionalization by dna that is theoretically applicable for a wide range of materials , which they applied to three model materials : gold B-nanoparticle nanoparticles I-nanoparticle with palladium B-material of three different shapes ( cube , octahedron and dodecahedron ) , iron B-material oxide I-material ( fe2o3 ) , and quantum B-nanoparticle dots I-nanoparticle ( cdse / cdte / zns and cdse / zns ) . [SEP]
[CLS] they identified three primary parameters that are crucial in establishing heterogeneous superstructures : ( i ) the role of the shape of the nanoparticle B-nanoparticle on crystallinity , ( ii ) the influence of non - specific interactions related to the presence of dna , and ( iii ) the emergence of disorder when manipulating multi - elemental structures . [SEP]
[CLS] this work follows the pioneering work by severac et al . , who first demonstrated the possibility of using dna as an assembly vector in the context of the synthesis of high energy performance materials from al and cuo nanoparticles B-nanoparticle . [SEP]
[CLS] in addition , fan et al . developed assemblies of five nanoparticles B-nanoparticle of different sizes involving a small nanoparticle B-nanoparticle surrounded by four larger nanoparticles B-nanoparticle . [SEP]
[CLS] such anisotropic assemblies have the advantage of having better defined optical properties . [SEP]
[CLS] in addition , tan et al . developed a general and simple single - step method for the functionalization and crystallization of anisotropic systems from 2 nanoparticle B-nanoparticle sizes . [SEP]
[CLS] beyond the grafting densities that strongly change with anisotropy , a predominant effect of the nanoparticle B-nanoparticle curve , which is dependent on the size of the nanoparticle B-nanoparticle , has been observed . [SEP]
[CLS] jones et al . have studied the crystallization of non - spherical nano B-nanoparticle - I-nanoparticle objects I-nanoparticle , such as octahedra , prisms and rods . [SEP]
[CLS] in general , the resulting structures of these objects remains the one that allows for the maximum hybridization of dna strands and involves a superposition of nanoprisms , 62 a 1d arrangement of nanorods B-nanoparticle , or a classical crystallization in cubic face centered and cubic centered . [SEP]
[CLS] however , only small aggregates are obtained , and it remains difficult to obtain crystals on large scales . [SEP]
[CLS] from a theoretical perspective , travesset and knorowski have studied the self - assembly of nanocubes , and a face - to - face orientation of the cubes was obtained when short strands were used , while several other structures could be obtained using longer dna strands with application of osmotic constraints . [SEP]
[CLS] finally , macfarlane et al . demonstrated the possibility of obtaining complex ternary systems by inserting a third size of nanoparticles B-nanoparticle into a pre - existing binary structure ( see figure 12 ) . [SEP]
[CLS] this process has made it possible to establish a wide range of crystalline structures in a completely reversible manner owing to the use of dna , opening the way for the development of a method that can be generalized to other materials . [SEP]
[CLS] the previously described work primarily focuses on the aggregation of nanoparticles B-nanoparticle , the diameter of which can vary from 10 to 150 or even 200 nm . [SEP]
[CLS] however , other teams have observed different behaviours using micrometric - sized nanoparticles B-nanoparticle , specifically with respect to their ability to stabilize colloidal solutions with increase in temperature . [SEP]
[CLS] indeed , interactions between particles vary with their size , especially with respect to weak van der waals interactions that can lead to non - specific and irreversible aggregation . [SEP]
[CLS] therefore , crystallization is much more difficult to achieve , and the aggregates are amorphous gels with a fractal structure . [SEP]
[CLS] crocker et al . demonstrated the possibility of crystallizing microspheres into short hexagonal crystals under extremely precise experimental conditions with respect to temperature and especially surface preparation . [SEP]
[CLS] indeed , only a specific method of functionalization by swelling - deflating micrometric organic particles ( such as polystyrene ) immersed in organic solvent in the presence of polyethylene glycol ( peg ) was sucessful . [SEP]
[CLS] beyond this example , the phenomenon of reversibility with temperature is rarely observed and strongly depends on the length of the dna bond and the temperature , which must be sufficient to de - hybridize double - stranded dna without irreversible aggregation of the nanoparticles B-nanoparticle . [SEP]
[CLS] although a recent review did not report significant progress in the self - assembly of micrometric particles , a team from the united states recently demonstrated the possibility of functionalizing micrometric organic particles [ polystyrene or poly ( methyl methacrylate ) ( pmma ) ] or inorganic ( titanium B-material oxide B-material or silicon B-material oxide I-material ) with single - stranded dna by a generic method of alkyne - azide cyclo - addition . [SEP]
[CLS] this method enables higher dna grafting densities to be obtained than methods using the biotin - streptavidin complex , consequently allowing for crystallization of the particles at room temperature or optimized rearrangement after annealing . [SEP]
[CLS] the most intriguing applications of dna nanotechnology , those that best take advantage of the small size , biocompatibility B-property and programmability of dna - based systems , lie at the interface with biology . [SEP]
[CLS] below , we highlight key successes in the development of dna - based imaging probes for biomolecular detection and prototypes of smart therapeutics and drug delivery systems . [SEP]
[CLS] several dynamic plasmonic systems have also been demonstrated for applications in energy harvesting , nanophotonics and imaging . [SEP]
[CLS] biomolecular detection - one of the first applications of dna nanotechnology put into practice was biomolecular detection based on the colorimetric principle . [SEP]
[CLS] for example , gold B-nanoparticle nanoparticles I-nanoparticle initially functionalized with complementary strands may be disintegrated in the presence of a target species , hybridizing with a portion of the dna strands ( see figure 13 ) . [SEP]
[CLS] disintegration caused by the de - hybridization of the two complementary strands is associated with a colour change from blue to red in the colloidal solution . this principle has been used by many teams to detect several species , such as thrombin , adenosine and cocaine , or cysteine B-material . [SEP]
[CLS] the possibilities are numerous but limited by the relatively high detection concentrations ( in the nanomolar range ) compared with the higher sensitivity that is attainable with fluorescence - based methods . [SEP]
[CLS] 77 optical and plasmonic devices - crystals composed of specific inorganic and semi - conductive nanoparticles B-nanoparticle , also called " quantum B-nanoparticle dots I-nanoparticle " , have the advantage of having luminescence B-property energies that have for biomedical applications to replace the traditional organic fluorophores used for imaging , exhibiting lower luminescence B-property and greater sensitivity to the spectrum of white light . [SEP]
[CLS] their association with a metal B-material such as gold B-material allows for further increase in the photoluminescence B-property released by the quantum B-nanoparticle dots I-nanoparticle . [SEP]
[CLS] thus , the organized assembly of gold B-nanoparticle nanoparticles I-nanoparticle and quantum B-nanoparticle dots I-nanoparticle allows the photoluminescence B-property of the structure to be controlled according to several parameters , such as the size of the constituents and the inter - particle distance , allowing for the monitoring of biological events and the development of improved plasmonic spectroscopies B-technique . [SEP]
[CLS] catalysis [SEP]
[CLS] - finally , control of the size , composition and density of particles is crucial for catalysis , where dna can provide novel possibilities . [SEP]
[CLS] a drawback is that dna requires a specific aqueous environment , limiting catalysis by passivating the surface of nanoparticles B-nanoparticle , making them less reactive . [SEP]
[CLS] to overcome these difficulties , auyeung et al . developed a threestep process , where after synthesizing a superstructure of gold B-nanoparticle nanoparticles I-nanoparticle , they freeze the structure in the silica and then calcine the whole structure , resulting in an organized porous structure [SEP]
[CLS] in summary , the authors predict a real utility for catalytic reactions using gold B-material , such as the oxidation of alcohol B-material or carbon B-material monoxide , or reduction via plasmonic effects . [SEP]
[CLS] while much of the literature describes the dna - directed assembly of gold B-nanoparticle nanoparticles I-nanoparticle , achieving the assembly of oxide B-material nanoparticles B-nanoparticle is a technically complex task due to increased surface chemistry issues . [SEP]
[CLS] this issue constitutes a formidable scientific challenge that well illustrates all possible routes to optimization of practical and efficient technological solutions and the promise of novel and practical applications in energy - generating materials . [SEP]
[CLS] this last section is devoted to such an application example , namely , thermite energetic materials . [SEP]
[CLS] interestingly , we present a series of methodological steps that have been performed to visualize and optimize the dna - directed assembly of al and cuo nanoparticles B-nanoparticle , which can be generalized to many other materials . [SEP]
[CLS] thermites are substances that store chemical energy that can be released after being submitted to an external electrical , optical , or pressure wave stimulus . . . [SEP]
[CLS] these substances are composed of a fuel ( most of the time aluminium B-material ) mixed with an oxidizer B-property , which can be varied to monitor reaction properties , and this metastable mixture undergoes exothermic oxidation - reduction reactions [SEP]
[CLS] the aluminium / copper B-material oxide I-material ( al / cuo ) system is among the most interesting as it offers high energy density and reactivity , particularly when mixing al and cuo nanoparticles B-nanoparticle , compared to their micronic counterpart [SEP]
[CLS] energetic materials are used in widespread applications , including in the space , military , automotive and civil security industries . [SEP]
[CLS] in the early 1990s , taking advantage of al and cuo compatibility with microelectromechanical systems ( mems ) fabrication techniques , we proposed that they be integrated into silicon B-material microsystems for the realization of local micro - actuations in extremely small volumes ( less than a cubic millimetre ) and relatively substantial forces ( ~ 0 . 1 n ) . [SEP]
[CLS] in this context , dna nanotechnologies open perspectives to create ad hoc materials that have high - performance , are safe and are reach legislation compatible . [SEP]
[CLS] the exothermic oxidation / reaction process , in which oxygen B-material atoms I-material are liberated from their cuo matrix ( reduction until pure copper B-material ) to oxidize aluminium , with formation of alumina , can be represented by the following overall chemical reaction : [SEP]
[CLS] the properties of these materials are highly dependent on the sizes of components and their distribution in the composite . [SEP]
[CLS] the use of nanoparticles B-nanoparticle allows for a significant improvement of energetic properties , but there remain important issues with respect to controlling the mixing of nanoparticles B-nanoparticle at the nanoscale . [SEP]
[CLS] in this context , the use of dna self - assembly shows great potential for the synthesis of new types of nanothermites with exquisite control of the contact surfaces between oxidizer B-property and reducer , thereby enabling the optimization and control of energetic performances . [SEP]
[CLS] our approach to using dna for the controlled assembly of al and cuo nps B-nanoparticle into thermite aggregates is outlined in figure 14 . [SEP]
[CLS] in the following section , we will successively detail and discuss : 1 . the fundaments and characterization of dna interactions and overall grafting protocol ; 2 . the characterization of dna - directed assembly ; and 3 . the performances of dna - assembled al / cuo energetic nanomaterials B-material compared with those of nanopowder mixtures . [SEP]
[CLS] to guide technological steps , we provided an atomic scale understanding of intrinsic physical and chemical interactions taking place between dna and oxide B-material surfaces . [SEP]
[CLS] to allow for experimental and atomic scale simulations of synergistic associations , we proposed to use a representative dtmp molecule that includes all dna subunits instead of a full dna fragment ( dtmp : 2 - desoxi - thymidine - 5 ' - monophosphate ) . [SEP]
[CLS] then , we studied the chemical interaction of dtmp molecules with controlled alumina surfaces through xps and infrared B-technique spectroscopy I-technique . [SEP]
[CLS] the experimental results were supported by density functional theory ( dft ) calculations . [SEP]
[CLS] in a second stage , we developed , characterized and optimized the overall grafting protocol quantifying all processing steps . [SEP]
[CLS] the dtmp molecule , as shown in figure 15 , is a rather small molecule compared to dna strands with multiple bases , but it still harbours all dna subunits , including a phosphate group , a sugar and a base , and in this case study harbours a thymine group . [SEP]
[CLS] the alumina oxide B-material surface is chosen as it is much simpler to manipulate and characterize at the atomic scale than cuo . [SEP]
[CLS] notably , to provide well - controlled model surfaces , we used flat alumina surfaces deposited by atomic B-technique layer I-technique deposition I-technique , which allows the thickness of alumina to be adjusted at the monolayer level . [SEP]
[CLS] we performed first principles calculations to evaluate the different possible chemical reactions taking place at surfaces , which was one of the objectives of the quantification of all dtmp vibrational modes of the different and most pertinent configurations ( either in the liquid or in contact with the surface ) for assignation of the complex infrared ir spectra obtained experimentally . [SEP]
[CLS] one such calculation is shown in figure 16 , corresponding to the most stable configuration of all trials , indicating chemical reactions at both the phosphate and base levels , with an energy gain in the order of 1 - 2 ev compared to dtmp in the liquid phase . [SEP]
[CLS] 100 to identify the bonding nature of dtmp with the alumina surface , we made use of infrared B-technique spectroscopy I-technique and extensively investigated deposition under multiple conditions ( concentration , temperature , nature of solvent … ) . [SEP]
[CLS] using first principles quantification of vibrational modes to assign different and reproducible peaks , we confirmed the creation of covalent bonds and multiple interactions on the surface , particularly through phosphate and thymine dissociation . [SEP]
[CLS] such assigned spectra are shown in figure 18 , in which the nature of the solvent was modified ( water B-material and methanol ) . [SEP]
[CLS] to direct the assembly of al and cuo , we decided to use a biotin / streptavidin grafting protocol . [SEP]
[CLS] with the knowledge that direct , irreversible and chaotic bonding would take place between dna and alumina or cuo nanoparticle B-nanoparticle surfaces , screening them as much as possible through streptavidin coverage would lead to subsequent specific interaction of the antigen / antibody B-material type with biotin terminated dna strands . [SEP]
[CLS] in the following section , we describe this grafting protocol and the process of generating the al and cuo colloidal solutions . [SEP]
[CLS] the overall dna functionalization procedure , which is conceptually equivalent for both the al and cuo nanoparticles B-nanoparticle , is shown in figure 14 . [SEP]
[CLS] the first step of the process consists of the dispersion and stabilization of al and cuo nanopowder B-nanoparticle . [SEP]
[CLS] for the experiment , we used 15 mg of 50 - nm cuo and 80 - nm al nanopowders of nominal size as provided by the suppliers . [SEP]
[CLS] these nanoparticles B-nanoparticle were dispersed in ultra - pure deionized B-material water I-material containing a surfactant B-property , tween - 20 ( or polyoxyethylene ( 20 ) sorbitan monolaurate ) , which acts as a stabilizing B-property agent I-property , and the solutions were buffered to neutral ph ( ph = 7 ) . [SEP]
[CLS] tween - 20 is a molecule comprising hydrophilic B-property and hydrophobic B-property regions on opposing sides . [SEP]
[CLS] in solution with nanoparticles B-nanoparticle , these molecules nonspecifically interact with nanoparticles B-nanoparticle and prevent aggregation . [SEP]
[CLS] during their dissolution , nanopowders do not spontaneously disperse , yielding numerous large aggregates , necessitating an ultrasound step followed by a sedimentation step . [SEP]
[CLS] the solutions are ultrasonicated for 8 min at 200 w , while maintaining the temperature below 40 °c . [SEP]
[CLS] the resulting solutions are then left overnight at room temperature , allowing large aggregates that have not been broken down to settle . [SEP]
[CLS] the supernatant of the solutions is then recovered and characterized by dynamic B-technique light I-technique scattering I-technique ( dls ) . [SEP]
[CLS] the results obtained for unmodified particles can be seen in figure 19 ( black curves ) . [SEP]
[CLS] we observed hydrodynamic diameters ( zh ) values of ~ 224 ± 7 and 187 ± 5 nm for al and cuo , respectively . [SEP]
[CLS] scanning electron B-technique microscopy I-technique results revealed that these colloidal solutions are composed of small indivisible aggregates of 2 to 4 nanoparticles B-nanoparticle , with diameters that are stable over hours . [SEP]
[CLS] note that a shell B-material layer of approximately 3 nm of oxide B-material covers the pure al core B-material of the al nanoparticles B-nanoparticle . [SEP]
[CLS] this protective shell B-material allows the particles to remain unoxidized and stable in solution under neutral ph conditions . [SEP]
[CLS] then , the grafting of streptavidin was performed after determining the optimal quantity that can be generated , which is derived from the total nanoparticle B-nanoparticle surface available in solution , considering a sphere approximation , and knowing that streptavidin covers approximately 5 nm 2 . [SEP]
[CLS] finally , for both al and cuo colloidal solutions , an excess of streptavidin was added . [SEP]
[CLS] in both cases , an incubation B-technique time of at least 4 h was used , and the solutions were rinsed multiple times to remove the excess of ungrafted streptavidin . [SEP]
[CLS] between each rinsing step , the solution was centrifuged and further dispersed in an ultrasonic bath after changing the solvent . [SEP]
[CLS] rinsing was performed out in a 0 . 1 % diluted phosphate buffered saline ( pbs ) solution containing 0 . 05 % vol . tween 20 , although the grafting of streptavidin tends to stabilize the colloids . [SEP]
[CLS] again , dls measurements were performed , shown as green curves in figure 19 , and could be compared to non - grafted particle diameters . [SEP]
[CLS] we observed a slight increase in the mean diameters due to the addition of streptavidin molecules . [SEP]
[CLS] notably , for the cuo nanoparticles B-nanoparticle , the inflation was more pronounced , which could be attributed to undesired interparticle interactions mediated by streptavidin or their accumulation of multiple layers . [SEP]
[CLS] the next step was to achieve biotin - terminated dna grafting owing to the biotin / streptavidin interaction . [SEP]
[CLS] for streptavidin , the first step was to estimate the amount of dna needed to functionalize all the available proteins B-material on the surface of the nanoparticles B-nanoparticle . [SEP]
[CLS] for both colloidal solutions of al and cuo streptavidin - functionalized particles , which were previously prepared , we added the dna solution at a concentration that was 5 - fold the amount necessary for the biotin - terminated dna to complement the optimal surface density of streptavidin sites . [SEP]
[CLS] again , after incubation B-technique , rinsing and centrifugation to remove excess dna in solution , the colloidal solution was redispersed after each centrifugation in an aqueous solution of 0 . 1× pbs and 0 . 05 % tween in an ultrasonic bath . [SEP]
[CLS] subsequently , the hydrodynamic diameter and zeta B-property potential I-property of the obtained functionalized nanoparticles B-nanoparticle were characterized by dls , the results of which are shown in figure 19 ( red curves , obtained with 30 - base dna strands ) . [SEP]
[CLS] shorter ( 15 bases ) and longer ( 45 bases ) dna strands have also been used , showing coherent results for cuo nanoparticles B-nanoparticle . [SEP]
[CLS] based on previous fluorescence based methodologies that were exclusively used for quantifying the grafting of dna strands on gold B-nanoparticle nanoparticles I-nanoparticle , we have derived the following quantification strategy , which allows the measurements of streptavidin and dna surface densities . [SEP]
[CLS] the end of the subsection describes the quantification of the grafted dna that is still functional for hybridization , which we termed the " hybridization efficiency " . [SEP]
[CLS] note that in this section , hybridization is performed with dna strands that are nanoparticle free . [SEP]
[CLS] because of the permanent immobilization of streptavidin on the surfaces of the nanoparticles B-nanoparticle , we developed a method for quantifying the amount of streptavidin grafted onto the nanoparticle B-nanoparticle surfaces ( al or cuo ) . [SEP]
[CLS] this process was performed in three steps , shown schematically in figure 20 that quantify a . streptavidin coverage , b . dna coverage and c . hybridization efficiency , respectively , which are the primary principles detailed in the following section . [SEP]
[CLS] for more technical details , see ref . . [SEP]
[CLS] quantification of streptavidin surface density - cy3 - labelled streptavidin is added to the colloidal suspensions . [SEP]
[CLS] after incubation B-technique , the solutions are centrifuged , and the fraction of unbound streptavidin [ figure 20 ( a ) ] is measured in the supernatant . [SEP]
[CLS] the difference in the streptavidin concentration before and after centrifugation is used to deduce the amount of bound streptavidin , which is then normalized by the quantity of nanoparticles B-nanoparticle determined by atomic absorption spectrometry ( aas ) . [SEP]
[CLS] quantification of dna surface density - fluorescent B-property fam - oligonucleotides are added to [UNK] [UNK] and [UNK] [UNK] colloidal suspensions . [SEP]
[CLS] after incubation B-technique , the solutions are centrifuged , and the fluorescence B-property intensity of the supernatant is measured . [SEP]
[CLS] the quantity of dna grafted onto the nanoparticles B-nanoparticle is then determined from the difference between the initial dna concentration ( using a control sample ) and the dna concentration in the supernatant , which is then normalized by the quantity of nanoparticles B-nanoparticle determined using aas . [SEP]
[CLS] this protocol is summarized in figure 20 ( b ) . [SEP]
[CLS] note that the reversibility of the streptavidin / biotin interaction can be evaluated by heating / cooling the solutions containing functionalized al and cuo nanoparticles B-nanoparticle . [SEP]
[CLS] quantification of dna hybridization efficiency - fam - labelled oligonucleotides are added to al and cuo colloidal suspensions previously functionalized with non - fluorescent B-property strands . [SEP]
[CLS] the nacl concentration is set to enable the hybridization of the fam - labelled strands with the nonfluorescent ones , grafted on the al and cuo nanoparticles B-nanoparticle . [SEP]
[CLS] after a few hours , the colloids are centrifuged and re - suspended in solution ( 0 . 1× pbs , tween 0 . 05 % and nacl ) to remove excess dna strands . [SEP]
[CLS] after rinsing , the solutions are finally heated to 80 °c for 5 min to de - hybridize dna double strands . [SEP]
[CLS] the fluorescence B-property intensity of the supernatant containing the released fluorescent B-property strands is then measured , and the quantity of dna strands grafted on the nanoparticles B-nanoparticle is calculated [ see figure 20 ( c ) ] . [SEP]
[CLS] overall , the results indicate that a careful handling of selected process parameters is required to maximize the dna surface density . [SEP]
[CLS] particularly , we observe that 15 - 30 mm nacl is required to avoid irreversible nanoparticle B-nanoparticle aggregation and that gentle sonication during the early stage of streptavidin incubation B-technique allows for increased streptavidin loading by approximately 175 and 135 % for the cuo and al nanoparticles B-nanoparticle , respectively . [SEP]
[CLS] the dna strand length has no noticeable impact on the dna grafting density . [SEP]
[CLS] we showed that direct grafting of dna onto al and cuo nanoparticles B-nanoparticle largely dominates the overall functionalization process , with dna immobilization on streptavidin sites representing less than 5 - 10 % of all dna strands ( see table 1 ) . [SEP]
[CLS] as streptavidin covers only a small portion of the al and cuo nanoparticle B-nanoparticle surfaces , a large surface area is prone to non - specific interactions with dna . [SEP]
[CLS] we experimentally confirmed the strong chemical affinity of dna bases with both copper B-material oxide I-material and alumina surfaces and the lack of control of the conformation of the immobilized strands onto the oxide B-material surfaces . [SEP]
[CLS] the higher density of grafted dna observed for cuo suggests a partially spread - out conformation compared to the completely spread - out strands on al . [SEP]
[CLS] finally , we quantitatively proved the crucial role of the antigen / antibody B-material protocol to preserve the ability of dna to hybridize , improving the hybridization efficiency by 2 - fold , ensured by the specific dna strands grafting onto streptavidin sites ( ~ 1 . 2 and 0 . 9 strands by streptavidin for cuo and al nanoparticles B-nanoparticle , respectively ) . [SEP]
[CLS] many parameters ( length and nature of the spacer B-material , nacl concentration . . . ) clearly affect the assembly and crystallinity of nanoparticle B-nanoparticle aggregates . [SEP]
[CLS] however , the role of the coding sequence selected to enable hybridization upon aggregation has hardly been addressed , despite being a crucial factor in determining the thermodynamics and kinetics of aggregation . [SEP]
[CLS] various hybridization strategies involving strands of variable coding sequences have been described in the literature . [SEP]
[CLS] in addition , the role of the length of the spacer B-material has been investigated . [SEP]
[CLS] however , the choice of the oligonucleotide sequence exclusively follows a priori empirical design and has not been studied nor commented on in an extensive manner . [SEP]
[CLS] in this section , we propose to design and optimize the sequence that will be used to obtain our energetic nanobiocomposites in silico , which will be validated experimentally in a second stage . [SEP]
[CLS] these results were published in ref . . [SEP]
[CLS] referring to the pioneering research by oleg gang , presenting a generic method for the functionalization of different types of nanoparticles B-nanoparticle ( gold B-material , palladium B-material , iron B-material oxide I-material or quantum B-nanoparticle dots I-nanoparticle ) , the chosen dna strand is composed of a repeated t - base spacer B-material to separate the coding portion that will be further hybridized to the surface of the particle , with a coding portion of 15 bases . [SEP]
[CLS] a close examination of the sequence allowed us to notice that partial hybridization between two complementary strands was possible due to the repetition of the four aggt bases twice in the sequence ( aat - agg - tga - agg - tta ) . [SEP]
[CLS] in addition , folding of the strand on itself is possible when the spacer B-material is composed of t bases , where the sequence becomes [ ( t ) 12 - ttt - aat - agg - tga - agg - tta ] . [SEP]
[CLS] this possible folding is crucial because it could prevent any hybridization with the complementary strand . [SEP]
[CLS] in addition , this folding can result in " stranded " interactions when a large number of strands of identical composition are in solution . [SEP]
[CLS] these parasitic interactions are schematically shown in figure 21 and should be avoided or minimized . [SEP]
[CLS] given the limited number of studies on the generation of an optimized dna strand sequence of a length close to standard requirements , we have developed an algorithm to define an optimized sequence by removing 4 types of undesired interactions : [SEP]
[CLS] - the generated dna strand must not fold back on itself to maintain its coding specificity . [SEP]
[CLS] - non - complementarity of the strand , total or partial , with a strand of identical composition . [SEP]
[CLS] based on the use of the dna strand in solution , it is indeed inevitable to identify many identical strands capable of interacting with each other . [SEP]
[CLS] - the considered strand must hybridize over the entire coding portion of the other strand to better control the hybridization process . [SEP]
[CLS] - the fourth type of constraints is based on the definition of hybridization criteria , where the user first chooses to consider the minimum number of adjacent bases allowing for hybridization ( denoted nm ) ( d ) , with the possibility of including one or two noncomplementary bases ( f ) between these adjacent bases . [SEP]
[CLS] the value of nm is set to 3 by default , according to the gibbs energies reported in the literature . [SEP]
[CLS] note also that constraint ( b ) is more restrictive than constraint ( a ) , because it does not require a loop for hybridization such that the application of constraint ( b ) includes constraint ( a ) . [SEP]
[CLS] considering all restrictions , the number of solutions drastically decreases from the intrinsic combination of dna coding possibilities , which is tractable by computer analysis . [SEP]
[CLS] the full details of our algorithm and obtained sequences are available in [SEP]
[CLS] this optimization process allows the sequences to be selected according to the type of constraint applied and for the results to be sorted according to the melting temperature of each selected dna strand . [SEP]
[CLS] such calculation results are shown in figure 22 ( a , c ) . [SEP]
[CLS] for each sequence length , the table presents the best sequence in term of melting temperature that also fits the restriction requirements . [SEP]
[CLS] the curve ( a ) of figure 22 shows the number of available sequences for each dna strand length that the operator wishes to implement in the assembly protocol and the associated average melting temperature . [SEP]
[CLS] note that while melting temperature continuously increases , it starts to become saturated at a nominal level for a dna length of approximately 6 to 10 bases . [SEP]
[CLS] - aggregation between functionalized al and cuo nanoparticles B-nanoparticle with complementary and optimized strands ( solid blue symbols ) , - aggregation between functionalized al and cuo nanoparticles B-nanoparticle with non - optimized complementary strands ( solid red symbols ) , - aggregation between functionalized al and cuo nanoparticles B-nanoparticle with optimized but identical strands , supposedly non - complementary ( empty blue symbols ) , - aggregation between functionalized al and cuo nanoparticles B-nanoparticle with optimized and identical strands , assumed to be non - complementary ( empty red symbols ) , - aggregation between non - functionalized al and cuo nanoparticles B-nanoparticle , i . e . without a strand of dna or streptavidin on the surface ( black symbols ) . [SEP]
[CLS] first , no or little aggregation was observed between non - functionalized al and cuo nanoparticles B-nanoparticle ( black curve ) . [SEP]
[CLS] therefore , this experiment enables the absence of non - specific interactions between particles to be verified under these conditions . [SEP]
[CLS] next , functionalized al and cuo nanoparticles B-nanoparticle with identical strands , supposedly non - complementary , are shown as blue and red empty symbols , respectively . [SEP]
[CLS] while a lack of aggregation is expected , an increase in the hydrodynamic diameter is observed over time in both cases that is greater than that shown in the black curve , corresponding to non - functionalized al and cuo particles . [SEP]
[CLS] when the sequence of the dna strands used has been optimized ( blue curve ) , the increase of the hydrodynamic diameter is limited , reaching 500 nm after 1 h 30 min . [SEP]
[CLS] in contrast , when the dna strands have not been optimized ( red curve ) , the hydrodynamic diameter reaches 725 nm after 1 h . [SEP]
[CLS] in both cases , the aggregation is caused by nonspecific interactions between nanoparticles B-nanoparticle functionalized with dna . [SEP]
[CLS] these interactions may result from various factors that can be investigated . [SEP]
[CLS] more importantly , one can note that these interactions are clearly limited when the sequence is optimized by our algorithm . [SEP]
[CLS] finally , a pronounced and rapid aggregation is observed when the nanoparticles B-nanoparticle are functionalized with complementary dna strands , optimized or not . [SEP]
[CLS] the aggregation regime differs according to the nature of the sequence , with faster kinetics observed when the sequence is optimized . [SEP]
[CLS] more precisely , the hydrodynamic diameter increases from 250 nm to 1 . 5 μm after 1 h 30 min of aggregation with optimized strands compared with 1 . 2 μm with non - optimized strands . [SEP]
[CLS] more information is available in on the nature of the nonspecific interaction , reversibility of the assembly , overall kinetics and role of temperature and salt B-material concentration . [SEP]
[CLS] this last section evaluates the energy performance of nanobiocomposites described above . [SEP]
[CLS] after evaporation of the solvent in a vacuum chamber for a few hours and drying , the aggregates are recovered and analysed by ( differential scanning calorimetry ( dsc ) to measure the exothermic and endothermic reactions with respect to changes in temperature . [SEP]
[CLS] the dried al - cuo nanobiocomposite is placed in a platinum B-material dsc crucible . [SEP]
[CLS] a similar but empty crucible is also considered for reference . [SEP]
[CLS] the heat exchanges between the sample and the reference are measured under a temperature ramp of 10 °c min - 1 from 30 to 700 °c under an inert atmosphere ( 99 . 999 % pure ar ) . [SEP]
[CLS] this characterization method is conventionally used for the analysis of nanothermite material B-material reactions . [SEP]
[CLS] in figure 23 the reaction enthalpies are annotated in the graphs , obtained from the area under the curve between 480 and 620 °c , highlighted by the coloured zone . [SEP]
[CLS] first , the three curves have a similar profile . [SEP]
[CLS] some exothermic peaks are detected from 200 °c , which are associated with pollutants related to the ionic aqueous solvent used for the aggregation of particles . [SEP]
[CLS] then , the primary exothermic peak is centred at 570 °c for the three nanocomposites . [SEP]
[CLS] finally , the initiation temperature , corresponding to the beginning of the reaction and highlighted in dashed lines in figure 23 , is located at approximately 490 °c . [SEP]
[CLS] nanocomposites obtained from the aggregation of nanoparticles B-nanoparticle without dna functionalization in aqueous solution and obtained from the assembly of nanoparticles B-nanoparticle functionalized with complementary dna strands initiated at a lower temperature than when the dna strands were not complementary ( 490 versus 500 °c ) , and the reaction enthalpies were also the highest observed at 1 . 46 and 1 . 35 kj g - 1 , respectively . [SEP]
[CLS] composites obtained by assembling functionalized nanoparticles B-nanoparticle with non - complementary dna strands are significantly less efficient , with an enthalpy of 1 . 00 kj g - 1 . [SEP]
[CLS] finally , note that the nanocomposites generated in hexane , corresponding to the most commonly used method in the literature , have a much lower initiation temperature , at approximately 440 °c , but a low reaction enthalpy at 0 . 76 kj g - 1 . [SEP]
[CLS] these results confirm the contribution of dna in the nanostructuring of al - cuo aggregates , as evidenced by the 135 % increase in reaction enthalpy when the dna strands are complementary . [SEP]
[CLS] thus , dna has a notable impact in the organization of nanoparticles B-nanoparticle , which was further on confirmed by structural and chemical analysis . [SEP]
[CLS] 24 presents tem images that allow for direct comparisons between aggregates assembled through dna or without . [SEP]
[CLS] the nanocomposite without dna indeed appears much thicker than that made with complementary dna strands , which appears spread on the surface . [SEP]
[CLS] there are also clear geometric patterns in the first case , revealing the presence of nacl crystals , which are not present in the nanobiocomposite . [SEP]
[CLS] this greater thickness in the case of the nanocomposite without dna shows that the aggregation of particles is equivalent in all spatial directions , retaining this structure at the time of drying . [SEP]
[CLS] the edx analysis in hr - tem of these two powders , presented in figure 25 , confirms that the aggregation processes differs in both cases . [SEP]
[CLS] the presence of chlorine B-material in the nanocomposite without dna clearly validates the presence of nacl crystals as previously pre - supposed . [SEP]
[CLS] notably , the distribution of al and cu is highly homogenous , with observed alterations in structural domains between 200 and 500 nm . [SEP]
[CLS] however , the nacl crystals are completely absent from the nanobiocomposite , and the dna assembly exhibits slightly greater homogeneity . [SEP]
[CLS] these results enable an understanding of how the nanocomposite is organized during aggregation and especially drying . [SEP]
[CLS] when there is no dna to direct the placement of particles , the organization is random and occurs around nacl crystals during their formation at the time of drying . [SEP]
[CLS] if the amount of nacl is sufficiently low , we have shown that the interaction between the particles is good and their alteration is highly homogenous , leading to higher thermal properties . [SEP]
[CLS] in contrast , when particles are functionalized with dna , their assembly in aqueous solution is directed and optimized . [SEP]
[CLS] at the time of drying , the composite is already created , and nacl does not crystallize within the material B-material . [SEP]
[CLS] the energy performances are also very good thanks to a strong association of the particles and a highly homogeneous distribution . [SEP]
[CLS] importantly , dna processing ensures a good reproducibility in the measured reaction enthalpies , with an average result of 1 . 25 ± 0 . 05 kj g - 1 obtained from four distinct samples . [SEP]
[CLS] this is not the case in the experiments performed without dna , for which large discrepancies were observed . [SEP]
[CLS] finally , the low energetic performance of the composite made in hexane is explained by a very poor distribution of al and cuo nanoparticles B-nanoparticle , pointing to the beneficial role of dna - directed assembly . [SEP]
[CLS] the tem observations and edx analysis results of these aggregates indeed show large clusters of cuo ( between 0 . 5 and 1 . 5 microns in diameter ) and a much lower al level ( 75 % cuo and 25 % al ) . [SEP]
[CLS] all the advances described in this review demonstrate the power and versatility of dna nanotechnology to control the structuring of materials at the nanoscale . [SEP]
[CLS] more than 30 years after suggesting the principle of using dna as a tool dedicated to nano - construction , major advances have been made in multiple directions . [SEP]
[CLS] among them , the assembly of nanoparticles B-nanoparticle has become a mainstream aspect of dna nanotechnology , which again has demonstrated the potential to propose advanced and multifunctional materials that would not otherwise exist : crystallization of an assembly of particles , nanoparticle B-nanoparticle decoration of functional superstructures … this review describes the existing research efforts to integrate dna nanotechnologies with nanoparticles B-nanoparticle . [SEP]
[CLS] we further focused on one example application , allowing us to provide an original example , namely , al / cuo nanoparticle B-nanoparticle assembly , in literature dominated by pure metallic B-nanoparticle nanoparticle I-nanoparticle manipulation . [SEP]
[CLS] this example also allowed us to provide key parameters and technological methodologies , making use of both experiment and theory , to optimize dnadirected assembly as a route to move from laboratory proof of principle to more applied if not industrial processes . [SEP]
[CLS] in particular , we highlighted the use of atomically precise techniques to evaluate fundamental interactions , the use of which has shown their multi - level complexity , including non - specific to specific dna interactions , grafting strategies , hybridization optimization and coding sequence choice … in this respect , we provided methodologies to quantify the performances of dna - mediated grafting strategies . [SEP]
[CLS] finally , we discussed the assembly performance in the specific scope of an application dedicated to energetic nanomaterials B-material . [SEP]
[CLS] indeed , on the long term , the prospects for mass production must be considered . [SEP]
[CLS] there are still many limiting factors due to scaling issues and the time and cost of manufacturing materials . [SEP]
[CLS] from a functional point of view , the robustness , ageing , performance and design complexity of such materials must be further evaluated . [SEP]
[CLS] nevertheless , all the efforts made by research teams will allow for these issues to be overcome , opening routes for dna technologies for the manufacture of the multifunctional materials of the future . [SEP]
[CLS] 1 : perspective of the relative scaling of the synthetic and biological worlds with respect to multiscale dna architecture . [SEP]
[CLS] from the dna molecule to nanotechnologiesdeoxyribonucleic acid I-material ( dna ) is a core B-material constituent of all living cells B-material , containing the genetic information from which all the functions of an organism are programmed . [SEP]
[CLS] the composition and properties of dna have been extensively studied , and the dna double helix is certainly one of the best - known structures in biology . [SEP]
[CLS] dna is composed of sequences of nucleotides that are themselves made up of three components ( figure2 ) : a phosphate group bonded to a sugar ( deoxyribose ) to which a nitrogenous base is linked . [SEP]
[CLS] it is therefore a true modular framework consisting of sugars and phosphate groups as a backbone that can harbour one of four different nitrogenous bases , including adenine ( a ) , thymine ( t ) , cytosine ( c ) and guanine ( g ) , ensuring the sequential signature of the dna molecule as well as the complementarity required to form the double helix . [SEP]
[CLS] 2 : schematic of the dna molecular substructure [SEP]
[CLS] 3 : double - double crossover ( ddx ) dna - based structure ( top schematics ) and afm image of a 2d crystal made of branched ddx motifs ( lower view ) . ( reprinted with permission from j am chem soc 2005 , 127 ( 50 ) , 17590 - 17591 . [SEP]
[CLS] copyright ( 2005 ) american chemical society ) [SEP]
[CLS] ( a ) ] called " staples " . [SEP]
[CLS] each staple is designed to bind to different locations of the long strand to induce local folding , which collectively cause the overall dna strand to acquire a defined shape . [SEP]
[CLS] rothemund illustrated the versatility of this concept by creating a number of different structures [ figure 4 ( b ) ] . [SEP]
[CLS] figure 4 ( et al . managed to create a periodic network of gold B-nanoparticle nanoparticles I-nanoparticle on a dna - modified surface [ [SEP]
[CLS] 4 : ( a ) design of a dna origami . [SEP]
[CLS] ( b ) different shapes obtained using the origami technique : a , square ; b , rectangle ; c , star ; d , smiley ; e and f , triangles . [SEP]
[CLS] the first two lines correspond to design images from computer simulation , while the two other lines correspond to experimental validations . [SEP]
[CLS] ( reprinted with permission from nature 2006 , 440 ( 7082 ) , 297 - 302 , copyright ( 2006 ) , springer nature ) ; ( c ) dna nanostructures used as templates for the assembly of a 2d network of gold B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] ( reprinted with permission from nano lett 2006 , 6 ( 2 ) , 248 - 251 , copyright ( 2006 ) , american chemical society ) ( d ) full origami dna box . [SEP]
[CLS] the box may be actuated for closing / opening through the introduction of an oligonucleotide ( the top image reconstructed from transmission electron cryomicroscopy experiment , and the bottom image is a schematic of the origami box . [SEP]
[CLS] ( reprinted with permission from nature 2009 , 459 ( 7243 ) , copyright ( 2009 ) , springer nature ) [SEP]
[CLS] 5 : ( a ) schematic diagram of the linker self - assembly of 13 - nm diameter gold B-nanoparticle nanoparticles I-nanoparticle ( left figure ) . [SEP]
[CLS] ( b ) measuring the absorbance at 260 and 700 nm of the colloidal solution of nanoparticles B-nanoparticle as a function of temperature ( 0 and 80 °c ) and demonstrating the reversibility of the hybridization of the dna strands . [SEP]
[CLS] ( c ) image of the colloidal solution heated to 80 °c ( red ) , and cooled to 0 °c ( blue ) . ( reprinted with permission from nature 1996 , 382 ( 6592 ) , 607 - 609 , copyright ( 1996 ) , springer nature ) [SEP]
[CLS] 7 : a . direct biotin / streptavidin - mediated nanoparticle B-nanoparticle aggregation ; b . protocol for the biotin / streptavidin - mediated dna - driven assembly of nanoparticles B-nanoparticle . [SEP]
[CLS] 32 ( reprinted with permission from j phys chem b 2003 , 107 ( 2 ) , 470 - 477 . copyright ( 2003 ) american chemical society ) [SEP]
[CLS] 8 : diagram summarizing the influence of the linker on the crystalline structure obtained after the self - assembly of gold B-nanoparticle nanoparticles I-nanoparticle . ( reprinted with permission from nature 2008 , 451 ( 7178 ) , 553 - 556 , copyright ( 2008 ) , springer nature ) [SEP]
[CLS] 9 : gold B-nanoparticle nanoparticle I-nanoparticle phase diagrams as a function of nanoparticle B-nanoparticle size ( rs ) or linker length ( rl ) for three different stoichiometries ( in a binary mixture , the ratio in terms of the population of nanoparticles B-nanoparticle functionalized with a given sequence versus another set of nanoparticles B-nanoparticle with the complementary sequence ) : ( a ) 1 : 1 , ( b ) 2 : 1 , ( c ) 3 : 1 . [SEP]
[CLS] the symbols represent the experimental points : the solid symbols represent the pure structures , while the voids represent polymorphic structures . [SEP]
[CLS] overall , there is good agreement between experimental points and theoretical results . [SEP]
[CLS] 10 : diagram showing the behaviour of nanoparticle B-nanoparticle aggregates formed upon dna selfassembly versus temperature and the length of dna strands used . ( reprinted with permission from nature 2008 , 451 ( 7178 ) , 549 - 552 , copyright ( 2008 ) , springer nature ) [SEP]
[CLS] 11 : comparison of the variation in the interparticle distance of 40 - nm diameter gold B-nanoparticle nanoparticles I-nanoparticle organized into dimers according to the use of hydrophilic B-property ( a ) or amphiphilic B-property ( b ) ligands for their stabilization after increases in the saline concentration ( i : 5 mm nacl ; ii : 800 mm nacl ) by dark field microscopy B-technique and spectral absorbance analysis [SEP]
[CLS] ( reprinted with permission from small 2015 , 11 ( 42 ) , 5696 - 5704 , copyright ( 2015 ) , wiley ) [SEP]
[CLS] 12 : diagram of the self - assembly of nanoparticles B-nanoparticle by dna in ternary crystalline structures . [SEP]
[CLS] a binary structure is first synthesized prior to insertion of a third size nanoparticles B-nanoparticle into a predetermined site ( a ) . [SEP]
[CLS] in ( b ) , the thermal analysis of the ternary structure is presented . [SEP]
[CLS] ( reprinted with permission from science 2013 , 341 ( 6151 ) , 1222 - 1225 , copyright ( 2013 ) , science american association for the advance of science ) [SEP]
[CLS] 13 : schematic diagram of the detection of adenosine using the colorimetric method . [SEP]
[CLS] functionalized gold B-material particles with dna strands are aggregated owing to the presence of a complementary aptamer with adenosine . [SEP]
[CLS] thus , in the presence of the target molecule , the aptamer hybridizes to the molecule , causing particle de - aggregation and a colorimetric change in the solution from blue to red . ( reprinted with permission from angew chem int edit 2006 , 45 ( 1 ) , 90 - 94 , copyright ( 2006 ) , wiley ) [SEP]
[CLS] 13 . illustration of the functionalization process developed to enable the dna - directed selfassembly of core - shell al ( al covered with al2o3 shell B-material ) and cuo nanoparticles B-nanoparticle . [SEP]
[CLS] 15 . schematic of the dtmp molecule with its subunits equivalent to those of dna . [SEP]
[CLS] 16 : chemisorption of dtmp on a hydrated al2o3 surface ( with an - oh termination ) in the most stable case , i . e . with formation of covalent bonds at the level of phosphate and thymine groups . [SEP]
[CLS] 17 : xps spectra for three primary elements in dtmp deposited on alumina surface [SEP]
[CLS] the black curve corresponds to the signal of the reference ( alumina substrate ) , while the red curve corresponds to the alumina surface with deposited dtmp . [SEP]
[CLS] 18 : influence of the nature of the solvent on the ir absorbance spectra of an alumina surface after dtmp deposition . [SEP]
[CLS] the incubation B-technique of the surface was performed using methanol ( top curve ) and water B-material ( bottom curve ) with the same concentration of dtmp to a clean alumina substrate in both cases . [SEP]
[CLS] related dtmp regions are reported on the top of the figure : t for thymine , s for sugar , and p for phosphate according to assignments resulting from dft calculations . [SEP]
[CLS] 19 : schematic overview of primary dna grafting steps and strategy for the al / cuo directed assembly . [SEP]
[CLS] 20 . schematic representation of the process developed for the determination of ( a ) streptavidin surface density on cuo or al nanoparticles B-nanoparticle , ( b ) the dna surface density on cuo or al nanoparticles B-nanoparticle and ( c ) the hybridization efficiency on cuo or al nanoparticles B-nanoparticle . [SEP]
[CLS] 21 : schematic of the constraints used in the programme . [SEP]
[CLS] oligonucleotides are represented by bars : red bars correspond to spacers B-material ( for example , repeated thymine bases ) , blue bars correspond to the sequence designed by the algorithm , and green bars correspond to the complementary strand of the designed oligonucleotide . [SEP]
[CLS] ( a ) avoidance of self - interactions for a single strand through the folding process , ( b ) hindered interactions ( total or partial hybridization ) between two strands with the same sequences , and ( c ) undesired partial hybridization of sticky - end sequences in the presence of their complementary counterpart . [SEP]
[CLS] ( d , e ) detailed and local sequence constraint : ( d ) a maximum of three [SEP]
[CLS] 22 : ( a ) number of sequences obtained according to the number of bases by considering three adjacent bases necessary for the hybridization and the stresses ( b , e of figure 21 ) with the inclusion [SEP]
[CLS] we show the distinct dsc signatures of three different nanocomposites differing in their assembly protocols : 1 . assembly of functionalized al and cuo nanoparticles B-nanoparticle with optimized and complementary dna strands ( blue curve ) . [SEP]
[CLS] 2 . assembly of functionalized al and cuo nanoparticles B-nanoparticle with optimized but noncomplementary dna strands ( red curve ) 3 . assembly of al and cuo nanoparticles B-nanoparticle without dna functionalization , mixed in aqueous solution ( black curve ) or in hexane - based organic solution ( dashed grey curve ) . [SEP]
[CLS] 23 : dsc curves of different al - cuo nanobiocomposites obtained by assembly of al and cuo nanoparticles B-nanoparticle with optimized and complementary dna strands ( blue curve ) , optimized and noncomplementary dna strands ( red curve ) and non - complementary functionalized dna strands in aqueous solution ( black curve ) or in hexane ( dashed grey curve ) . [SEP]
[CLS] 24 : tem images of nanocomposites made by direct mixing of al and cuo nanoparticles B-nanoparticle ( a ) , and assembled with optimized and complementary dna strands ( b ) . [SEP]
[CLS] 25 : dark film tem images ; elemental maps of al ( blue ) , cu ( red ) and cl ( green ) ; and elemental composition of a slice of al - cuo nanocomposites obtained by aggregation of al and cuo nanoparticles B-nanoparticle without dna ( a ) and by assembly with optimized and complementary dna strands ( b ) [SEP]
[CLS] quantification [SEP]
[CLS] an azide - functionalized 12 - armed buckminster fullerene B-nanoparticle has been monosubstituted in organic media with a substoichiometric amount of cyclooctyne - modified oligonucleotides . [SEP]
[CLS] exposing the intermediate B-property products then to the same reaction ( i . e . , strain - promoted alkyne−azide cycloaddition , spaac ) with an excess of slightly different oligonucleotide constituents in an aqueous medium yields molecularly defined monofunctionalized spherical nucleic B-material acids I-material ( snas ) . [SEP]
[CLS] this procedure offers a controlled synthesis scheme in which one oligonucleotide arm can be functionalized with labels or other conjugate groups ( 1 , 4 , 7 , 10 - tetraazacyclododecane - 1 , 4 , 7 , 10 - tetraacetic acid , dota , and alexa - 488 demonstrated ) , whereas the rest of the 11 arms can be left unmodified or modified by other conjugate groups in order to decorate the snas ' outer sphere . [SEP]
[CLS] extra attention has been paid to the homogeneity and authenticity of the c 60 - azide scaffold used for the assembly of full - armed snas . [SEP]
[CLS] spherical nucleic B-material acids I-material ( snas , introduced originally by the chad mirkin laboratory ) consist of an appropriate core B-material ( gold B-material , silica , liposomes B-nanoparticle , proteins B-material ) and densely packed oligonucleotide ( on ) chains . [SEP]
[CLS] they share many beneficial properties that overcome some of the major shortcomings perceived for therapeutic ons : they have efficient free cellular uptake via class a scavenger receptor - I-event mediated I-event endocytosis B-event ( which correlates with the density and chemistry of the component ons ) , they have muted innate immune responses and resistance to nuclease degradation ( due to steric reasons ) , and they are large enough to avoid renal clearance . [SEP]
[CLS] the cellular uptake occurs via an endosomal pathway , and snas are able to silence target rnas via steric blocking , once they enter into the cytoplasm . [SEP]
[CLS] also , small interfering rnas ( sirnas ) can work in spherical formulation . [SEP]
[CLS] dicer is able to cleave sirnas from snas and release them for the canonical rna interference pathway . [SEP]
[CLS] most of the reported snas are polydisperse structures . [SEP]
[CLS] the polydispersity may be a shortcoming as the data shows a similar behavior , but still a population of particles lacking thorough characterization of the molecules exists , something that is readily accessible for covalent on conjugates . [SEP]
[CLS] recently , a molecular sna , based on a buckminster c 60 - fullerene B-nanoparticle core B-material , has been described . [SEP]
[CLS] this structure , consisting of antisense on sequences on the c 60 core B-material , was dense enough to induce scavenger receptor - I-event mediated I-event endocytosis B-event . [SEP]
[CLS] the internalization of these particles to breast cancer ( mcf7 ) cells B-material was determined to be ca . 500 - fold compared to the free component ons . [SEP]
[CLS] regulation of protein B-material expression by an antisense on that targeted human epidermal growth B-material factor I-material receptor I-material 2 ( her2 ) mrna transcripts B-event was also demonstrated . [SEP]
[CLS] our interest in molecularly defined snas is to apply them as delivery vehicles B-material together with the covalent conjugation strategy . [SEP]
[CLS] the rationale of this idea is that radial formulation could be a simple option to emphasize the ligand - specific effect on the outer sphere of the snas and at the same time hide the unfavorable distribution properties of negatively charged ons . [SEP]
[CLS] for the monitoring of cellular uptake and biodistribution of these decorated snas , appropriate labeling is needed , which may cause a misleading distribution and cellular delivery of the actual structure ( if all arms are labeled ) . [SEP]
[CLS] therefore , to keep the label effect minimal , an established method that allows controlled monofunctionalization of the snas is valuable . [SEP]
[CLS] furthermore , the controlled monofunctionalization can be utilized to integrate snas specifically with other delivery vehicles B-material . [SEP]
[CLS] in the present study , an azidefunctionalized 12 - armed buckminster fullerene B-nanoparticle ( 1 ) is exposed to a substoichiometric amount of cyclooctyne - modified and - labeled ( dota and alexa 488 ) ons , which gave the monofunctionalized fullerene B-nanoparticle in relatively high yields . [SEP]
[CLS] the isolated intermediate B-property products were then exposed to an excess of slightly different on constituents in an aqueous medium , which gave the monolabeled full - armed snas . [SEP]
[CLS] this two - step process was noticed to be crucial , not only for the controlled assembly but also for the preparation of the c 60 - based snas in more general , as the solubility B-property properties of the lipophilic B-property c 60 core B-material and hydrophilic B-property ons severely retard the full decoration in one reaction medium only . [SEP]
[CLS] reverse - phase high - performance liquid B-technique chromatography I-technique ( rp - hplc ) , native polyacrylamide B-technique gel I-technique electrophoresis I-technique ( page ) , capillary B-technique electrophoresis I-technique ( ce ) , ms spectroscopy B-technique , dynamic B-technique light I-technique scattering I-technique ( dls ) , and size exclusion chromatography B-technique equipped with a multiple - angle light - scattering detector ( sec - mals ) were used to analyze the end products . [SEP]
[CLS] we also paid extra attention to the homogeneity and authenticity of the initial c 60 - azide scaffold ( 1 ) as it is readily contaminated by a hardly distinguishable azide - c 60 [ 3 + 2 ] cycloaddition side product that would hamper the assembly , purification , and identification of the target snas . [SEP]
[CLS] overall , this procedure allows a controlled synthesis scheme in which one of the on arms of snas can be selectively functionalized with labels or other conjugate groups . [SEP]
[CLS] in one case , d - galactose - conjugated ons were used to decorate the outer sphere . [SEP]
[CLS] in addition , radiolabeling of a dota - labeled sna has been demonstrated . [SEP]
[CLS] synthesis and purification of the c 60 - azide core B-material ( 1 ) . [SEP]
[CLS] the c 60 - azide scaffold ( 1 ) was synthesized following a previously published procedure ( scheme 1 ) . [SEP]
[CLS] however , in our hands , bingel ' s cyclopropanation between buckminster fullerene B-nanoparticle ( c 60 ) and bis ( 2 - ( 2 - ( 2 - ( 2 - azidoethoxy ) ethoxy ) ethoxy ) ethyl ) malonate gave a mixture of compounds ( 1 and 2 , ca . 1 : 1 , n / n ) with equal molecular masses and similar nmr data ( 2 with markedly broader resonances , a vs b in scheme 1 ) . [SEP]
[CLS] repeated column chromatography B-technique and rp - hplc purification were needed to obtain the homogenized 1 in 15 % overall isolated yield , which was used for the preparation of snas . [SEP]
[CLS] in order to provide further understanding of the products ' identity and applicability for the snas ' assembly , preliminary spaac trials were carried out : both 1 and 2 were exposed to an excess of bicyclo [ 6 . 1 . 0 ] non - 4 - yn - 9 - ylmethanoland 5 ′ - 2 - ( bicyclo [ 6 . 1 . 0 ] non - 4 - yn - 9 - yl ) ethylphosphate ( bcn ) modified t 6 sequence ( on1 , following the two - step process in scheme 2 ) . [SEP]
[CLS] ms ( esi - tof ) analysis verified that all 12 arms of 1 could be readily functionalized , but reactions with 2 stacked to undecafunctionalized products ( figures s6 and s7 ) . [SEP]
[CLS] h− 15 n heteronuclear multiple bond correlation ( hmbc ) analysis was used to further verify the authenticity of 2 , which revealed that part of the nitrogen B-material signals was characteristic to triazol and not entirely to alkylazide ( figures s8 and s9 ) . the fact that the [ 3 + 2 ] cycloaddition occurred upon bingel ' s cyclopropanation is understandable , as this reaction has been used to functionalize the c 60 core B-material in very similar conditions . [SEP]
[CLS] synthesis of oligonucleotides . [SEP]
[CLS] for the assembly of snas ( s1−s6 ) , bcn - modified ons ( on1−on3 , on5 , and on7−on10 , scheme 2 ) were synthesized using an automated dna / rna synthesizer . [SEP]
[CLS] a standard phosphoramidite coupling cycle and commercially available 2 ′ - o - methylribonucleotide and 2 ′ - deoxyribonucleotide building blocks were used for the assembly . [SEP]
[CLS] 3 - phenyl 1 , 2 , 4 - dithiazoline - 5 - one ( pos ) was used as a sulfurization B-event reagent for the synthesis of on5 . [SEP]
[CLS] our previously reported customized solid supports [SEP]
[CLS] were utilized for the synthesis of appropriately 3 ′ - modified ons : on7 and on10 with a 1 , 4 , 7 , 10 - tetraazacyclododecane - 1 , 4 , 7 , 10 - tetraacetic acid ( dota ) and a d - galactose moiety , respectively . [SEP]
[CLS] while on1 and on2 are short sequences , on4 ) is an antisense sequence that targets her2 mrna transcripts B-event . [SEP]
[CLS] its biological activity in sna formulation has previously been demonstrated . [SEP]
[CLS] the phosphorothioate ( ps ) sequence of on5 ( and on6 ) is a splice switching on that prevents expression of an androgenic receptor B-material variant ( ar - v7 ) in prostate cancer cells B-material . [SEP]
[CLS] the 2 ′ - o - methylated sequence found in on7 , on9 , and on10 is complementary to micro - rna 15b that is involved in hepatocyte apoptosis B-event . [SEP]
[CLS] we have previously 68 ga labeled this same on and its glycoconjugates and studied their biodistribution by in vivo positron B-technique emission I-technique tomography I-technique / I-technique computed I-technique tomography I-technique ( pet / ct ) imaging . [SEP]
[CLS] controlled assembly of monofunctionalized snas on the c 60 - azide core B-material . [SEP]
[CLS] in initial trials , the c 60 - azide core B-material 1 was dissolved in a minimum volume of dmso and treated with an excess ( > 12 equiv ) of bcn - modified oligonucleotides in an aqueous solution containing 1 . 5 m nacl . [SEP]
[CLS] however , the drastically different solubility B-property properties between the lipophilic B-property c 60 - azide core B-material ( 1 ) and the hydrophilic B-property ons retarded the spaac conjugation , and complex mixtures of products were obtained ( reactions using different dmso−h 2 o ratios , different spacers B-material between the ons and the core B-material , bcn - vs dibenzocyclooctyne - modified ons , and different temperature were attempted ) . [SEP]
[CLS] this guided us to try a two - step process in which 1 was first conjugated with ons in dmso , and once the partially functionalized more hydrophilic B-property intermediate B-property products were obtained , the reaction was changed to an aqueous medium to yield full - armed snas . [SEP]
[CLS] interestingly , monofunctionalization proceeded in dmso ( figure 1a , scheme s2 ) with a reasonable excess of 1 , which could be utilized for the controlled assembly of heteroantennary snas ( s1 - - s6 , scheme 2 ) . [SEP]
[CLS] in optimized conditions ( scheme 2 ) , bcn - modified ons ( on1−3 , on5 , and on7 ) were treated with 5 equiv of 1 in dmso to yield monofunctionalized c 60 − on conjugates ( c1−c3 , c5 , and c7 ) in relatively high rp - hplc - isolated yields ( 45−50 % ) . [SEP]
[CLS] the remaining excess of 1 could be reisolated ( cf . figure 1a ) and reused . [SEP]
[CLS] the aminomodified conjugates ( c3 and c5 ) were labeled with alexa - 488 - n - hydroxysuccinimide ( nhs ) ester , and the conjugates ( c1 , c2 , c4 , c6 , and c7 ) were then dissolved in aqueous 1 . 5 m nacl solution and mixed with a slight excess ( 12 equiv ) of bcn−ons ( on1 , on5 , and on8−on10 ) . [SEP]
[CLS] after incubation B-technique for 2 days at room temperature , one additional equivalent of bcn−ons was added to confirm the completion of the full decoration ( in overall 1 . 2 equiv / arm ) . [SEP]
[CLS] the reaction mixtures were then incubated B-technique one more day ( 3 days total ) and subjected as such to rp - hplc ( figure 1c , scheme s2 ) . [SEP]
[CLS] the obtained snas ( s1−s6 ) were isolated in 40−57 % yield according to uv absorbance at 260 nm ( s1 , s2 , s5 , and s6 ) and 488 nm ( s3 and s4 ) . [SEP]
[CLS] characterization of the snas . [SEP]
[CLS] the homogeneity and identity of the rp - hplc - isolated snas were evaluated by page , ce , sec - mals , and ms - esi spectroscopy B-technique . [SEP]
[CLS] as seen on page ( figure 2b ) , each sna resulted in a distinct and relatively sharp band . [SEP]
[CLS] the stereoisomeric phosphorothioate backbone of s4 caused a blurred band compared to other snas . [SEP]
[CLS] on the electrophoregrams of larger snas ( s5 and s6 ) , faster eluting trace products ( < 5 % of the total intensity ) could be additionally observed that indicated incomplete decoration . [SEP]
[CLS] fractionation by rp - hplc ( figure 1c ) did not affect the results on page , neither did the prolonged reaction time nor a higher excess of bcn - ons ( on9 and on10 ) used for the spaac conjugation . [SEP]
[CLS] despite these traces of side products ( most likely 11 - armed snas ) , the overall purity of the assembled snas after single rp - hplc purification proved relatively high on page . [SEP]
[CLS] next , the applicability of ce to evaluate the homogeneity of the snas was demonstrated ( figure 2a and scheme s2 ) . [SEP]
[CLS] ce could not discriminate traces of incomplete ( 11 - armed ) products from the full - armed snas , but it proved to be a valuable tool to confirm the absence of smaller component ons . [SEP]
[CLS] sec - mals is a common technique employed to estimate the homogeneity , aggregation tendency , and molecular weight of biomolecules . [SEP]
[CLS] 36 this is particularly useful for large molecular weight compounds ( > 100 kda ) , characterization of which is often hardly accessible by ms spectroscopy B-technique . [SEP]
[CLS] this was the case also with snas . [SEP]
[CLS] acceptable m / z data ( a spectrometer equipped with a hybrid quadrupole orbitrap and nano esi ionization B-property was used ) could be obtained for small model snas s1 and s2 ( figure s12 ) , whereas humps of overlapping multiply charged ion B-material patterns , unsuitable for reliable ms characterization , were obtained for s3−s6 . [SEP]
[CLS] in fact , even s1 and s2 were prone to form stable multiple sodium B-material adducts ( figure s12 ) , and the observed molecular masses were 0 . 1 and 0 . 2 kda higher compared to the calculated values ( table 1 , entries 1 and 2 ) . [SEP]
[CLS] therefore , we applied sec - mals to estimate the molecular weights of s3−s5 . [SEP]
[CLS] the samples of s3−s5 were eluted with 150 mm phosphate buffer ( ph 7 ) through a 300 a , 2 . 7 μm , 4 . 6 × 300 mm sec column . [SEP]
[CLS] as seen in figure 2c and figure s12 , each sna resulted in a major peak ( retention time ca . 7 min ) that represented the 70−80 % mass fraction of the sample . [SEP]
[CLS] the mals - based estimation of the molecular weights extracted from the major peaks matched relatively well with the expected calculated values ( table 1 , entries 3−6 ) . [SEP]
[CLS] it may be worth mentioning that the errors of the observed molecular masses were less than the molecular mass of the component ons . [SEP]
[CLS] in each case , faster eluting fractions ( retention time ca . 5 . 0−6 . 5 min ) were observed also , the molecular weight of which ( > 200 kda ) may be attributed to aggregation of the snas ( figure 2c and figure s12 ) . [SEP]
[CLS] together with the molecular masses obtained for small model snas s1 and s2 and the sec - mals - based characterization of snas s3−s6 , the authenticity of the products could be verified . [SEP]
[CLS] finally , the hydrodynamic diameter of the snas was determined by dls . [SEP]
[CLS] the diameters ( ranging from 9 . 2 to 21 . 4 nm , table 1 ) of s1−s6 correlated with the lengths of the component ons . [SEP]
[CLS] melting analysis ( t m ) and titration of sna with a complementary rna strand . [SEP]
[CLS] in order to evaluate the hybridization properties of the c 60 - based snas , uv - melting profile experiments ( t m ) and titration of s4 with a short model sequence of ar - v7 pre - mrna were carried out . [SEP]
[CLS] the s4− rna duplex resulted in a −3 °c decrease in the t m value when compared to the corresponding free duplex ( figure 3a ) . [SEP]
[CLS] furthermore , gentler melting profiles were observed , which was more obvious , when fully hybridized s4 was compared to a partially hybridized one ( 12 vs 6 equiv of complementary strands ) . [SEP]
[CLS] this indicates electrostatic repulsion / steric crowding between the duplexes on fully loaded sna ( s4 + 12 equiv of rna ) . [SEP]
[CLS] this observation is consistent with the previous findings in which a retarded loading of sirnas onto snas has been observed . 12 , 37−39 however , it is notable that the fully loaded sna ( s4 + 12 equiv of rna ) was virtually stable below the physiological temperature and the observed " ' premature ' " partial denaturation occurred at a higher temperature . [SEP]
[CLS] titration of s4 with the same complementary rna verified the correct stoichiometry of the melting profiles ( figure 3b ; note , the concentration of s4 was determined according to the alexa content ) . [SEP]
[CLS] gradual addition of the complementary rna strand increased the overall absorbance with a constant slope ( hypochromic effect due to the hybridization compensates for the increased absorbance ) , and a turning point of the slope was observed once the amount of the complementary rna exceeded the fully occupied sna . [SEP]
[CLS] the observed turning point at 11 . 9 equivalents matched well with the correct 12armed sna structure . [SEP]
[CLS] radiolabeling . [SEP]
[CLS] to evaluate the applicability of dota as a 68 ga - chelating agent on snas , s5 was used as a model in s12 ) . [SEP]
[CLS] b the values were obtained by sec - mals - based estimation of the molecular mass ( cf . figure 2c ) . [SEP]
[CLS] preliminary radiolabeling experiments . [SEP]
[CLS] with these small - scale trials ( 1−2 nmol of s5 ) we also tried to find conditions that minimize precursor loading at the expense of yield . [SEP]
[CLS] radiochemical B-property yields of up to 68 mbq were achieved ( 24 % decay corrected yield ) . [SEP]
[CLS] radiochemical B-property purity was measured at up to 69 % as measured by page and 73 % by ultrafiltration ( figure 4 ) . [SEP]
[CLS] we observed that size exclusion purification of the reaction mixture could not sufficiently remove all unbound 68 ga , even when two successive column purifications were used . [SEP]
[CLS] also , commonly used solid - phase extraction columns with c8 , c18 , and hydrophobic lipophilic balance ( hlb ) solid phases were attempted . [SEP]
[CLS] due to the difficulty of separating unbound 68 ga and the low performance of the size exclusion purification , we suspected unspecific binding of 68 ga to the sna structure . [SEP]
[CLS] this was confirmed by incubating B-technique the end product ( s5 [ 68 ga ] ) in 50 mm ethylenediaminetetraacetic acid / phosphate - buffered saline ( edta / pbs ) at ph 7 . 4 in 37 °c . [SEP]
[CLS] as expected , we observed a further 10 % increase in the unbound activity fraction after 1 h edta challenge , as measured by ultrafiltration . [SEP]
[CLS] this relatively stable unspecific 68 ga binding to the densely packed on construct is in agreement with the behavior of the snas in ms in which relatively stable multiple sodium B-material adducts were observed ( table 1 , entries 1 and 2 ) . [SEP]
[CLS] although the small - scale ( 1−2 nmol of precursor s5 loading ) experiments did not produce acceptable purities , valuable information was obtained , which will aid future in vivo pet / ct imaging studies of snas . [SEP]
[CLS] due to the unspecific 68 ga binding observed , an indirect labeling method , such as a click reaction with a reactive agent prelabeled with either 68 ga or 18 f , may prove a more suitable choice for snas . [SEP]
[CLS] ■ conclusion [SEP]
[CLS] a two - step procedure that allows controlled assembly of monofunctionalized c 60 - based snas has been described . [SEP]
[CLS] this preliminary data opens the door for controlled decoration of snas with labels or tissue / organ - specific ligands and possibility integrating them specifically to other delivery vehicles B-material . [SEP]
[CLS] despite the multicomponent spaac - based assembly and two rp - hplc purifications , the overall yield of these snas proved to be relatively high , ca . 20−30 % . [SEP]
[CLS] we also paid extra attention to the homogeneity of the c 60 - azide scaffold ( 1 ) . [SEP]
[CLS] 1 was noticed to be readily contaminated by a hardly distinguishable azide - c 60 [ 3 + 2 ] cycloaddition side product ( 2 ) that would hamper the assembly , purification , and identification of the snas . [SEP]
[CLS] the homogeneity and authenticity of the snas were evaluated by various methods , including rp - hplc , native page , ce , sec - mals , and ms - esi spectroscopy B-technique . [SEP]
[CLS] while page was a superior technique to evaluate the homogeneity of these c 60 - based snas , sec - mals could be used for rough evaluation of the molecular weights . [SEP]
[CLS] preliminary radiolabeling experiments suggested that the chelation - based techniques should be replaced by a covalent radiolabeling strategy to prevent unspecific metal B-material ion B-material ( 68 ga ) binding to the densely packed on shell B-material of these nanoparticles B-nanoparticle . [SEP]
[CLS] the described procedure with detailed analytical control of each single step will expectedly promote design of new molecularly defined monofunctionalized snas , which may find interesting therapeutic and diagnostic applications . [SEP]
[CLS] synthesis and purification of the c 60 - azide core B-material ( 1 ) . [SEP]
[CLS] 1 was synthesized by bingel cyclopropanation following the previously published procedure : buckminster fullerene B-nanoparticle c 60 ( 0 . 40 g , 0 . 56 mmol ) was dissolved in dry and degassed ( oxygen B-material removed by bubbling with argon B-material ) o - dichlorobenzene ( 140 ml ) . bis ( 2 - ( 2 - ( 2 - ( 2 - azidoethoxy ) ethoxy ) ethoxy ) ethyl ) malonate ( 2 . 8 g , 5 . 6 mmol , 10 equiv ) , cbr 4 ( 19 g , 56 mmol , 100 equiv ) , and 1 , 8 - diazabicyclo [ 5 , 4 , 0 ] undec - 7 - ene ( dbu , 1 . 7 ml , 11 mmol , 20 equiv ) were added , and the mixture was stirred 3 days at room temperature under argon B-material . [SEP]
[CLS] the mixture was purified by silica gel column chromatography B-technique ( pore size 60 a , 230−400 mesh particle size , 40−63 μm particle size , isocratic elution with 2 % meoh in dichloromethane , twice ) to yield the desired product 1 ( 0 . 66 g , 32 % ) and a near identically behaving side product 2 ( 0 . 65 g , 31 % , for further characterization , see figures s2−s9 ) . [SEP]
[CLS] a sample ( 30 mg ) of columnpurified 1 was further purified by rp - hplc to yield homogenized 1 ( 7 mg , 23 % , overall yield 15 % ) , which was used for the preparation of snas . [SEP]
[CLS] slight differences in yields between the different conjugates observed ) of c1−c7 were determined by uv absorbance at 260 nm . [SEP]
[CLS] synthesis of alexa - 488 - labeled conjugates c4 and c6 . [SEP]
[CLS] af488 nhs ester ( 0 . 4 μmol in 6 μl of dmso ) was added to a buffered mixture of c 60 −on conjugate ( 50 nmol of c3 or c5 in 60 μl of 0 . 1 m sodium B-material borate , ph 8 . 5 ) . [SEP]
[CLS] the reaction mixture was gently shaken overnight at room temperature and subjected to rp - hplc . [SEP]
[CLS] an analytical rp - hplc column ( 250 × 4 . 6 mm , 5 μm ) , a gradient elution from 40 % to 100 % mecn in 50 mmol l −1 triethylammonium acetate over 30 min , and detection at 260 nm were used . [SEP]
[CLS] the product fractions were collected and lyophilized to dryness . [SEP]
[CLS] the authenticity of the products ( c4 and c6 ) was verified by ms ( esi - tof ) ( figure s11 ) . [SEP]
[CLS] isolated yields ( c4 , 32 % ; c6 , 62 % ) of the products were determined by uv absorbance at 488 nm . [SEP]
[CLS] assembly of snas ( s1−s6 ) . [SEP]
[CLS] general procedure : c 60 −on conjugate ( c1−c7 , 20 nmol in 55 μl of h 2 o ) was mixed with bcn−on ( on1 , on3 , on5 , and on8−on10 , 240 nmol in 145 μl of h 2 o ) , and 100 μl of 4 . 65 m nacl was added . [SEP]
[CLS] the reaction mixture was gently shaken for 2 days at room temperature , and then one additional equivalent of bcn−on was added . [SEP]
[CLS] the mixture was then incubated B-technique one more day ( 72 h total using 1 . 2 equiv of bcn−on / azide arm ) and subjected to rp - hplc . [SEP]
[CLS] an analytical rp - hplc column phenomenex , aeris 3 . 6 μm widepore xb - c18 200 a , 150 × 4 . 6 mm , a linear gradient from 5 % to 60 % mecn in 50 mmol l −1 triethylammonium acetate over 40 min , a flow rate of 1 . 0 ml min −1 , and detection at 260 nm were used for purification ( figure 1c and scheme s2 ) . [SEP]
[CLS] the product ( s1−s6 ) fractions were collected and lyophilized to dryness . [SEP]
[CLS] isolated yields ( 40− 57 % ) of the products were determined by uv absorbance at 260 nm ( s1 , s2 , s5 , and s6 ) and 488 nm ( s3 and s4 ) . [SEP]
[CLS] the obtained snas were characterized by ms - esi ( equipped with a hybrid quadrupole orbitrap and nano - esi ionization B-property ) ( s1 and s2 ) and sec - mals ( s3−s6 ) ( table 1 , figure 2c , and figure s12 ) . [SEP]
[CLS] for the homogeneity and particle size evaluation of s1−s6 , see the page ( figure 2b ) , ce ( figure 2a and scheme s2 ) , sec - mals ( figure 2c , scheme s2 ) , and dls experiments described below . [SEP]
[CLS] page analysis of snas . [SEP]
[CLS] native 6 % tris base , boric acid , edta , and acrylamide ( tbe ) gel were used to check snas ' purity . [SEP]
[CLS] a precast gel cover ( 10 cm × 10 cm in size , thermo fisher scientific ) was fixed into a vertical electrophoresis B-technique chamber , and the running buffer ( 90 mm tris , 90 mm borate , and 2 mm edta , 8 . 3 ph ) was filled into the chamber . [SEP]
[CLS] sna samples ( 5 μl of 0 . 1 μm snas mixed with 5 μl of tbe sample buffer ) and a dna ladder ( 100 , 200 • • • 1000 bp ; note , the ladder is just used to confirm the quality and comparability of the runs and cannot be used for size evaluation of the snas ) were loaded and electrophoresed at constant 200 v ( 45 ma ) for approximately 30 min . [SEP]
[CLS] after completion of electrophoresis B-technique , gel was removed from the chamber and the sna bands were monitored either directly by uv or after staining by sybrtm gold B-material nucleic B-material acid I-material stain ( thermo fisher scientific ) . [SEP]
[CLS] capillary B-technique electrophoresis I-technique experiments . [SEP]
[CLS] samples were analyzed by capillary zone electrophoresis B-technique in a fused silica capillary of 75 μm i . d . and 57 cm effective length . [SEP]
[CLS] the background electrolyte was 0 . 3 m citrate buffer , ph 3 . 1 . [SEP]
[CLS] a voltage of 15 kv and pressure of 0 . 3 psi were applied . [SEP]
[CLS] uv detection at λ = 260 nm was used . [SEP]
[CLS] sec - mals experiments . [SEP]
[CLS] sec - mals was performed using an agilent technologies 1260 infinity ii hplc system ( sampler , pump , and uv−vis detector ) equipped with a wyatt technologies minidawn light scattering detector and wyatt technologies optilab refractive B-property index I-property detector . [SEP]
[CLS] an agilent advancebio sec 300 a 2 . 7 μm 4 . 6 × 300 mm column and 150 mm sodium B-material phosphate , ph 7 . 0 , as mobile B-property phase eluting at a rate of 0 . 2 ml min −1 and run time of 20 min were used for each experiment . [SEP]
[CLS] for each run , 4 μl of sample with a sna concentration of 1 mg ml −1 in milli - q water B-material was loaded onto the pre - equilibrated column . [SEP]
[CLS] detector signals were aligned with a bovine serum albumin ( bsa ) standard , which was analyzed prior to sna samples . [SEP]
[CLS] the ri and mals signals were used for the mw calculations using an average refractive B-property index I-property increment ( dn / dc ) of 0 . 1703 ml / g . [SEP]
[CLS] melting experiments ( t m ) and titration of s4 with a complementary rna . [SEP]
[CLS] the melting curves ( absorbance vs temperature ) were measured at 260 nm on a uv−vis spectrometer equipped with a multiple cell B-material holder and a peltier temperature controller . [SEP]
[CLS] the temperature was changed at a rate of 0 . 5 °c min −1 between 10 and 80 °c . [SEP]
[CLS] the measurements were performed in 10 mmol l −1 sodium B-material cacodylate ( ph 7 . 0 ) with 0 . 1 mol l −1 nacl and 1 . 0 μmol l −1 on . [SEP]
[CLS] t m values were determined as the maximum of the first derivative of the melting curve . [SEP]
[CLS] the uv titration of s4 ( absorbance vs equivalents of complementary rna oligonucleotide added ) was performed in 10 mmol l −1 sodium B-material cacodylate ( ph 7 . 0 ) with 0 . 1 μmol l −1 nacl and 63 nmol l −1 s4 . [SEP]
[CLS] rna oligonucleotide with a complementary sequence to s4 was added gradually in the solution , and the total absorbance at 260 nm was monitored with an uv−vis spectrometer . [SEP]
[CLS] the sequence of complementary rna was c aau guc ucu cuu uca uac uag . [SEP]
[CLS] for the formation of free duplex , cua gua uga aag aga gac auu g ( 2 ′ - omethyl rna phosphorothioate ) was used . [SEP]
[CLS] dls experiments . [SEP]
[CLS] the size of the snas was measured at room temperature using a zetasizer nano zs90 ( malvern instruments ltd . , uk ) . [SEP]
[CLS] the settings and conditions for the measurements were as follows : material B-material protein B-material ( ri , 1 . 450 ; absorption , 0 . 001 ) , dispersant B-property water B-material ( viscosity B-property , 0 , 8872 cp ; ri , 1 . 330 ) temperature was 20 °c , and equilibration time was 60 s . [SEP]
[CLS] each sample ( 10 μg of sna in 100 μl of aqueous 10 mmol l −1 pbs , 2 . 7 mmol l −1 m kcl , 0 . 137 mol l −1 nacl , ph 7 . 4 ) was measured three times . [SEP]
[CLS] radiolabeling experiments . [SEP]
[CLS] [ 68 ga ] gacl 3 was eluted from an igg - 100 68 ge / 68 ga generator with 0 . 1 m hcl through a strata scx cartridge into a waste container . [SEP]
[CLS] the cartridge was then eluted with 300 μl of 1 . 0 m sodium B-material chloride / 0 . 1 m hcl solution , and an aliquot ( 200 μl ) was transferred to a reaction vial preloaded with a mixture of hepes ( 12 mg ) and s5 ( 1−2 nmol ) in 50 μl of water B-material . [SEP]
[CLS] gentisic acid ( 10 μl , 0 . 1 m in water B-material ) was added as a radical scavenger to counter possible radiolysis . [SEP]
[CLS] the reaction mixture was then incubated B-technique at 70 °c for 15 min . [SEP]
[CLS] the mixture was cooled on ice after incubation B-technique and purified using consecutive illustra nap - 5 and nap - 10 size - exclusion columns ( cytiva , usa ) equilibrated with phosphate - buffered saline ( pbs , ph 7 . 4 ) . [SEP]
[CLS] this afforded the end product formulation in 1 . 2 ml of phosphate - buffered saline ( pbs ) . [SEP]
[CLS] radiochemical B-property purity was determined by native page and ultrafiltration . [SEP]
[CLS] page was carried out with tbe - buffered 6 % polyacrylamide gels in a biorad miniprotean ii system ( bio - rad laboratories , hercules , ca , usa ) ran at 250 v . the gels were developed on bas - bioconjugate chemistry tr2025 phosphor imaging plates and analyzed with a bas - 5000 scanner ( fuji , tokyo , japan ) . [SEP]
[CLS] ultrafiltration was done in triplicate by loading 0 . 5 ml , 30 kda microcon filters ( millipore , bedford , ma , usa ) , with 100 μl of pbs and 1 μl of reaction mixture . [SEP]
[CLS] the filters were centrifuged three times for 5 min at 14 000g , with addition of 100 μl of pbs between spins . [SEP]
[CLS] the activities of the filter and the filtrate were measured with a 1480 wizard gamma counter ( perkinelmer / wallac , turku , finland ) , and the purity was calculated by dividing the filter activity by the total activity . [SEP]
[CLS] * sı supporting information [SEP]
[CLS] the supporting information is available free of charge at https : / / pubs . acs . org / doi / 10 . 1021 / acs . bioconjchem . 1c00187 . [SEP]
[CLS] for further evidence , ad hoc - synthesized triazolino fullerenes B-nanoparticle were synthesized by treating c 60 with 2 - ( 2 - ( 2 - ( 2 - azidoethoxy ) ethoxy ) ethoxy ) ethanol . [SEP]
[CLS] the nmr signals of the triazolino fullerenes B-nanoparticle ( di - , tri - , and tetrafunctionalized products obtained , scheme s1 ) were comparable to trace signals of 2 . [SEP]
[CLS] 1 h nmr ( 500 mhz , cdcl 3 ) spectrum of 1 ( a ) and 2 ( b ) a [SEP]
[CLS] 2 a [SEP]
[CLS] ( a and c ) examples of rp - hplc profiles of crude product ( c7 and s5 ) mixtures . [SEP]
[CLS] ( b ) example of the ms - esi spectrum of purified monosubstituted c 60 - on - conjugate ( c7 ) . [SEP]
[CLS] ( d ) example of the rp - hplc profile of purified sna ( s5 ) . [SEP]
[CLS] ( a ) example of capillary electrophoregrams ( ce ) of purified sna ( s5 ) . [SEP]
[CLS] ( b ) polyacrylamide gel electrophoregrams ( page ) of purified snas ( s1−s6 ) . [SEP]
[CLS] ( c ) example of the sec - mals profile of purified sna ( s5 ) used to evaluate the molecular mass . [SEP]
[CLS] for the conditions , see experimental section . [SEP]
[CLS] melting profile analysis and titration of s4 with complementary rna : c aau guc ucu cuu uca uac uag . for the formation of free duplex cua gua uga aag aga gac auu g ( 2 ′ - o - methyl rna phosphorothioate ) was used . [SEP]
[CLS] 1 : 1 h nmr ( 500 mhz , cdcl 3 ) δ 4 . 43 ( t , 24h , j = 4 . 1 hz ) , 3 . 75 ( t , 24h , j = 4 . 1 hz ) , 3 . 69 ( t , 24h , j = 4 . 3 hz ) , 3 . 67 ( s , 48h ) , 3 . 64 ( s , 48h ) , 3 . 40 ( t , 24h , j = 4 . 2 hz ) ; 13 c nmr ( 125 mhz , cdcl 3 ) δ 163 . 5 , 145 . 8 , 141 . 0 , 70 . 7 , 70 . 0 , 69 . 0 , 68 . 6 , 65 . 8 , 50 . 7 , 45 . 2 ; ms ( esi - tof ) molecular mass for c 174 h 192 n 36 o 60 na 3 3816 . 6 , found 3816 . 4 ( calculated from [ ( m + 3na ) / 3 ] 3 + [SEP]
[CLS] synthesis of c 60 - on conjugates c1−c3 , c5 , and c7 . [SEP]
[CLS] general procedure : bcn - modified oligonucleotide ( on1−3 , on5 , or on7 , 0 . 1 μmol in 60 μl of h 2 o ) was slowly added to a mixture of c 60 core B-material 1 ( 0 . 5 μmol in 540 μl of dmso ) in a microcentrifuge tube . [SEP]
[CLS] the reaction mixture was gently shaken overnight at room temperature and subjected to rp - hplc ( figure 1a and scheme s2 ) . [SEP]
[CLS] an analytical rp - hplc column ( 250 × 4 . 6 mm , 5 μm ) , a gradient elution from 40 % to 100 % mecn in 50 mmol l −1 triethylammonium acetate over 30 min , and detection at 260 nm were applied . [SEP]
[CLS] the c 60 −on conjugate ( c1−c7 ) and unreacted fullerene B-nanoparticle core B-material 1 were collected individually and lyophilized to dryness . [SEP]
[CLS] the authenticity of the products was verified by ms ( esi - tof ) ( figure 1b and figure s11 ) . [SEP]
[CLS] isolated yields ( 45−50 % , only [SEP]
[CLS] 4 . representative polyacrylamide B-technique gel I-technique electrophoresis I-technique ( page ) autoradiography image and quantification of radiolabeled sna s5 [ 68 ga ] . [SEP]
[CLS] further characterization data of 1and 2 , monosubstituted c 60 - conjugates c1−c7 , and snas ( s1−s6 ) ( pdf ) ■ author information corresponding author pasi virta − department of chemistry , university of turku , fi - 20014 turku , finland ; department of biologics , orion pharma , 20101 turku , finland ; orcid . org / 0000 - 0002 - 6218 - 2212 ; [SEP]
[CLS] phone : + 358 503285719 ; email : pamavi @ utu . fi [SEP]
[CLS] molecular masses of snas s1−s6 [SEP]
[CLS] in 2015 , he defended a ph . d . thesis on the synthesis of regular polymeric biomaterials using microfluidics at [SEP]
[CLS] during the course of human history , the evolution of diseases has brought about a constantly rising demand for novel effective medical treatments . [SEP]
[CLS] scientists working in different fields have made a huge effort to pioneer brand - new strategies useful for fighting against severe medical conditions and developing new biomedical therapies . [SEP]
[CLS] material B-material scientists , supported by biotechnologists and medical doctors , have focused on acquiring a thorough understanding of molecular interactions in the human body to create new biomedical " weapons " . [SEP]
[CLS] their activities have significantly stimulated the design and synthesis of bioactive molecules with innovative functionalities . [SEP]
[CLS] from a historical perspective , drug research has concentrated on small molecules , that is , all the bioactive molecules with a molecular weight lower than 900 [SEP]
[CLS] in recent years , the main quest of science has been the pioneering of the groundbreaking biomedical strategies needed for achieving a personalized medicine . [SEP]
[CLS] ribonucleic B-material acids I-material ( rnas ) are outstanding bioactive macromolecules identified as pivotal actors in regulating a wide range of biochemical pathways . [SEP]
[CLS] the ability to intimately control the cell B-material fate and tissue activities makes rna - based drugs the most fascinating family of bioactive agents . [SEP]
[CLS] however , achieving a widespread application of rna therapeutics in humans is still a challenging feat , due to both the instability of naked rna and the presence of biological barriers B-property aimed at hindering the entrance of rna into cells B-material . [SEP]
[CLS] recently , material B-material scientists ' enormous efforts have led to the development of various classes of nanostructured carriers customized to overcome these limitations . [SEP]
[CLS] this work systematically reviews the current advances in developing the next generation of drugs based on nanotechnology - assisted rna delivery . [SEP]
[CLS] the features of the most used rna molecules are presented , together with the development strategies and properties of nanostructured vehicles B-material . [SEP]
[CLS] also provided is an in - depth overview of various therapeutic applications of the presented systems , including coronavirus disease vaccines and the newest trends in the field . [SEP]
[CLS] lastly , emerging challenges and future perspectives for nanotechnologymediated rna therapies are discussed . [SEP]
[CLS] daltons , which can modify biochemical processes and prevent , identify , or cure diseases . [SEP]
[CLS] such molecules are easily synthesizable with well - optimized chemical processes , scalable from the laboratory to the manufacturing level . [SEP]
[CLS] however , due to their simple working mechanism and low specificity , they can be less than efficient in reaching the desired effects . [SEP]
[CLS] in addition , the effectiveness of these bioactive molecules has been greatly hampered by the resistance developed by cancer cells B-material and pathogens . [SEP]
[CLS] over the past decades , a new class of bioactive molecules , called biological drugs , has achieved an increasingly important role in fighting human body diseases . [SEP]
[CLS] the food and drug administration ( fda ) has defined biological drugs as large , complex molecules derived from living cells B-material or biological processes which are used to diagnose , prevent , treat , and cure a broad spectrum of diseases and medical conditions . [SEP]
[CLS] biologics comprise a wide range of substances , including carbohydrates B-material , proteins B-material , nucleic B-material acids I-material , and elaborated composites of these substances . [SEP]
[CLS] unlike small molecules , biological drugs have sophisticated structures , from a few hundred to more than one thousand times larger than the classic marketed agents . [SEP]
[CLS] they boast exceptional therapeutic properties , with an excellent and unique specificity for targeting a precise biological process . [SEP]
[CLS] compared with small molecules , bioactive molecules are superior in terms of biomedical efficiency , off - target toxicity B-property , and safety for patients , making them ideal candidates for personalized medicine . [SEP]
[CLS] despite all these considerations , small molecules are still leading the drug market due to both the high cost of biologics and the conservative implementation strategy of pharmaceutical companies . [SEP]
[CLS] in any case , the pharmaceutical industry has recognized the possibility of developing new drug - based treatments using biological molecules , employable as vaccines , gene and cellular therapy drugs , hormones , monoclonal antibodies B-material , and recombinant therapeutic protein B-material allergens , cytokines , growth factors , along with others . [SEP]
[CLS] in 2016 and 2017 , small drugs and biological molecules equally split the top ten positions of the best - selling drug list , while the number of approved biological drugs is progressively rising ( figure 1a ) . [SEP]
[CLS] in addition , the advent and application of the fascinating geneediting tool crispr - cas9 and the new , enormous healthcare needs imposed by the coronavirus disease ( covid - 19 ) outbreak are watershed events in the field of drug development , which now lead to a common belief that the next generation of medical treatments will be dominated by biological drugs . [SEP]
[CLS] among biological molecules used as therapeutic agents , ribonucleic B-material acids I-material ( rnas ) stand out thanks to their unique properties and diverse impacts on the biological processes of the human body . [SEP]
[CLS] rna is a family of complex biological molecules made up of linear chains of monomeric nucleotides , playing a fundamental role in different biochemical cellular mechanisms . [SEP]
[CLS] rna was discovered a few decades ago , and in the beginning , it was simply considered as an intermediate B-property product in the genetic information transmission from deoxyribonucleic B-material acid I-material the number of published articles on rna delivery during the period 2001 - 2020 , obtained from web of science database . [SEP]
[CLS] c ) unlike small molecules , rnas cannot overcome the lipid B-material bilayer I-material barrier B-property developed to protect cells B-material from rna entering . [SEP]
[CLS] naked rnas are too large and too charged to pass through the cell B-material membrane ; therefore , they require a delivery agent to invade cells B-material . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2017 , nature publishing group . d ) rna protection from rnase - mediated degradation and capability of rna internalization into the cell B-material are guaranteed by the use of nanoplatforms as rna carriers . [SEP]
[CLS] fulfilling these objectives allows rna molecules to exploit their therapeutic effect , directly influencing biochemical cell B-material processes . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , wiley - vch . [SEP]
[CLS] ( dna ) [SEP]
[CLS] to ribosomes [SEP]
[CLS] over the past decades , various rna roles have been discovered , indicating involvement in almost all biochemical paths . [SEP]
[CLS] these exciting findings have drawn the attention of a large number of scientists toward testing rna as therapeutic molecules , thus triggering a constant increase in outcomes and scientific discoveries in this field ( figure 1b ) . [SEP]
[CLS] all these efforts have also led to the approval of a few rnabased drugs in the past 2 years ( figure 1a ) . [SEP]
[CLS] indeed , rna has gained a central role in pharmacotherapy , achieving a level of complexity and efficiency that would have been unimaginable in the beginning . [SEP]
[CLS] however , the transition from prospective biomolecules to effective therapeutic agents is ambitious and arduous , since the systemic delivery of naked rna molecules to a specific target ( e . g . , cells B-material or tissues ) is extremely challenging . [SEP]
[CLS] naked rna are negatively charged large molecules , and cells B-material have a robust defense system to keep exogenous rnas out of their membranes ( figure 1c ) . [SEP]
[CLS] an additional issue is that some naked rna might trigger an inflammatory response . [SEP]
[CLS] lastly , naked rna is prone to degradation and needs to be protected during the delivery process . [SEP]
[CLS] for these reasons , there has been a rapid development in groundbreaking nanoplatforms for drug delivery applications during the past 5 years , triggering a remarkable progress in rna therapy . [SEP]
[CLS] a vast number of nanostructured vehicles B-material that guarantee the efficient delivery of rna , while protecting their cargo from the threat of the immunological system , have been designed , manufactured , and tested . [SEP]
[CLS] nanotechnology - based systems make it possible for rna to overcome human body barriers B-property and exploit their own biochemical functions into the target ( figure 1d ) . [SEP]
[CLS] the merging of the striking advances in the nanoscience and bioactive molecule discovery fields opens a new era in drug development , which is bringing about a revolution in the pharmacological treatment of a broad range of diseases . [SEP]
[CLS] this paper offers a systematic summarization of the most significant advancements in the field of rna delivery . [SEP]
[CLS] first , we briefly introduce rna - based drugs and their impact on the ongoing revolution in drug development . [SEP]
[CLS] in the second part , we present the essential features of the key rna families , focusing on messenger rna ( mrna ) , small interfering rna ( sirna ) , micro rna ( mirna ) , and short hairpin rna ( shrna ) . [SEP]
[CLS] next , we highlight the recent advances in using nanostructured platforms for delivering rnas to the defined targets , including carbonaceous nanomaterials B-material , inorganic B-nanoparticle nanoparticles I-nanoparticle , polymer B-material nanomaterials B-material , virus - like particles , and lipid B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] in the next section , an in - depth review of the recent progress in applying biomedical treatments based on the delivery of rna drugs is extensively discussed . [SEP]
[CLS] the most advanced rna application methods , including wound healing , treatments of different classes of cancers , various nervous system therapies , and the development of covid - 19 vaccines are reported and enlightened . [SEP]
[CLS] the newest trends , such as the use of both lightactivated nanoplatforms for the on - demand delivery of bioactive molecules and rna for guiding the pluripotent stem cell B-material differentiation and reprogramming , are then illustrated . [SEP]
[CLS] the review concludes with a valuable discussion of future prospects , challenges , and opportunities in designing brand - new healthcare materials and developing innovative therapies based on rna , while emphasizing the enormous potential impact of artificial intelligence ( ai ) in this field . [SEP]
[CLS] this review will provide a comprehensive panorama of rna - based therapies ; most importantly , it will inspire and offer guidance for developing nextgeneration biomedical treatments that combine the unique properties of rna molecules and nanostructured B-material materials I-material . [SEP]
[CLS] gene therapy using rnas is a promising treatment because of its therapeutic effect on several types of diseases . [SEP]
[CLS] it permits the delivery of targeted nucleic B-material acid I-material sequences to edit ( e . g . , downregulate , augment , or correct ) specific genetic anomalies or mutations . [SEP]
[CLS] therapies may be broken down into coding and noncoding rna approaches . [SEP]
[CLS] coding rna therapeutics introduce rna sequences into the host body to stimulate the synthesis of coded - protein B-material antigens , which resemble antigens of the targeted disease . [SEP]
[CLS] this stimulates a specific immunological response in terms of antibody B-material and cytotoxic B-property lymphocyte production . [SEP]
[CLS] noncoding rna approaches aim not to encode a protein B-material , thus silencing one single gene or multiple related genes and inhibiting protein B-material production . [SEP]
[CLS] rna therapies offer enormous advantages , including simplicity of sequence design and synthesis , functional versatility , safety and cost , and the possibility of patient - specific treatments . [SEP]
[CLS] moreover , rnas can initiate the translation of proteins B-material into the cellular cytoplasm without requiring nuclear entry ( as in dna therapies ) , and do not integrate with the host genome , thus ensuring the safety of these treatments . [SEP]
[CLS] in light of this , the development of more accurate and customized therapeutic treatments for various chronic diseases is possible . [SEP]
[CLS] in this section , the main rna therapeutics - including messenger rnas ( mrnas ) , small interfering rnas ( sirnas ) , short hairpin rnas ( shrnas ) , and micro rnas ( mirnas ) are described and discussed . [SEP]
[CLS] mrnas are single - strand structures made of a sequence of nucleotides ( figure 2a ) . [SEP]
[CLS] they participate in the translation of genetic information from genes to proteins B-material . [SEP]
[CLS] mrnas are coding rnas which act as an intermediated B-property agent in the transport of information from the nuclear dna to the cellular cytoplasm , where the ribosomal translation into functional proteins B-material occurs . [SEP]
[CLS] more specifically , mrnas are produced by the gene transcription B-event of complementary dna , resulting in a transcribed genetic sequence . [SEP]
[CLS] the dimensions of mrnas are 300 - 5000 kda , while nucleotide sequences are organized into codons formed by three ribonucleotides . [SEP]
[CLS] codons encode for a specific structural unit of a protein B-material ( i . e . , amino B-material acid I-material ) , resulting in protein B-material synthesis . [SEP]
[CLS] at the end of the process , the mrna is naturally degraded in the cell B-material body . [SEP]
[CLS] as mentioned earlier , mrnas do not have to cross the cellular nuclear barrier B-property to initiate the encoding process , thus offering a high efficacy per dose . [SEP]
[CLS] moreover , the mrna is not inserted into the genome , thus ensuring safety of the treatment . [SEP]
[CLS] another advantage of mrna therapeutic strategies is the fast , cost - effective , and efficient development because of its easy in vitro production . [SEP]
[CLS] the limitations of mrna therapies , however , concern mrna ' s large size , stability , biological activity , and immunogenicity B-property , as well as its translational and delivery efficiency . [SEP]
[CLS] for these reasons , mrna modifications have been studied . [SEP]
[CLS] within this framework , chemical treatments and the architectonic stabilization of mrnas ( e . g . , circularization of mrnas ) can significantly improve the sensitivity to enzymatic degradation and immunity . [SEP]
[CLS] moreover , the introduction of self - replication functions may prolong and extend the protein B-material synthesis and ameliorate the mrna immunogenicity B-property . [SEP]
[CLS] the therapeutic strategies based on mrnas have been explored for several applications , such as protein B-material replacement , vaccine design , cancer immunotherapy , genomic editing , genetic engineering , and regenerative medicine . [SEP]
[CLS] more specifically , cancer immunogenic B-property therapies provide the encoding of tumor B-material antigens by mrnas , in order to promote specific immune figure 2 . [SEP]
[CLS] schematic illustration of mrna - and sirna - based therapies . [SEP]
[CLS] a ) mrna structure and design for engineering vaccines : mrna single - strands are loaded into nanoparticle B-nanoparticle carriers and delivered to the host body , encoding virus antigens and inducing the immunological response by producing antigen - specific antibodies B-material . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , wiley - vch . [SEP]
[CLS] b ) sirna structure and design for cancer therapy : sirna double strands are loaded into nanoparticle B-nanoparticle carriers and delivered into the host body in order to silence the tumor - associated gene and induce cancer cell B-material apoptosis B-event . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , american chemical society . [SEP]
[CLS] responses against tumors B-material [SEP]
[CLS] in addition , mrna - based vaccines have been developed using specific mrna sequences to induce the production of proteins B-material , which mimic the infectious antigens of the targeted disease , thus leading to the synthesis of antibodies B-material and lymphocytes . [SEP]
[CLS] mrna - based vaccines have been developed recently for treating viral diseases ( figure 2a ) . [SEP]
[CLS] some studies have shown that mrnas can induce the antibody B-material and t cell response to attack and neutralize mutants of virus , thus defending the host body against the viral infection . [SEP]
[CLS] furthermore , mrna - based therapy has been investigated for liver regeneration purposes . [SEP]
[CLS] the delivery of mrnas by injection induced a high hepatocyte proliferation rate , while the liver function was successfully restored and tissue regeneration was accelerated . [SEP]
[CLS] in the same manner , mrna therapeutics which encode the vascular endothelial growth factor a ( vegfa ) have been researched and developed for the treatment of type 2 diabetes mellitus . [SEP]
[CLS] results show a successful upregulation of vegfa expression and the subsequently improved skin blood flow , thus evidencing the potential of this therapy for angiogenesis B-event . [SEP]
[CLS] 2 . 2 . sirnas sirnas are double - stranded rnas which act during rna interference ( rnai ) pathways in gene silencing mechanisms ( figure 2b ) . [SEP]
[CLS] sirnas have 21 - 23 nucleotides with 3 ′ two - nucleotide overhangs and size of 13 - 15 kda . [SEP]
[CLS] sirnas are noncoding rnas which modulate the expression of a specific gene at the post - transcriptional stage by silencing targeted mrnas . [SEP]
[CLS] more specifically , sirnas can silence a gene by interaction with a fully complementary mrna gene sequence , inducing mrna degradation and translation suppressions . [SEP]
[CLS] this prevents the encoding of selected genes into proteins B-material and inactivates the gene expression . [SEP]
[CLS] sirnas can potentially be designed to silence any targeted gene , since they are likely to inhibit the progression of any genetic diseases , attack any viral infections , and prevent cell B-material degeneration processes . [SEP]
[CLS] however , each sirna can successfully target only one specific gene . [SEP]
[CLS] for this reason , sirna therapeutic approaches appear particularly suitable for singlegene disorders ( e . g . , hemophilia and hereditary amyloidosis ) . [SEP]
[CLS] on the other hand , sirna - based strategies are not expected to have a high therapeutic potential for complex multigene - related diseases . [SEP]
[CLS] the efficiency of sirna gene silencing is strictly dependent on the target complementary mrnas that should be cleaved . [SEP]
[CLS] within this framework , it has been demonstrated that the length of the double - strand and its thermodynamic symmetry can positively influence the gene inhibition potency . [SEP]
[CLS] sirnas are also widely studied in molecular biology and pharmacology . [SEP]
[CLS] however , some disadvantages of the application of sirna therapeutics include their hydrophobicity B-property and relatively large dimensions , which result in a difficult diffusion of the sirna through the extracellular membrane and its rapid renal excretion . [SEP]
[CLS] in addition , the high sensitivity of sirnas to ribonucleases leads to an ineffective delivery as well as an inefficient targeting of specific cells B-material . [SEP]
[CLS] to overcome these limitations , some chemical modifications of sirna nucleotides have been introduced , thus improving sirna potency , stability , and safety . [SEP]
[CLS] more specifically , chemical modification geometries and convenient delivery systems have been proven to enhance treatment potency , cellular target specificity , and delivery efficacy , while reducing sirna potential immunogenicity B-property and toxicity B-property . [SEP]
[CLS] sirna conjugates have also been demonstrated to improve the target delivery efficiency and the higher durability of the silencing treatments . [SEP]
[CLS] enhanced long - term activity and organ specificity were found in sirna conjugated with trivalent n - acetylgalactosamine ( galnac ) therapeutics , allowing a continuous activity of rnai for months in vitro and in vivo . [SEP]
[CLS] in light of this , several sirna - based drugs for gene control during rnai have been investigated and developed for anticancer B-property treatments , gene mutations and disorders , and viral infection therapies . [SEP]
[CLS] within this framework , several studies have demonstrated high sirna silencing efficiency in anticancer B-property therapies . [SEP]
[CLS] authors have reported a successful sirna inhibition of targeted gene expression and the induction of cancer cell B-material apoptosis B-event , while also detecting a reduction in tumor B-material size ( figure 2b ) . [SEP]
[CLS] furthermore , the effect of sirnas in neurological disorders has been studied to effectively inhibit targeted gene expression in the central nervous system by specific gene silencing . [SEP]
[CLS] additionally , galnac - sirna conjugates are exploited for efficient sirna delivery to liver hepatocytes , inducing targeted gene silencing while guaranteeing the duration of the effect . [SEP]
[CLS] lastly , the suitability of sirna - therapeutics in cardiovascular diseases has also been proved . [SEP]
[CLS] the beneficial silencing of a specific gene ( i . e . , monocyte chemotactic protein B-material 1 , mcp1 ) led to the specific cell B-material inhibition and decreased leucocyte production , thus promoting tissue healing . [SEP]
[CLS] 2 . 3 . shrnas shrnas are stem - loop rnas which have a mediating role in rnai processes ( figure 3a ) . [SEP]
[CLS] they are noncoding rnas which perform specific gene silencing functions like those of sirnas . [SEP]
[CLS] shrnas are characterized by a slow degradation and low turnover rate , resulting in a sustained efficacy on cellular functions . [SEP]
[CLS] the significantly longer effect of shrnas compared to sirnas is , most probably , their greatest advantage . [SEP]
[CLS] however , it should be mentioned that longevity knockdown effect of shrna appears less significant if compared to galnac - sirna conjugates ( e . g . , inclisiran ) . [SEP]
[CLS] chemically synthesized shrnas are considered a promising therapeutic alternative for the treatment of genetic disorders and viral infectious diseases . [SEP]
[CLS] however , shrnas ' insufficient silencing potency and off - targets are considered the main limitations of this approach . [SEP]
[CLS] another limitation of shrna therapeutics is the necessity for viral - based delivery systems . [SEP]
[CLS] indeed , even though viral vectors are considered more effective with superior delivery and transfection B-property efficiency I-property than non - viral vectors , their safety for medical applications has been strongly argued because of their potential immunogenicity B-property and oncogenicity . [SEP]
[CLS] moreover , viral vectors have small insert size and the additional disadvantages of being more costly and less reproducible compared to non - viral vectors . [SEP]
[CLS] today shrna - based therapy research is mainly focused on cancer treatment , where chemically synthesized shrnas are introduced to downregulate the expression of specific tumor - related genes . [SEP]
[CLS] within the framework of cancer gene therapy , shrnas are delivered using oncolytic or adeno - viruses and act by attacking tumor B-material cancer cells B-material and inhibiting specific gene expressions . [SEP]
[CLS] positive results on both the shrna inhibition of tumor B-material cell B-material proliferation and the high rate of cell B-material apoptosis B-event have been reported . [SEP]
[CLS] besides , it is worth pointing out that the prolonged duration of shrna activity has not always been considered an advantage . [SEP]
[CLS] indeed , since the long - term effects are still unknown , the benefits related to long - lasting therapies remain questionable . [SEP]
[CLS] mirnas are double - stranded stem - loop rna structures with a length of 21 - 25 nucleotides and dimensions of 13 - 15 kda ( figure 3b ) . [SEP]
[CLS] mirnas are noncoding rnas which take part in rnai mechanisms , playing a crucial role in gene modulation and editing . [SEP]
[CLS] the mirna structure is partially complementary to a targeted mrna sequence and can regulate the gene expression at the post - transcriptional level . [SEP]
[CLS] mirnas act selectively by inhibiting the mrna sequence via mediation of its translational repression , and cleaving or inducing its degradation . [SEP]
[CLS] more specifically , only 2 - 8 nucleotides of mirnas can actually interact with the targeted mrnas via imperfect base - pairing interactions . [SEP]
[CLS] this short nucleotide sequence of mirna is called " seed " region and usually pairs with the 3 ′ - untranslated region ( utr ) of the target mrna , inducing post - transcriptional silencing . [SEP]
[CLS] less frequently , mirna can bind to other mrna sites , such as the 5 ' - utr and the coding region . [SEP]
[CLS] because of the short binding region and the non - perfect complementarity with mrna , the specificity of the mirna action copyright 2019 , elsevier b . v . b ) schematic representation of mirna structure ( double - stranded stem - loop rna ) , functions and activity . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , john wiley and sons . [SEP]
[CLS] is lower than that of other rna therapeutics ( e . g . , sirna ) . [SEP]
[CLS] additionally , the low target specificity of mirnas results in a great ability to target a broader range of mrnas , by simultaneously modulating the expression of multiple genes . [SEP]
[CLS] moreover , since mirnas are not required to be perfectly complementary to the mrna target sequence , the design of mirnas appears straightforward and much easier to develop than that of sirnas ( which requires the full complementarity of the strands ) . [SEP]
[CLS] the therapeutic approaches of mirnas may be broken down into two main classes : mirna inhibition and mirna replacement . [SEP]
[CLS] the first strategy is based on the silencing of endogenous mirnas using a synthetic mirna antagonist ; the latter provides the introduction of synthetic mirnas to mimic the activity of endogenous mirnas . [SEP]
[CLS] however , some limitations of mirna therapies , such as off - target and immunological effects and a less than optimal delivery efficiency , have been pointed out . [SEP]
[CLS] to overcome these disadvantages , the chemical modification of mirnas has been studied , leading to improved mirna properties while preserving its silencing activity . [SEP]
[CLS] additionally , mirnas ' hydrophilicity B-property , high molecular weight , and negative charge result in their poor in vivo stability and consequent difficulties in crossing cellular barriers B-property . [SEP]
[CLS] thus , in order to guarantee the mirna clinical application , several delivery systems have been investigated and developed . [SEP]
[CLS] due to their multiple gene targeting ability , mirnas can modulate one third of mrnas . [SEP]
[CLS] therefore , mirnas are involved in a large number of biological processes , including cellular activities and functions , as well as degeneration processes and the defense from viral pathogens . [SEP]
[CLS] within this framework , mirna - based therapies can target a wider range of diseases than other rna therapeutics . [SEP]
[CLS] more specifically , the mirna approach can have significant therapeutic effects on complex multigenic diseases ( e . g . , cancers and neurodegenerative and cardiovascular disorders ) which require the control over the expression of entire gene families . [SEP]
[CLS] thus , the silencing of undesirable and mutated genes as well as the inhibition of pathological cellular pathways can be targeted by mirna activity and the recovery of a regular gene expression can be achieved . [SEP]
[CLS] in light of all the characteristics and features described , we believe that mirna therapeutics would be the most promising alternatives for some specific applications , for example , within the wound healing framework . this is because of some significant mirna advantages , including its relatively simple synthesis and the possibility to simultaneously regulate the expression of multiple genes to accelerate the cure of wounds . [SEP]
[CLS] on the other hand , the high efficacy of mrna - based treatments in terms of encoding processing , as well as their safety and cost - efficiency , make mrna , in our opinion , the most relevant candidate to produce vaccines for the treatment of viral diseases . [SEP]
[CLS] the use of safe delivery platforms can ensure the efficient delivery of therapeutic rna and protect it from degradation , and compensate for its inherent hydrophilicity B-property and negatively charged nature when crossing the cellular membrane . [SEP]
[CLS] rna carriers should be carefully engineered to adjust to physiological conditions and overcome the obstacles posed by the human immune system . [SEP]
[CLS] to this end , a perfect rna carrier with a high loading capacity should be nontoxic and undetectable by the immune system , retain rna stability , and protect it from being digested by the nuclease enzymes in living organisms . [SEP]
[CLS] lastly , an ideal carrier should be taken up into the desired cell B-material and demonstrate good transfection B-property efficiency I-property in order to ensure a successful endosomal escape of the cargo . [SEP]
[CLS] recent advances in the field of nanotechnology have shown that nanostructured platforms have remarkable rna delivery perspectives . [SEP]
[CLS] these nanocarriers can overcome various biological constraints and spread into the body . [SEP]
[CLS] in this section , we summarize recent advances in this field and describe the main delivery platforms including carbonaceous nanomaterials B-material , inorganic B-nanoparticle nanoparticles I-nanoparticle , polymer B-material nanoplatforms , virus - like particles , and lipid B-nanoparticle nanoparticles I-nanoparticle ( figure 4 ; table 1 ) . [SEP]
[CLS] ideally , the effectiveness of nanotechnology - assisted strategies is based on the capability to remain in the bloodstream for a considerable time and overcome the physical body barriers B-property , represented respectively by i ) the first - pass hepatic effect , ii ) the spleen sieve activity , and iii ) the gap junction between endothelial cells B-material ( ecs ) . [SEP]
[CLS] key advantages of nanotechnology - assisted therapies consist in increasing the intracellular concentration of drugs and reducing dose - limiting toxicities B-property simultaneously . [SEP]
[CLS] to tackle these challenges , nanocarriers ' size and surface are the principal features that should be considered during their design . [SEP]
[CLS] the size of these vectors , which can be easily adjusted , should be sufficiently small to escape from macrophages present principally in the reticuloendothelial system of the liver and spleen and , at the same time , should be large enough to prevent their extravasation from the capillaries . [SEP]
[CLS] a broad literature has identified the best nanocarrier size to be below 100 nm . [SEP]
[CLS] additionally , the nanocarriers lifespan and destiny in the bloodstream can be increased by modifying nanocarrier surface . [SEP]
[CLS] hydrophilic B-property polymers B-material , such as poly ( ethylene glycol ) ( peg ) , and repelling plasma proteins B-material - that prevent the opsonization and therefore their phagocytosis B-event - are the principal strategies used to achieve the surface modification . [SEP]
[CLS] further , the initial interaction of nanocarriers with the plasma membrane of the target cells B-material can be promoted by introducing positive charges or active targeting B-material ligands I-material on the outer nanocarrier surface , which can interact with the negatively charged cell B-material membrane or specific proteins B-material present on the surface of the target cells B-material . [SEP]
[CLS] nanocarriers should also be designed to prevent non - specific uptake . [SEP]
[CLS] this feature is particularly important in all the applications - for instance delivery of chemo drugs - where the nanocarrier content must be delivered selectively to certain targeted cells B-material . [SEP]
[CLS] passive and active targeting are the two approaches that can be used to tune nanocarrier uptake . [SEP]
[CLS] the enhanced permeability and retention effect ( epr effect ) , characteristic of the blood vessels committed with the tumor B-material mass , can be used in passive strategies to allow the infiltration of nanocarriers into the cancer tissues . [SEP]
[CLS] in this case , it has been reported that liposomes B-nanoparticle up to ≈400 nm are suitable for correct extravasation while those having size lower than 200 nm offer higher cell B-material uptake . [SEP]
[CLS] however , several disadvantages occur with passive targeting approaches . [SEP]
[CLS] tumor B-material cells B-material can develop acquired resistance to chemotherapies , overexpress the transporter proteins B-material that actively expel drugs from cancer cells B-material , or do not release factors that induce epr effect , hence limiting the passive strategy . [SEP]
[CLS] alternatively , active approaches , in which the nanocarrier surface is modified with specific ligands , can be used to tune the cellular uptake . [SEP]
[CLS] these ligands can recognize specific receptors present on the surface of the host target cells B-material , thus enhancing their selectivity . [SEP]
[CLS] further , to prevent undesired , non - specific uptake , the receptors recognized by the ligands should be overexpressed on target cells B-material . [SEP]
[CLS] interestingly , the progress in the nanomaterials B-material field has allowed developing nanocarriers labeled with specific ligands that - after the interaction with the target protein B-material - can be engulfed into the cytoplasm by receptor - mediated internalization machinery . [SEP]
[CLS] the physical properties of nanocarriers and the internalization machinery of targeted cells B-material govern the uptake of nanoparticles B-nanoparticle into the cytoplasm . [SEP]
[CLS] remarkably , the same nanocarriers can be internalized by different mechanisms in different cell B-material types , which underline the relevance to identify the endocytic pathways involved , especially in in vivo models . [SEP]
[CLS] the direct fusion with the plasma membrane and endocytosis B-event are the two main routes of entry of nanocarriers into the cells B-material . [SEP]
[CLS] however , the latter is the principal pathway involved in the internalization process . [SEP]
[CLS] so far , five major types of endocytosis B-event were investigated : i ) clathrincoated pit - mediated endocytosis B-event ( cme ; clathrin and dynamin dependent ) , ii ) fast endophilin - mediated endocytosis B-event ( feme , a clathrin - independent but dynamin dependent pathway for rapid ligand - driven endocytosis B-event of specific membrane proteins B-material ) , iii ) clathrin - independent carrier ( clic ) / glycosylphosphatidylinositol - anchored protein - enriched early endocytic compartment ( geec ) endocytosis B-event ( clathrin and dynamin independent ) , iv ) macropinocytosis , and v ) phagocytosis B-event . [SEP]
[CLS] a thorough description of all these mechanisms is out of the scope of this review . [SEP]
[CLS] for more information , the reader can refer to the following references in which these mechanisms are described in detail . [SEP]
[CLS] all these mechanisms show early common trafficking . [SEP]
[CLS] after the interaction with the target cells B-material , the nanocarriers - independently from the internalization pathways - are conveyed to the early endosomes ( ees ) and successively can be recycled back to the plasma membrane or moved forward to the late endosomes ( les ) and consecutively to the lysosomes . [SEP]
[CLS] however , these pathways can share common components . [SEP]
[CLS] for example , clic / geec endocytosis B-event and micropinocytosis boundaries can be unclear because most of the proteins B-material involved are the same . [SEP]
[CLS] internalization of nanocarriers is only the first step for the delivery of therapeutic nanoparticles B-nanoparticle . [SEP]
[CLS] several studies suggest that the delivery of the nanocarriers to the ees , independently from the internalization pathways , is mediated by the rab5 / eea1 - dependent trafficking pathway . [SEP]
[CLS] the fate of nanocarriers and subsequent cellular trafficking can be affected by several factors that relate to the mechanism of internalization . [SEP]
[CLS] however , so far , it is not clarified if the internalization pathway or the signaling from the receptor B-material drives the nanocarriers trafficking . [SEP]
[CLS] from the ees , the cargo can be recycled back to the cell B-material surface via the rab11 - positive recycling endosome or can proceed to les compartment . [SEP]
[CLS] it is important to underline that the cargo endocytosed by the same pathway can be delivered into different ees and that nanocarriers endocytosed by different signaling can be sorted into the same ees compartment . [SEP]
[CLS] so far , the internalization of rna - loaded lipid - based nanocarriers has not been fully clarified . [SEP]
[CLS] however , several evidences revealed that the principal uptake mechanism is the clathrindependent endocytosis B-event followed by micropinocytosis . [SEP]
[CLS] further , it has been determined that only a tiny fraction ( 1 - 2 % ) of lipid B-nanoparticle nanoparticles I-nanoparticle can evade the endosomal pathway . [SEP]
[CLS] the recent advances in our understanding of endocytosis B-event pathways have shown that cme , feme , and clic / geec are all involved in the processing of nanocarriers with a diameter smaller than 200 nm , which means it is unlikely that these routes can internalize particles larger than 200 nm . [SEP]
[CLS] importantly , the nanocarriers , after the resuspension in cell B-material medium or injection in vivo , are quickly adsorbed on the surface of serum proteins B-material ( for example , vitronectin ) , forming a protein B-material corona I-material . [SEP]
[CLS] this interaction , in turn , can influence the binding B-event to I-event specific I-event receptors I-event on the target cells B-material and lead to the selection of the uptake pathway . [SEP]
[CLS] unfortunately , the formation of protein B-material corona I-material can favor the aggregation of the nanocarriers and affect the real size of the particles that will be larger than the size measured ex vivo . [SEP]
[CLS] therefore , improving our knowledge regarding the fate and the remodeling of nanocarriers in vivo is essential . [SEP]
[CLS] to face this aspect , the standardization of the synthesis , analysis , and follow - up of in vivo nanocarrier administration represents crucial aspects to be considered . [SEP]
[CLS] the minimum information reporting in bio - nano experimental literature ( mirabel ) has been recently proposed to introduce guidelines to improve reproducibility , increase quantitative comparison of bio - nano materials , and facilitate meta - analyses and in silico modeling . [SEP]
[CLS] to provide data standardization , these guidelines are focalized onto three main categories : i ) material B-material characterization , ii ) biological characterization , and iii ) experimental protocols [SEP]
[CLS] unfortunately , nowadays , few data are available on the magnitude of different endocytic pathways in physiological contexts and on different tissues and how these alternative pathways can impair / improve the delivery of nanocarriers into the cells B-material of interest . [SEP]
[CLS] the unique structural properties of carbonaceous nanomaterials B-material make them promising in many fields , including energy storage and electrochemistry . [SEP]
[CLS] the encouraging potential of these nanomaterials B-material , such as graphene oxide B-material ( go ) and carbon B-nanoparticle nanotubes I-nanoparticle ( cnts ) , has been recently explored in biomedicine and tissue engineering . [SEP]
[CLS] therapeutic molecules can bind noncovalently to go and cnts through π−π stacking . [SEP]
[CLS] cnts are particularly advantageous since they can benefit from the high surface area . [SEP]
[CLS] in this frame , for the successful integration of cnts in a biological system , surface functionalization of nanotubes B-nanoparticle should be practiced to break the relatively high number of nanotube B-nanoparticle agglomerates in suspension and to improve their low biocompatibility B-property . [SEP]
[CLS] the addition of cell B-material - targeting agents to the design of cnt carriers is another requirement of rna delivery for high cell B-material recognition and efficient internalization . [SEP]
[CLS] cao [SEP]
[CLS] a combination of a poly ( ethylenimine ) ( pei ) - betaine conjugate and a targeting peptide B-material was further reacted with oxidized cnts to grant cell B-material penetration and ph - sensitive endosomal escape characteristics to the carrier . [SEP]
[CLS] the undesirable size of pristine cnts was decreased to 250 nm after polymeric modification . [SEP]
[CLS] a similar approach was used in the study conducted by edwards et al . in their report , polyamidoamine dendrimer B-nanoparticle and cnt suspension were placed under sonication to modify the surface of cnts and improve their functional properties . [SEP]
[CLS] on the other hand , go is a sheet of oxidized carbon B-material atoms I-material with a hexagonal conformation resembling a honeycomb , which has a higher specific surface area , suspension stability , and biocompatibility B-property than cnts . [SEP]
[CLS] the carrying capacity of bare go was evaluated via the intracellular delivery of sirna . [SEP]
[CLS] the small interfering rna was complexed at different mass ratios with pristine go , maintaining an average lateral size of one µm and thickness of two nm . [SEP]
[CLS] go accumulated and isolated in large vesicles and the intracellular trafficking was hindered , most probably due to the formation of go agglomerates . [SEP]
[CLS] furthermore , the charges between cargo and carrier were cancelled out when the complex was introduced into the cell B-material culture medium , which led to low transaction efficiency . [SEP]
[CLS] in another study , go with an average diameter of approximately 200 nm was exfoliated under sonication and mixed with a complementary strand of mir - 21 and doxorubicin B-material hydrochloride as an anticancer B-property drug . [SEP]
[CLS] results showed a quick cellular uptake of the carrier and desirable gene silencing by the cargo in cancer cells B-material . [SEP]
[CLS] these findings show that although go benefits from several functional groups , effective delivery of rna can be further enhanced by polymer B-material and cationic B-material lipid B-material coating B-material of the sheets . [SEP]
[CLS] these coatings B-material have the capability to extend the presence of nanoparticles B-nanoparticle in blood circulation , by circumventing immune system recognition , and improving functionalization for a targeted delivery of therapeutic agents . [SEP]
[CLS] dense polymer B-material brushes were fabricated from fluorescent B-property conjugated polyelectrolyte macro initiators on the surface of go sheets with a thickness of 1 . 3 nm , which permitted the cellular tracking of the nanocarrier . [SEP]
[CLS] qu et al . constructed a composite of go and poly ( amidoamine ) dendrimer B-nanoparticle incorporated with a peg - modified glycyrrhetinic acid as targeting B-material ligand I-material , and complexed it with sirna to demonstrate an active targeting of cancer liver cells B-material . [SEP]
[CLS] a satisfactory cell B-material uptake of nanocomplex by hepg2 cells B-material and a decreased expression of vegfa in mrna and protein B-material levels were observed , and the effective in vitro gene silencing was demonstrated . [SEP]
[CLS] in a recent study , saravanabhavan et al . introduced a functionalized chitosan go nanoparticle B-nanoparticle into traditional pristine go carriers and developed a suitable tumor - targeted material B-material with good biocompatibility B-property and the potential to regulate the b - cell lymphoma - 2 ( bcl - 2 ) expression . [SEP]
[CLS] in this experiment , chitosan B-material was mixed with sirna prior to go addition . [SEP]
[CLS] a composite of loaded chitosan B-nanoparticle nanoparticles I-nanoparticle and go , formed at the weight ratio of 1 : 1 , were complexed with sirna , and used to reduce the survivability of I-property tumor I-property cells I-property ( figure 5a ) . [SEP]
[CLS] it was apparent that reproduced with permission . [SEP]
[CLS] copyright 2019 , elsevier . [SEP]
[CLS] b ) glucose - linked gold B-nanoparticle nanoparticles I-nanoparticle for targeted sirna delivery to breast cancer stem - like cells B-material . [SEP]
[CLS] glucose ligands endow the nanoparticles B-nanoparticle with target ability toward the breast . [SEP]
[CLS] adapted with permission . [SEP]
[CLS] copyright 2019 , elsevier . c ) green nanoparticles B-nanoparticle for sirna delivery . [SEP]
[CLS] natural polyphenol from green tea catechin was complexed with sirna to form negatively charged nanoparticles B-nanoparticle , followed by surface coating B-material with pll . [SEP]
[CLS] reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2020 , the authors . published by springer nature . [SEP]
[CLS] d ) higher - molecular weight , bioreducible , cationic B-material polymer enhanced sarna delivery . [SEP]
[CLS] high - molecular weight pabols were achieved by improved aza - michael addition . [SEP]
[CLS] complexation with sarna happened via titration method and transfection efficacy of the pabol - 100 polyplexes were compared to jetpei and peimax . [SEP]
[CLS] reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2020 , the authors . [SEP]
[CLS] published by american chemical society . [SEP]
[CLS] chitosan B-material prevented immunogenicity B-property , while the lateral size of go reduced the inflammatory response . [SEP]
[CLS] the fickian ph - sensitive diffusion of sirna from the carrier complex showed the controlled release required to target tumor B-material cells B-material . [SEP]
[CLS] in addition , an advanced structure of go and a porous zeolitic imidazolate framework were designed to enhance rna - delivery efficiency . [SEP]
[CLS] the complex of sirna and positively charged go - zeolite composite improved the cell B-material transfection and demonstrated adequate in - vitro gene knockdown . [SEP]
[CLS] a prime obstacle in the delivery of nanomaterials B-material to the targeted cells B-material is represented by the liver and spleen sequestration of carriers . [SEP]
[CLS] carbonaceous platforms are not exempt from this process . [SEP]
[CLS] the yet unclear cnt toxicity B-property at the molecular and cellular level makes a future endorsement of this material B-material uncertain and doubtful . [SEP]
[CLS] specifically , adopting cnt as a carrier of genetic molecules may carry more risks of malignant transformation , dna damage , and mutation . [SEP]
[CLS] on the other hand , the adverse effects of accumulated sequestrated cnts and go on zonation , epigenetic changes , and liver function should be studied and addressed . [SEP]
[CLS] wu et al . set out to find that the controlling mechanisms related to go liver retention and nano - bio interaction stemmed from the unavoidable liver sequestration of go nanosheets . [SEP]
[CLS] go oxidation level , average lateral size , and the frequency and type of surface B-nanoparticle functional I-nanoparticle groups I-nanoparticle dictated the in vivo behavior of go . [SEP]
[CLS] the pattern of liver functional zonation appeared to be strikingly disrupted by go and some notable changes in representative liver gene expression were found . [SEP]
[CLS] in spite of the minute changes in liver function , the study showed that the transcription B-event and epigenetics of liver cells B-material were largely affected by go . [SEP]
[CLS] these pieces of evidence prompted researchers to take cautionary steps toward the consideration of carbonaceous nanomaterials B-material as rna carriers . [SEP]
[CLS] inorganic B-nanoparticle nanoparticles I-nanoparticle are another group of materials for rna delivery which feature several advantages , including their simple synthesis and functionalization , tunable size , distinctive optical and electrical properties , as well as good biocompatibility B-property and low cytotoxicity B-property . [SEP]
[CLS] the advantages of gold B-nanoparticle nanoparticles I-nanoparticle are their unique optical properties , ease of functionalization , and tunable size and shape . [SEP]
[CLS] gold B-material ( au ) nanoparticles B-nanoparticle can be engineered to protect rna against degradation through steric hindrance . [SEP]
[CLS] furthermore , au nanoparticles B-nanoparticle benefit from the compton effect , facilitating radical production and making them promising vehicles B-material for cancer therapy . [SEP]
[CLS] the surface plasmon resonance of these nanoparticles B-nanoparticle , tuned to their size and shape , bestows a property of light - controlled release of the cargo . [SEP]
[CLS] the quantum - based parameters of the thiolation of a nucleotide and its adsorption on metallic B-nanoparticle nanoparticles I-nanoparticle were studied using the density functional theory ( dft ) . [SEP]
[CLS] seed - mediated growth , a simple and low - cost process , was used to synthesize au nanoparticles B-nanoparticle from haucl 4 • 3h 2 o and cetyltrimethylammonium bromide B-material ( ctab ) solutions . ctab molecules were later exchanged with a peptide B-material to decorate au with the regulatory protein B-material hiv - 1 tat and enhance the cellular uptake . the carrier was noncovalently complexed with receptor B-material tyrosine B-material kinase - like orphan receptor 1 ( ror1 ) sirna and the apoptosis B-event in mda - mb - 231 breast cancer cells B-material was evaluated . [SEP]
[CLS] thanks to their likeness to carbonaceous nanomaterials B-material and enhanced cargo delivery , the trimming of inorganic B-nanoparticle nanoparticles I-nanoparticle with polymer B-material brushes or natural substances has been the topic of many studies . [SEP]
[CLS] yi et al . introduced glucose - installed peg - block - poly ( l - lysine ) ( pll ) ( peg - block - pll ) modified with lipoic acid complexed with a single sirna into a 20 nm gold B-nanoparticle nanoparticle I-nanoparticle through sulfur B-material - gold bonding in the presence of sucrose , and a two - step bottom - up self - assembly method . [SEP]
[CLS] to obtain nanocarriers with a < 50 nm size , it was necessary to add sucrose to the buffer during the construction of glucose - modified particles , probably due to the hydrogen B-material bonding hindrance between glucose on the carrier surface . [SEP]
[CLS] the carrier system profited from stealth , targetability , and uniform size . [SEP]
[CLS] the unimer polyion complex helped the targeted carrier show a high cellular uptake of payloads in a spheroid breast cancer ( figure 5b ) . [SEP]
[CLS] an efficient internalization into the glutathione ( gsh ) - rich environment of the stem - like cancer cells B-material was observed and gene silencing was notably enhanced in a cancer cell B-material orthotopic mda - mb - 231 . [SEP]
[CLS] in vivo investigations showed significant suppression of tumor B-material growth . [SEP]
[CLS] the accumulation of modified nanoparticles B-nanoparticle in the brain was reported to be significantly small . [SEP]
[CLS] the authors attributed this finding to both the low probability of an effective transcytosis of glucose - gold B-nanoparticle nanoparticles I-nanoparticle into the brain tissue and the low density of nanoparticles B-nanoparticle on the luminal plasma membrane of brain capillary endothelial cells B-material ( bcecs ) in the normal glycemic condition . [SEP]
[CLS] cytotoxicity B-property , cellular uptake , and downregulation efficiency of chitosan B-material - coated gold B-nanoparticle nanoparticles I-nanoparticle were evaluated . [SEP]
[CLS] the positively charged carrier was subsequently complexed with sirna and modified by a final layer of chitosan B-material over the therapeutic molecules . [SEP]
[CLS] yang et al . designed an updated nanosystem by decorating amine - terminated generation 5 dendrimers B-nanoparticle with 1 , 3 - propanesultone , and then entrapped it with au nanoparticles B-nanoparticle to take advantage of the antifouling and serum - enhanced transfection properties . [SEP]
[CLS] the carrier was then complexed with hypoxia - inducible factor 1 - alpha ( hif1a ) sirnas for a dual sensitization - boosted radiotherapy ( rt ) of tumors B-material . [SEP]
[CLS] other advantageous inorganic platforms are iron B-nanoparticle oxide I-nanoparticle nanoparticles I-nanoparticle which are promising biomaterials for gene delivery and can be bound covalently to nucleic B-material acids I-material . [SEP]
[CLS] it is worth mentioning that iron B-material oxide I-material particles can be promptly degraded and join the iron B-material stores in the body . [SEP]
[CLS] to guide nanocarriers toward the desired tissue by an external magnetic B-property field , cristofolini et al . proposed a hybrid of superparamagnetic B-nanoparticle iron I-nanoparticle oxide I-nanoparticle nanoparticles I-nanoparticle with caffeic acid for sirna delivery to breast cancer cells B-material . [SEP]
[CLS] iron B-nanoparticle oxide I-nanoparticle nanoparticles I-nanoparticle were synthesized by a co - precipitation method from degassed solutions of fecl 3 • 6h 2 o , fecl 2 , and nh 4 oh , with the subsequent addition of caffeic acid in naoh . [SEP]
[CLS] spherical particles had an average diameter of 14 nm and a hydrodynamic diameter of 93 nm . [SEP]
[CLS] a hierarchical nanostructure was formed by the agglomeration of 70 magnetic B-property particles when iron B-material oxides I-material were stabilized by layers of calcium B-material phosphate and peg - polyanion block copolymer . [SEP]
[CLS] au and iron B-nanoparticle oxide I-nanoparticle nanoparticles I-nanoparticle are homogeneous nanoscale particles suitable for avoiding biological barricades and for effective rna delivery . [SEP]
[CLS] selenium B-material is also a promising chemotherapeutic gene carrier . [SEP]
[CLS] the biocompatibility B-property , easy surface modification , and low toxicity B-property of these nanoparticles B-nanoparticle can be compared with that of au . [SEP]
[CLS] in a recent study , a positively charged peptide B-material was installed on the surface of selenium B-material to forge a tumor - targeted sirna delivery carrier . [SEP]
[CLS] selenium B-material was synthesized from a solution of na 2 seo 3 and vitamin c . the modified 80 nm selenium B-nanoparticle nanoparticles I-nanoparticle obtained were able to form a stable suspension , significantly bind sirna , and protect it from degradation . [SEP]
[CLS] in cases of bone - related delivery of drugs and genes , calcium B-material phosphate can be considered a good candidate for in vitro and in vivo transfection . [SEP]
[CLS] the advantages of these nanoparticles B-nanoparticle are biocompatibility B-property , low toxicity B-property , remarkable surface - to - volume ratio , stability in the extracellular space , and a strong affinity for binding to nucleic B-material acids I-material . [SEP]
[CLS] recently calcium B-material phosphate nanoparticles B-nanoparticle were stabilized using a conjugate of hyaluronic acid ( ha ) and 3 , 4 - dihydroxy - l - phenylalanine . [SEP]
[CLS] the inorganic carrier was synthesized from a cacl 2 and na 2 hpo 4 solution and coated with marine mussel - derived amino B-material acids I-material and anionic non - sulfated glycosaminoglycan in an acidic mixture . [SEP]
[CLS] the surface - stabilized nanoparticles B-nanoparticle offered an orderly cellular delivery of microrna to human mesenchymal stem cells B-material followed by the down - regulation of noggin . [SEP]
[CLS] among various types of inorganic carriers , mesoporous silica B-nanoparticle nanoparticles I-nanoparticle ( msnps ) have been proven suitable for various uses . [SEP]
[CLS] their excellent biocompatibility B-property , large surface area , tunable porosity , and ability to encapsulate and protect nucleic B-material acids I-material turned the focus of many studies toward the development of engineered silica carriers that can ferry different sizes and types of therapeutic molecules . [SEP]
[CLS] for photodynamic therapy , sirna and photosensitizers B-property were simultaneously delivered using amine - functionalized mesoporous silica . [SEP]
[CLS] nanoparticles B-nanoparticle were synthesized using a ha - catalyzed sol - gel procedure . [SEP]
[CLS] another report showed that sirna - loaded haassembled msnps were effective in controlling the drug release and internalization in cal27 cancer cells B-material . [SEP]
[CLS] modified nanoparticles B-nanoparticle of an average size of 184 nm were synthesized from a solution of ctab , nh 4 oh , and tetraethyl orthosilicate ( teos ) . [SEP]
[CLS] the design of effective amine - functionalized msnps was investigated , and the pore size - dependent thermodynamic driving force of the interactions of double - stranded rna ( dsrna ) with msnps depending on dsrna length was evaluated through isothermal titration calorimetry . [SEP]
[CLS] during the synthesis of silica B-nanoparticle nanoparticles I-nanoparticle with 8 nm pore sizes , triisopropylbenzene ( tipb ) was used as a ctab pore expanding agent . [SEP]
[CLS] it was shown that the efficient design of dsrna nanocarriers offers the advantage of creating small pores to protect the rnas from degradation , while a sufficient space is required for dsrna threading into pores . [SEP]
[CLS] a layer of lipid B-material conjugated with irgd peptide B-material was used to stabilize silica B-nanoparticle nanoparticles I-nanoparticle by copper - free clickchemistry in a tumor - penetrating sirna and mirna codelivery investigation . [SEP]
[CLS] the carrier efficiency in cytosolic rna delivery was further enhanced by loading a near - infrared - responsive photosensitizer B-property into nanoparticles B-nanoparticle for the local generation of reactive oxygen B-material species ( ros ) . [SEP]
[CLS] during zeta B-property potential I-property assay , silica particles kept their gross positive charge - an indication of an undersaturated particle surface by the negatively charged molecules leading to high loading capacity . [SEP]
[CLS] the disruption of the endolysosomal membrane was facilitated by the light - triggered photodynamic effect of reactive oxygen B-material production on the surface of silica B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] the large specific surface area of inorganic particles ensures the easy adsorption of rna . [SEP]
[CLS] as far as loading , complexing efficiency , protection , and release of rna are concerned , these results suggest the suitability of the design of inorganic particles as nanocarriers . [SEP]
[CLS] polymer B-material nanoplatforms are popular materials for rna delivery due to their great diversity , structural flexibility , and low immunogenicity B-property . [SEP]
[CLS] many studies have adopted new strategies to engineer a new class of synthesized polymers B-material or copolymers with lower toxicity B-property , higher transfection B-property efficiency I-property , and stability . [SEP]
[CLS] this endeavor involves chemical modification of traditional polymers B-material used to form polyplexes and studying large polymeric libraries through the implementation of high - throughput strategies , which ease the assessment of the relationship between structure and activity . [SEP]
[CLS] moreover , recent investigations on the architectural design of polymeric nanoplatforms can facilitate tissue integration , promote localized delivery of genetic molecules , and finally affect the development of fibrous capsules . [SEP]
[CLS] finally , and in order to address the unbalanced relationships between cytotoxicity B-property and transfection B-property efficiency I-property of polymeric nanocarriers , the emergence of green nanoparticles B-nanoparticle will be discussed in this section . [SEP]
[CLS] considered one of the most potent carriers , pei is a cationic B-material polymer B-material currently widely used in clinical trials with different formulations . [SEP]
[CLS] unfortunately , the high toxicity B-property and low colloidal stability of this cationic B-material polymer B-material cause several limitations in physiological conditions . [SEP]
[CLS] the high positive charge and non - biodegradable B-property nature of pei can be corrected with peg grafting . [SEP]
[CLS] grafted polymeric B-nanoparticle nanoparticles I-nanoparticle offer a low toxicity B-property and high colloidal stability in correlation with peg chain length and peg grafting degree . [SEP]
[CLS] ke et al . outlined the synthesis of a pei - g - peg library for mrna delivery and examined the effects of complexing volume , efficiency B-property of I-property encapsulation I-property , cell B-material penetration , and endosomal escape , with various peg assemblies and different grafting ratios . [SEP]
[CLS] an ideal carrier should have a straightforward and consistent manufacturing process . [SEP]
[CLS] taking this into account , ke et al . also assessed a scalable flash nano complexation production method to manufacture nanoparticles B-nanoparticle in a reproducible manner for future clinical translations . [SEP]
[CLS] another scientific work dealt with various pei nanoparticles B-nanoparticle such as dexamethasone - conjugated pei ( 2 kda , pei2k ) and deoxycholic acid - conjugated pei2k for mrna delivery into the brain . [SEP]
[CLS] among the different pei derivatives , branched pei has the highest transfection B-property efficiency I-property in serum - free conditions owing to both its ability to form stable polyplexes and its buffering capacity . [SEP]
[CLS] the latter ensures the endosomal escape via the proton sponge effect . [SEP]
[CLS] the grafting of a branched pei with poly ( lactide - co - glycolide ) ( plga ) has been reported to be a viable method for increasing the transfection B-property efficiency I-property of the carrier in the presence of serum , specifically when carried out in vivo . [SEP]
[CLS] for example , a cationic B-material amphiphilic B-property copolymer of plga - graft - pei nanocarriers of different shapes and sizes was used for nucleic B-material acid I-material delivery . [SEP]
[CLS] to improve the site - specific intracellular delivery of rna , endolysosomal escape , and temporal delivery of the cargo , a photochemical B-property internalization technology has been devised . [SEP]
[CLS] this method , however , suffers from both its reduced reactive oxygen B-material generation in the microenvironment of the tumor B-material and the low efficiency of rna and photosensitizer B-property co - loading . [SEP]
[CLS] to address these issues , zhang et al . designed a nanoparticle B-nanoparticle system featuring a photoactivated polyprodrug for the light - controlled codelivery of sirna for cancer synergistic therapy . [SEP]
[CLS] the stability and tumor B-material - targeting ability of this carrier were later enhanced by coating B-material the nanoparticles B-nanoparticle with peg - grafted ha . [SEP]
[CLS] the polymeric protective I-material shell B-material and active tumor B-material - targeting B-material ligand I-material protected sirna from degradation and facilitated the carrier accumulation by the tumor B-material . [SEP]
[CLS] chitosan B-material is another interesting polymeric carrier . [SEP]
[CLS] copolymeric nanoparticlessirna - loaded chitosan B-material - cysteine B-material nanoparticles B-nanoparticle were devised by crosslinking cationic B-material polyplexes with various amounts of anionic cross - linkers : tripolyphosphate , ha , and a copolymer of methacrylic acid - methyl methacrylate . [SEP]
[CLS] to reduce cross - linker cytotoxicity B-property , amine B-material groups I-material of low molecular weight chitosan B-material were covalently reacted with ha dialdehyde , while ha bound to the nanoparticle B-nanoparticle surface . [SEP]
[CLS] to enhance the colloidal stability and lengthening the circulation time of the polymeric carrier , the pegylation of chitosan B-material was also reported . [SEP]
[CLS] periodate oxidation in an ethanol - water B-material mixture was used to prepare ha dialdehyde . [SEP]
[CLS] monodisperse chitosan B-material polyplexes ranging from 100 to 120 nm , with spherical morphology , were achieved . [SEP]
[CLS] it was demonstrated that the presence of conjugated ha did not affect the particle size . [SEP]
[CLS] carrier - cargo complexes accumulated specifically in the tumor B-material site and inhibited the targeted oncogene . [SEP]
[CLS] since chitosan B-material can only dissolve in acidic solutions , other derivatives of this natural polymer B-material , such as carboxymethyl chitosan B-material and chitosan B-material hydrochloride , have been tested for water B-material - I-property soluble B-property chitosan - based delivery systems . [SEP]
[CLS] these systems show advantages for the oral route of delivery . [SEP]
[CLS] an intestinal - targeted sirna was efficiently delivered by using an external stimulus , while sirna was released from carboxymethyl chitosan B-material - fluorescein B-material isothiocyanate I-material - chitosan B-material hydrochloride nanoparticles B-nanoparticle to inhibit the β - catenin protein B-material expression . [SEP]
[CLS] for a targeted delivery , sirna microparticles were condensed with cationic B-material pll and modified using electrostatic deposition and click chemistry . [SEP]
[CLS] tetrazine - conjugated ha was deposited on condensed nanoparticles B-nanoparticle via electrostatic interactions , forming a carrier with a single - targeting moiety . [SEP]
[CLS] then , trans - cyclooctene - n - hydroxysulfosuccinimide ( nhs ) ester - modified her2 antibody B-material was conjugated onto the ha layer of nanoparticles B-nanoparticle via click chemistry to achieve a dual - targeting complexed carrier . [SEP]
[CLS] synthesis and the assessment of transfection B-property efficiency I-property , toxicity B-property , and polyplex stability of polymeric materials have been carried out . [SEP]
[CLS] in this context , ulkoski et al . synthesized a library of copolymers with a balance of ionizable B-property diethyl aminoethyl methacrylate or dimethyl aminoethyl acrylate and hydrophobic B-property alkyl methacrylate monomers B-material to disassociate and electrostatically interact with genetic molecules under acidic conditions . [SEP]
[CLS] in this study , ph - responsiveness copolymers self - assembled into micellar nanoparticles B-nanoparticle and entrapped the cargo once titrated to ph 7 . 4 to ensure a controlled ionizability B-property that promoted membrane destabilization within endosomal compartments . [SEP]
[CLS] another strategy for reducing the unwanted carrier uptake and accumulation in tissues is the surface - mediated delivery of genetic molecules via electrospun nanofibers B-nanoparticle . [SEP]
[CLS] therefore , rna - loaded tissue engineering scaffolds , fabricated by electrospinning , have recently been receiving much attention . [SEP]
[CLS] to minimize systemic side effects and enhance gene transfection B-property efficiency I-property , pinese et al . achieved a sustained delivery of sirna - msnp complex , while incorporated into a nanofibrous scaffold . [SEP]
[CLS] poly ( caprolactone ) ( pcl ) was chosen as the main material B-material for scaffold fabrication and two methods had been tested for sirna loading : the first method was the direct fiber surface absorption of the therapeutic molecule using a mussel - inspired bioadhesive , while the second was the direct encapsulation within poly ( caprolactone - co - ethyl ethylene phosphate ) ( pcleep ) nanofibers B-nanoparticle . [SEP]
[CLS] using a similar method , randomly oriented pcleep nanofibers B-nanoparticle were fabricated to encapsulate mirna and sirna for modulating macrophage phenotypes . [SEP]
[CLS] the presence of genetic molecules in the electrospinning solution did not affect the average diameter of the fibers and a sustained release was achieved for at least 30 days . [SEP]
[CLS] it was also demonstrated that the minute changes in average fiber diameter had negligible effects on the variation of mirna release . [SEP]
[CLS] several strategies exist to compensate for the inherent hydrophobicity B-property of pcl fibers . [SEP]
[CLS] specifically , and inspired by mussel adhesion chemistry , polydopamine - coated pcl nanofibers B-nanoparticle showed enhanced cell - substrate interactions and biomolecule immobilizations . [SEP]
[CLS] zhang et al . successfully fabricated a layer - by - layer self - assembling peptide B-material coated on pcl nanofibers B-nanoparticle . [SEP]
[CLS] the proposed system was able to mediate the localized delivery to promote neural regeneration . [SEP]
[CLS] in another study , mirna - gelatin polyplexes were introduced into pcl nanofibers B-nanoparticle . [SEP]
[CLS] as a result , the cell B-property viability I-property and osteogenic differentiation were improved . [SEP]
[CLS] in order to address the shortcomings of common polycationic nanoparticles B-nanoparticle , such as unfavorable charge density , lack of specific cell B-material targeting , and low rna uptake , several strategies have been devised to alleviate these limitations through functionalizing the constructional polymers B-material of nanoparticles B-nanoparticle or polymerizing a new advanced category of bioreducible polymers B-material . [SEP]
[CLS] by using a simple high - throughput synthetic scheme , blersch et al . developed and assessed a nanoparticle B-nanoparticle library of one hundred sixty polymers B-material for the controlled delivery of rna therapeutics . [SEP]
[CLS] michael - type addition chemistry was selected to synthesize polymers B-material with a photocleavable linker . [SEP]
[CLS] the set was further introduced to bisacrylamide and monomers B-material of amine B-material . [SEP]
[CLS] the unreacted acrylamide groups were terminated with an amine B-material to obtain high transfection efficiencies . [SEP]
[CLS] the advantages of the library were a large variety of disulfide bonds that rendered nanoparticles B-nanoparticle stable in physiological conditions and promptly degradable in intracellular reductive environments . [SEP]
[CLS] moreover , polymeric B-nanoparticle nanoparticles I-nanoparticle , with a size ranging from 100 to 500 nm , showed different solubility B-property and hydrophilicity B-property in an aqueous solution . [SEP]
[CLS] six polyplexes of this library were more efficient than commercial lipofectamine in knocking down gfp expression . [SEP]
[CLS] a mixture of di - acrylate and amine B-material monomers B-material was suggested for another library of acrylate - terminated poly ( β - amino ester ) . [SEP]
[CLS] polymers B-material were synthesized by mixing monomers B-material in a dioxane solution with 1 , 8 - diazobicylcoundecene as a catalyst B-property and then precipitated in diethyl ether B-material . [SEP]
[CLS] the result was centrifuged , dried in a vacuum , and dissolved in anhydrous tetrahydrofuran to react with a methoxy - peg amine . [SEP]
[CLS] from this library , a polyplex formulation was detected that increased type i interferon production by 13 times , compared to that of naked dsrna . [SEP]
[CLS] following vaccination , the mentioned carrier - cargo complex enhanced the magnitude , duration , and affinity maturation of antigen - specific antibodies B-material . [SEP]
[CLS] furthermore , the terminal group modification with oligopeptides may suggest a targeted rna therapy . [SEP]
[CLS] to investigate the hydrolysis rate effect on the ph - independent mechanism , another library of poly ( dimethyl aminoethyl ) acrylate ( pdmaea ) copolymers was synthesized with differences in the co - monomer B-material lipophilicity B-property . self - immolative chargealtering carriers can be engineered from the polymerization of dimethyl aminoethyl monomers B-material which can effectively condense rna in its positively charged state , followed by selfcatalyzed hydrolysis and charge inversion for the repelling and final release of the cargo . [SEP]
[CLS] while degrading in an aqueous media , the charge of pdmaea polymeric chains can alter from positive to negative and the polymer B-material releases the nonacrylated monomers B-material . [SEP]
[CLS] with this knowledge , gurnani et al . prepared a library and analyzed the effects of monomer B-material ratio in the copolymeric library and the alteration of charge on mrna delivery . [SEP]
[CLS] examining the biological properties and structure - activity relationship in large polymeric libraries is timeconsuming and costly . [SEP]
[CLS] therefore , automated high - throughput screening is beneficial for the production of multivariate delivery carriers . [SEP]
[CLS] a library of poly ( cba - co - 4 - amino - 1 - butanol ( abol ) ) ( pabol ) polymers B-material was also prepared . [SEP]
[CLS] ranging from 5 to 167 kda , the prepared category contained pabol with different molecular weights , synthesized using an optimized aza - michael polyaddition protocol , a common method for making poly ( amidoamine ) . [SEP]
[CLS] to facilitate the reaction rate , triethylamine was used as a lewis base catalyst B-property . [SEP]
[CLS] the double - bond conversion rate exceeded 99 . 9 % after 4 days of reaction . [SEP]
[CLS] the cytotoxicity B-property and in vitro transfection B-property efficiency I-property of the library were characterized and compared to the commercially available pei ( figure 5c ) . [SEP]
[CLS] the molecular weight - dependent pabols cytotoxicity B-property was lower than that of pei . [SEP]
[CLS] due to the presence of hierarchical structures and steric hindrances , which cause a reduced accessibility of binding sites on high molecular weight pabols to complex self - amplifyng rna ( sarna ) , the range of polymer B-material / rna weight ratios adopted was different from commonly used values . [SEP]
[CLS] in spite of the weight ratios , the nano polyplexes formed were in the range of 100 to 400 nm , while pabol with a molecular weight of more than 5 kda showed a positive surface charge that was adequate for maintaining a sufficient colloidal stability and good cell B-material permeability . [SEP]
[CLS] it is easier to obtain a successful endosomal escape when the desired surface charge is reached with a low polymer B-material loading . [SEP]
[CLS] advanced polymeric nanoparticlespabols with higher molecular weight make this attainable by means of more effective binding sites per chain , which can then increase the binding constant between polymer B-material chains and sarna . [SEP]
[CLS] as a result , more polymers B-material are incorporated into nanoparticles B-nanoparticle , while the surface charge increases . [SEP]
[CLS] transfection B-property efficiency I-property was also enhanced at high pabol molecular weights and plateaued for pabols with molecular weights between 72 and 167 kda . [SEP]
[CLS] unlike pei , pabol is bioreducible and can release sarna via the intracellular gsh reduction of disulfide bonds on its backbone , where the fast release mechanism facilitated a rapid transgene expression . [SEP]
[CLS] the in vivo protein B-material expression of pabol polyplexes was 100 times higher than commercially available peis , while higher percentages of cells B-material expressing sarna both ex vivo in human skin explants and in vivo in mice after intramuscular ( im ) and intradermal ( id ) injections were achieved . [SEP]
[CLS] bioreducibility can also be induced by adding a cystine unit into each repeating B-material unit I-material of the polymer B-material backbone . cystine - containing architectures ensure a low cytotoxicity B-property and facilitate the intracellular disassembly of the cargo . [SEP]
[CLS] all these strategies , although promising , may not yet be able to counteract either the high cytotoxicity B-property and poor biodistribution of carriers , which may affect rna therapy , or the adverse effects of polymer B-material accumulation in cells B-material and impaired tissue functions . [SEP]
[CLS] the undesirable compromise between delivery efficiency and cytotoxicity B-property should be addressed . [SEP]
[CLS] looking at nature for inspiration , shen et al . discovered that a catechin derivative from green tea could ease sirna condensation in carriers made of low molecular - weight polycations with minimum cytotoxicity B-property . [SEP]
[CLS] since these materials are regarded as nontoxic and are produced naturally , " green nanoparticles B-nanoparticle " was the name chosen for these carriers . [SEP]
[CLS] in their work , natural polyphenol ( - ) - epi - gallocatechin gallate ( egcg ) was complexed with sirna to form negatively charged nanoparticles B-nanoparticle and a shell B-material of low molecular weight ε - pll was used as a coating B-material . [SEP]
[CLS] it was postulated that egcg could protect sirna from nuclease degradation , while bacterial manufactured pll ensures an efficient internalization with low cytotoxicity B-property ( figure 5d ) . [SEP]
[CLS] coated green nanoparticles B-nanoparticle maintained an average hydrodynamic size of 127 nm with a 78 % complexing efficiency . [SEP]
[CLS] virus - like particles ( vlps ) account for another group of materials that are attractive for rna delivery due to their suitable size , uniform structure , controllable assembly , and easy modification . [SEP]
[CLS] this method is based on an artificial pseudovirus progress , using multiprotein structures that resemble viruses in many characteristics but lack any genetic material B-material . [SEP]
[CLS] by using the natural infectious potential of viruses for gene delivery to living cells B-material , vlps are excellent vaccine carriers . [SEP]
[CLS] the remnant of the helical or icosahedral envelope and capsids of the virus in the vlp structure can efficiently encapsidate the genetic molecule within the virions of the host and deliver it to the targeted cells B-material . [SEP]
[CLS] viral infection or transfection , recombinant techniques , and cell - free systems are the three main categories of viral capsid production . [SEP]
[CLS] the first category describes a method for harvesting the empty capsids being fabricated as by - products of the infected cells B-material . [SEP]
[CLS] one interesting vlp , hepatitis b vlp ( hbv vlp ) , is used for the development of vaccine and drug delivery platforms . [SEP]
[CLS] the exterior shell B-material of hbv vlp can be easily modified with targeted moieties , such as site - specific protein B-material conjugation with spycatcher , while surface spikes are fourhelix bundles of the hepatitis dimer . [SEP]
[CLS] the core B-material component maintains an icosahedral structure with a size of 34 nm and assembles from 240 copies of hepatitis b proteins B-material . [SEP]
[CLS] hartzell et al . proposed a modular nanocarrier platform using hbv vlp and inserted spycatcher into the c / e1 loop . [SEP]
[CLS] the high - yield production of particles took place in escherichia coli ( e . coli ) shake flasks . [SEP]
[CLS] to achieve a modular platform capable of an easy customization with different targeting and detection components , hbv - spycatcher was decorated with 240 functional moieties for different applications . [SEP]
[CLS] in another study , the conjugated form of truncated hepatitis b core B-material antigen ( thbcag ) vlp with folic B-material acid I-material was used to benefit from attained precise targeting property in comparison with the native form of this viral capsid . [SEP]
[CLS] the conjugation process was performed by the mediation of sulfo - nhs and 1 - ethyl - 3 - ( 3 - dimethylaminopropyl ) carbodiimide ( edc ) that activates the carboxylate B-material groups I-material of folic B-material acid I-material and couples the surface primary B-material amine I-material groups of vlp to activated ligands . [SEP]
[CLS] the urea dissociation and association technique was used prior to folic B-material acid I-material modification in order to load the genetic molecules inside the icosahedral capsid . [SEP]
[CLS] the internal c - terminal of hepatitis b capsid has an arginine - rich section with positive charge that can complex the negative therapeutic molecules . [SEP]
[CLS] folate B-material receptors I-material are abundant in cancerous tissues , and nanocarriers modified with folic B-material acid I-material are able to transfect the malignant cancers with high efficiency . [SEP]
[CLS] therefore , the targeted delivery of short hairpin rna , encapsidated in thbcag vlp , to hela cells B-material was able to downregulate anti - apoptotic bcl - 2 expression and hampered the proliferation of cancerous cells B-material . [SEP]
[CLS] to generate type 1 helper t ( th1 ) immune responses , activate the desired intracellular signaling pathways , and shift dendritic cell B-material ( dc ) responses to prompt a specific outcome , alam et al . investigated a dc - targeting strategy using vlps . [SEP]
[CLS] synthetic glycans were linked to a vlp to harness different factors using a structurally well - controlled platform . moreover , to address the selection method of a proper dc targeting agent , different criteria - including identity optimization , spatial distribution , and valency - were taken into account for an efficient antigen uptake and dc activation . [SEP]
[CLS] therefore , a highly stable particle , derived from the bacteriophage qβ , was used and its surface lysine B-material was modified for ligand conjugation . [SEP]
[CLS] it was demonstrated that the density and the nature of vlp ' s binding ligands play a crucial role in signaling , especially in aryl mannoside - induced proinflammatory signalling pathways and cytokine expression , leading to the induction of th1 cells B-material in vivo . [SEP]
[CLS] another well - studied vlp is a derivative of the ms2 bacteriophage ( ms2 vlp ) , which takes advantage of a chemically robust genetic assembly . [SEP]
[CLS] ms2 vlp comprises 180 copies of identical coat B-material proteins B-material ( cp ) . [SEP]
[CLS] the coat B-material protein B-material is harvested from bacterial media , such as e . coli . [SEP]
[CLS] the c - terminus of q - beta vlp was engineered to display binding peptides B-material that can evoke an immune response when it resides in its native viral form . [SEP]
[CLS] furthermore , to present epitopes on vlps , a nonfusion technique was investigated to increase the diversity of ms2 vlp peptide B-material insertion sites . [SEP]
[CLS] a mutation - prompt region known as the " fg loop " was detected . [SEP]
[CLS] by inserting all possible three - residue peptides B-material into the fg loop of ms2 cp , the " systematic mutagenesis and assembled particle selection " method was adopted to elicit the sequencing of the selected peptide B-material insertion library . [SEP]
[CLS] positive controls for molecular assays are used in reverse transcription B-event - polymerase chain reactions ( rt - pcrs ) , which serve as a detection tool for covid - 19 infection to validate each test with high accuracy . recently , to overcome the related limitations of positive controls , including cold - chain distribution requirements , a biomimetic virus - like particle was devised from a bacteriophage and a plant virus to encapsidate a sars - cov - 2 detection module . [SEP]
[CLS] two different perpetrations , namely , i ) a module capable of target detection during the in vivo coexpression and ii ) module assembly , were used to obtain chimeric vlps . [SEP]
[CLS] the vlp positive controls produced were able to mimic sars - cov - 2 packaged rna and remained noninfectious . [SEP]
[CLS] this important consideration was demonstrated by the deletion of the ribosome binding site and appendage of qβ hairpin to achieve an efficient in vivo reconstitution . [SEP]
[CLS] furthermore , the scalability , stability , and the broad use of these positive controls were demonstrated . [SEP]
[CLS] qβ vlp was also used to eradicate ovarian cancer . [SEP]
[CLS] by implementing an mg - based micromotor as an active and dynamic qβ vlp delivery platform in the weak acidic peritoneal cancer ovarian fluid , a motor - based therapy was applied . [SEP]
[CLS] interestingly , motor propulsion was engineered to be a result of the spontaneous reaction of mg in acidic environments , which generates hydrogen B-material bubbles . [SEP]
[CLS] it was postulated that motor propulsion could enable the delivery of the payload in the tumor B-material microenvironment , while enhancing the local distribution and retention time of the carrier ( figure 6a ) . [SEP]
[CLS] in this study , qβ vlp was obtained from e . coli bl21 ( de3 ) cells B-material using a well - documented method and labeled with cyanine3 dye using nhs - activated esters which target surface lysine B-material on the viral coat B-material proteins B-material . [SEP]
[CLS] magnesium B-material microparticles were used to construct micromotors and surface coated with tio2 and two layers of plga for further protection of the cargo via atomic deposition method . [SEP]
[CLS] chitosan B-material coating B-material was used as another protective shield to coat B-material the qβ - loaded mg - based micromotors as an outermost layer . [SEP]
[CLS] using this autonomous propulsion system , the qβ vlp ' s cargo possessed immunostimulatory characteristics , and qβ vlps distribution and particle - macrophages interactions enhanced [SEP]
[CLS] nanomedical vlp based on plant viruses is another burgeoning area . [SEP]
[CLS] their easy and inexpensive production , inability to infect mammals , immunogenic B-property properties , and the ability to induce antitumor responses in the tumor B-material microenvironment are some of the advantages of adopting plant viruses by molecular farming in plants . [SEP]
[CLS] plant molecular farming uses transgenic plants to capture naturally occurring empty capsids of plant viruses or particles consisting of the reassembled coat B-material protein B-material to produce plant virus - derived carriers . [SEP]
[CLS] by using this method and selecting a plant - derived cowpea chlorotic mottle virus ( ccmv ) , cpg - oligodeoxynucleotides ( odns ) ( cpg - odns ) were target - delivered to tumor B-material - associated macrophages . [SEP]
[CLS] it was demonstrated that ccmv is more able to package rna than odns . [SEP]
[CLS] biddlecome et al . investigated the encapsidation of a self - amplifying mrna in ccmv vlps and its delivery to activate the dcs . [SEP]
[CLS] the premature dcs antigen - presenting cells B-material of immune system activated after being internalized by vlps and expressed maturation markers . [SEP]
[CLS] furthermore , and in order to increase the uptake and activation of dcs , ccmv vlps , carrying non - translated rna , were first injected into mice and the blood serum of immunized animals was collected prior to exposing the immune cells B-material with mrna . [SEP]
[CLS] the results of dcs incubated B-technique with anti - vlp suggested improved maturation and increased level of eyfp - replicon mrna . [SEP]
[CLS] bromoviruses are another plant vlps that can be used in biomedical applications due to their ease of production and handling . [SEP]
[CLS] a brome mosaic virus ( bmv ) , which falls in this category , has a simple structure with the size of 28 nm , 180 identical proteins B-material , and an icosahedral shell B-material . [SEP]
[CLS] bmv has a region at its n - terminal which provides a positive internal surface and through electrostatic interactions is capable of rna encapsidation inside the vlp . [SEP]
[CLS] in a study , the potentials of bmv vlp as a nanocarrier of sirna were explored , and different aspects of this plant bromoviruses such as biocompatibility B-property , internalization into breast tumor B-material cells B-material and gene silencing efficiency were compared against ccmv . [SEP]
[CLS] it is noteworthy to mention that the immediate resemblance of size , shape , and protein B-material quantity of bmv and ccmv should be considered . the lower immunogenicity B-property observed for bmv was promising , and the in - vivo results indicated a successful inhibition of gfp expression and tumor B-material growth . [SEP]
[CLS] moreover , both carriers were internalized by the targeted cells B-material short of the necessity of adding cell targeting ligands to vlps and despite the low amount of vimentin receptor B-material on the surface of the cancerous cells B-material . [SEP]
[CLS] these findings hold important prospects for vaccine delivery , when the vlp design is optimized with optimal dynamic immune responses . [SEP]
[CLS] rna delivery based on virus - like and lipid B-material nanocarriers . [SEP]
[CLS] virus - like particlesa ) active delivery of vlps by qβ - motors for enhanced therapy of ovarian cancer : fabrication , in vivo administration , and in vivo actuation of qβ - motors . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , wiley - vch . [SEP]
[CLS] b ) deconvoluting the structure of lipid B-nanoparticle nanoparticles I-nanoparticle for mrna delivery . [SEP]
[CLS] different morphologies of the unilamellar perimeter , " onion " or multilamellar perimeter , bilamellar perimeter , polymorphic or faceted , and polymorphic and multilamellar were achieved . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , american chemical society . [SEP]
[CLS] lipid B-nanoparticle nanoparticles I-nanoparticle ( lnps ) are a large , popular class of nonviral carriers for rna delivery . [SEP]
[CLS] first introduced in 1989 , 1 , 2 - di - o - octadecenyl - 3 - trimethylammonium - propane ( dotma ) became one of the two most widely used cationic B-material liposomes B-nanoparticle for therapeutic delivery , the other being 1 , 2 - dioleoyl - 3 - trimethylammonium - propane ( dotap ) . [SEP]
[CLS] these synthetic lipids B-material differ from their classical structure , which consists of an ester - linked glycerol head and hydrocarbon tail . [SEP]
[CLS] for instance , dotma has two unsaturated aliphatic hydrocarbon chains , ether B-material - linked to a quaternary amine B-material . [SEP]
[CLS] the new class of synthetic lipids B-material is more efficient in encapsulating genetic molecules when forming spherical - shaped liposome B-nanoparticle vesicles or lipoplexes . [SEP]
[CLS] while conventional liposomes B-nanoparticle are composed of only lipid B-material bilayers I-material , the hybrids of lipids B-material and polymers B-material ( lpns ) can form more stable nanoparticles B-nanoparticle with less leakage of the cargo . [SEP]
[CLS] benefiting from stealth property , longer in vivo circulation time can also be provided if an outer lipid - peg layer is incorporated in the design of lpns . [SEP]
[CLS] quite recently , and as an illustration , new lipoplexes made of sirna and dotap have been loaded into plga hybrid nanoparticles B-nanoparticle for the pulmonary delivery of genetic molecules to treat severe lung disorders . [SEP]
[CLS] defined by their hydrophobic B-property or amphiphilic B-property nature , lnp formulation is versatile when using different molecular structures of lipids B-material . [SEP]
[CLS] an ionizable B-property - cationic B-material lipid B-material or lipidoid compound , cholesterol , a phospholipid ( helper lipid B-material ) , and a peg - conjugated lipid B-material are the four primary components of lnps with the role of complexing rna , enhancing the stability of particles , facilitating endosomal escape , and preventing particle aggregation , respectively . [SEP]
[CLS] peg - conjugated lnps benefit from reduced protein B-material opsonization and increased circulation times where the peg layer provides hydrophilic B-property steric hindrance and acts as a stealth coating B-material . [SEP]
[CLS] unfortunately , the issues related to peg activity and safety are still unclear . [SEP]
[CLS] low transfection potency and cellular uptake are two of the drawbacks associated with the presence of peg in lnp formulation . [SEP]
[CLS] to deal with this contradiction , a dynamic pegylation strategy is being used via nanoparticle B-nanoparticle surface decoration with cleavable ph - responsive peg . [SEP]
[CLS] a notable feature of this strategy is sequential - targeting which involves a two - stage consequent passive and active targeting . [SEP]
[CLS] the latter occurs when the ligands of a peg - surface - functionalized nanoparticle B-nanoparticle are being exposed as a result of ph - triggered de - pegylation in acidic media . [SEP]
[CLS] the exposed ligands , at that point , perform an active targeting . [SEP]
[CLS] nevertheless , this strategy is well suited for cancer therapies , where the tumor B-material microenvironments are slightly acidic which has prompted many studies considering peg substitution with amino acid - derived polypeptides B-material . [SEP]
[CLS] for instance , nogueira et al . selected polysarcosine ( psar ) as a peg substitute due to its stealth - like characteristic . [SEP]
[CLS] lipid B-material nanoparticlespsar is made of endogenous amino B-material acid I-material sarcosine ( n - methylated glycine B-material ) repetitive units and shows no acute immune response and low immunogenicity B-property . [SEP]
[CLS] other amino B-material acid I-material - based peg substitute peptides B-material are gemini surfactants B-property derived from serine B-material . [SEP]
[CLS] three variants of this surfactant B-property , namely ( 12ser ) 2n12 ( amine B-material derivative ) , ( 12ser ) 2coo12 ( ester derivative ) , and ( 12ser ) 2con12 ( amide B-material derivative ) , were investigated for sirna delivery in combination with monoolein , a neutral single - tailed unsaturated lipid B-material . [SEP]
[CLS] the selection of monoolein was based on its potential to increase the cationic B-material surfactant B-property transfection B-property efficiency I-property by improving both the system ' s stability in physiological conditions and the endosomal escape ability . [SEP]
[CLS] despite all the reported shortcomings of peg in lnp formulation , research into the chemical structure of this polymer B-material has continued . [SEP]
[CLS] linear - dendritic peg lipids B-material can boost the in vivo delivery of sirna and increase the presence of lnps in blood circulation . [SEP]
[CLS] the unusual topology of peg lipids warrants diverse chemical and biophysical properties of materials . [SEP]
[CLS] to perform a systemic investigation into the effects of the hydrophobic B-property domains of peg lipids B-material on lnp formulation , cellular uptake and trafficking , and in vivo rna delivery , a series of linear - dendritic peg lipids B-material with different lipid B-material lengths and generation was synthesized . [SEP]
[CLS] with a sirna encapsulation B-property efficiency I-property of up to 90 % and a size ranging from 50 to 100 nm , the similarities between lnps and synthesized peg lipids B-material were established , while only first - generation and second - generation peg lipids B-material were able to deliver sirna effectively , in vitro and in vivo . [SEP]
[CLS] bioreducible lnps were formulated for the systematic simultaneous delivery of crispr / cas9 for an efficient and very rapid genome editing in vitro and in vivo . disulfide bondcontaining hydrophobic B-property tails of these bioreducible lnps were synthesized by heating amine B-material and acrylates or acrylamides . [SEP]
[CLS] for the delivery of sirnas into leukocytes , different linker moieties such as hydrazine , hydroxylamine , and ethanolamine were selected to synthesize novel ionizable B-property amino lipids . [SEP]
[CLS] the transfection of leukocytes is reported to be a difficult endeavor . [SEP]
[CLS] therefore a lipid B-material , beta7 integrin , was formulated into lnps as a leukocyte - selective targeting B-property agent I-property . [SEP]
[CLS] the next - generation branched - tail , ionizable B-property , lipid - like ( lipidoid ) material B-material was also used in a recent study . [SEP]
[CLS] lipidoids were synthesized from amine 3 , 3 ′ - diamino - n - methyldipropylamine and reacted with the tail isodecyl acrylate and further combined with dioleoylphosphatidylethanolamine ( dope ) , cholesterol , and c14 - peg2000 to form lnps of a size of 124 nm prior to complexation with mrna . [SEP]
[CLS] the loaded carriers were compared with the lnps organized by ionizable B-property lipid B-material dlin - mc3 - dma , the first fda - approved lipid B-material in lnp formulation , and achieved a threefold higher total organ expression while their efficacy was sustained with repeated dosing . [SEP]
[CLS] the key finding was that antibodies B-material were not formed in response to the proposed lnps , as this issue prevented the repeated dosing of other potent materials . [SEP]
[CLS] the delivery of a small rna , in the range of 30 or fewer nucleotides , has been extensively studied by conventional lnps . [SEP]
[CLS] unfortunately , for larger rna containing more than 100 nucleotides , the lnp formulation should be revised . [SEP]
[CLS] this can be done through the substitution of cholesterol in lnp with other types of cholesterol derivatives . [SEP]
[CLS] the deconvolution of the size , shape , and internal structure of lnps can be achieved by the replacement of phospholipids , peg - lipids B-material , and ionizable B-property lipids B-material . [SEP]
[CLS] a major component of lnps , cholesterol , contributes to nanoparticle B-nanoparticle morphology and affects gene delivery . [SEP]
[CLS] eygeris et al . formulated lnps with natural phytosterols and observed different degrees of rigidity and crystallinity ( figure 6b ) . [SEP]
[CLS] these c24 alkyl derivatives of cholesterol gave rise to a polymorphic shape with various degrees of multi - lamellarity , lipid B-material partitioning , thermal response , and more than 90 % encapsulation B-property efficiency I-property . [SEP]
[CLS] selected phytosterol analogs were fucosterol ( fuco ) , beta - sitosterol ( sito ) , campesterol ( camp ) , and stigmastanol ( stig ) , all of which have at least one additional carbon B-material atom I-material in the c24 aliphatic chain compared to cholesterol . [SEP]
[CLS] the intriguing multi - lamellar morphology can be tuned by the addition of methyl and ethyl groups to the c24 alkyl tail of the cholesterol backbone where lipid B-material partitioning is induced by the addition of a double bond . [SEP]
[CLS] this study demonstrated that even minute changes in the chemical structures of cholesterol counterparts could significantly affect the lipid B-material packing capability . [SEP]
[CLS] considering that small - sized lnps have theoretically a better tissue penetration , microfluidic mixing made possible the generation of small - sized lnps . [SEP]
[CLS] the idea is to create the smallest thermodynamically stable aggregates of lnp achievable . [SEP]
[CLS] unfortunately , these small - sized lnps are highly sensitive to serum or biological fluids . [SEP]
[CLS] to overcome the poor stability and weak intracellular trafficking of small - sized lnps , sato et al . studied the hydrophobic B-property scaffolds of ph - sensitive cationic B-material lipids B-material with various lengths and shapes . [SEP]
[CLS] the decreased potency of small - sized lnps is due to both the diffusion of lipid B-material components from lnps and the adsorption of proteins B-material on their surface . [SEP]
[CLS] interestingly , in this study , the helper lipid B-material was replaced with egg sphingomyelin and formed 22 nm smaller lnps . [SEP]
[CLS] a series of examinations concerning the properties of scaffolds with higher molecular weights possessing 18 carbons B-material and more were conducted . [SEP]
[CLS] it was demonstrated that long - chain , linear scaffolds with hexanol linkers conjugated to fatty acids improved the strength of small - sized lnps . [SEP]
[CLS] a greater potency can also be achieved by combining a ph - sensitive cationic B-material lipid B-material and a phosphocholine - containing phospholipid . [SEP]
[CLS] moreover , different scaffold lengths can reinforce a weak endosomal escape . [SEP]
[CLS] to further assess the complexing role of amino lipids B-material in lnp formulation for the delivery of sirna , anderluzzi et al . designed four lipid - based nanocarriers including liposomes B-nanoparticle , solid lnps , polymeric B-nanoparticle nanoparticles I-nanoparticle , and emulsions . [SEP]
[CLS] all nanoparticles B-nanoparticle had either dotap or dimethyl dioctadecyl ammonium bromide B-material ( dda ) in their formulation , while microfluidic mixers or microfluidization were used to prepare them . [SEP]
[CLS] these scalable manufacturing methods have the benefit of producing synthetic particles with consistent size and biophysical properties . [SEP]
[CLS] it is worth mentioning that the in vitro antigen expression result was not in correlation with the in vivo immune response , thus indicating the insufficiency of performing in vitro assays exclusively . [SEP]
[CLS] this highlighted how reaching the clinic stage requires taking careful and admonitory steps . [SEP]
[CLS] researchers should acquire a greater understanding of the factors controlling the correlation between in vivo and in vitro assays , because the efficacy of lnp carriers may vary in either condition . [SEP]
[CLS] as was the case with the recent focus on natural substances in polymeric B-nanoparticle nanoparticles I-nanoparticle , lnp formulation can also benefit from natural cues , especially for targeted delivery . [SEP]
[CLS] these natural signals diversify chemical and biological aspects such as membrane fluidity , permeability , and cell B-material signalling . [SEP]
[CLS] therefore , mrna was delivered to the lungs by using natural lipids B-material originating from the cell B-material membrane of plants and microorganisms . [SEP]
[CLS] a conventional structural lipid B-material , distearoylsn - glycero - 3 - phosphocholine ( dspc ) , was replaced with glycolipids , which have sugar moieties in their head groups and originate from the chloroplast of plant cells B-material . [SEP]
[CLS] in vivo and in vitro lnp transfection results were poorly correlated . [SEP]
[CLS] it was demonstrated that natural cholesterol analogs enhance the endosomal escape of lnps , while the presence of natural structural lipids B-material on nanoparticle B-nanoparticle surfaces forms a corona when dispersed in biological fluids . [SEP]
[CLS] mrna delivery to t cells B-material was studied by creating a vast library of lnps . [SEP]
[CLS] structural analogs of immune - cell targeted ionizable B-property lipid B-material materials were synthesized via michael addition chemistry . [SEP]
[CLS] alkyl chains terminated with epoxide groups were reacted with polyamine cores B-material of varying lengths . [SEP]
[CLS] using microfluidic chips , a combination of 24 ionizable B-property lipids B-material , cholesterol , helper lipids B-material , and peg - lipids B-material were used to encapsulate mrna and form lnp complexes ranging from 51 to 97 nm . [SEP]
[CLS] screening of the process detected improved mrna delivery for seven lnp formulations , compared to commercially available lipofectamine . [SEP]
[CLS] no significant correlation was observed among lnp size , concentration of the genetic molecules , or the pka of ionizable B-property lipid B-material with the enhanced delivery of the cargo . [SEP]
[CLS] furthermore , lnps containing purified saturated ionizable B-property lipids B-material improved mrna delivery over that of crude lipid B-material formulations . [SEP]
[CLS] lnps were also used to deliver mrna for hemophilia treatment . in this investigation , lipid B-material components consisting of ionizable B-property lipid B-material distearoylphosphatidylcholine , cholesterol , and peg - lipid B-material were mixed with synthesized mrnas to synthesize mrna lnps . [SEP]
[CLS] lnp complexes of sizes smaller than 100 nm , more than 80 % rna encapsulation B-property efficiency I-property , and less than 10 endotoxin units were obtained . [SEP]
[CLS] thanks to a bioluminescence B-property signal in the liver and a weak or absence of signal in the spleen or other organs , it was suggested that lnps efficiently delivered mrna to the liver . [SEP]
[CLS] repeated injections of lnps , however , induced the inhibitor formation of short - lived factors . [SEP]
[CLS] in the case of long - term storage of lnps carriers , specifically covid - 19 mrna vaccines , little is known about the physical stability of lipid B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] zhao et al . set out to perform a systematic study of various conditions , such as temperature and physical state ( aqueous , freezing , or lyophilized ) , for the long - term storage of lipidoid nanoparticles B-nanoparticle complexed with rna . [SEP]
[CLS] it was demonstrated that the addition of 5 % sucrose or trehalose to lnp formulation could extend mrna delivery efficiency for at least 3 months in the liquid nitrogen B-material storage condition . [SEP]
[CLS] research on lnp carriers achieved a significant milestone after patisiran gained fda approval . [SEP]
[CLS] a new wave of inspiring studies dedicated to embracing the natural substances in nanocarrier formulation was started , kindling new hope for the future . [SEP]
[CLS] the lesson learned from covid - 19 vaccine development evokes the reorganization and improvement of nanocarriers . [SEP]
[CLS] the current progress rate of the academic and industrial world in the design of suitable delivery systems ensures the development of an impactful rna therapy in the near future . [SEP]
[CLS] the development of advanced lnps with state - of - the - art ionizable B-property lipids B-material components helped to break down the tradeoff between toxicity B-property and transfection efficacy , further resulting in a series of compatible lnps with a variety of rna cargoes . [SEP]
[CLS] simultaneously , automated high - throughput screening combined with modern synthesis reduced the assessment and evaluation period of lnp libraries and allowed rapid and active responses to the crisis such as the covid - 19 pandemic . [SEP]
[CLS] in our opinion , all these promising features endorse lnps as the primary choice for rna delivery . [SEP]
[CLS] cutaneous wound healing is of paramount importance in restoring the integrity and proper function of injured skin . [SEP]
[CLS] impaired wound healing can be harmful to human health or even life - threatening . [SEP]
[CLS] it represents a critical global healthcare issue due to an aging population and sharp rise in the incidence of diabetes and obesity worldwide . [SEP]
[CLS] wounds heal following a specific , sequenced process : i ) the inflammatory phase , which is characterized by hemostasis and inflammatory response ; ii ) the proliferation phase , including the formation of new blood vessels , granulation tissue , epithelialization , and collagen deposition ; iii ) the remodeling phase , in which the organized wound matrix breakdown and synthesis of the new extracellular matrix take place . [SEP]
[CLS] these stages can overlap over time , while the remodeling phase takes longer , additional months even after the wound has closed . [SEP]
[CLS] as said , the above - mentioned phases may overlap ; however , not all phases will be reached if any of these stages are disrupted . [SEP]
[CLS] despite the development of various wound dressings which , for example , provide moisture balance in the wound and protect it from infection , research in this field is still ongoing and very intense . [SEP]
[CLS] one of the emerging technologies that may have a significant impact on the healing process is gene therapy . [SEP]
[CLS] at its origin , gene therapy aimed to modify the human genome to obtain gene improvement . [SEP]
[CLS] the first clinical trials in the 1990s brought about an eruption of subsequent research , accompanied by the discovery of mirnas in 1993 , first identified in the nematode caenorhabditis elegans . [SEP]
[CLS] these short endogenous non - coding molecules mediate the post - transcriptional regulation of gene expression . [SEP]
[CLS] unlike growth factors with a short half - life , rna delivery offers an alternative that promotes cellular activity for an extended period . [SEP]
[CLS] other genetic therapies explored for wound healing include sirna and plasmid dna delivery . [SEP]
[CLS] as many groups showed , the loss of the dicer enzyme , which plays a vital role in short regulatory rna biogenesis , leads to delayed wound healing . [SEP]
[CLS] moreover , the wound healing process involves changes in the expression of individual mirnas in a specific phase of wound healing . [SEP]
[CLS] the abnormal regulation of mirnas plays a crucial role in transforming a wound into a chronic condition . [SEP]
[CLS] despite significant progress in this field , our knowledge of non - coding genes and their effect on wound healing is still limited . [SEP]
[CLS] several mirnas were identified as playing an important role in each phase of wound healing and scar formation . [SEP]
[CLS] during the inflammatory response , mir - 146a , mir - 155 , mir - 132 , mir - 21 , mir - 125b , and mir - 223 , among others , were identified as playing significant roles . [SEP]
[CLS] for example , mir - 21 , mir - 155 , mir - 99 , and mir - 210 expressions in the proliferation phase , and mir - 29a , mir - 29b , mir - 29c , and mir - 192 / 215 expressions in remodeling , were found to be upregulated or downregulated . [SEP]
[CLS] since mirnas were identified as promising mediators in wound healing , they are attractive candidates for a broad range of innovative applications . [SEP]
[CLS] the most significant advantage of rna therapy is that , even though rna is absent in the plasma , it can exert its functions in cells B-material due to the high biological half - lives . [SEP]
[CLS] mir - 146a and mir - 146b are negative regulators of immune and inflammatory processes in both tissue - resident and specialized immune cells B-material , through the regulation of toll - like receptor B-material ( tlr ) signalling and cytokine responses . [SEP]
[CLS] one of the strategies includes targeting mir - 146a downregulation , which possibly contributes to chronic inflammation and , lastly , to delayed healing in diabetic wounds . [SEP]
[CLS] this mir ' s impact is multidirectional and includes the stimulation of macrophages to reduce the production of ros and promote the m2 phenotype . [SEP]
[CLS] other important rnas impacting the inflammatory phase are mir19 and mir - 20 , which regulate keratinocyte inflammatory response . [SEP]
[CLS] these two mirs decreased the tlr3 - mediated nf - κb activation by targeting shcbp1 and sema7a , respectively , reducing the production of inflammatory chemokines and cytokines by keratinocytes . [SEP]
[CLS] in vivo mouse models of type 2 diabetes showed significantly accelerated wound closure ( figure 7a ) and low detection of both mirs in other organs ( figure 7b ) . [SEP]
[CLS] mir19b is also involved in the regulation of the tissue factor , which is a primary initiator of blood B-event coagulation I-event . [SEP]
[CLS] a high level of hypoxia in chronic ischemic wounds induces the expression of mir - 210 . [SEP]
[CLS] this was found to inhibit keratinocyte proliferation and impair ischemic wound closure in a murine model by targeting cell B-material - cycle regulatory protein B-material e2f transcription B-event factor 3 ( e2f3 ) . [SEP]
[CLS] in ecs , mir - 210 promotes angiogenesis B-event by targeting ephrin - a3 ( efna3 ) . [SEP]
[CLS] after delivering lipid B-nanoparticle nanoparticles I-nanoparticle encapsulating the mir - 210 inhibitor into murine ischemic skin wounds , the time for wound closure was significantly reduced , thus demonstrating that mir - 210 is a promising therapeutic target for improving wound healing . [SEP]
[CLS] the microrna - 200b / c - 3p expression is abundant in the intact epidermis ; in skin wounds ; however , it decreases to a considerable extent . [SEP]
[CLS] as identified in silico and confirmed by luciferase reporter assay , rac1 is a mir - 200b / c - 3p target . [SEP]
[CLS] forced mir - 200b / c - 3p expression repressed rac1 and inhibited keratinocyte migration and re - epithelialization in a mouse back skin full - thickness wound healing model . [SEP]
[CLS] the few examples described above suggest that micrornas are fine - tuning regulators that contribute to the highly coordinated wound healing process . [SEP]
[CLS] in most studies devoted to finding the role and mechanism of single mirna , direct injections of naked mirna are used in selected wound areas , for example , the wound edge . [SEP]
[CLS] as was previously described , however , such administration routes entail many limitations , such as low transfection efficacy and degradation . [SEP]
[CLS] therefore , most gene therapy systems use vectors to facilitate the access of nucleic B-material acid I-material into target cells B-material . [SEP]
[CLS] two significant challenges in applying rna are i ) the prevention of degradation by nucleases , and ii ) delivery to specific target sites . [SEP]
[CLS] gene delivery systems may be classified as either viral or non - viral . [SEP]
[CLS] both approaches have been extensively investigated in various models of wound repair . [SEP]
[CLS] however , nonviral vector delivery systems for wound treatment have many advantages over viral - based systems . [SEP]
[CLS] these advantages include no inflammation or infection risk , simplicity , and low cost . [SEP]
[CLS] although non - viral vectors are considered promising vehicles B-material for gene therapy , they are not without faults . [SEP]
[CLS] for example , the low transfection B-property efficiency I-property is a significant disadvantage for clinical use . [SEP]
[CLS] this results from the limited ability of nucleic B-material acids I-material to penetrate cell B-material membranes , due to their negative charge and high molecular weight . [SEP]
[CLS] however , numerous attempts are being made to develop nucleic B-material acid I-material delivery systems that are efficient , safe , and specific for targeting cells B-material to induce the desired longlasting therapeutic effect . [SEP]
[CLS] chitosan B-material is a natural cationic B-material polysaccharide B-material that is used extensively in the formulation of wound dressings due to its antimicrobial B-property properties . [SEP]
[CLS] its functional properties , such as bioactivity , solubility B-property , swelling ratio , and biodegradation B-property , are influenced by the degree of acetylation . [SEP]
[CLS] in particular , chitosan B-material has been studied as a carrier for gene delivery due to its positive charge that permits easy complexing with negatively charged mirna or sirna . [SEP]
[CLS] castleberry et al . developed a self - assembled wound dressing made of a nylon bandage with alternating metalloproteinase - 9 sirna ( simmp - 9 ) matrix and chitosan B-material coatings B-material . [SEP]
[CLS] the sustained delivery of sirna lasted up to 2 weeks in vitro and in vivo . [SEP]
[CLS] released sirna downregulated mmp - 9 levels to 20 % and reduced mmp - 9 activity by 60 % compared to untreated tissue in a diabetic mice model . [SEP]
[CLS] hydrogels have shown great potential in biomedical applications , thanks to their high - water B-material content . [SEP]
[CLS] they are non - toxic B-property and non - inflammatory I-property ; moreover , they are biodegradable B-property , and have viscosity B-property and elasticity properties comparable to those of the surrounding soft tissues . [SEP]
[CLS] finally , in the frame of rna or dna delivery , they can be injected to provide a local and sus - tained release . [SEP]
[CLS] for these reasons , hydrogels have been extensively studied in vitro and in vivo . [SEP]
[CLS] cell B-material - penetrating peptides B-material ( cpps ) are a broad group of short peptides B-material that translocate across cell B-material membranes and can make non - covalent complexes with double - stranded nucleic B-material acids I-material driven by ionic interactions . [SEP]
[CLS] cpps can be used to deliver potentially therapeutic molecules , including dna , sirna , and also mirna [SEP]
[CLS] yan et al . developed collagen / gag scaffolds containing mmp - 9 - targeting sirna ( simmp - 9 ) to advance diabetic foot ulcer ( dfu ) healing . [SEP]
[CLS] a novel cell B-material - penetrating peptide B-material ( cpp ) - rala was used to protect the simmp - 9 from degradation and prolong the effects of this therapy by forming rala - simmp - 9 complexes . [SEP]
[CLS] the cellular uptake of the cell - penetrating cationic B-material peptide B-material complex with simmp - 9 exceeded 60 % . [SEP]
[CLS] after that , it contributed equally to the reduction in mmp - 9 gene expression in a low glucose culture . [SEP]
[CLS] in an in vitro model of dfu , mmp - 9 was downregulated by around 90 % ( figure 7c ) . [SEP]
[CLS] taken together , combining the performance of rala - simmp - 9 complexes with the proven tissue regeneration capacity of collagen / gag scaffolds , the presented scaffold could be a powerful candidate for dfu healing . [SEP]
[CLS] nickfect ( nf ) and pepfect ( pf ) types of cell B-material - penetrating peptides B-material were used to deliver mir - 146a , which is a negative regulator of inflammatory response in both tissue - resident and specialized immune cells B-material . [SEP]
[CLS] both peptides B-material supported the delivery of fluorescently B-property labeled mir - 146a into keratinocytes ( kcs ) and dendritic cells B-material ( dcs ) . [SEP]
[CLS] while in case of kcs they were equally effective , the nfs were more efficient in dcs as assessed by measuring downregulation of mir - 146a - influenced genes . [SEP]
[CLS] in an in vivo mouse model of irritant contact dermatitis , injected nf71 : mir - 146a nano complexes confirmed the suppression of inflammatory responses . [SEP]
[CLS] this was evidenced by the reduced ear swelling and the downregulation of il - 1β , il - 6 , il - 33 , and tnf - α . [SEP]
[CLS] copyright 2021 , the authors . published by elsevier . [SEP]
[CLS] c ) in vitro 3d transfection of human fibroblasts with rala - simmp - 9 complexes visualized with a confocal fluorescence B-property microscope . [SEP]
[CLS] these images clearly confirm that the rala - simmp - 9 complexes are able to associate with and enter the cells B-material . [SEP]
[CLS] green , actin ; blue , cell B-material nucleus ; red , rala - simmp - 9 complexes . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , elsevier . [SEP]
[CLS] another natural drug carrier system with a promising outlook in wound healing is provided by extracellular vesicles ( evs ) . [SEP]
[CLS] these cell - derived natural products mediate cell - to - cell communication by transporting various bioactive molecules , including nucleic B-material acids I-material . [SEP]
[CLS] their intravenous administration leads to their rapid elimination from blood circulation ; however , it is possible to encapsulate them in other biomaterials such as hydrogels , thus achieving a prolonged retention time , up to even 5 days in the wound . [SEP]
[CLS] a human adipose stem cellderived extracellular vesicle ( hasc - exos ) - based mirna delivery strategy was described by lv et al . [SEP]
[CLS] mir - 21 - 5p mimics were loaded into hasc - exos by electroporation to treat diabetic wounds . [SEP]
[CLS] the in vitro studies showed increased proliferation and migration of keratinocytes due to application of engineered extracellular vesicles ( e - exos ) . [SEP]
[CLS] the regenerative potential was also assessed in diabetic wounds resulting in increased reepithelialization , collagen remodeling , new blood - vessel formation , and vessel maturation in vivo . [SEP]
[CLS] for the treatment of diabetic wounds , li et al . developed human epidermal keratinocyte extracellular vesicles incorporating mir - 21 mimics . [SEP]
[CLS] these evs significantly promoted skin wound healing in diabetic rats by promoting fibroblast migration , differentiation , and contraction . [SEP]
[CLS] mir - 21 mimics containing evs also induced a pro - angiogenic process in endothelial cells B-material and mediated a proinflammatory response . [SEP]
[CLS] after cardiovascular diseases , cancer is the second leading cause of death in the united states . [SEP]
[CLS] conventional cancer treatments , such as rt , surgery , chemotherapy , and proton therapy have aided in reducing the death rate . [SEP]
[CLS] for instance , approximately 50 % of all cancer patients receive radiation therapy , which accounts for approximately 40 % of the total curative cancer treatments . [SEP]
[CLS] so far , these traditional therapies continue to entail unsolved healthcare challenges . [SEP]
[CLS] for example , conventional therapies suffer from a high level of toxicity B-property and a variety of long - term complications . [SEP]
[CLS] in addition , complex factors such as unpredictable metastasis B-event and mutations in the cancer gene pose new challenges for cancer treatment . [SEP]
[CLS] consequently , there is an urgent need to develop a new strategy for cancer treatment by taking more factors into account . [SEP]
[CLS] disordered gene expression is a major hallmark of cancer ; therefore , much effort has been devoted to altering the activity of the genes related to cancers . [SEP]
[CLS] rna plays a key role in participating in and regulating transcription B-event , thus providing a new opportunity to treat cancer by altering the activity of rna . [SEP]
[CLS] nevertheless , naked sirna is susceptible to degradation by nucleases in blood serum and unable to cross through the cell B-material membrane due to its anionic charge . [SEP]
[CLS] therefore the need to develop feasible rna vectors for cancer therapy has become urgent . [SEP]
[CLS] remarkable progress in nanocarriers has led to advances in drug delivery systems for rna delivery to target locations in vivo . [SEP]
[CLS] accordingly , we are providing an overview of the applications of rna - based drug delivery in several typical cancer treatments targeting glioblastoma , pancreatic , liver , prostate , lung , and breast cancers . [SEP]
[CLS] glioblastoma is one of the most common , aggressive , and poorly treated brain tumors B-material . [SEP]
[CLS] the average survival rate of glioblastoma patients is still low ( < 2 years ) . [SEP]
[CLS] exposure to ionizing B-property radiation and rare genetic syndromes , including li - fraumeni syndrome and lynch syndrome , are among the main factors associated with the onset of glioblastoma , which represents a huge hurdle due to the blood - brain barrier B-property ( bbb ) . [SEP]
[CLS] rnai has been considered as a promising strategy for the treatment of various cancers ; however , the applications were limited by its easy degradation . [SEP]
[CLS] in order to enhance the safety and efficiency of sirna delivery , kong et al . used pei - entrapped gold B-nanoparticle nanoparticles I-nanoparticle , which were modified with arginine - glycineaspartic peptides B-material as a carrier to deliver bcl - 2 sirna to glioblastoma cells B-material , showing a positive effect on gene silencing in specific cells B-material . [SEP]
[CLS] in another study , zheng et al . constructed a polymeric sirna nanomedicine stabilized by triple interactions , including electrostatic interaction , hydrogen B-material bond , and hydrophobic B-property interaction I-property . [SEP]
[CLS] however , it is exceptionally difficult for conventional drugs or biological agents to target brain tumor - initiating cells B-material , due to the heterogeneous inheritance and epigenetic aberrations . [SEP]
[CLS] multiple rnai delivery via nanoparticles B-nanoparticle could effectively hinder tumor B-material growth in the body and improve survival rates . [SEP]
[CLS] the subcutaneous injection of an rna nanomedicine is a common route . [SEP]
[CLS] sukumar et al . explored the potential of the nose - to - brain direct transport pathway for hybrid polymer particle - loaded mirna , which would permit the pre - sensitization of glioblastoma cells B-material . [SEP]
[CLS] however , in view of their practical application , the new therapies and challenges of glioblastoma need to be studied further . [SEP]
[CLS] pancreatic cancer is one of the most highly malignant tumors B-material . [SEP]
[CLS] its rapid progression , high metastasis B-event rate , and profound chemoresistance result in a low survival rate of pancreatic cancer patients . [SEP]
[CLS] previous reports have demonstrated that the activation of the mutant kras ( in codons 12 , 13 , and 16 ) is involved in most pancreatic cancers . [SEP]
[CLS] as a result , the mutant kras is a major target for the treatment of pancreatic cancers . [SEP]
[CLS] based on the extraordinary sequence specificity of rnai , zeng et al . developed a nanomedicine system made of pegblock - pll and sirna for directing kras oncogene silencing and arsenic B-material - induced apoptosis B-event in vitro and in vivo . [SEP]
[CLS] similarly , uchida et al . investigated peg - polycation block copolymerscholesterol ( peg - pasp ( tep ) - chol ) nano - micelle B-material as a carrier of mrna for the treatment of pancreatic cancer , finding that the mrna nanomicelle generated an efficient protein B-material expression in tumor B-material tissues . [SEP]
[CLS] han et al . constructed a tumor B-material microenvironment - responsive nanosystem with activated pancreatic stellate cells B-material as a potential target ( figure 8a ) . [SEP]
[CLS] in this nanosystem , all - trans retinoic acid ( an inducer of psc quiescence ) and sirna targeting heat shock protein B-material 47 ( hsp47 , a collagenspecific molecular chaperone ) could re - educate pscs and promote drug delivery to pancreatic tumors B-material , leading to a significant enhancement of the anti - tumor efficacy of chemotherapeutics . [SEP]
[CLS] although liposome - based carriers offer advantages for rna delivery over viral - based delivery systems , they are characterized by low efficiency and a rapid clearance from the blood circulation . [SEP]
[CLS] kamerkar et al . investigated engineered exosomes as carriers of sirna or shrna , which are specific to oncogenic kras . [SEP]
[CLS] it was found that the exosomes suppressed pancreatic cancers in multiple mouse models and significantly increased overall survival . [SEP]
[CLS] liver cancer , with a 5 - year survival rate of 18 % , was the second leading cause of cancer death ( 8 . 3 % of total cancer deaths ) worldwide in 2020 . [SEP]
[CLS] hepatocellular carcinoma ( hcc ) , representing 75 - 85 % of all cases , and intrahepatic cholangiocarcinoma ( 10 - 15 % ) are the two main forms of primary liver cancer , which are commonly caused by chronic liver damage due to cirrhosis from hepatitis virus infection , alcohol B-material abuse , or nonalcoholic fatty liver disease . [SEP]
[CLS] many small - molecule drugs for hcc treatment failed in phase iii human clinical trials , partly because late - stage liver dysfunction amplifies drug toxicity B-property . [SEP]
[CLS] still , the tremendous progress in rna - based drugs has shown a huge potential for liver cancer therapy . [SEP]
[CLS] here we report on several studies of rna - based treatments for liver diseases . [SEP]
[CLS] the initial stage of liver fibrosis begins with hscs fibrosis , which is generally reversible , avoiding the onset of liver cirrhosis . [SEP]
[CLS] sato et al . cured liver fibrosis in rats by delivering sirna against gp46 , the homolog of human hsp47 , via vitamin a - coupled liposomes B-nanoparticle to hscs . [SEP]
[CLS] unfortunately , hepatitis virus infection may cause cirrhosis without any initial clinical signs , thus making the diagnosis difficult . [SEP]
[CLS] wooddell et al . used a polymer B-material - based peptide B-material with a liver - tropic cholesterol - conjugated sirna to knock down the expression of viral rnas in hbv - infected mouse models , resulting in the multilog repression of viral rna , proteins B-material , and viral dna with longlasting effects . [SEP]
[CLS] from theory to clinical application , the efficiency and nontoxicity of delivery vehicles B-material to organs are inevitable hurdles to be overcome . [SEP]
[CLS] khan copyright 2018 , the authors . [SEP]
[CLS] published by springer nature . [SEP]
[CLS] b ) vaccine - based sirna delivery to initiate innate immunity for breast cancer treatment . [SEP]
[CLS] targeting capability of hpvp on tumor - bearing mice . [SEP]
[CLS] in vivo tumor - targeting capacity of hpvp on 4t1 tumor - bearing mice after injection . [SEP]
[CLS] immunofluorescence images showing pdl1 inhibition effect . [SEP]
[CLS] in vivo anticancer B-property capacity of sirna - based system on breast tumor B-material model . [SEP]
[CLS] reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2020 , the authors . [SEP]
[CLS] published by springer nature . [SEP]
[CLS] ionizable B-property dendrimer B-nanoparticle cores B-material , which enhanced the efficient complexation with negatively charged sirna under an acidic micro - environment , thus demonstrating that the target gene in sirna could target specific cells B-material , such as ecs and hepatocytes . [SEP]
[CLS] dendrimers B-nanoparticle are other powerful delivery vectors . [SEP]
[CLS] the main challenge is how to design dendrimers B-nanoparticle with low toxicity B-property and high potency to reduce tissue damage . [SEP]
[CLS] zhou et al . reported that modular degradable dendrimers B-nanoparticle with small rnas showed a pronounced survival benefit in an aggressive genetic cancer model , owing to a high anti - tumor B-material potency and the low hepatotoxicity of dendrimers B-nanoparticle . [SEP]
[CLS] some rna - based drugs or delivery vehicles B-material were studied in clinical trials . [SEP]
[CLS] as an example , voutila et al . developed small activating rnas to upregulate the transcription B-event factor ccatt / enhance binding protein B-material alpha . [SEP]
[CLS] this drug has been undergoing a phase i clinical trial for patients with liver cancer . [SEP]
[CLS] rna - based chemotherapy has shown an extremely high value in the treatment of liver cancer . [SEP]
[CLS] one of the future goals is to reduce the side effects produced by the degradation of drug - carriers . [SEP]
[CLS] in men all over the world , prostate cancer is the second most common cause of cancer - related mortality after lung cancer . [SEP]
[CLS] common risk factors for developing prostate cancer include age , race , and family history , mostly related to genetic factors . [SEP]
[CLS] because of the relatively limited understanding of these genes , their clinical management is difficult . [SEP]
[CLS] although conventional therapiesincluding the surgical removal of the prostate , radiation therapy , and hormone therapy - have shown to be useful for prostate cancer , the life quality of patients can be seriously impacted by surgical or chemical castration . [SEP]
[CLS] thanks to its ability to specifically silence the target gene expression , rnai technology is emerging as a promising therapeutic procedure for prostate cancer . [SEP]
[CLS] with the development of nanotechnology , various efforts have been devoted to developing rna nanocarriers for prostate cancer therapy . [SEP]
[CLS] hasan et al . reported on plga / sirna nanoparticles B-nanoparticle prepared via a unique soft lithography , leading to a high sirna encapsulation B-property efficiency I-property of 32 - 46 % . [SEP]
[CLS] as another example , chen et al . synthesized tertiary amine - functionalized cationic B-material polylactides nanoplexes with a remarkable hydrolytic degradability , while interleukin - 8 sirna can be released by thiol - ene click functionalization . [SEP]
[CLS] in addition , researchers have moved a step forward to design rna carriers with a stimulus - responsive function for cancer therapy . [SEP]
[CLS] for instance , xu et al . proposed a multifunctional envelope - type rna - nanoparticle B-nanoparticle , obtained by the modification of acupa , a small molecular ligand specifically recognizing the prostate - specific membrane antigen receptor B-material , resulting in an efficient silencing of the prohibitin1 expression . [SEP]
[CLS] further research should focus on the study of specific genes for prostate cancer , to improve the effectiveness of targeted therapy . [SEP]
[CLS] lung cancer is the leading cause of cancer death and ranks second in incidence ( 11 . 4 % of new cases ) , its limited successful treatment can be attributed to its heterogeneity and adaptability . [SEP]
[CLS] lung cancers can be broken down into two classes : i ) non - small - cell B-material lung carcinoma , accounting for ≈85 % of all lung cancer cases , and ii ) small - cell B-material lung carcinoma . [SEP]
[CLS] rna - based therapeutics have recently come into focus as an emerging therapeutic class with great potential for fighting cancer . [SEP]
[CLS] it is worth noting that nanoparticles B-nanoparticle represent an advanced delivery platform for rna therapeutics due to their high surface areas and easy processability . [SEP]
[CLS] as spherical vesicles , liposomes B-nanoparticle have been widely used in rna - based delivery systems . [SEP]
[CLS] as the earliest liposomal B-nanoparticle delivery system , zhao et al . developed a lipid - polycation - ha nanoparticle B-nanoparticle for vascular endothelial growth factor ( vegf ) sirna delivery for vegf knockdown in a human lung cancer xenograft . [SEP]
[CLS] nanoparticles B-nanoparticle were found to induce antitumor efficacy through the activation of the amp - activated protein B-material kinase and inhibition of the rapamycin ; their function was equivalent to that of metformin , an anticancer B-property drug . [SEP]
[CLS] another powerful delivery system is represented by msnps , thanks to good biocompatibility B-property , tunable pore size , and customizable properties . [SEP]
[CLS] for instance , dilnawaz et al . explored the efficacy of the codelivery of a complex with an anticancer B-property drug - such as etoposide B-material or docetaxel - loaded msnps and surviving sirna for lung cancer . [SEP]
[CLS] this delivery system demonstrated pronounced apoptosis B-event effects with a high - dose drug in vitro . [SEP]
[CLS] in another study , sirna / msnp - pei immobilized on electrospun nanofibers B-nanoparticle - prepared by nascimento et al . provided a longer release period , which exhibited a great potential to achieve a local and sustained release of cancer therapeutics [SEP]
[CLS] another strategy for the suppression of cancer is to disrupt the proliferation process of cancer cells B-material . [SEP]
[CLS] although the growth of cancer cells B-material can be tuned by different sirna , adverse side effects usually increase , as happens with normal cells B-material . [SEP]
[CLS] therefore , developing sirna delivery systems with low adverse side effects is one of the further lines of research . [SEP]
[CLS] breast cancer is one of the most frequently occurring cancers , with an estimated 2 . 3 million new cases ( 11 . 7 % of the new cases ) in 2020 , and the leading cause of cancer - related deaths among women worldwide . [SEP]
[CLS] many factors are associated with an increased risk of breast cancer , such as obesity , use of estrogen and progestin , advanced maternal age at first birth and alcohol B-material consumption . [SEP]
[CLS] in addition , genetic mutations and epigenetic mechanisms are closely related to the tumorigenesis of breast cancer . [SEP]
[CLS] therefore , there is still a long way to go for the treatment of breast cancer . [SEP]
[CLS] fortunately , the past few years have witnessed great strides in breast cancer treatment . [SEP]
[CLS] one example is triple - negative breast cancer : it was defined as a type of breast cancer with negative expression of estrogen , progesterone , and human epidermal growth B-material factor I-material receptor I-material - 2 , and its mortality rate is 40 % within the first 5 years after diagnosis . [SEP]
[CLS] more efforts based on conventional chemotherapy were proven to be effective in reducing side effects , including drug resistance and organ dysfunction . novel nanocomplex carriers and oncogenic mirna have been widely used for a specific and efficient delivery in cancer treatment to reduce collateral damage to healthy cells B-material or organs . [SEP]
[CLS] the combination of multimodal therapeutics shows a good performance in terms of inhibiting breast cancer growth . [SEP]
[CLS] juneja et al . developed a porphyrin - based polysilsesquioxane platform to deliver rna , and this platform was proven to affect silencing when tested in mdamb - 231 / gfp cells B-material . [SEP]
[CLS] in another study , paclitaxel B-material , an anti - cancer drug , can be solubilized and co - loaded with rna into nanoparticles B-nanoparticle , which showed an ultra - thermodynamic stability for targeted cancer therapy . [SEP]
[CLS] the treatment of cancers involves the combination of multiple technologies , through which some potential complications could be resolved . [SEP]
[CLS] advanced antiangiogenic therapy has gradually become a new means of preventing and treating cancer metastasis B-event , and it has been successfully used clinically in the treatment of various cancers . [SEP]
[CLS] both anti - vegf sirna and nogo - b receptor sirna are reported to be effective for suppressing ec migration and tubule formation . [SEP]
[CLS] in the future , more research should focus on how to balance the effectiveness and biosafety of rna delivery systems . [SEP]
[CLS] also , it has become increasingly clear that the treatment of cancer , a multifactorial disease , cannot be entrusted to a single molecule or gene editing . [SEP]
[CLS] therefore , future research may focus on developing combined strategies including chemotherapy / cancer gene therapy , chemotherapy , rt and immunotherapy . [SEP]
[CLS] in addition to surgery , targeted pathway inhibition , chemotherapy , radiation therapy , and immunotherapy have emerged as alternative modalities of cancer treatment since they can fight aggressive diseases relying on the body ' s own immune system . [SEP]
[CLS] nowadays , cancer immunotherapy comes in a variety of forms , including cancer vaccines , cell B-material therapy , tumor - infecting viruses , immune checkpoint , cytokines , targeted antibodies B-material , immunogenic B-property cell B-event death I-event , and adjuvants . [SEP]
[CLS] impressively , rnabased therapy has made a huge stride forward for cancer treatment since many kinds of rnai drugs ( patisiran , phase iii ; envision , phase iii ) entered clinical trials . [SEP]
[CLS] as a typical immunotherapy method , rna - based immunotherapy has been increasing interest in elucidating the function of rna in the regulation of anticancer B-property immune responses and different cancer therapeutics . [SEP]
[CLS] therefore , we will discuss and summarize the rna - based immunotherapy currently applied in the cancer therapy field . [SEP]
[CLS] vaccines as an attractive and effective option have been widely used in treating many infectious diseases , such as polio , measles , and even covid - 19 . [SEP]
[CLS] nowadays , there are four kinds of vaccines , including viral vector , tumor B-material / immune cell B-material , peptides B-material , and nucleic B-material acid I-material , among which rna - based vaccines are usually designed to translate tumor - associated antigens in antigen - presenting cells B-material and trigger antigen - specific cells . [SEP]
[CLS] in 1999 , ying et al . utilized self - replicating rna vectors to enhance the immunogenicity B-property of nucleic vaccines . [SEP]
[CLS] the experimental mice survived from tumor B-material under the self - replicating rna protection , successfully demonstrating that rna may be an excellent candidate for the development of new cancer vaccines . [SEP]
[CLS] the rna - based vaccine field is developing extremely rapidly in recent years ; sahin et al . adopted individualized mutanome vaccines and implemented an rna - based poly - neoepitope approach to mobilize immunity against a spectrum of cancer mutations , and the results indicated that the personal rna vaccine has a positive effect on melanoma . [SEP]
[CLS] in another study , it has been proven that human papillomavirus pseudovirus ( hpvp ) nanoparticles B-nanoparticle loaded with sirna , forming a human papillomavirus ( hpv ) vaccine , have effective immunotherapy effects , a high response rate , and good biological safety ( figure 8b ) . [SEP]
[CLS] the mrna vaccine is the highest promising candidate in cancer immunotherapy as it can encode tumor - associated antigens without potential dangers . [SEP]
[CLS] in order to deliver mrna to antigen - presenting cells B-material effectively , the lipopolyplex mrna vaccine incorporated with a lipid B-material shell B-material or polymer B-material hydrogel has been studied , which demonstrated that the antigen - specific cells B-material increased and tumor B-material growth was inhibited relatively . [SEP]
[CLS] the optimal mrna stability , cytosolic delivery , and mrna expression are needed for the efficient mrna vaccine . [SEP]
[CLS] at the end of this part , we summarized the significance and challenges of cancer immunotherapy based on the following three therapy methods . [SEP]
[CLS] immune checkpoint blockade ( icb ) therapies can induce durable tumor B-material control and extend the survival time of cancer patients by reactivating tumor B-material - associated cells B-material . [SEP]
[CLS] recently , long noncoding rnas show a vital function of immune response , which can predicate survival and immune checkpoint blockade in hepatocellular carcinoma . [SEP]
[CLS] however , the promising results were difficult to be demonstrated due to the complex immune system . [SEP]
[CLS] in order to enhance efficiency of immune checkpoint therapy , a more powerful combination with rna has been studied . [SEP]
[CLS] immune checkpoint inhibitors , such as t lymphocyte - associated protein B-material 4 , programmed B-event cell I-event death I-event protein B-material , and programmed B-event cell I-event death I-event ligand 1 , have been applied in many malignant cancers . [SEP]
[CLS] in the typical study , bialkowski et al . has demonstrated that icb combined with antibodies B-material targeting ( il - 6 and tgf - β ) inhibitions could prolong the survival of treated mice . [SEP]
[CLS] due to multiple immunosuppressive mechanisms in the tumor B-material microenvironment , the dysfunctional immune system was the barrier B-property to cancer treatment . [SEP]
[CLS] sheng et al . studied ablation of the histone demethylase lsd1 in cancer cells B-material , and the results demonstrated that it led to double - stranded rna stress and activation of type 1 interferon , which stimulates anti - tumor cells B-material immunity and restrains tumor growth . [SEP]
[CLS] the cell B-material ' s molecular composition , signalling activity , and metabolic determine the cell B-material fate and function , making rnasequencing an effective method for cancer diagnosis . [SEP]
[CLS] recently , more advanced technologies were utilized to characterize complex cell B-material responses . [SEP]
[CLS] katzenelenbogen et al . studied ins - seq for recording scrna - seq and intracellular protein B-material activity . [SEP]
[CLS] genetic ablation in mice models showed a marked decrease in dysfunction cd8 + cells B-material and reduced tumor B-material growth . [SEP]
[CLS] nissim et al . designed an rna - based and gate with de novo synthetic promoters to enhance specificity . [SEP]
[CLS] it showed a huge potential for killing cancer cells B-material and significant tumor B-material reduction , and prolonged mouse survival in vitro and in vivo . [SEP]
[CLS] the delivery of mitotic checkpoint sirna - loaded nanoparticles B-nanoparticle reported in some recent studies showed that the essential mitotic checkpoint gene ' s silencing could induce cell B-event death I-event . [SEP]
[CLS] to some degree , a collaboration between traditional and novel therapies is crucial to achieving further advances in cancer treatment . [SEP]
[CLS] except for the three immune therapies mentioned a , b ) reproduced with permission [SEP]
[CLS] copyright 2018 , royal society of chemistry . [SEP]
[CLS] c , d ) sem images of pcl fiber - collagen hydrogel system . [SEP]
[CLS] above , the other methods , like cytokines and viruses , are key components of rna - based therapies . [SEP]
[CLS] how to develop immunotherapies with safe and effective antitumor immunity is unsettled . [SEP]
[CLS] based on previous research , rna has been applied as clinical biomarkers B-property for cancer prognosis , diagnosis , and treatment response . [SEP]
[CLS] rna - based therapeutics for cancer treatment have great potential to enhance immunotherapy by combining the current treatment methods . [SEP]
[CLS] an acute trauma affecting the central nervous system often results in an irreversible loss of neural functions . [SEP]
[CLS] in this context , over the years , several approaches have been experimented to replace damaged neurons and foster axonal regeneration . [SEP]
[CLS] thanks to their relative abundance and easy accessibility , mesenchymal stem cells B-material ( mscs ) are a clinically viable cell B-material source , whose potential for cross - lineage neuronal differentiation in the presence of proteins B-material and biochemical cocktails is widely reported in the literature . [SEP]
[CLS] within this framework , low et al . proposed the absorption of sirnas onto the surface of dopa - melanin ( dm ) coated electrospun nanofibrous scaffolds with the aim of obtaining materials characterized by i ) a prolonged silencing of inhibitory factors against the desired lineage commitment , and ii ) a fibrous morphology with a high surface - to - volume ratio in order to maximize the exposure of sirnas to mscs , thus making it possible to efficiently induce the msc differentiation to neural cells B-material . [SEP]
[CLS] dm - coated random and aligned ε - pcl electrospun nanofibers B-nanoparticle were subjected to the adsorption of sirna complexed with the transfection reagent transit - tko ( sirna / tko ) , in the same way as in the previously reported procedure published by the same authors . [SEP]
[CLS] the studies performed on release kinetics highlighted that the presence of dm significantly reduces the initial sirna burst ( from 9 . 7 ± 0 . 5 % to 6 . 6 ± 0 . 2 % for random nanofibers B-nanoparticle and from 16 . 6 ± 1 . 5 % to 12 . 3 ± 0 . 8 % for the aligned ones ) and increases its maximum cumulative release ( from 59 . 4 ± 3 . 6 % to 30 . 1 ± 3 . 1 % for random nanofibers B-nanoparticle and from 57 . 3 ± 3 . 7 % to 43 . 3 ± 3 . 2 % for aligned nanofibers B-nanoparticle ) . [SEP]
[CLS] the reduction of the burst release can be ascribed to electrostatic attractions between the positively charged sirna / tko complexes and the negatively charged dm layer . [SEP]
[CLS] the enhanced sirna release in the presence of dm coating B-material might be explained by taking into account i ) the non - covalent and non - specific interactions between sirnas and transit - tko molecules , and ii ) the fact that the highly negatively charged surface of dm coated fibers promotes the release of free sirna into the external aqueous environment . [SEP]
[CLS] under non - specific differentiation conditions , at day 7 a significant rest knockdown was observed only in the presence of dmcoated aligned pcl scaffolds . [SEP]
[CLS] in a different study , the same authors employed functionalized electrospun pcl nanofibers B-nanoparticle properly optimized to induce the rest knockdown in human induced pluripotent stem cell - derived neural progenitor cells B-material ( hipsc - npcs ) in order to enhance their neuronal differentiation . [SEP]
[CLS] in this one , two distinct mussel - derived coatings B-material , that is , polydopamine and polydopa - melanin , lipofectamine rnaimax and transit - tko , complexed with sirna , as transfection reagents , were tested ( figure 9a ) . [SEP]
[CLS] with the aim of assessing the interactions between the scaffolds seeded with cells B-material and the damaged tissues in terms of implant integration , an organotypic spinal cord slice culture , considered a bridge between the in vitro and the in vivo experiments , was used . [SEP]
[CLS] interestingly , unlike mscs , with the same drug dosage transit - tko proved to be cytotoxic B-property for hipsc - npcs . [SEP]
[CLS] despite a similar sirna loading B-property efficiency I-property , a lower sirna release was observed from polydopa - melanincoated nanofibers B-nanoparticle than from polydopamine - coated ones : in the former , this was probably due to the greater amount of carboxyl B-material groups I-material interacting with positively - charged sirna complexes . [SEP]
[CLS] in light of these results , cellular tests were performed only for polydopa - melanin - coated scaffolds functionalized with rnaimax lipofectamine - sirest . [SEP]
[CLS] these results documented : i ) the successful integration of the scaffold with the transected spinal cord tissues after 7 days of culturing ( figure 9b ) ; ii ) the ability of sirest - absorbed scaffolds to provide differentiation signals to direct cell B-material fate in post - injured tissues ; iii ) the ability of the scaffold to enable neurite extension across the 2 mm injury gap , without relevant differences the response of the glial cells B-material . [SEP]
[CLS] the lack of glial response led to the conclusion that the scaffolds did not aggravate spinal cord injuries after in vivo transplantation . [SEP]
[CLS] one of the possible obstacles to the spontaneous recovery of traumatic nerve injuries is the lack of surviving oligodendrocytes ( ols ) after the injury , often resulting in an inefficient myelin regeneration and , therefore , in the insurgency of defects c , d ) reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2019 , nanyang technological university . published by wiley - vch [SEP]
[CLS] e ) effect of injection of lentiviral vectors encoding nogo - 66 receptor B-material 1 ( ngr1 ) - short hairpin rna ( shrna ) in three groups of rats : ln ( lv - ngr1 shrna injection ) , lc ( lv - control shrna injection ) and sham ( laminectomy only ) ; marker cnpase was used to detect oligodendrocytes , mbp immunostaining and lfb staining were used to detect myelinated fibers . [SEP]
[CLS] reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2018 , the authors . published by springer nature . [SEP]
[CLS] f ) magnetofection of striatal cells B-material by fluorescein B-material isothiocyanate I-material ( fitc ) oligonucleotides complexed with neuromag , magnetic B-property polymeric B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2020 , the authors . published by multidisciplinary digital publishing institute . [SEP]
[CLS] g ) composition and physical properties of plga B-nanoparticle nanoparticles I-nanoparticle ( nps B-nanoparticle ) containing perfluoro - 1 , 5 - crown ether B-material , a fluorine B-material compound trackable non - invasively by fluorine B-material ( 19 f ) - magnetic B-property resonance imaging , and coated with protamine sulfate in order to complex mir - 12 . [SEP]
[CLS] h ) photomicrographs of svz stem / progenitor cells B-material ( scale bar : 10 µm ) . [SEP]
[CLS] g , h ) reproduced with permission . [SEP]
[CLS] copyright 2016 , elsevier . [SEP]
[CLS] i ) scheme for the fabrication of gal - np B-nanoparticle @ sirna . j ) transmission electron micrographs of gal - np B-nanoparticle @ sirna . k ) in vitro gene silencing effects of gal - np B-nanoparticle @ sibace1 and controls at day 3 post - transfection ( n = 3 , * * * p < 0 . 001 ) . l ) representative image for cy5 signal in the brain of np B-nanoparticle @ sirna and gal - np B-nanoparticle @ sirna groups 1 h after injection . m ) behavioral evaluation of gal - np B-nanoparticle @ sibace1 nanomedicine therapy in app / ps1 mice : representative swimming track for probe test in the mwm . [SEP]
[CLS] i - m ) reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2020 , the authors , some rights reserved . [SEP]
[CLS] published by american association for the advancement of science . [SEP]
[CLS] in the neuronal signal transduction [SEP]
[CLS] among micro - rna , mir - 219 and mir - 338 have been recognized as playing a pivotal role in the regulation of ol development , being able to silence the expression of negative regulators for ol differentiation . [SEP]
[CLS] within this framework , diao et al . designed a fiber - mediated strategy for the delivery of mir - 219 and mir - 338 to control opc development . [SEP]
[CLS] similarly to the above - described strategies , dopa - coated pcl nanofibers B-nanoparticle were used for mirna absorption . [SEP]
[CLS] the authors carefully investigated the effect of both fiber topography and their coupling with mir on the gene silencing outcome . [SEP]
[CLS] fiber topography was studied in terms of orientation ( random and aligned fibrous scaffolds were taken into account ) and diameter ( aligned fibers with a diameter varying from 200 nm to 2 µm were produced ) . [SEP]
[CLS] fiber topography ( with scrambled mir ) resulted in a downregulation of the expression of inhibitory regulators with respect to 2d culture . [SEP]
[CLS] more specifically , the smaller aligned fibers ( 200 nm in diameter ) induced the poorer pdgfr - α knockdown , while 2 µm diameter random fibers inhibited gene expression more than 2 µm diameter aligned fibers did . [SEP]
[CLS] when coupled with mir - 219 or mir - 219 / mir - 338 , 2 µm diameter random fibers continued to induce the lowest expression of inhibitory regulators . [SEP]
[CLS] although the highest expression of rip ( the early - stage marker of opc differentiation ) was registered for 2 µm diameter random fibers , the coupling with mir - 219 and the mir - 219 / mir - 338 cocktail had more influence on aligned fibers , with a significant increase in the rip signal , while the fiber diameter decreased . [SEP]
[CLS] with regard to the expression of mbp ( a late myelin marker of ol maturation ) , independently from the coupled mir , large - diameter fibers induced more mbp expression than 200 nm diameter fibers . [SEP]
[CLS] this study made it possible to conclude that fiber topography significantly affects the gene expression and , when coupled with mir , both random and aligned large - diameter fibers promoted opc differentiation , although the small - diameter aligned fibers seemed to be the most promising substrate ; on the other hand , the large - diameter aligned fibers seemed more conducive to ol maturation . [SEP]
[CLS] in a more recent study , the authors translated the fibrous 2d substrate into a 3d system for direct in vivo micro - rna screening . [SEP]
[CLS] four micro - rnas ( mir - 21 , mir - 222 , mir - 132 , and mir - 431 ) and their cocktails , selected for their ability to regulate neurogenesis and axon growth , were systematically screened . [SEP]
[CLS] a properly designed fiber - hydrogel system ( figure 9c ) was used to enable the implantation of the aligned nanofibers B-nanoparticle ( figure 9d ) into the damaged spinal cord tissue . [SEP]
[CLS] in the system employed , fibers were immersed in a collagen matrix , enabling the delivery of multiple biochemical factors , while supporting and retaining the aligned fibers in a 3d configuration , in order to guide the direction of axon growth as in the in vitro situation . [SEP]
[CLS] furthermore , according to the in vitro results , mir - 132 / mir - 222 / mir - 431 were shown to provide the best regeneration outcomes with respect to mir - 21 , mir - 222 / mir - 431 , and neg - mir treatments . [SEP]
[CLS] regeneration of injured spinal cord tissue : zhao et al . [SEP]
[CLS] exploited the local injection of lentiviral vectors encoding nogo - 66 receptor B-material 1 ( ngr1 ) - shrna to induce nerve regeneration and functional recovery after spinal cord injury . [SEP]
[CLS] a lentiviral vector was used due to its documented high transfection rate and long - term and stable gene modifications ; ngr1 shrna was chosen because of its capability in knocking down the expression of ngr1 gene , which is responsible for suppressing neurogenesis due to its high affinity with myelin - associated inhibitors . [SEP]
[CLS] the results , achieved from in vivo experiments on rat models , demonstrated that the local injection of lentiviral vectors encoding ngr1 shrna ( lv - ngr1 shrna ) was able to promote nerve regeneration and functional recovery after spinal cord injury furthermore , studies performed with cnpase and mdb markers to assess , respectively , the presence of oligodendrocytes and remyelination at the injury site demonstrated both the survival of more oligodendrocytes and the presence of a higher number of myelinated fibers in the rats subjected to lv - ngr1 shrna injection , compared to those in the lv - control shrna group , as reported in figure 9e . [SEP]
[CLS] another strategy for inducing the regeneration of injured spinal cord tissue by exploiting sirna delivery is based on silencing nischarin , a protein B-material that inhibits neurite outgrowth and neural cell B-material regeneration , in order to increase the protein B-material expression of the growth - associated protein B-material - 43 ( gap - 43 ) . [SEP]
[CLS] nischarin - targeted sirna ( nis - sirna ) was delivered by poly ( ethylenimine ) - alginate ( pei - alg ) nanoparticles B-nanoparticle to the spinal cord tissue with the aim of promoting motor function recovery in rats . [SEP]
[CLS] pei - alg nanoparticles B-nanoparticle were selected as carriers since they had been demonstrated to effectively deliver sirnas , while preventing enzymatic hydrolysis and facilitating the entry of nucleic B-material acids I-material into cells B-material . [SEP]
[CLS] as expected , 3 weeks after the injection , a dramatic decrease in nischarin expression was registered in the lesion site for the rats treated with nis - sirna , whereas in the region 1 cm distal from the lesion , no significant differences were observed compared to the ctl - sirna group . [SEP]
[CLS] furthermore , a motor function recovery , in terms of right hindlimb movement , was observed in spinal cordinjured rats treated with pei - alg / nis - sirna . [SEP]
[CLS] in addition to the approaches based on nervous tissue regeneration after a traumatic injury , strategies focused on blocking or decreasing inflammasome activation after an injury have also been investigated . [SEP]
[CLS] de rivero vaccari et al . , in an attempt to achieve this aim , exploited neuronal - derived exosomes , loaded with sirna - gfp against apoptosis - associated speck - like protein B-material containing a caspase recruitment domain ( asc ) , as a therapeutic vector to block inflammasome activation after a spinal cord injury . [SEP]
[CLS] in vivo tests were mainly devoted to assessing the ability of exosomes , injected into the femoral vein , to cross the blood - spinal cord barrier B-property , penetrate the spinal cord parenchyma , and deliver their cargo . [SEP]
[CLS] the results showed that cells B-material in the epicenter of the lesion were positive to gfp , while they were negative to gfp in the regions distant from the epicenter , thus indicating an effective delivery of the cargo . [SEP]
[CLS] the potential of rna for the treatment of neurodegenerative diseases is under constant investigation , as demonstrated by very recent studies in this field . [SEP]
[CLS] positive outcomes have motivated researchers to identify strategies for aiding the delivery of rna , injected locally into the brain or at a systemic level , for the treatment of these diseases . [SEP]
[CLS] to be suitable for this specific application , a proper design of delivery systems is required to provide them in vivo stability and transcytotic potential . [SEP]
[CLS] in this context , haroon et al . developed a strategy in which a peptide B-material known to target specific gangliosides was fused to a double - stranded rna binding protein B-material to deliver sirna to the brain parenchyma of rat models . [SEP]
[CLS] the resulting protein B-material was labeled as tarbp - btp and it was able to enter neuro 2a , imr32 , and hepg2 cells B-material in the presence of monosialoganglioside gm1 . [SEP]
[CLS] the delivery of tarbp - btp bound to sirna led to a significant knockdown in the brain of bace1 , whose activity is associated with neurodegeneration and accretion of amyloid precursor protein B-material products . [SEP]
[CLS] another approach exploiting peptides B-material is based on the production of peptide B-material nanofibers B-nanoparticle ( pnfs ) , intended as the assembly of amphiphilic B-property peptides B-material in a single - dimension cylindrical geometry . [SEP]
[CLS] these pnfs can be engineered with amino B-material acids I-material showing a positive charge in order to electrostatically link and deliver the negatively charged sirna . [SEP]
[CLS] mazza et al . realized a surfactantlike peptide B-material ( palmitoyl - gggaaakrk ) showing the capability to self - assemble into pnfs . [SEP]
[CLS] the authors demonstrated that the complex , administrated intracranially , was uptaken intracellularly and promoted the increase in sirna ' s residence time ( from 48 h for sirna to 7 days for pnf - sirna ) in the brain . [SEP]
[CLS] more specifically , the complex was explored as a nanovector to the target and silenced bcl2 , responsible for inhibiting apoptosis B-event in the neuronal population , in the subthalamic nucleus . [SEP]
[CLS] results demonstrated that the complex remained localized in close proximity to the injection site , without migrating into the brain ' s distal region even 1 week after the administration . [SEP]
[CLS] furthermore , the complex turned out to successfully silence the expression of bcl2 in the subthalamic nucleus compared to the noninjected hemisphere . [SEP]
[CLS] according to this result , a significant loss of nissl - stained cells B-material was registered only in the hemisphere subjected to the treatment , thus highlighting the localization of neuronal tissue ablation . [SEP]
[CLS] peptides B-material were also used to produce polymer - peptide nanoparticles B-nanoparticle capable of linking sirna to be released into neuro 2a neuronal cells B-material . [SEP]
[CLS] nanoparticles B-nanoparticle were obtained by chemoselectively conjugating peg to the chitosan B-material ' s c2 hydroxyl B-material group I-material and to a cell B-material - penetrating tat peptide B-material . [SEP]
[CLS] the domain of the selected tat peptide B-material was composed of residues of arginine B-material and lysine B-material amino B-material acids I-material that played a relevant role in translocation across the biological membrane . [SEP]
[CLS] the potential of chitosan B-material - peg - tat nanoparticles B-nanoparticle complexed with sirna to silence ataxin - 1 protein B-material was assessed by using an in vitro model of spinocerebellar ataxia , a neurodegenerative disease . [SEP]
[CLS] results indicated a suppression of the ataxin - 1 protein B-material after 48 h of transfection with negligible toxicity B-property effects . [SEP]
[CLS] a different approach was adopted by dai et al . , who proposed a library of multiblock copolymer structures for nucleic B-material acid I-material delivery . [SEP]
[CLS] the obtained copolymers , produced from peg , poly ( propylene glycol ) ( ppg ) , and pll , were the following : peg - pll ( p1 ) , pll - peg - pll ( p2 ) , and pll - ppg - peg - ppg - pll ( p3 ) . [SEP]
[CLS] the resulting particles , obtained from these block copolymers , showed an amphiphilic B-property nature which enabled the ready formation of micelle B-material - complex with sirna . [SEP]
[CLS] the block copolymer p2 was the largest in size and showed the highest cation B-material charge , proving to induce a great gene knockdown but also the highest cytotoxicity B-property . [SEP]
[CLS] compared to p1 and p2 , p3 can induce an effective gene knockdown due to the presence of ppg units in its structure . [SEP]
[CLS] furthermore , while p1 and p2 copolymers could complex sirna , forming micelle - like aggregates of large size and poor density , p3 - sirna complex , with a smaller volume and compact micelle B-material - architecture , could enter the cells B-material more easily . [SEP]
[CLS] in light of these considerations , p3 complex turned out to be the most efficient among the tested complexes for sirna delivery and gene silencing , permitting achieving an in vitro decrease in gfp expression in gfp - expressing neuro 2a cells B-material of around 28 % . [SEP]
[CLS] polymeric B-nanoparticle nanoparticles I-nanoparticle , made of gelatin B-material and cross - linked with glutaraldehyde , were tested for the intranasal delivery of encapsulated sirna silencing inos in post - ischemic rat brain . [SEP]
[CLS] inos - rna was selected for its ability in silencing inos - derived no , which contributes to neurotoxicity after an ischemic stroke , since inos inhibition could reduce the infarct volume . [SEP]
[CLS] gelatin B-material was selected for its biocompatibility B-property and the non - toxicity B-property of its degradation products . [SEP]
[CLS] moreover , the drug release can be controlled by manipulating the kinetics of gelatin B-material degradation , that is , by controlling the crosslinking degree . [SEP]
[CLS] as for the intranasal administration , this was preferred over systemic injection in order to bypass the bbb . [SEP]
[CLS] the delivery of gelatin B-nanoparticle nanoparticles B-nanoparticle incorporating inos - sirna 6 h after middle cerebral artery occlusion resulted in suppressing the infarct volumes , with a maximum reduction of around 42 % . [SEP]
[CLS] very recently , krienke et al . proposed an approach based on the delivery of mrna for the treatment of neurodegenerative diseases . [SEP]
[CLS] the study suggested a non B-property - I-property inflammatory I-property mrna vaccine for the treatment of autoimmune encephalomyelitis . [SEP]
[CLS] in particular , the authors showed that the delivery at the systemic level of nanoparticle - formulated 1 - methylpseudouridine - modified messenger rna coding for disease - related autoantigens resulted in the presentation of the antigen on splenic cd11c + antigen - presenting cells B-material in absence of costimulatory signals . [SEP]
[CLS] in vivo tests performed on multiple sclerosis mouse models highlighted suppression of the disease by treatment with this mrna ; this result was ascribed to both the decrease of effector t cells B-material and the development of the regulatory t cell populations . [SEP]
[CLS] parkinson ' s disease : alpha - synuclein ( α - syn ) deposition in lewy bodies ( lb ) is one of the most characteristic neuropathological hallmarks of parkinson ' s disease ( pd ) . [SEP]
[CLS] schlich et al . investigated anionic liposomes B-nanoparticle , functionalized with rabies virus glycoprotein ( rvg ) as a target agent , loaded with a sirnaprotamine complex for the silencing of α - syn gene . [SEP]
[CLS] the potential of the proposed approach was tested in terms of in vitro effects on primary cortical and hippocampal cells B-material . [SEP]
[CLS] the achieved results on primary cortical neurons showed a significant decrease in the α - syn immunopositive signal in cells B-material subjected to decorated liposomes B-nanoparticle containing sirna , compared to those of controls and rvg liposomes B-nanoparticle not containing sirna . [SEP]
[CLS] data were in line with those obtained on cultures of primary hippocampal neuronal cells B-material , selected as usually affected by α - syn deposition in dementia lb . [SEP]
[CLS] also in this case , a reduced α - syn - immunopositive signal in neurons subjected to decorated liposomes B-nanoparticle containing sirna was registered , with respect to the other two groups of cells B-material . [SEP]
[CLS] as documented in the above - mentioned study , sirna can be successfully applied for the treatment of pd ; however , the inefficient delivery of sirna into neurons hampers its in vivo use . [SEP]
[CLS] as in other neurodegenerative diseases , strategies enabling the in vivo delivery of sirna are required . [SEP]
[CLS] in this context , sirna , against α - sin , complexed with low molecular weight pei , was delivered across the central nervous system down to the lumbar spinal cord after a single intracerebroventricular ( icv ) infusion in mice overexpressing human wild - type α - sin . [SEP]
[CLS] pei complexes were selected for their non - pathogenic characteristics and in order to overcome the issues related to viral vectors . [SEP]
[CLS] furthermore , pei complexation facilitated the entry of sirna into brain tissues . [SEP]
[CLS] the results achieved demonstrated that sirna delivered in pei nanoparticles B-nanoparticle resulted in an efficient target knockdown already after a single icv injection . [SEP]
[CLS] more specifically , a single icv infusion of 0 . 75 mg pei / sirna resulted in a 67 % reduction of α - syn mrna in the striatum , while a 50 % reduction in α - syn protein B-material was registered in all investigated regions ( striatum , medial septum , and cortex ) . [SEP]
[CLS] such an extensive knockdown caused by the proposed approach is expected to reduce the buildup of toxic B-property species , thus facilitating a slowdown in the progression . [SEP]
[CLS] an alternative strategy implemented to inhibit α - syn accumulation in lb is based on magnetic B-property fe 3 o 4 nanoparticles B-nanoparticle coated with oleic acid as nanocarriers . [SEP]
[CLS] these nanoparticles B-nanoparticle were functionalized with n - isopropylacrylamide derivative ( nipam - aa ) hydrogel and subjected to the absorption of shrna and nerve growth factor ( ngf ) . [SEP]
[CLS] nipam - aa , being characterized by a low critical solution temperature , thermo - responsiveness and ph sensitivity , made it possible to control and target the release ; ngf was used with the aim of promoting pc12 cellular uptake through ngf receptor - mediated endocytosis B-event . [SEP]
[CLS] since pd patients are affected by a degenerative apoptosis B-event in the substantia nigra pars compacta with a ph of the cytoplasmatic matrix in apoptotic cells B-material lower than that of normal tissue cells B-material , the ph sensitivity of nipam - aa hydrogel made it possible to accumulate nanoparticles B-nanoparticle on the apoptotic cell B-material surface , thus enabling them to reach their therapeutic target and release shrna via a change in hydrogel volume . [SEP]
[CLS] in vivo , the results obtained by observing the morphological changes in the fur of mice and performing the walking gait test indicated that nanoparticles B-nanoparticle ( nps B-nanoparticle ) could improve pd motor dysfunction . [SEP]
[CLS] prussian blue staining confirmed the ability of nps B-nanoparticle to cross the bbb and reach the substantia nigra . [SEP]
[CLS] lastly , the upregulation of th and the downregulation of α - syn confirmed the potential of the strategy used to prevent dopamine neuron degeneration . [SEP]
[CLS] a completely different approach from the strategies described so far is based on the regulation of mirna expression . [SEP]
[CLS] mirnas regulate gene expression at the post - transcriptional level by triggering an rna interference ; therefore , aberrant expressions of mirnas are one of the causes of numerous neurodegenerative disorders , including pd . [SEP]
[CLS] within this framework , neurologists are focusing on the identification of possible strategies for controlling mirna expression . [SEP]
[CLS] a possible strategy is based on the delivery of oligonucleotides into brain cells B-material . [SEP]
[CLS] titze de almeida et al . proposed the injection of mirna inhibitor complexed with neuromag - magnetic B-property polymeric B-nanoparticle nanoparticles I-nanoparticle capable of delivering nucleic B-material acids I-material in vivo into rat brains - by stereotaxic surgery into the lateral ventricles next to the striatum of rats . [SEP]
[CLS] to assess whether the intracerebroventricularinjected neuromag - complexed oligonucleotides reached the striatum , the oligonucleotides were labeled with fluorescein B-material isothiocyanate I-material ( fitc ) . [SEP]
[CLS] the collected microscope images demonstrated that green fluorescent B-property fitc - labeled oligonucleotides were next to the cell B-material nucleus of striatal neurons stained by neun and also present in gfap - positive glial cells B-material ( figure 9f ) . [SEP]
[CLS] conversely , the non - injected hemisphere showed no fitc - oligonucleotide green fluorescence B-property ( figure 9f ) . [SEP]
[CLS] the injected oligonucleotides proved to be effective in terms of mirna inhibition ; indeed , the injection of 0 . 36 nmol of neuromag - structured mir - 134 antimir was able to induce a 0 . 35 - fold decrease in striatal mir - 134 . [SEP]
[CLS] saraiva et al . investigated an approach to enhance brain repair in several neurodegenerative disorders , including pd . [SEP]
[CLS] this strategy consists of using biocompatible B-property and traceable plga nps B-nanoparticle containing perfluoro - 1 , 5 - crown ether B-material - a fluorine B-material compound trackable non - invasively by fluorine B-material ( 19 f ) - magnetic B-property resonance imaging - coated with protamine sulfate to complex mir - 12 , whose overexpression in the subventricular zone niche increased the number of newborn neurons without affecting their migratory capability . [SEP]
[CLS] mir - 124 nps B-nanoparticle , obtained according to the procedure shown in figure 9g , were demonstrated to efficiently deliver mir - 124 , while the internalization of mir - dy547 occurred in neural stem / progenitor cells B-material and neuroblasts ( figure 9h ) . [SEP]
[CLS] in vivo , the intracerebral administration of mir - 124 nps B-nanoparticle was shown to induce the migration of neurons into the lesioned striatum of 6 - ohda - treated mice . [SEP]
[CLS] alzheimer ' s disease : alzheimer ' s disease ( ad ) triggers a neurodegenerative process involving the pathological formation of plaques in the brain made of a mis - folded β - amyloid peptide B-material ( aβ ) . [SEP]
[CLS] the ad therapeutic treatments are mainly concentrated on the reduction of amyloid plaques by preventing aβ formation and blocking its aggregation . [SEP]
[CLS] β - secretase ( bace1 ) is the rate - limiting enzyme responsible for producing aβ peptides B-material from the amyloid precursor protein B-material ( app ) . [SEP]
[CLS] therefore , bace1 is considered one of the top drug targets for cerebral aβ plaque reduction in ad . [SEP]
[CLS] sirnas offer promising therapeutics for alzheimer ' s disease treatment via the specific silencing of bace1 . however , the effective and safe systemic delivery of sirna to the brain is challenging due to the bbb , short circulation lifetime , enzymatic degradation , and limited tissue penetration , among other factors . [SEP]
[CLS] as a result , gene therapy applied to ad requires the development of delivery technologies capable of targeting specific tissues or cell B-material types through a systemic administration , while avoiding nonspecific delivery , while at the same time being safe , biocompatible B-property , physiologically stable , and easy to administer . [SEP]
[CLS] in 2011 , alvarez - erviti et al . proposed controlling bace1 expression levels with exosome - mediated sirna delivery . [SEP]
[CLS] they demonstrated that sirna can be successfully encapsulated in naturally produced exosomes and that the modification of the exosomal membrane with rvg peptide B-material - the central nervous system - specific rabies viral glycoprotein - makes it possible to deliver the sirna cargo to the brain region by avoiding in vivo tissue non - specificity . [SEP]
[CLS] these experiments showed that , in vitro , the exosome - mediated delivery of sirna to neuronal cells B-material ( neuro2a ) can be as efficient as state - of - the - art transfection reagents , and that gene knockdown was cell B-material - type specific , by demonstrating that exosomes were successfully endowed with cell - targeting abilities . [SEP]
[CLS] similar results were achieved in vivo by a systemic administration : when naked gapdh sirna www . advancedsciencenews . com www . small - methods . com was injected , gapdh silencing was detected in the spleen , liver , and kidneys ; conversely , exosome - encapsulated sirnas were resistant to nonspecific uptake , but resulted in a significant knockdown of gapdh mrna in several brain regions . [SEP]
[CLS] the authors then focused on bace1 . [SEP]
[CLS] two validated bace1 sirnas were applied in vitro to neuro2a cells B-material in increasing doses , resulting in a dose - dependent knockdown , whereas when the acetylcholine receptor B-material ( the cellular target of rvgpeptide ) was blocked by α - bungarotoxin , a reduced bace1 knockdown occurred . [SEP]
[CLS] in vivo , the administration to normal c57bl / 6 mice of bace1 sirna encapsulated in rvg exosomes resulted in a significant protein B-material knockdown . [SEP]
[CLS] lastly , the authors demonstrated a significant decrease in the total β - amyloid 1 - 42 levels - a main component of the amyloid plaques in alzheimer ' s disease - which was greater than that reported after the intraventricular injection of bace1 inhibitors in normal mice . [SEP]
[CLS] guo et al . used nanocomplexes made of cationic B-material polymers B-material , pegylated poly ( 2 - ( n , n - dimethylamino ) ethylmethacrylate ) ( peg - pdmaema ) , loaded with sirnas against bace1 and modified on the surface by cgn and qsh peptides B-material for targeting bbb and amyloid plaques , respectively . [SEP]
[CLS] they demonstrated that nanocomplexes ( diameter of 70 nm ) efficiently cross the bbb monolayer without being damaged and that they can be internalized by neuronal cells B-material ( neuro2a ) in vitro , while in vivo , they are mainly concentrated on the neurons surrounding amyloid plaques , and are useful for gene accumulation in the ad lesion , consequently achieving a better therapeutic effect . [SEP]
[CLS] moreover , the presence of sirna in nanocomplexes efficiently inhibited the expression of bace1 in vivo , with a consequent reduction in the amyloid plaque formation , an increased synaptophysin level , and an improvement in the cognitive performance of ad transgenic mice . [SEP]
[CLS] zhou et al . developed a glycosylated polymeric nanomedicine containing sirna stabilized by a triple - interaction ( gal - np B-nanoparticle @ sirna ) to target bace1 in transgenic ad mouse models ( figure 9i ) . [SEP]
[CLS] the triple interaction strategy consisted of using a salt B-material of guanidinium - phosphate ( gu + / po 3 4− ) to stabilize the electrostatic and hydrogen B-material bond interactions , and a hydrophobic B-property interaction I-property due to the complexation between sirna and the galactose - modified fluorinated polymer B-material . [SEP]
[CLS] this strategy determines an effective encapsulation of sirna and a higher stability performance in blood circulation than with nanomedicines based on cationic B-material polymers B-material , where only electrostatic interactions stabilize sirna . [SEP]
[CLS] moreover , the gal - np B-nanoparticle @ sirna ( diameter of 118 nm , figure 9j ) can effectively penetrate the bbb via glycemia - controlled glucose transporter - 1 ( glut1 ) mediated transport . [SEP]
[CLS] after verifying that in neuro2a cells B-material the gal - np B-nanoparticle @ sirna determines bace1 gene silencing in vitro ( figure 9k ) , the authors investigated in vivo pharmacokinetics . [SEP]
[CLS] they found that gal - np B-nanoparticle @ sirna has a longer blood circulation time than in controls , with an elimination half - lifetime ( t1 / 2 ) of 39 . 2 min ; the brain targeting by the glycemia - controlled glut1mediated transport was verified ( figure 9l ) . [SEP]
[CLS] the therapeutic effect of gal - np B-nanoparticle @ sibace1 was investigated in the app / ps1 double transgenic mouse model with several behavioral tests , such as the novel object recognition ( nor ) and the morris water B-material maze ( mwm ) tests ( figure 9m ) , thus confirming that i ) the gal - np B-nanoparticle @ sirna significantly improves cognitive performances in app / ps1 mice , and ii ) both hippocampal and cor - tical bace1 protein B-material levels were relevantly reduced with respect to other app / ps1 control groups . [SEP]
[CLS] a different strategy for the treatment of alzheimer ' s disease consists of regulating mir132 , which plays a key role in the neural development and regulation of neuronal activity . [SEP]
[CLS] in recent years , a strategy based on the nose - to - brain delivery of nps B-nanoparticle complexed with mir132 to prevent the decrease of mir132 levels was experimented . [SEP]
[CLS] this intranasal injection was preferred over systemic injection with the aim of inducing a faster transport of the drug to the brain , while minimizing systemic exposure and bypassing the bbb . [SEP]
[CLS] samaridou et al . proposed the formation of electrostatically driven nanocomplexes between a lauric acid chemically conjugated octa - arginine B-material and the mirna . [SEP]
[CLS] the resulting nanocomplexes ( encps ) were enveloped with different protective polymers B-material , that is , peg - polyglutamic acid ( peg - pga ) or ha , in order to enhance their stability across the olfactory nasal mucosa . [SEP]
[CLS] the results , obtained in vivo on an app nl−g - f knock - in mouse model of alzheimer ' s disease , demonstrated the suitability of the designed nps B-nanoparticle in reaching the hippocampus area - which is involved in memory formation and one of the first areas affected in alzheimer ' s disease - while leading to increased mir - 132 endogenous levels after 24 h from the administration . [SEP]
[CLS] furthermore , additional studies in vivo demonstrated that thanks to the presence of encps , mir - 132 in its active form was successfully delivered to the hippocampus , as demonstrated by the reduced levels of two mirna targets , gata2 and rb1 . [SEP]
[CLS] in order to prevent the decrease of mir132 levels in the blood , su et al . delivered wheat germ agglutinin ( wga ) - nps B-nanoparticle - mir132 intranasally in mouse and rat models of alzheimer ' s disease with ischemic brain injury , to treat neurological damages after cerebral ischemia . [SEP]
[CLS] wga - nps B-nanoparticle were selected as effective carriers for the intranasal delivery to the central nervous system of the brain , as demonstrated in a previous study . [SEP]
[CLS] the potential of intranasally delivered wga - nps B-nanoparticle - mir132 was evaluated through in vivo analyses on wild - type c57bl / 6 mice , by investigating the aβ plaques and synaptic - related protein B-material ( syn and psd95 ) levels . [SEP]
[CLS] the results demonstrated that aβ protein B-material expression , upregulated in ad mice , significantly decreases after wga - nps B-nanoparticle - mir132 treatment . [SEP]
[CLS] conversely , a significantly increased expression of syn and psd - 95 was detected after the inhalation . [SEP]
[CLS] in mcao model rats , the authors evaluated the effect of np B-nanoparticle inhalation in terms of apoptosis B-event around brain lesions after cerebral ischemia . [SEP]
[CLS] the results demonstrated that wga - nps B-nanoparticle ( not containing mirna ) had no protective effect against ischemic brain damages ; mirna delivered alone was easily degraded and / or cleared by nasal cilia ; wga - nps B-nanoparticle - mir132 proved to be effective in reducing the area of cerebral infarction , the number of microglia , and the number of apoptotic cells B-material after cerebral hemorrhage . [SEP]
[CLS] the emergence of global disease outbreaks has been one of the most significant challenges to be faced for the human society . [SEP]
[CLS] at irregular intervals , novel flu viruses against which most people were not immune have naturally appeared , thus resulting in global pandemics . [SEP]
[CLS] moreover , increased international traveling , trading , and ethnic integration , together with a greater urbanization and massive exploitation of environmental resources , have sustained the spread of infections worldwide . [SEP]
[CLS] since early in the 21 st century , three viruses - specifically coronaviruses - have crossed the species barrier B-property , causing severe pneumonia disease in humans : i ) the severe acute respiratory syndrome coronavirus ( sars - cov ) broke out in 2002 in guangdong province ( china ) ; ii ) the middle - east respiratory syndrome coronavirus ( mers - cov ) was discovered in 2012 on the arabian peninsula ; iii ) the sars - cov - 2 , a novel coronavirus , broke out in december 2019 in wuhan ( hubei province , china ) . [SEP]
[CLS] sars - cov - 2 was associated with an outbreak of atypical pneumonia , named coronavirus disease 2019 ( covid - 19 ) , that rapidly spread from china to all continents , unleashing a global infection . [SEP]
[CLS] coronaviruses consist of a positivesense , non - segmented , single - stranded mrna [ ( + ) ssrna ] of ≈30 kb , enveloped in a helical nucleocapsid . the viral rna encodes a set of 28 specific proteins B-material grouped into three main categories : i ) non - structural proteins B-material ( nsp1 to nsp16 ) ; ii ) structural spike ( s ) , nucleocapsid ( n ) , membrane ( m ) , and envelope ( e ) proteins B-material ; and iii ) the accessory proteins B-material ( orf3a , orf6 , orf7a , orf7b , orf8 , orf9b , orf9c , and orf10 ) . [SEP]
[CLS] nsp proteins B-material lead to the formation of the replication - transcription B-event complex ( rtc ) . [SEP]
[CLS] this complex promotes the synthesis of a set of nested subgenomic minus - strands of rna [ ( - ) sgrna ] in specific doublemembrane vesicles . [SEP]
[CLS] these [ ( - ) sgrna ] serve as templates and encode subgenomic mrnas from which all the structural and accessory proteins B-material are synthesized . [SEP]
[CLS] bioinformatic analyses showed that sars - cov - 2 belongs to the coronaviridae family ( order nidovirales , subfamily orthocoronaviridae ) . furthermore , sars - cov - 2 is a beta - coronavirus ( 2b lineage ) showing a relationship with the sars - like coronavirus strain in bats , batcov ratg13 ( 96 % homologies ) , which can effectively infect a wide range of vertebrates , including bats , pangolins , and humans . [SEP]
[CLS] therefore , at the present time , sars - cov - 2 seems to have a zoonotic origin and to have acquired human - to - human spreading capacity . [SEP]
[CLS] several studies suggest that horseshoe bats ( rhinolophus affinis ) seem to be a natural reservoir of both sars - cov and sars - cov - 2 , while pangolins ( manis javanica ) and american minks ( neovison vison ) appear to be the intermediate B-property hosts of sars - cov - 2 for animal - to - human infection . in a recent study , it has been posited that the beta - coronavirus isolated from pangolins ( pangolin - cov ) has a 100 % , 98 . 6 % , 97 . 8 % , and 90 . 7 % amino B-material acid I-material similarity with human sars - cov - 2 e , m , n , and s proteins B-material , respectively . furthermore , the receptorbinding domain ( rbd ) of the s protein B-material of pangolin - cov practically matches the rbd of sars - cov - 2 , with only one difference in a non - critical amino B-material acid I-material . [SEP]
[CLS] unfortunately , humans remain the principal source of transmission for other humans and domestic , farm , and zoo animals . [SEP]
[CLS] interestingly , dogs , pigs , and poultry have shown resistance to sars - cov - 2 . [SEP]
[CLS] the primary access mode of sars - cov - 2 is through the upper respiratory tract ( via droplets or aerosol suspension ) , where the nasal and bronchial epithelial cells B-material and pneumocytes are its targets . [SEP]
[CLS] coronavirus recognizes the angiotensin - converting enzyme 2 ( ace2 ) , a cell B-material membrane enzyme present in epithelial cells B-material of the airway , lungs , heart , blood vessels , kidneys , liver , and intestine ( figure 10a ) . [SEP]
[CLS] in detail , the s1 transmembrane subunit of s protein B-material promotes the virus attachment to host ace2 proteins B-material , while the s2 subunit is responsible for the fusion with the host cell B-material membrane . [SEP]
[CLS] ace2 is a crucial factor in the renin - angiotensinaldosterone system ( raas ) pathway , since it regulates processes such as blood pressure , wound healing , and inflammation . [SEP]
[CLS] furthermore , ace2 , by counteracting the angiotensin ii ( ang ii ) activity and reducing the pathological effects associated with its high levels , plays a crucial role in preventing tissue damage . [SEP]
[CLS] therefore , the binding of sars - cov - 2 with ace2 hinders ang ii downregulation and sustains tissue injuries . [SEP]
[CLS] covid - 19 presents a wide range of symptoms , including fever ( 88 . 7 % ) - more commonly in adults than in children - dry cough ( 67 . 8 % ) , fatigue / tiredness ( 38 . 1 % ) , sputum production ( 33 . 4 % ) , dyspnea ( 18 . 6 % ) , sore throat ( 13 . 9 % ) , and headache ( 13 . 6 % ) . [SEP]
[CLS] so far , more than 110 million confirmed covid - 19 cases and 2 . 4 million deaths have been reported . [SEP]
[CLS] it is important to stress that most adult patients have had an asymptomatic infection or mild flu - like symptoms , while 14 % of covid patients have suffered severe health issues that require hospitalization and / or oxygen B-material support , and 5 % have been treated in intensive care units . [SEP]
[CLS] while several treatments have been adopted to deal with the impact of the disease , vaccination represents the primary means to effectively control this pandemic . [SEP]
[CLS] to stimulate the human immune response against a virus , a vaccine usually contains an antigen made from its weakened or killed forms , toxins , or one of its surface proteins B-material . [SEP]
[CLS] most covid - 19 vaccine candidates are in the development stages , and the so - called s spike protein B-material of sars - cov - 2 is their main target . [SEP]
[CLS] multiple approaches are being deployed to identify a safe and effective vaccine . [SEP]
[CLS] among them , those based on nucleic B-material acids I-material represent the most important contemporary drug discovery platform , since they enable rapid vaccine development , authorization , and manufacturing . [SEP]
[CLS] this method permits the release of small rna fragments that encode specific viral proteins B-material into the host cells B-material ; then they will be internally processed and loaded onto the host cell B-material membrane . [SEP]
[CLS] immune cells B-material can recognize them and lead to the synthesis of antibodies B-material . [SEP]
[CLS] remarkably , these vaccines are self - adjuvant and promote a humoral and cellular immune response against the engendered proteins B-material ; they can deliver multiple antigens with one immunization . [SEP]
[CLS] furthermore , their safety , tolerance , and high potency have been demonstrated in numerous studies . [SEP]
[CLS] kariko et al . laid the foundation for rna - based vaccines in 2008 . [SEP]
[CLS] in their research , the authors demonstrated that the nucleoside - modified mrna ( modrna ) containing pseudouridines is translated more efficiently than unmodified mrna in vitro , particularly in primary mammalian dendritic cells B-material and in vivo in mice . [SEP]
[CLS] moreover , the modrna effectively improved rna biological stability , while reducing its immunogenicity B-property in vivo . [SEP]
[CLS] several other advantages have been demonstrated , including : i ) improved safety ( modrna cannot be integrated into chromatin ) , ii ) lack of host inflammatory response ; iii ) efficient transduction and rapid protein B-material expression using the host ribosomal machinery ; iv ) unlimited engineered nucleotide - sequence size ; v ) closely controllable half - life ; and vi ) high manufacturability for largescale continuous production . [SEP]
[CLS] as discussed earlier , lnp represents a robust strategy for delivering the modrna to host cells B-material ( figure 10b ) . [SEP]
[CLS] the lnp system is usually made of cholesterol , which plays various structural roles , including filling gaps in the particle , preventing lnp - protein B-material interactions , and promoting fusion with the cell B-material membrane . [SEP]
[CLS] in addition , the ionizable B-property lipids B-material and functional lipids B-material - which play a crucial role in maintaining the physiological ph and promote endosomal release - are essential components of the modrna vaccine . [SEP]
[CLS] currently , there are two kinds of mrna - based vaccines : non - amplifying mrna - based , and self - amplifying mrna - based . [SEP]
[CLS] to date , there are 181 candidates in the pre - clinical evaluation stage and 69 candidates in the clinical stage as covid - 19 vaccines . [SEP]
[CLS] among them , 30 are based on rna technologies . [SEP]
[CLS] so far , the fda and the european medicines agency ( ema ) have granted the emergency use and marketing of two mrna - based vaccines : comirnaty ( tozinameran or bnt162 - 02 ) , produced by pfizer - biontech , and the mrna - 1273 developed by moderna . [SEP]
[CLS] remarkably , a research group of the departments of pathology , genetics , pediatrics , and medicine at stanford university has recently provided the putative sequence information for the two synthetic rna molecules of pfizer / biontech and moderna covid - 19 vaccines and has released them online on github . [SEP]
[CLS] the rnas samples were obtained from vaccine drops that remained in vials - and destined to be otherwise discarded - after patients immunization and were analyzed under fda authorization for research use . [SEP]
[CLS] pfizer - biontech covid - 19 vaccine : comirnaty vaccine , based on a cell - free in vitro transcription B-event approach , was designed from sars - cov - 2 dna templates . [SEP]
[CLS] it contains a modrna encoding the sars - cov - 2 full - length viral spike ( s ) protein B-material , in which two proline mutations have been introduced to lock it in the prefusion conformation ( figure 10c ) . [SEP]
[CLS] the exact formulation of the pfizer - biontech lnp was recently disclosed . [SEP]
[CLS] this contains dspc , cholesterol , and two novel functional lipid B-material excipients B-property , alc - 0315 and alc - 0159 . [SEP]
[CLS] alc - 0315 is a physiological ph cationic B-material synthetic lipid B-material ( ( 4 - hydroxybutyl ) azanediyl ) bis ( hexane - 6 , 1 - diyl ) bis ( 2 - hexyldecanoate ) essential for the efficient copyright 2020 , elsevier . b ) modrna - lnp structure in which mrna is bound to the ionizable B-property lipids B-material ( central core B-material ) while peg lipids and dspc form the surface of the lipid B-nanoparticle nanoparticle I-nanoparticle [SEP]
[CLS] reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2021 , the authors . published by multidisciplinary digital publishing institute . [SEP]
[CLS] c ) rna vaccine vectors that include a cap , 5 ′ utr , 3 ′ utr , and poly ( a ) tail of variable length , and modrna target sequence . [SEP]
[CLS] reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2021 , the authors . [SEP]
[CLS] published by multidisciplinary digital publishing institute . [SEP]
[CLS] self - assembly and encapsulation of the modrna within the lnp and its delivery into host cells B-material . [SEP]
[CLS] alc - 0159 is a pegylated lipid B-material designed to exchange out of the lnps . [SEP]
[CLS] polack et al . reported that two 30 µg doses of vaccine ( given 3 weeks apart ) into the muscle of the upper arm promote the development of high sars - cov - 2 antibody B-material titers and robust antigen - specific cd8 + and th1 - cd4 + t cell responses with an overall efficacy of 95 % . [SEP]
[CLS] the first benefit has been observed from day 12 after the first injection , indicating an early onset of immunization , reaching a 52 % efficacy in the interval between the two injections . [SEP]
[CLS] furthermore , complete protection against covid - 19 was observed 7 days after the second dose . [SEP]
[CLS] adverse reactions were classified as mild or moderate in particular in the oldest adults , and interestingly , the reactogenicity after both injections was similar . [SEP]
[CLS] unfortunately , despite this promising data , the study is affected by several limitations , including i ) the low cold temperatures ( −80 °c ) required for shipping and storage , ii ) the low number of participants for each of the selected groups , iii ) the limited follow - up time ( approx . 2 months ) after the second dose , and iv ) the long - term monitoring that remains to be determined . [SEP]
[CLS] regarding the last item , ethical barriers B-property can give rise to a heated debate . [SEP]
[CLS] over the next 2 years , the planned follow - up should also monitor the placebo recipients to collect long - term data , but on the other hand , it is not ethical to deprive these people from receiving the covid - 19 vaccine before the conclusion of the trial . [SEP]
[CLS] moderna covid - 19 vaccine : moderna covid - 19 vaccine ( mrna - 1273 ) encodes for the full - length sars - cov - 2 spike protein B-material modified with two proline substitutions , k986p and v987p , within the heptad repeat 1 domain ( s - 2p ) , to block the spike protein B-material into a prefusion conformation . [SEP]
[CLS] in detail , the mrna sequence includes a 5 " cap , the 5 " untranslated region ( utr ) , the open reading frame ( orf ) , the 3 " utr , and the 3 " polya tail . [SEP]
[CLS] the modrna is encapsulated into lnps , and the exact formulation of the moderna mrna - 1273 - lnp was publicly released recently . [SEP]
[CLS] it is made of dspc , cholesterol , and two innovative excipients B-property , peg2000 - dmg , and the ionisable B-property lipid B-material heptadecan - 9 - yl - 8 - ( ( 2 - hydroxyethyl ) ( 6 - oxo - 6 - ( undecyloxy ) - hexyl ) amino ) - octanoate ( sm - 102 ) . [SEP]
[CLS] sm - 102 , a molecule highly soluble B-property in several organic solvents and insoluble B-property in I-property water I-property , was selected for its vaccine potency , tolerability , and biodegradability B-property . [SEP]
[CLS] interestingly , the storage temperature of the finished product is −20 °c , since only a slight degradation was observed . [SEP]
[CLS] baden et al . reported that two 100 µg doses of the mrna - 1273 vaccine , given 28 days apart into the deltoid muscle , lead to a 94 . 1 % efficacy in covid - 19 illness prevention , including its severe forms . [SEP]
[CLS] moreover , the local adverse vaccination reactions were mild . [SEP]
[CLS] however , moderate - to - severe systemic side effects ( fatigue , myalgia , arthralgia , and headache ) were observed in around 50 % of the participants in the mrna - 1273 group after the second dose . [SEP]
[CLS] additionally , anderson et al . showed that the mrna - 1273 vaccine elicited a remarkable cd4 cytokine response , including th1 cells B-material . [SEP]
[CLS] moreover , the tumor B-material necrosis factor α ( tnfα ) responses were more significant than the interleukin - 2 ( il2 ) ones , which in turn were greater than the interferon - γ ( infγ ) ones . [SEP]
[CLS] unfortunately , only after the second dose low levels of cytotoxic B-property cd8 t cell B-material responses were observed [SEP]
[CLS] automatic feature learning , supported by the increased computational capabilities , has contributed immensely to the successful application of machine learning ( ml ) - a branch of the broader artificial intelligence ( ai ) sector in almost every research field . [SEP]
[CLS] among the outstanding examples demonstrated so far , one of the most impactful areas consists of vaccine and drug discovery , in which ml has offered several advantages , including compound , activity and reactivity prediction , and ligand - protein B-material interaction . [SEP]
[CLS] an example is " reverse vaccinology " ( rv ) , a genome - based vaccine design approach that has revolutionized vaccine research , introducing a very efficient method for identifying the target . [SEP]
[CLS] vaxijen , the first integrated ml and rv approach , has improved antigen prediction for vaccine development , while vaxign - ml , a web - based rv program , enables bacterial antigen prediction . [SEP]
[CLS] additionally , artificial intelligence has led to the development of tools such as the vaccine adverse event reporting system ( vaers ) and vaccine safety databank ( vsd ) , for the prediction of safety and reliability . [SEP]
[CLS] at present , the most powerful ai tools used to counteract covid - 19 provide for the virtual screening of vaccine candidates , investigate the biological pathways involved , predict the off - targets , and discover new chemical compounds . [SEP]
[CLS] two of the most promising ai - based approaches are maria and netm - hcpan4 , which are supervised neural network - driven tools capable of finding putative t - cell epitopes for the sars - cov - 2 spike receptor - binding domain ( rbd ) . [SEP]
[CLS] furthermore , in a recent study , malone et al . , using the long short - term memory ( lstm ) , ineo , nec immune profiler , iedb , and bepipred tools , provided a comprehensive vaccine design blueprint for sars - cov - 2 . [SEP]
[CLS] these tools can predict simulated sequences which offer a useful guideline for further vaccine discoveries to fight covid - 19 and novel zoonoses that may arise in the future . [SEP]
[CLS] malone et al . presented the first computational approach that uses a large - scale epitope database of sars - cov - 2 to improve t - cell mediated immune responses , thus leading to more effective and comprehensive vaccine blueprints . [SEP]
[CLS] this two - step analysis first provides the identification of statistically significant epitope hotspot regions by monte carlo simulations . [SEP]
[CLS] afterward , " digital twin " person - specific simulation ranks these epitope hotspots to identify the candidate that best promotes a robust t cell B-material immune response on a global scale . [SEP]
[CLS] lastly , several mutations that lead to a genetic drift and escape from immune control were recently recognized in the coronavirus genome . [SEP]
[CLS] ai is a suitable tool for facing the spreading of sars - cov - 2 variants , detecting its mutations , predicting the efficacy of the vaccines approved , and developing new candidates . [SEP]
[CLS] notably , among the mutations discovered , the most worrisome ones are the united kingdom and south africa variants , which share a common mutation , the n50y , in the spike domain . [SEP]
[CLS] this mutation is found in the viral receptor B-event binding site for cell B-material entry , resulting in an increased affinity for the ace2 . [SEP]
[CLS] leung et al . discovered that the 501y variant 2 ( also named b . 1 . 1 . 7 , 20b / 501y . v1 and voc - 202012 / 01 and assigned clade gr ) is 75 % more transmissible than the 501n strain . [SEP]
[CLS] in conclusion , ai can contribute to designing a " one size fits all " universal vaccine to protect against sars - cov - 2 mutations and future coronavirus variants . [SEP]
[CLS] current progresses in nanoparticle B-nanoparticle design and synthesis open up unprecedented opportunities for the development of stimuli - responsive , on - demand delivery systems that provide great spatiotemporal control over therapeutics release . [SEP]
[CLS] stimuli - responsive nanosystems are nanostructured B-material materials I-material that produce a precise behavior in response to a specific instruction incoming from a chemical or a physical stimulus such as ph , redox potential , temperature variation , light , magnetic B-property field , radiofrequency , or ultrasounds . [SEP]
[CLS] in particular , once a stimulus ( instruction ) is received , smart nanosystems can modify the surrounding environments , change wettability , convert chemical and biochemical signals into optical , electrical , thermal , and mechanical signals , and control the transport of ions B-material and molecules of different species . [SEP]
[CLS] light is one the most valuable of physical stimuli , since it is a safe and useful energy source that can be controlled with great precision and a high degree of freedom ; indeed , light can be controlled in terms of wavelength , intensity , and light - spot dimensions . [SEP]
[CLS] an excellent overview of photoactive nanocarriers for controlled delivery is reported in reference . [SEP]
[CLS] in particular , applications in the biomedical field require preferably near - infrared light ( nir ) in order to be beneficial and safe . [SEP]
[CLS] indeed , electromagnetic radiation ranges from 700 to 900 nm and from 1000 to 1300 nm correspond , respectively , to the first and second biological windows , where the living tissue absorption is low . [SEP]
[CLS] therefore , the penetration depth of light is high : a light source emitting at 800 nm can penetrate tissues up to 3 mm in depth . [SEP]
[CLS] furthermore , nir causes less damage to cells B-material than uv and visible light because hemoglobin , water B-material , and lipids B-material barely absorb light in this region . [SEP]
[CLS] for this reason , nir is often used to trigger the selective release of oligonucleotides and molecules from suitably functionalized photoactive nanoparticles B-nanoparticle , keeping the cargo inactive until its release is triggered on - demand , and providing a high local concentration of therapeutic molecules in a specific site , while maintaining the overall systemic dosage relatively low . [SEP]
[CLS] the two main mechanisms involved in controlled nir - triggered payload release entail the activation of photochemical B-property reactions and the conversion of light into heat , respectively . [SEP]
[CLS] the former mechanism relies on using upconversion nanoparticles B-nanoparticle ( ucnps ) , and on promoting non - linear photoconversion by switching nir light into visible light or uv light . [SEP]
[CLS] at the same time , the latter makes use of nanoparticles B-nanoparticle capable of absorbing nir - light and efficiently converting it into localized heat , thus inducing the localized and controlled release of the cargo molecules . [SEP]
[CLS] among the huge number of nanoparticlebased photothermally induced delivery systems , this section is mainly focused on rna delivery systems triggered by thermoplasmonic - assisted gold - based nanostructures , selected because of their unique properties in terms of low toxicity B-property , in vivo stability , and enhanced tumor B-material uptake , and for their ability to convert nir light into heat , making them extremely promising in forefront nanomedicine fields . [SEP]
[CLS] nanoconstructs based on rna functionalized gold B-nanoparticle nanoparticles I-nanoparticle can be effectively used for promoting on - demand gene regulation in several applications , including basic cell B-material research , regenerative medicine , and cancer therapy . [SEP]
[CLS] the nir - triggered release of rna mediated by gold B-nanoparticle nanoparticles I-nanoparticle relies on the efficiency of gold B-nanoparticle nanoparticles I-nanoparticle of a suitable morphology to absorb nir light and convert it into thermal energy , highly localized in the proximity of the nanoparticles B-nanoparticle . [SEP]
[CLS] when such a phenomenon , known as thermoplasmonic heating , is promoted by rna functionalized gold B-nanoparticle nanoparticles I-nanoparticle internalized in cells B-material and located in the endocytic vesicles , the heating of water B-material surrounding the nanoparticles B-nanoparticle generates localized nanobubbles that disrupt the endosomal barrier B-property , promoting the release of rna without damaging the cell B-material . [SEP]
[CLS] importantly , as the optical properties of gold B-nanoparticle nanoparticles I-nanoparticle are strongly related to their morphology , the nir - light - triggered release of rna can obviously occur only by irradiating gold B-nanoparticle nanoparticles I-nanoparticle capable of effectively absorbing nir light lying in the first and the second biological water B-material windows , such as gold B-material nanorods B-nanoparticle , nanoshells B-nanoparticle , and hollow nanoshells . [SEP]
[CLS] this issue has been tackled by morgan et al . , who prepared a set of nir - absorbing gold B-nanoparticle nanoparticles I-nanoparticle differing in shape , but with a similar average size of 45 nm , including hollow gold B-material nanoshells B-nanoparticle ( hgns ) , hollow gold nanocages I-nanoparticle ( hgncs ) , and gold B-material nanorods B-nanoparticle ( gnrs ) . [SEP]
[CLS] the authors have evaluated sirna release ability under an 800 nm laser irradiation , demonstrating how rna release ability is affected by nanoparticle B-nanoparticle morphology , irrespective of their specific spectroscopic features . [SEP]
[CLS] the amount of released rna is independent of the laser power for gnrs and hgncs , while the release ability of hgns is affected by the laser power . [SEP]
[CLS] moreover , provided that the rna release is normalized to the rna loading for each nanoparticle B-nanoparticle morphology , the rna release rate from hgns appears relatively low if compared to the hgn loading capability . [SEP]
[CLS] however , the three gold B-nanoparticle nanoparticle I-nanoparticle morphologies have shown a comparable relative rna release rate when a laser power of 900 mw has been used for an irradiation time of 3 s . [SEP]
[CLS] it is important to point out that the efficient protein B-material knockdown effect is linked not only to nanoparticle B-nanoparticle photoinduced release ability but also to nanoparticle B-nanoparticle internalization effectiveness . [SEP]
[CLS] from a chemical - physical standpoint , thermal energy produced by rna - functionalized gold B-nanoparticle nanoparticles I-nanoparticle upon nir irradiation can trigger rna release by inducing the dehybridization of double - stranded nucleic B-material acids I-material on nanocarriers , by dissociating the gold B-material - thiol B-material bond ( responsible for the rna linkage B-property to gold B-material nanocarriers ) , or by altering the thermally sensitive moiety involved in the assembly of the nanoconstruct . [SEP]
[CLS] importantly , photo - thermal - assisted rna delivery is not strictly related to a temperature increase . [SEP]
[CLS] wang et al . have reported hgns conjugated with a sirna against hsp70 ( sihsp70 ) as a thermoplasmonic platform able to generate heat and at the same time inhibit hsp70 : a heat shock protein B-material ( hsp ) , expressed under hyperthermia , that produces a cytoprotective effect that contrasts ptt - induced apoptosis B-event . [SEP]
[CLS] the nanoconstruct promotes a 90 % sirna release ( estimated based on the sihsp70 loading ) when it is irradiated with a continuous wave ( cw ) laser emitting at the resonant wavelength of 765 nm for 90 s , at a power density greater than 4 w cm −2 , resulting in a temperature increase of up to 48 °c . [SEP]
[CLS] in particular , the nanoconstruct proposed by wang et al . can release 80 % of sihsp70 when the nir irradiation produces a temperature of 35 °c . [SEP]
[CLS] in contrast , when the temperature increase is obtained without nir irradiation ( using a water B-material bath ) , no obvious sihsp70 release is observed . [SEP]
[CLS] therefore , nir irradiation ' s thermoplasmonic heating , interacting with sihsp70 - functionalized hgns , is sufficient to promote the au - s bond cleavage , being extremely localized in the proximity of the nanocarrier . [SEP]
[CLS] in particular , irradiation of 90 s is sufficient to trigger sihsp70 release in cells B-material . [SEP]
[CLS] still , an irradiation time of 6 min is required to induce a temperature increase suitable for ptt ' s tumor B-material ablation , with an inhibited hsp70 expression in the treated cells B-material . [SEP]
[CLS] this result demonstrates the possibility to achieve a precise gene strategy without affecting unirradiated cells B-material . [SEP]
[CLS] irradiation conditions are a crucial parameter governing the rna release efficiency . [SEP]
[CLS] indeed , irradiation with a continuous wave ( cw ) laser produces a different effect than irradiation with a femtosecond ( fs ) pulsed laser . [SEP]
[CLS] as shown in figure 11a , a cw laser produces a temperature increase and a temperaturedependent release of both single - strand rna and dsrna . [SEP]
[CLS] in contrast , the fs pulsed laser does not produce a temperature increase because of the extremely short duration of the pulsed irradiation , but promotes an efficient release of duplex rna . [SEP]
[CLS] such a feature is relevant , as the duplex rna can enable ondemand gene regulation , recognized by the induced silencing complex . [SEP]
[CLS] furthermore , for gene regulation - related applications , a temperature increase is not desirable , as it can induce nucleic B-material acid I-material denaturation , and therefore the loss of the protein B-material knockdown effect . [SEP]
[CLS] a fs pulsed laser , irradiating sirna functionalized silica - gold ( sio 2 @ au ) nanoparticles B-nanoparticle in a core @ shell geometry , can induce a 72 % release of duplex sirna , which can mediate on - demand gene silencing , demonstrated by the measurement of a 33 % decrease in green fluorescence B-property protein B-material expression , as shown in the histogram of figure 11a . [SEP]
[CLS] however , suitable plasmonic nanocarriers can be created to promote both a gene silencing effect ( by sirna ) and ptt , as per the paper by zhang et al . which proposes a nanocomposite consisting of a biodegradable B-property and thermosensitive polymer folate / pei - conjugated poly ( organophosphazene ) polymer nanocapsule B-nanoparticle , containing both sirna and au - fe 3 o 4 nanoparticles B-nanoparticle . [SEP]
[CLS] as shown in figure 11b , this nanocapsule B-nanoparticle provides triple tumor B-material targeting ( active , passive , and magnetic B-property ) , resulting in a 12 % reproduced with permission . [SEP]
[CLS] copyright 2018 , american chemical society . b ) intravenous injections can administer hydrogel nanocapsule B-nanoparticle containing plasmonic and magnetic B-nanoparticle nanoparticles I-nanoparticle for the sirna release under nir light irradiation . reproduced with permission . [SEP]
[CLS] copyright 2019 , american chemical society . c ) gold B-material nanoshells B-nanoparticle loaded with mir - 34a for triple - negative breast cancer ( tnbc ) treatment can effectively suppress tnbc cells B-material under irradiation with nanosecond pulsed laser mir - 34 . reproduced with permission . [SEP]
[CLS] copyright 2021 , american chemical society . d ) upconversion nanoparticles B-nanoparticle functionalized with a sirna for silencing the polo - like kinase 1 ( plk1 ) and the photosensitizer B-property chlorin B-material e6 for the combinatorial cancer treatment through gene and photodynamic therapy . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2014 , royal society of chemistry . [SEP]
[CLS] intravenous delivery in vivo , with a consequent tumor B-material elimination in vivo via the combination of gene therapy and ptt [SEP]
[CLS] nir - light - controlled rna release , mediated by plasmonic nanoparticles B-nanoparticle , has also been successfully used to suppress triple - negative breast cancer ( tnbc ) cells B-material . in this case , the nanoformulation consists of sio 2 @ au conjugated with mir - 34a , that is , a tumor - suppressive microrna which is extremely promising in the treatment of tnbc ( figure 11c ) . [SEP]
[CLS] the authors have proposed the irradiation of this nanoformulation with an 810 nm - emitting cw laser and a nanosecond ( ns ) pulsed laser , using a power density of less than 0 . 4 w cm −2 to prevent the cell B-material damage caused by consequent thermoplasmonic heating . [SEP]
[CLS] the mirna rate linked to the sio 2 @ au , as depicted in the histogram of figure 11c , is higher when cw laser irradiation is applied . [SEP]
[CLS] therefore , mir - 34a - conjugated sio 2 @ au releases a greater rate of mirna under ns pulsed laser irradiation , irrespective of the power density , preserving the mirna functionality ( see the gel B-technique electrophoresis I-technique of the released mirna shown in figure 11c ) . [SEP]
[CLS] such an efficient release is associated with a greater amount of localized heating and higher energy available to cleave the au - thiol B-material bond , responsible for nanoparticle B-nanoparticle conjugation with the mirna . [SEP]
[CLS] nir - induced controlled rna release can also be achieved by plasmonic heterostructures such as gold B-material @ silver B-material @ gold B-nanoparticle nanoparticles B-nanoparticle in a core @ shell @ shell array and upcns ( nagdf 4 : ybeb ) . [SEP]
[CLS] the latter was used by wang et al . to build a multifunctional nanocomplex that assembles a photosensitizer B-property ( chlorin B-material e6 , ce6 ) suitable for photodynamic therapy ( pdt ) and a sirna for silencing the polo - like kinase 1 ( plk1 ) to trigger apoptosis B-event in treated cells B-material . [SEP]
[CLS] as shown in figure 11d , the nanocomplex has been prepared by a multistep approach that involves the surface functionalization by a polymer B-material layer - by - layer assembly to promote the loading of both the ce6 and the plk1sirna . [SEP]
[CLS] when ucnps are irradiated with a laser emitting at 980 nm for 20 min ( 0 . 8 w cm −2 ) , the occurrence of a resonance energy transfer triggers the excitation of the ce6 , which generates singlet oxygen B-material ( cytotoxic B-property to cancer cells B-material ) and the release of the plk1sirna , whose silencing activity is not hindered by the pdt . [SEP]
[CLS] overall , the spatiotemporal controlled release of rna triggered by nir light and mediated by plasmonic nanoparticles B-nanoparticle can be regarded as a powerful tool for developing effective protocols for combinatorial biomedical treatments . [SEP]
[CLS] this is due to the multifold properties of plasmonic nanoparticles B-nanoparticle which , according to the nanoconstruct design , can merge stimuliresponsive nanocarrier properties for combined gene therapy , photothermal , and / or photodynamic therapy . [SEP]
[CLS] among other types of cells B-material , pluripotent stem cells B-material ( pscs ) have an interesting unlimited self - renewal capacity and can differentiate into multiple cell B-material lineages . [SEP]
[CLS] the fate of pscs , including psc renewal and psc differentiation , is strongly regulated and modulated by rnas exploiting the expression or inhibition of several transcription B-event factors . [SEP]
[CLS] indeed , rnas influence several biological processes and functions , including cell B-material embryogenesis , metabolism , division , proliferation , self - renewal , differentiation , organ development , and apoptosis B-event . [SEP]
[CLS] due to the high efficiency B-property of I-property transfection I-property , rna therapeutics are considered one of the most promising approaches for directing the fate of pscs in many tissue engineering and regenerative medicine applications . [SEP]
[CLS] within this framework , the ability of rnas to induce psc differentiation into lineage - specific cells B-material , cell reprogramming into pscs , and transdifferentiation have gained the attention of scientific researchers . [SEP]
[CLS] several studies have demonstrated that rna - based therapies can effectively direct the psc differentiation into fully differentiated myocytes , cardiomyocytes , neurons as well as epithelial and renal cells B-material . [SEP]
[CLS] more specifically , pscs have been successfully induced to differentiate into lineage - specific neurons by introducing rna therapeutics that act by upregulating a specific cell B-material transcription B-event factor ( figure 12a , b ) . [SEP]
[CLS] in another study , differentiated neurons are obtained from pscs by the rna coding of a specific factor that knocks down the expression of a targeted gene ( figure 12c ) . [SEP]
[CLS] in the same manner , a study conducted by hiratsuka et al . on the rna coding of transcriptional B-event factors reports the successful induction of psc differentiation into nephron - like organoids belonging to kidney tissue ( figure 12d ) . [SEP]
[CLS] differentiation protocols have distinguished a high efficiency ( > 90 % ) of differentiation in a relatively short time , revealing the higher potential of rna therapeutics compared to growth factor - based treatments . [SEP]
[CLS] on the other hand , as a differentiation reverse process , cell B-material reprogramming is considered the most promising approach in the psc field for the regeneration of tissues and the treatment of diseases . [SEP]
[CLS] indeed , this process aims to reprogram differentiated cells B-material into pscs , regaining a self - renewal ability and the capacity to differentiate into multiple cell B-material lines . [SEP]
[CLS] within this framework , the research on rna coding reprogramming factors has highlighted several valuable results , including the reprogramming of differentiated cells B-material into pscs . [SEP]
[CLS] zhao et al . performed the reprogramming of epiblast stem cells B-material by using rna transcripts B-event to silence a specific key gene . [SEP]
[CLS] the same method has also evidenced the self - renewal of mouse embryonic stem cells B-material . [SEP]
[CLS] in addition , the successful reprogramming of human fibroblasts into pscs has been reported in a study by kogut et al . the authors established a very efficient ( approximately 90 . 7 % ) protocol by the introduction of combined rna encoding reprogramming factors and silencing rnas . [SEP]
[CLS] however , it is worth mentioning that each cell B-material type requires the establishment of a specific protocol for the successful reprogramming process . [SEP]
[CLS] for these reasons , scientists are continuously involved in the challenging development of efficient methods for reprogramming differentiated cells B-material into pscs . [SEP]
[CLS] recent years have seen a vast and rapid development of novel rna - based technologies in biomedical treatments . [SEP]
[CLS] the applicability of different nanostructured B-material materials I-material used as nanoplatforms for biological drug delivery has been greatly improved , paving the way for the clinical translation of rna therapeutics . [SEP]
[CLS] the achievement of effective rna targeting has been possible thanks to some crucial advantages given by novel nanocarriers , including their ability to overcome biological barriers B-property , prevent bioactive molecule degradation , and avoid unwanted inflammatory response of the body tissues . [SEP]
[CLS] this enormous progress has confirmed the bright future for these nanotechnologyassisted therapies , thus inducing the pharmaceutical industry to move toward scaling up and marketing the next generation of drugs based on rna molecules . [SEP]
[CLS] therefore , significant effort has been devoted recently by researchers working in different fields - including chemists , physicists , biologists , material B-material engineers , and medical doctors - to rationally designing , developing and testing nanoplatforms with exceptional features for targeting rna to specific cells B-material and tissues . [SEP]
[CLS] even though tremendous progress and outstanding achievements have been recently reported , major challenges in developing ideal therapeutic agents still remain . [SEP]
[CLS] in this frame , we believe that the unique characteristics of mrna therapeutics will be continuously explored in the near future for vaccine production , while mirna - based therapies will be researched and developed for wound healing applications . [SEP]
[CLS] on the other hand , much work is still necessary to reduce nanocarrier bioaccumulation , aggregation in the body fluids , and non - specific adsorption , to optimize these nanovehicles and achieve the desired goal . [SEP]
[CLS] for instance , though considerable developments have been made on carbonaceous nanomaterials B-material and inorganic B-nanoparticle nanoparticles I-nanoparticle , several issues such as potential toxicity B-property , carcinogenicity , and dna damage still hinder the broad applications of these vectors as carriers of genetic molecules . [SEP]
[CLS] undoubtedly , novel nanovehicles with enhanced biocompatibility B-property , especially lnps , represent the most advanced and promising nanocarriers for the effective delivery of rna molecules . [SEP]
[CLS] despite the already described new trends , future directions of research on rna - based healthcare materials are continuously explored . [SEP]
[CLS] within this framework , the use of fascinating ai tools has been recently considered for investigating the potential of rna as a therapeutic agent capable of revolutionizing the current pharmacological therapies and opening up many new biomedical applications . [SEP]
[CLS] ai approaches aim at significantly enhancing the timing and accuracy of medical diagnostics and therapeutics , while predicting the results of potential treatments . [SEP]
[CLS] it consists of enormous , organized , structured data sets and algorithms . [SEP]
[CLS] its power lies in the capacity to process , integrate , interpret , and find patterns in extremely large volumes of data with high accuracy , sensitivity , and specificity , and within a relatively fast timeline . [SEP]
[CLS] the possibility of including additional complexities and several variabilities also helps guarantee reliable results and problem solutions . [SEP]
[CLS] for these reasons , artificial intelligence tools are particularly suitable for analyzing and predicting rna - related mechanisms and alterations , such as rna structure and folding . [SEP]
[CLS] this permits a better and faster recognition of complex gene expression patterns , thus permitting the detection of gene disorders or mutations , as well as infectious disease characteristics . [SEP]
[CLS] hence , ai can be useful for designing rnas therapeutics for silencing , correcting , and inhibiting patient - specific gene mutations as well as genome editing . [SEP]
[CLS] copyright 2020 , the authors . [SEP]
[CLS] published by john wiley and sons . [SEP]
[CLS] c ) brightfield microscopic images of pscs during the culture time , illustrating the change in cell B-material morphology due to the induction of psc differentiation into neuron - specific lineages ( scale bar : 100 µm ) . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , john wiley and sons . d ) induction of psc differentiation into nephron progenitor cells B-material : analysis of expression of nephron specific markers , showing the efficient differentiation of pscs into renal cells B-material ( scale bar : 100 µm ) . reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2019 , the authors . published by springer nature . [SEP]
[CLS] additionally , artificial intelligence tools are also quite powerful within the framework of rna - based cancer treatments . [SEP]
[CLS] during the past few years , the rna therapeutics field has , without a doubt , experienced an enormous growth . [SEP]
[CLS] today , novel requests for the development of rna - based vaccines emerged during the covid - 19 pandemic have brought this technology into the spotlight . [SEP]
[CLS] it follows that today ' s tremendous interest in rna will ensure an explosive growth of rna therapeutics over the next few decades . [SEP]
[CLS] overall , there is still plenty of room in nanotechnology - assisted rna delivery . [SEP]
[CLS] we believe that this review will play a crucial role in the ongoing revolution in patients ' pharmacological treatment and personalized medicine , inspiring and guiding a large audience of scientists . [SEP]
[CLS] 1 . impact of nanotechnology - based delivery of biologics and rnas on drug development and biomedical fields . [SEP]
[CLS] figure 1 . a ) biological drugs approved by fda in the last 16 years . [SEP]
[CLS] asterisks represent the approved rna - based pharmaceutical products . [SEP]
[CLS] b ) the number of published articles on rna delivery during the period 2001 - 2020 , obtained from web of science database . [SEP]
[CLS] c ) unlike small molecules , rnas cannot overcome the lipid B-material bilayer I-material barrier B-property developed to protect cells B-material from rna entering . [SEP]
[CLS] naked rnas are too large and too charged to pass through the cell B-material membrane ; therefore , they require a delivery agent to invade cells B-material . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2017 , nature publishing group . [SEP]
[CLS] d ) rna protection from rnase - mediated degradation and capability of rna internalization into the cell B-material are guaranteed by the use of nanoplatforms as rna carriers . [SEP]
[CLS] fulfilling these objectives allows rna molecules to exploit their therapeutic effect , directly influencing biochemical cell B-material processes . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , wiley - vch [SEP]
[CLS] 3 . schematic illustration of the shrna - and mirna - based therapies . [SEP]
[CLS] figure 3 . a ) schematic representation of shrna structure ( stem - loop ) , activity , and function . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , elsevier b . v . b ) schematic representation of mirna structure ( double - stranded stem - loop rna ) , functions and activity . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , john wiley and sons [SEP]
[CLS] 4 . schematic representation of nanocarriers for rna delivery . [SEP]
[CLS] fueled by investigation and innovation , nanocarrier functionalities develop progressively as the pursuit continues for achieving higher transfection B-property efficiency I-property balanced with lower immunogenicity B-property . [SEP]
[CLS] 5 . rna delivery based on carbonaceous , inorganic , and polymer B-material nanocarriers . [SEP]
[CLS] figure 5 . a ) graphene oxide B-material functionalized with chitosan B-nanoparticle nanoparticles I-nanoparticle as a carrier of sirna for regulating the bcl - 2 expression . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , elsevier . [SEP]
[CLS] b ) glucose - linked gold B-nanoparticle nanoparticles I-nanoparticle for targeted sirna delivery to breast cancer stem - like cells B-material . [SEP]
[CLS] glucose ligands endow the nanoparticles B-nanoparticle with target ability toward the breast . [SEP]
[CLS] adapted with permission . [SEP]
[CLS] copyright 2019 , elsevier . [SEP]
[CLS] c ) green nanoparticles B-nanoparticle for sirna delivery . [SEP]
[CLS] natural polyphenol from green tea catechin was complexed with sirna to form negatively charged nanoparticles B-nanoparticle , followed by surface coating B-material with pll . [SEP]
[CLS] reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2020 , the authors . published by springer nature . [SEP]
[CLS] d ) higher - molecular weight , bioreducible , cationic B-material polymer enhanced sarna delivery . [SEP]
[CLS] high - molecular weight pabols were achieved by improved aza - michael addition . [SEP]
[CLS] complexation with sarna happened via titration method and transfection efficacy of the pabol - 100 polyplexes were compared to jetpei and peimax . [SEP]
[CLS] reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2020 , the authors . [SEP]
[CLS] published by american chemical society . [SEP]
[CLS] 7 . rna - based wound healing systems . [SEP]
[CLS] figure 7 . a ) days 0 to 6 wounds in control and mirs - treated group ( n = 6 ) . [SEP]
[CLS] wound closure was quantified and presented as a healing rate with visible faster wound closure in the mirs - treated group . [SEP]
[CLS] b ) mir - 19b and mir - 20a exhibit therapeutic potential for chronic wounds . [SEP]
[CLS] the mixture of mir - 19b and mir - 20a mimics or control oligos was injected into the wound - edges of db / db mice after injury . [SEP]
[CLS] qrt - pcr detected mir - 19b ( left ) and mir - 20a ( right ) in wounds and inner organs . [SEP]
[CLS] a , b ) reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2021 , the authors . [SEP]
[CLS] published by elsevier . [SEP]
[CLS] c ) in vitro 3d transfection of human fibroblasts with rala - simmp - 9 complexes visualized with a confocal fluorescence B-property microscope . [SEP]
[CLS] these images clearly confirm that the rala - simmp - 9 complexes are able to associate with and enter the cells B-material . [SEP]
[CLS] green , actin ; blue , cell B-material nucleus ; red , rala - simmp - 9 complexes . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , elsevier . [SEP]
[CLS] 8 . rna delivery for cancer treatment . [SEP]
[CLS] figure 8 . a ) nanoparticles based - sirna delivery system for pancreatic tumors B-material . [SEP]
[CLS] schematic of the preparation of gold B-material nanoparticle - based atra and hsp47 sirna codelivery system . [SEP]
[CLS] tumor B-material growth curves during treatment . [SEP]
[CLS] reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2018 , the authors . published by springer nature . [SEP]
[CLS] b ) vaccine - based sirna delivery to initiate innate immunity for breast cancer treatment . [SEP]
[CLS] targeting capability of hpvp on tumor - bearing mice . [SEP]
[CLS] in vivo tumor - targeting capacity of hpvp on 4t1 tumor - bearing mice after injection . [SEP]
[CLS] immunofluorescence images showing pdl1 inhibition effect . [SEP]
[CLS] in vivo anticancer B-property capacity of sirna - based system on breast tumor B-material model . [SEP]
[CLS] reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2020 , the authors . [SEP]
[CLS] published by springer nature . [SEP]
[CLS] 9 . rna - based treatment for neural tissue applications . [SEP]
[CLS] figure 9 . a ) flowchart for preparation of electrospun pcl nanofibers B-nanoparticle functionalized with polydopamine and polydopa - melanin mussel - derived coatings B-material and lipofectamine rnaimax and transit complexed with sirna , as transfection reagents and b ) integration with transected spinal cord tissue of polydopa - melanin coated scaffold functionalized with rnaimax lipofectamine - sirest . [SEP]
[CLS] a , b ) reproduced with permission . [SEP]
[CLS] copyright 2018 , royal society of chemistry . [SEP]
[CLS] c , d ) sem images of pcl fiber - collagen hydrogel system . [SEP]
[CLS] 10 . covid - 19 rna vaccine . [SEP]
[CLS] figure 10 . a ) side view of sarbecovirus s glycoproteins plotted on the sars - cov - 2 s structure . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , elsevier . [SEP]
[CLS] b ) modrna - lnp structure in which mrna is bound to the ionizable B-property lipids B-material ( central core B-material ) while peg lipids and dspc form the surface of the lipid B-nanoparticle nanoparticle I-nanoparticle . [SEP]
[CLS] reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2021 , the authors . [SEP]
[CLS] published by multidisciplinary digital publishing institute . [SEP]
[CLS] c ) rna vaccine vectors that include a cap , 5 ′ utr , 3 ′ utr , and poly ( a ) tail of variable length , and modrna target sequence . [SEP]
[CLS] reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2021 , the authors . [SEP]
[CLS] published by multidisciplinary digital publishing institute . [SEP]
[CLS] 11 . [SEP]
[CLS] nanostructures for the spatiotemporal controlled nir - triggered release of rna . [SEP]
[CLS] figure 11 . a ) gold B-material nanoshells B-nanoparticle functionalized with sirna can induce an efficient release of sirna duplex and an effective gene silencing activity without a temperature increase under irradiation with femtosecond pulsed laser . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , american chemical society . [SEP]
[CLS] b ) intravenous injections can administer hydrogel nanocapsule B-nanoparticle containing plasmonic and magnetic B-nanoparticle nanoparticles I-nanoparticle for the sirna release under nir light irradiation . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , american chemical society . [SEP]
[CLS] c ) gold B-material nanoshells B-nanoparticle loaded with mir - 34a for triple - negative breast cancer ( tnbc ) treatment can effectively suppress tnbc cells B-material under irradiation with nanosecond pulsed laser mir - 34 . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2021 , american chemical society . [SEP]
[CLS] d ) upconversion nanoparticles B-nanoparticle functionalized with a sirna for silencing the polo - like kinase 1 ( plk1 ) and the photosensitizer B-property chlorin B-material e6 for the combinatorial cancer treatment through gene and photodynamic therapy . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2014 , royal society of chemistry . [SEP]
[CLS] 12 . [SEP]
[CLS] differentiation and reprogramming of pscs by rnas - based therapies . [SEP]
[CLS] figure 12 . a ) induction of psc differentiation into neurons : immunostaining microscope images and b ) quantitative real - time pcr analysis of neuronal specific markers , showing the efficient neural differentiation of pscs ( scale bar : 50 µm ) . [SEP]
[CLS] a , b ) reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2020 , the authors . published by john wiley and sons . [SEP]
[CLS] c ) brightfield microscopic images of pscs during the culture time , illustrating the change in cell B-material morphology due to the induction of psc differentiation into neuron - specific lineages ( scale bar : 100 µm ) . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , john wiley and sons . [SEP]
[CLS] d ) induction of psc differentiation into nephron progenitor cells B-material : analysis of expression of nephron specific markers , showing the efficient differentiation of pscs into renal cells B-material ( scale bar : 100 µm ) . [SEP]
[CLS] reproduced under the terms of the creative commons attribution 4 . 0 international license ( http : / / creativecommons . org / licenses / by / 4 . 0 / ) . [SEP]
[CLS] copyright 2019 , the authors . [SEP]
[CLS] published by springer nature . [SEP]
[CLS] selected nanocarrier platforms for rna delivery . [SEP]
[CLS] continued . [SEP]
[CLS] table 1 . continued . www . advancedsciencenews . com www . small - methods . comet al . developed functionalized single - walled cnts for the codelivery of surviving sirna and 4 - substituted - 2 , 5 - dimethoxyamphetamines ( dox ) . [SEP]
[CLS] our brain is protected by physio - biological barriers B-property . [SEP]
[CLS] the blood - brain barrier B-property ( bbb ) main mechanism of protection relates to the abundance of tight junctions ( tjs ) and efflux pumps . [SEP]
[CLS] although bbb is crucial for healthy brain protection against toxins , it also leads to failure in a devastating disease like brain cancer . [SEP]
[CLS] recently , nanocarriers have been shown to pass through the bbb and improve patients ' survival rates , thus becoming promising treatment strategies . [SEP]
[CLS] among nanocarriers , inorganic nanocarriers , solid B-nanoparticle lipid I-nanoparticle nanoparticles I-nanoparticle , liposomes B-nanoparticle , polymers B-material , micelles B-material , and dendrimers B-nanoparticle have reached clinical trials after delivering promising results in preclinical investigations . [SEP]
[CLS] the size of these nanocarriers is between 10 and 1000 nm and is modified by surface attachment of proteins B-material , peptides B-material , antibodies B-material , or surfactants B-property . [SEP]
[CLS] multiple research groups have reported transcellular entrance as the main mechanism allowing for these nanocarriers to cross bbb . [SEP]
[CLS] transport proteins B-material and transcellular lipophilic B-property pathways exist in bbb for small and lipophilic B-property molecules . [SEP]
[CLS] nanocarriers cannot enter via the paracellular route , which is limited to water B-property - soluble B-property agents due to the tjs and their small pore size . [SEP]
[CLS] there are currently several nanocarriers in clinical trials for the treatment of brain cancer . [SEP]
[CLS] this article reviews challenges as well as fitting attributes of nanocarriers for brain tumor B-material treatment in preclinical and clinical studies . [SEP]
[CLS] structural or functional impairments to the brain or spinal cord including trauma , infection , inflammation , tumors B-material , degeneration , and autoimmune disorders are classified as central nervous system ( cns ) disorders . [SEP]
[CLS] these conditions can lead to serious cognition and physiological impairments and may prove fatal in certain cases . [SEP]
[CLS] brain tumors B-material have a high fatality rate and can seriously affect and devastate lives . [SEP]
[CLS] despite many improvements in treatment protocols , drug delivery remains a major challenge , and treatment options are limited . [SEP]
[CLS] the most malignant brain tumors B-material begin with genetic mutations that impair and dysregulate cell B-material function and division . [SEP]
[CLS] brain tumors B-material are classified as malignant and benign , and further subcategorized as primary and secondary . [SEP]
[CLS] whereas primary tumors B-material are caused by the division B-event of I-event brain I-event cells I-event only , secondary tumors B-material develop from the metastasis B-event of other organs to the brain and are also hardly treatable . [SEP]
[CLS] primary tumors B-material unlike secondary types start and involve the brain cells B-material . [SEP]
[CLS] the glioblastoma multiform ( gbm ) , with a treatment - refractory disorder with a patients ' short lifespan , cognitive impairments , and high mortality rate , is one of the most common primary brain tumors B-material . [SEP]
[CLS] the patient ' s survival percentage with grade iv glioblastoma is reported to be 4 . 5 % for 5 years by world health organization ( who ) . [SEP]
[CLS] metastatic brain tumor B-material as the secondary tumor B-material type has almost 6 months overall survival . [SEP]
[CLS] the median survival was at its highest with surgery which was followed by radiation therapy . [SEP]
[CLS] the surgical resection plus radiotherapy , as well as adjuvant chemotherapy frequently , has been comprised by the treatment guidelines ( figure 1 ) . [SEP]
[CLS] with the lack of clear tumor B-material margins , precise anatomical location , and biases that exist during the surgery , chemotherapy has been one of the most effective approaches . [SEP]
[CLS] despite recent progress in the development of effective chemotherapy agents and efforts on improved drug formulations , the tumor B-material ' s molecular heterogeneity has made the treatment process and the resulting outcomes more complicated . [SEP]
[CLS] the delivery of chemotherapy agents to the brain tissue faces the challenge of crossing the anatomical and physiological barrier B-property , the blood - brain barrier B-property ( bbb ) being the most important one . [SEP]
[CLS] the bbb is a highly selective barrier B-property ( hardly permeable ) that protects the brain cells B-material against blood - circulating agents such as pathogens and toxins . [SEP]
[CLS] many approaches to overcoming bbb have been suggested in the literature . [SEP]
[CLS] one of the promising applied approaches lies in the nanotechnology . [SEP]
[CLS] nanotechnology , particularly nano - drug delivery systems , is emerging as promising tools in the cancer diagnostics and therapeutics . [SEP]
[CLS] based on the drug - loading methods and nanoparticle B-nanoparticle surface , size , and zeta B-property potential I-property modifications , the effector molecules can be encapsulated , adsorbed , or attached to the nano - drug delivery system . [SEP]
[CLS] to date , the most successful nanocarriers based on clinical trials are inorganic B-nanoparticle nanoparticles I-nanoparticle ( such as metal B-material , metal oxide I-material , carbon B-material , and silica particles ) , liposomes B-nanoparticle , micelles B-material , dendrimers B-nanoparticle , and polymers B-material . [SEP]
[CLS] assuming that blood capillaries and cells B-material range between 6 to 9 and 10 to 20 μm , respectively , almost all types of nano - drug delivery systems can reach and deliver therapeutics into organs and cells B-material . [SEP]
[CLS] specifically targeting the bbb receptors by surface - functionalized nano - drug delivery systems makes them ideal candidates for the brain cancer drug delivery . [SEP]
[CLS] this study aims to discuss the challenges regarding brain tumor B-material treatment and to review the current application of nanotechnology in preclinical and clinical studies for this disease category . [SEP]
[CLS] with careful examination of the literature , the physiological and biological aspects of the bbb were summarized along with the nanodrug delivery strategies that have been reviewed in detail based on the nanostructures . [SEP]
[CLS] finally , the majority of all ongoing nanodrug delivery systems which have made their way through the clinical trials were reviewed as the realistic perspective of nanomedicine for brain cancer drug delivery systems . [SEP]
[CLS] in 1885 , ehrlich and his colleagues demonstrated the presence of barriers B-property between cns and the periphery using brain parenchyma staining via intravenous injection . [SEP]
[CLS] one of ehrlich ' s students , edwin goldmann completed ehrlich ' s dye experiments by directly administrating trypan blue directly into the cerebrospinal fluid ( csf ) . [SEP]
[CLS] the term bbb generally refers to the distinct characteristics of continuous nonfenestrated brain microvasculature . [SEP]
[CLS] this unique feature is the consequence of physical barrier B-property properties , molecular barrier B-property properties , as well as specific transporters . [SEP]
[CLS] the cells B-material , molecules , and ions B-material entrance are suppressed by the protective role of the bbb . [SEP]
[CLS] furthermore , it is almost an impermeable barrier B-property for drugs . [SEP]
[CLS] even though the bbb makes uniform coverage between the brain parenchyma and blood vessels interface , the circumventricular organs link the cns and peripheral blood vessels . [SEP]
[CLS] blood vessels are made up of 2 cell B-material types : endothelial cells B-material ( ecs ) that build up blood vessels , and mural cells B-material including vascular smooth muscle cells B-material and pericytes ( pcs ) , which are placed on the outer layer of the ecs . while ecs are primarily responsible for the bbb characteristics , the function and maintenance of the bbb depend on interactions between ecs , pcs , and astrocytes ( figure 2 ) . [SEP]
[CLS] simple squamous epithelial cells B-material are the bricks of the blood vessel walls . [SEP]
[CLS] cns microvascular ecs are 39 % thinner than muscle ecs and also there is a 200 nm diameter between the luminal and abluminal surface . [SEP]
[CLS] the main role of ecs is to limit the entry of cells B-material , molecules , and ions B-material to the brain . the ec tight junctions not only block the paracellular pathway but also vesicle - mediated transcellular flux too . [SEP]
[CLS] as a result of paracellular and transcellular restrictions , blood - brain transportation is highly controlled . [SEP]
[CLS] higher amounts of mitochondria and very low levels of leukocyte adhesion molecules are other specific limiting features . [SEP]
[CLS] the pcs incompletely cover the abluminal microvascular side that is attached to the vascular basement membrane . [SEP]
[CLS] pc cells B-material with their contractile proteins B-material control the capillary diameter . [SEP]
[CLS] in comparison with other tissues like muscles , in the cns pcs provide the most coverage . [SEP]
[CLS] the ec - to - pc content ratio is between 1 : 1 and 1 : 3 in cns microvascular versus 100 : 1 in muscles . [SEP]
[CLS] astrocytes are polarized glial cells B-material that cover the entire vessel ' s tube . [SEP]
[CLS] they not only connect neuronal cells B-material with the blood vessels but also reflect the neuronal signals on the microvessels ' blood flow . [SEP]
[CLS] this includes contraction / dilation regulation of the vascular smooth muscle cells B-material next to capillaries and arterioles , respectively . [SEP]
[CLS] the main cns endothelial cell B-material transporters are efflux and nutrient types . [SEP]
[CLS] efflux transporters , including mrps , mdr1 , and bcrp take advantage of atp hydrolysis to actively transport different biological membranes . [SEP]
[CLS] nutrient transporters facilitate the transportation of nutrients against their concentration gradient . [SEP]
[CLS] most of these belong to the family of solute carrier transporters , including slc16a1 ( lactate , pyruvate ) , slc2a1 ( glucose ) , slc7a5 ( neutral amino B-material acids I-material , l - dopa ) , and slc7a1 ( cationic B-material amino B-material acids I-material ) . [SEP]
[CLS] most of the small lipophilic B-property molecules , less than 400 to 500 da , can pass through the bbb . [SEP]
[CLS] of the greater than 7000 drugs for insomnia , depression , and schizophrenia that were assessed in comprehensive medicinal chemistry ( cmc ) database study , only 5 % reached the cns and averaged 357 da . [SEP]
[CLS] in another study , 12 % of drugs were activated upon entering the cns while only 1 % of them were of use for nonpsychotic disorders ( neurosis ) . [SEP]
[CLS] antibiotics , antineoplastics , and neuropeptides are common examples of compounds with limited transfer rates through the bbb . [SEP]
[CLS] to that end , the objective for many brain drug delivery systems is concerned with targeting the bbb receptors . [SEP]
[CLS] generally , invasive and noninvasive methods are 2 major interventions to allow passage through bbb . [SEP]
[CLS] the invasive procedures occur with transient disruption of bbb by chemical , biological , and physical stimuli . [SEP]
[CLS] these methods are expensive and have proven to be highly risky . [SEP]
[CLS] as a result , they are not preferable for the brain drug delivery enhancement . [SEP]
[CLS] in contrast , noninvasive methods have proven more effective at providing a relatively harmless drug - to - brain delivery system . [SEP]
[CLS] furthermore , a blood - to - brain strategy that improves the bbb permeability and facilitates drug - carrier conjugation minimizes the mentioned drawbacks . [SEP]
[CLS] it has been demonstrated that the transcytosis mechanism by the bbb cells B-material supports active drug transportation due to the sizable presence of mitochondria . various nanocarriers have been thought to be able to overcome the bbb , potentiating drug delivery to ischemic lesions and various tumors B-material in cns . many research and review papers have aimed to comprehensively examine the effectiveness of brain drug delivery systems . [SEP]
[CLS] generally , the paracellular pathway is considered the common route of entry for small hydrophilic B-property molecules , and the transcellular pathway is the preferable route for the transport of small nutritional or therapeutic compounds . [SEP]
[CLS] unfortunately , due to the physiologic limitations of the bbb , both pathways apply to a small selection of compounds . [SEP]
[CLS] other more feasible and commonly used modes of transportation include a carrier or receptor - mediated transcytosis . [SEP]
[CLS] briefly , the endosomal formation following carrier conformational change due to the concentration gradient elucidates pathways . [SEP]
[CLS] these established pathways to get through the bbb are illustrated in figure 3 . [SEP]
[CLS] current methods in the treatment of brain tumors B-material . [SEP]
[CLS] these methods are including surgical resection plus radiotherapy , as well as adjuvant chemotherapy . [SEP]
[CLS] inorganic nanomaterials B-material . [SEP]
[CLS] these types of nanomaterials B-material based on silica , carbon B-material , metal B-material , and metal oxide I-material particles are widely used in imaging B-technique techniques I-technique . [SEP]
[CLS] stabilized size and monodispersed formation in the bloodstream , high surface area , and for this reason , ease of functionalizing are just a few positive aspects of inorganic nanomaterials B-material . [SEP]
[CLS] silica mesoporous nanoparticles I-nanoparticle , carbon B-nanoparticle nanotubes I-nanoparticle with an ultrahigh surface area , gold B-material and iron B-nanoparticle oxide I-nanoparticle nanoparticles I-nanoparticle , especially superparamagnetic B-nanoparticle iron I-nanoparticle oxide I-nanoparticle nanoparticles I-nanoparticle ( spions ) , are typical inorganic B-nanoparticle nanoparticle I-nanoparticle examples ( table 1 ) . [SEP] B-nanoparticle
[CLS] in addition to chemical modification by peg , lactoferrin , cationic B-material serum albumin , poly ( isobutylene - alt - maleic anhydride [ pma ] ) s can be used as chemical modifications . [SEP]
[CLS] these improve the hydrophilicity B-property and decrease both blood aggregations and the reticuloendothelial system ( res ) clearance . [SEP]
[CLS] additionally , physical approaches like magnetism B-property as a novel drug delivery mechanism have been used to facilitate passing through bbb . [SEP]
[CLS] furthermore , it has been demonstrated that external magnetic B-property forces can effectively cross spions through the bbb . [SEP]
[CLS] although numerous investigations have found inorganic B-nanoparticle nanoparticles I-nanoparticle to be efficient enough to pass through the bbb , hard degradability and its following toxicities B-property , and undesirable drug delivery , have excluded them from tangible clinical studies . [SEP]
[CLS] solid B-nanoparticle lipid I-nanoparticle nanoparticles I-nanoparticle ( slns ) . [SEP] B-nanoparticle
[CLS] sln refers to nanodispersions ranging from 10 to 1000 nm of biocompatible B-property lipids B-material including fatty acids ( eg , stearic acid ) , triglycerides ( eg , tristearin ) , waxes ( eg , cetyl palmitate ) , and steroids ( eg , cholesterol ) that are stabilized with surfactants B-property ( table 1 ) . [SEP]
[CLS] a combination of surfactants B-property with the hydrophilic - lipophilic B-property balance ( hlb ) values less than 12 , such as poloxamer 188 and pluronic ® f68 , are used in the sln structure . [SEP]
[CLS] the core B-material of sln is made of solid lipids B-material , which makes them ideal for hydrophobic B-property drug loading . [SEP]
[CLS] slns have attracted growing attention as potential nano - based anticancer B-property drug delivery formulations for gliomas and glioblastoma . [SEP]
[CLS] similar to other nanocarriers , the surface of slns is modified and functionalized via the attachment of various targeting B-material ligands I-material including proteins B-material , peptides B-material , small molecules , and antibodies B-material . [SEP]
[CLS] this results in increased antitumor activities and reduced adverse effects by targeting specific receptor - I-event mediated endocytosis B-event . [SEP]
[CLS] along with the aforementioned advantages , prolonged retention time in the serum and brain can be increased by improving the hydrophilicity B-property of slns via pegylation . [SEP]
[CLS] biocompatibility B-property , biodegradability B-property , and surface modifications are the main advantages of slns . [SEP]
[CLS] however , they can be easily eliminated by the res from the blood due to their lipophilicity B-property , which can present a potential challenge . [SEP]
[CLS] polymeric B-nanoparticle nanoparticles I-nanoparticle . [SEP] B-nanoparticle
[CLS] polymeric B-nanoparticle nanoparticles I-nanoparticle including nanospheres B-nanoparticle and nanocapsules B-nanoparticle are thermodynamically stable structures made of natural or synthetic polymers B-material , with a range of sizes between 1 and 1000 nm . [SEP]
[CLS] as a result of considering an optimum nanoformulation that is biodegradable B-property for over a few days , the nondegradable formulations are excluded including quantum B-nanoparticle dots I-nanoparticle , nonorganic nanocarriers ( silica and metal B-material particles ) , and needle - shaped carbon B-nanoparticle nanotubes I-nanoparticle . [SEP]
[CLS] consequently , 3 types of nanostructured B-material materials I-material are considered including polylactic acid ( pla ) or its copolymer poly lactide - co - glycolide acid ( plga ) , poly butyl cyanoacrylate acid ( pbca ) or poly iso hexyl cyanoacrylate acid ( pihca ) , and human serum albumin ( hsa ) . [SEP]
[CLS] it has been reported that 80 % of pbcas are degraded 24 h after iv injection . therefore , poly alkyl cyanoacrylates with approximately 2000 to 3000 da molecular weights have the fastest degradability among polymers B-material . [SEP]
[CLS] higher poly alkyl cyanoacrylates toxicity B-property rates were observed with the slowest or fastest degradability rates . [SEP]
[CLS] the intermediate B-property rates showed lower toxicities B-property . [SEP]
[CLS] interestingly , a formulation of pbca has achieved clinical trial phase iii and now is purchased by the trade name of livatag ® ( doxorubicin B-material transdrug [UNK] ) . [SEP]
[CLS] livatag ® is the product of pbca and dox hcl attachment , which increases the pbca ' s molecular weight and substantially improves hepatocarcinoma drug delivery . [SEP]
[CLS] the enzymatic cleavage by lipases and esterases is thought to be the prevailing degradation mechanism for plga and cyanoacrylates , respectively . [SEP]
[CLS] furthermore , albumin nanoparticles B-nanoparticle are degraded within 3 days in macrophages . [SEP]
[CLS] natural polymers B-material are prospective candidates for brain drug delivery over synthetics due to the lessened toxicity B-property , improved biodegradability B-property , and lowered costs . [SEP]
[CLS] chitosan B-material with a linear structure and randomly distributed n - acetyl - d - glucosamine ( acetylated unit ) and β - ( 1→4 ) - linked d - glucosamine ( deacetylated unit ) units are the product of chitin extraction from shrimp shells B-material . [SEP]
[CLS] as a stabilized , biodegradable B-property , and biocompatible B-property formulation with lower toxicity B-property among natural polymers B-material , it can be prepared by simple techniques . [SEP]
[CLS] as the hydrophobic B-property structures have increased permeability , chitosan B-material - based nanoformulations have been modified to enhance their hydrophobicity B-property and thus their ability to penetrate the bbb . [SEP]
[CLS] wang et al have shown that trimethylated chitosan B-material loading on the surface of plga has enhanced brain uptake . [SEP]
[CLS] some polymeric surfactants B-property have been used as the bbb ' s permeation enhancer such as polysorbate 20 , 40 , 60 , and 80 and also poloxamer 188 in contrast with the polymers B-material such as poloxamine 908 , cremophor ® , and brij ® 35 . [SEP]
[CLS] moreover , pegylated or the so - called stealth nanoparticles B-nanoparticle are characterized by lower liver uptake and better blood circulation time and tissue distribution . [SEP]
[CLS] the elevated brain concentrations of nanopolymers B-material in tumorbearing animals versus nontumoral animals indicate that diseases such as brain cancer considerably increase the delivery of nanoformulations to target sites ( table 1 ) . [SEP]
[CLS] although pegylation has improved many aspects of nano - drug delivery , it is not sufficient for an optimum brain delivery system . [SEP]
[CLS] several studies have revealed the substantial effect of the adsorbed drug on the nanoparticle B-nanoparticle ' s charge . [SEP]
[CLS] in some , tumor B-material accumulation reduction has been referred to as ionic interactions especially the therapeutic agents ' positive charge which causes higher reticuloendothelial system accumulation . [SEP]
[CLS] the polymeric B-nanoparticle nanoparticles I-nanoparticle are penetration enhancers with the ability to link with a peptide B-material as a targeting B-material ligand I-material which improves brain drug delivery . [SEP]
[CLS] apolipoprotein targeting B-material ligands I-material have been used on the surface of polymer - based nanocarriers to target the low - density lipoprotein receptor B-material and scavenger receptors on the bbb . [SEP]
[CLS] additionally , a few studies have investigated targeting the transferrin receptors with antitransferrin or transferrin antibodies B-material ( ox26 or r17217 ) . [SEP]
[CLS] likewise , the insulin receptors have been targeted with just 100 μm amounts of insulin receptor B-material antibody B-material which improves nanopolymer B-nanoparticle ' s drug delivery . [SEP]
[CLS] optimistically , polymeric nanocarriers can reach the clinic for brain cancer treatment only if they and their catabolites are biodegradable B-property , and less toxic B-property and immunogenic B-property . [SEP]
[CLS] unfortunately , as none of the bbb ' s receptors are specific or ubiquitously expressed , the adverse effects might be a limiting factor . [SEP]
[CLS] dendrimers B-nanoparticle . [SEP] B-nanoparticle
[CLS] dendrimers B-nanoparticle are monodisperse , symmetric , and spherical compounds with a chemical core B-material . [SEP]
[CLS] they are classified based on their molecular weight . [SEP]
[CLS] their properties are mostly determined by their surface B-nanoparticle functional I-nanoparticle groups I-nanoparticle . [SEP]
[CLS] the particle size growth starts from a nucleation site in the center of dendrimers B-nanoparticle , from where many consecutive branches develop . [SEP]
[CLS] consequently , hundreds of reaction sites are available on the surface of the particles . [SEP]
[CLS] poly - amidoamine ( pamam ) , poly - l - lysine B-material ( pll ) , and polypropylene imine B-material ( ppi ) are the most common types of dendrimers B-nanoparticle that have been applied for drug delivery . [SEP]
[CLS] they can be loaded by treatment agents and targeted as novel drug delivery systems . [SEP]
[CLS] another advantage of these types of formulations is dual targeting by different agents . [SEP]
[CLS] in one study , modified dendrimers B-nanoparticle via serine - arginine - leucine ( srl ) peptide B-material were used as gene delivery systems for the brain . [SEP]
[CLS] it was shown that srl peptide B-material was bound to endothelial cell B-material membrane receptors on the bbb and lipoprotein receptor B-material - I-material related protein B-material ( lrp ) . [SEP]
[CLS] consequently , this enhanced the dendrimers B-nanoparticle uptake by brain parenchyma tissue . [SEP]
[CLS] furthermore , transferrin and wheat - germ agglutinin ( wga ) dual targeting on the pegylated 7 to 10 nm dendrimers B-nanoparticle can serve as brain tumor B-material therapeutic agents because of well penetration and accumulation . [SEP]
[CLS] cationic B-material dendrimers B-nanoparticle electrostatically bind to the negatively charged genes for gene delivery applications . [SEP]
[CLS] the reason why gene delivery through the dendrimers B-nanoparticle seems to be so promising is this mechanism in which they destroy endosome storage in the cytoplasmic environment . [SEP]
[CLS] furthermore , exogenous genes , microrna ( mrna ) , and small interfering rna ( sirna ) delivery to tumor B-material sites have been shown in a few studies . [SEP]
[CLS] in conclusion , dendrimers B-nanoparticle have specific advantages such as uniform size distribution , high drug loading capacity , multiple targeting B-material ligand I-material conjugations , and high stability . [SEP]
[CLS] in contrast , complex synthesis and formulation development , toxicity B-property , and safety issues ( especially the amino - functional groups ) have restricted the clinical application of dendrimers B-nanoparticle ( table 1 ) . [SEP]
[CLS] furthermore , their positive amino groups can interact with the blood cells B-material which are negatively charged and structurally disrupt and erode cells B-material . [SEP]
[CLS] this can lead to hematologic toxicities B-property and nano - drug eliminations . [SEP]
[CLS] even though the dendrimer B-nanoparticle cationic B-material groups cause toxicity B-property , their chemical modifications generally minimize these effects . [SEP]
[CLS] micelles B-material . [SEP]
[CLS] spheroidal nanomicelles are made by the aggregation of amphiphilic B-property block copolymers in an aqueous environment . [SEP]
[CLS] in micelles B-material , the hydrophobic B-property core B-material and hydrophilic B-property surface have provided low soluble B-property drug loading , modification , and conjugation for targeted brain drug delivery . [SEP]
[CLS] the hydrophilic B-property outer layer prolongs both blood circulation and stability . [SEP]
[CLS] micelles B-material range between 10 and 100 nm , which is ideal for the enhanced permeation and retention ( epr ) mechanism at the tumor B-material site . [SEP]
[CLS] these nano - drug delivery systems are prioritized over other types of novel drug delivery approaches due to simple , stable , and reproducible formulations , as well as simple filtration techniques for sterilization . [SEP]
[CLS] their small size and hydrophilic B-property shell B-material help them evade the res and consequently have a longer circulation time . [SEP]
[CLS] therefore , nanomicelles are the potential candidates for the treatment of brain cancer . [SEP]
[CLS] however , the potential of immunogenicity B-property , premature drug release , poor stability , and lack of appropriate methods for formulation scale - up have limited their application . [SEP]
[CLS] in addition to the aforementioned disadvantages , the dissociation of micelles B-material at concentrations lower than the critical micelle B-material concentration ( cmc ) is one of the most significant challenges . [SEP]
[CLS] exosomes . [SEP]
[CLS] natural nanovesicles with a diameter of 30 to 100 nm have gotten a lot of attention for potential drug delivery applications because of their ability to carry targeted ligands on their surface . [SEP]
[CLS] they can escape the immune system entrapments due to their production from body cells B-material , resulting in better blood circulation , biodegradability B-property , and biocompatibility B-property . [SEP]
[CLS] they are also considered intriguing nano - drug delivery systems due to their inherent aptitude for passing across biological membranes and barriers B-property including the bbb without affecting its integrity . [SEP]
[CLS] additionally , some investigations have reported glioma - secreted exosomes circulating in the blood , which indicates their capacity to cross the bbb . [SEP]
[CLS] the abovementioned characteristics have made them promising candidates for delivery for brain cancer treatment . [SEP]
[CLS] many studies have utilized exosomes for the delivery of nucleic B-material acids I-material , proteins B-material , and small molecules . [SEP]
[CLS] many studies have been carried out to take advantage of cutting - edge exosome research , including rna therapeutics . [SEP]
[CLS] exosomes containing mrna inhibited and reduced vasculogenic mimicry , migration , and angiogenesis B-event , leading to glioma tumor B-material suppression . [SEP]
[CLS] the administration of brain endothelial cell - derived exosomes that were loaded with sirna was used to treat brain cancer in another investigation . despite their limited cell B-material absorption , sirnas have shown intriguing therapeutic promise . [SEP]
[CLS] the nanosized exosomes were successful in delivering sirnas for the treatment of brain diseases . [SEP]
[CLS] interestingly , the main mechanism by which exosomes acquired the ability to cross through the bbb was unraveled in vitro . [SEP]
[CLS] the findings suggested active transcytosis under the impression of inflammation factors ( eg , tnf - α ) . [SEP]
[CLS] for simultaneous glioma imaging and treatment study , the neuropilin - 1 - targeted exosomes were co - loaded with spions and curcumin by electroporation method . [SEP]
[CLS] natural nanostructures were given in vitro and in vivo , and their therapeutic and diagnostic benefits were greatly improved . [SEP]
[CLS] potent synergistic anticancer B-property effects were attributed to the effect of spion - mediated magnetic B-property flow hyperthermia and curcumin - mediated treatments . [SEP]
[CLS] exosomes have also been employed to decrease brain malignancies in some innovative and exciting studies . [SEP]
[CLS] chaperone - rich cell B-material lysates ( crcls ) in particular may play a key role in the development of antitumor vaccinations . [SEP]
[CLS] dendritic cells B-material ( dcs ) are activated by tumor - derived crcls , resulting in potential anti - tumor B-material efficacy . [SEP]
[CLS] dc - derived exosomes were produced in this study using dcs loaded with crcls obtained from gl261 glioma cells B-material . [SEP]
[CLS] they made antitumor t cell B-material immune responses more robust and effective . [SEP]
[CLS] the notion that brain endothelial cell - derived exosomes can transfer anticancer B-property medicine across the bbb for the treatment of brain cancer in a zebrafish model has been tested in new findings on anticancer B-property drug delivery . [SEP]
[CLS] the findings show that exosomes supplied to tumors B-material reduced tumor B-material growth markers in a significant manner . [SEP]
[CLS] as a result , brain endothelial - derived exosomes could be a promising new nano - drug delivery system that could be investigated further in the clinical development of brain cancer therapy . [SEP]
[CLS] in a 3d glioblastoma model , exosomes generated from human endometrial stem cells B-material harboring the apoptotic drug atorvastatin , which inhibits cancer growth through a variety of mechanisms , dramatically reduced tumor B-material growth . [SEP]
[CLS] a number of researchers have looked into the possibility and mechanism of exosomal surface changes . [SEP]
[CLS] surface adjustments are implemented to exosomal surface proteins B-material and functional groups . [SEP]
[CLS] the presence of exosomal membrane proteins B-material such as cytoskeletal components ( actin , tubulin , etc ) , intracellular fusion proteins B-material ( annexin and rab ) , and heat shock proteins B-material have been decoded by proteomic research . [SEP]
[CLS] mhc class i and ii , cd86 proteins B-material , integrins , as well as other proteins B-material were also listed . [SEP]
[CLS] in conclusion , covalent and noncovalent alterations , as well as genetic engineering , are being investigated as techniques to improve exosome efficacy and reduce their drawbacks in the targeted drug delivery . [SEP]
[CLS] considering all the facts , exosomes have natural biocompatibility B-property , greater chemical stability , and the ability to control intercellular interactions when compared to synthesized nanoparticles B-nanoparticle . [SEP]
[CLS] they are also regarded as nonimmunogenic , nontoxic , and nonspecific nanocarriers . [SEP]
[CLS] however , there are challenges to exosome use , such as a lack of standardized exosome separation and purification criteria , an uncertain mechanism for exosome uses in cancer treatment , heterogeneity , and difficulty maintaining exosomes . [SEP]
[CLS] they must be analyzed and adjusted to get a more desirable and controllable release and scalation in clinical scenarios . [SEP]
[CLS] minicells . [SEP]
[CLS] minicells are anucleated , nano - sized ( 100 - 300 nm ) , neither alive nor dividing cells B-material due to mutation in genes involved in the normal bacterial cell B-material cycle . [SEP]
[CLS] ribosomes , peptidoglycans , plasmids , rna , and bacterial proteins B-material are all kept intact . [SEP]
[CLS] as a result , they are metabolically active and capable of carrying out cell B-material processes such as mrna translation , transcription B-event , and translational activities , as well as atp synthesis . [SEP]
[CLS] therefore , various minicell - producing bacterial strains ( such as e coli or others ) have been brought into attention or are being researched for possible adoption . [SEP]
[CLS] chemotherapeutics , si / shrna , drugs , and chemotherapeutics can all be administered to malignant tissue with pinpoint accuracy . [SEP]
[CLS] additionally , multiple targeting B-material ligands I-material , such as bi - specific antibodies B-material , can modify their surface to improve their clinical applications . [SEP]
[CLS] the biocompatibility B-property and biodegradability B-property of these nanocarriers are equivalent to that of other nanocarriers . [SEP]
[CLS] furthermore , they appear to be one of the most unique and appealing nano - drug delivery approaches due to increased encapsulation B-property efficiency I-property , overcoming drug leakage , enhancing targeting specificity , and improving treatment agents ' therapeutic index . [SEP]
[CLS] many studies are being conducted in order to develop optimal minicell - based drug delivery systems for therapeutic purposes . minicells that administered the mir - 34a greatly increased the temozolomide effects in vivo in a study as adjuvant therapy . [SEP]
[CLS] mir - 34a regulates signaling pathways implicated in intratumoral heterogeneity and , as a result , temozolomide resistance . [SEP]
[CLS] in another experiment , late - stage brain cancer dogs were treated with egfr - targeted minicells carrying doxorubicin B-material and coated with bsabs . [SEP]
[CLS] the nanoparticles B-nanoparticle were found in the brain tumor B-material ' s center and had a high median survival rate . [SEP]
[CLS] there have been no reports of particular toxicity B-property . [SEP]
[CLS] a phase 1 clinical trial employing egfr - targeted , doxorubicin - loaded minicells for the management of patients with relapsed glioblastoma was designed on this premise . [SEP]
[CLS] despite the benefits and advantages of minicell structures , more research is needed to interpret them in the clinic , notably on their stability and release profile , as well as their release mechanism in the tumor B-material microenvironment and intratumoral . [SEP]
[CLS] according to the experiments that have been reported so far , they had no significant detrimental impacts . [SEP]
[CLS] before they are extensively employed , however , their immunogenicity B-property due to bacterial sources must be thoroughly investigated . [SEP]
[CLS] additionally , the risk of organ damage from long - term organ accumulations adds to the concerns regarding these novel nanoformulations . [SEP]
[CLS] liposomes B-nanoparticle . [SEP]
[CLS] liposomes B-nanoparticle are attractive vesicles for the delivery of various drugs and compounds such as antibiotics , therapeutic proteins B-material , antineoplastics , and peptides B-material . [SEP]
[CLS] these vesicles , consisting of phospholipids , are spontaneously formed and comprise of single or multiple layers . [SEP]
[CLS] liposomes B-nanoparticle are categorized based on the following classes : ( ι ) multi - lamellar vesicles ( mlvs ) , ( ιι ) large B-material unilamellar I-material vesicles I-material ( luvs ) , and ( ιιι ) small B-material unilamellar I-material vesicles I-material ( suvs ) . [SEP]
[CLS] the sizes of mlvs , luvs , and suvs are approximately 500 , 100 to 500 , and 25 to 100 nm , respectively . [SEP]
[CLS] mlvs are commonly used for extended drug release objectives , while suvs are optimal for drug delivery through the endothelial cells B-material lining the blood vessels and tissue epithelium . [SEP]
[CLS] luvs are medium - sized structures for vaccine and gene delivery purposes . [SEP]
[CLS] due to the recent improvements in encapsulation B-property efficiency I-property , drug loading , stabilized formulation preparations , decoration for the molecules targeting delivery , and co - delivery , liposomes B-nanoparticle are promising drug delivery systems . [SEP]
[CLS] however , some limitations affect the liposomal B-nanoparticle formula application and restrict its utilization for intended purposes . [SEP]
[CLS] these particulate systems have high clearance and low blood circulation and are cleared by the res . [SEP]
[CLS] the blood circulation time can be improved by decreasing the particle size to less than 100 nm , and liposomal B-nanoparticle surface pegylation . [SEP]
[CLS] additionally , active targeting is accomplished by ligand attachment for a specific receptor B-material ( like monoclonal B-material antibody I-material ) on the liposomes B-nanoparticle , preferentially to the end of the peg moieties as the targeted liposomes B-nanoparticle have been demonstrated to be much more effective if they are sterically stabilized . [SEP]
[CLS] many attempts have been made to reach an optimal liposomal B-nanoparticle formulation . [SEP]
[CLS] improving liposomal B-nanoparticle transportation to the tumor B-material site has been the main objective of several previous studies . [SEP]
[CLS] most of these studies examined either cationic B-material or pegylated liposomes B-nanoparticle . [SEP]
[CLS] based on recent studies , the main mechanism for liposomal B-nanoparticle entrance into the brain remains unclear , however , it has been suggested that tight junction disruption may be the most probable mechanism . [SEP]
[CLS] some ph - sensitive cell B-material - penetrating peptides B-material such as tr peptides are surface conjugated to the drug - loaded liposomes B-nanoparticle and were developed and utilized for glioma treatment . [SEP]
[CLS] this type of nanoformulation was shown to enhance the drug efficacy toward gliomas , both in vitro and in vivo . moreover , adriamycin ( adm ) - encapsulated thermosensitive liposomes B-nanoparticle enhanced dox delivery to the brain and prolonged the survival of gliomabearing mice . [SEP]
[CLS] receptor - mediated endocytosis B-event is a general mechanism of cells B-material to import particles . [SEP]
[CLS] the endocytosis B-event mechanism has been targeted by nanosystems especially liposomes B-nanoparticle . [SEP]
[CLS] for instance , the transferrin receptor B-material is over - expressed on the brain capillary endothelial cells B-material . [SEP]
[CLS] therefore , the efficacy of dual - targeted doxorubicin B-material ( dox ) liposomes B-nanoparticle conjugated concomitantly to folate and transferrin was investigated in a study . [SEP]
[CLS] this dual - targeting dox liposome B-nanoparticle enhanced the therapeutic efficacy of dox toward gliomas and reduced the off - target side effects as compared to dox solution . [SEP]
[CLS] in another study , the tf - specific targeting B-material ligand I-material and tat , a nonspecific cellpenetrating peptide B-material ( a small positive charged variant derived from trans - activating transcription B-event activator peptide B-material of hiv ) were attached to the paclitaxel B-material and dox containing liposomes B-nanoparticle ( tf / tat - lp ) . [SEP]
[CLS] they exhibited an effective antitumor B-event activity I-event against gliomas and enhanced the median survival time of glioma - bearing mice . [SEP]
[CLS] moreover , targeting is an approach that enhances the liposomal B-nanoparticle formulations ' therapeutic and antitumor effects . [SEP]
[CLS] it is ascribed to their median particle size and an extent to the homogeny particle size distribution . [SEP]
[CLS] the epr effect is the main mechanism for liposomal B-nanoparticle accumulation and accordingly , 100 nm liposomes B-nanoparticle can merely reach specific regions . [SEP]
[CLS] in one study , the noninvasive focused ultrasound method accompanied bubble liposome B-nanoparticle ( with 55 - 299 nm diameter range ) injection into the circulatory system . [SEP]
[CLS] the results showed that smaller liposomes B-nanoparticle serve as more effective drug delivery systems than larger ones . [SEP]
[CLS] in another study , cationic B-material liposomes B-nanoparticle were used to transfect tumor B-material cells B-material with an interferon gene - expressing vector ( plasmid ) , which resulted in tumor B-material regression . [SEP]
[CLS] likewise , antisense epidermal growth factor was entrapped into cationic B-material liposomes B-nanoparticle and tested in human malignant glioma cell B-material lines . [SEP]
[CLS] the possible cationic B-material liposomal B-nanoparticle cell B-material uptake mechanism can be explained by negatively charged phospholipid head group interactions with the positively charged liposomes B-nanoparticle ( absorption ) and therefore is called adsorptive mediated transcytosis . [SEP]
[CLS] in this regard , cationic B-material bovine serum albumin as a positive ligand has shown increased cell B-material absorption and enhanced drug delivery through the aforementioned mechanism . [SEP]
[CLS] the difficulties in surface conjugation and small number of functional groups can be the main liposomal B-nanoparticle drawbacks ( table 1 ) . [SEP]
[CLS] moreover , other disadvantages exist , such as poor and low reproducibility of nanoparticle B-nanoparticle size , stability , and insoluble agent I-property loading . [SEP]
[CLS] optimum sterilization and uncontrolled drug release are other challenges associated with the liposomes B-nanoparticle . [SEP]
[CLS] nanotechnology clinical approaches for brain cancer drug delivery [UNK] . sponsored by lawrence recht , nektar therapeutics has designed a polymeric version of irinotecan as the first long - acting topoisomerase i inhibitor . [SEP]
[CLS] irinotecan molecules are attached via an ester bond to the peg polymer B-material . [SEP]
[CLS] carboxylesterase and other enzymes react with the ester bond and cause the release of irinotecan and consequently , producing 7 - ethyl - 10 - hydroxy - camptothecin or sn38 as the active metabolite . [SEP]
[CLS] sn38 attacks dna and causes its damage through topoisomerase inhibition . [SEP]
[CLS] the main objective of the etirinotecan pegol ( nktr - 102 or onzeald ) design is to eliminate or attenuate the irinotecan side effects and also improve its efficacy by drug distribution modifications . [SEP]
[CLS] in preclinical studies , it demonstrated a 300 - fold tumor B-material concentration in comparison with a first - generation topo i - inhibitor . [SEP]
[CLS] the nktr is larger than normal vessel pores , helping it reach the tumor B-material microenvironment by enhanced permeation and retention . [SEP]
[CLS] onzeald efficacy has been assessed in ovarian , breast , brain , colorectal , and lung cancer . [SEP]
[CLS] the pharmacokinetic characteristics of the onzeald metabolite ( sn38 ) differ significantly from others by providing maximal tumor B-material exposure of the active drug ( table 2 ) . [SEP]
[CLS] for example , the c max has been reduced 5 to 10 times and also the half - life has been increased to almost 50 days . [SEP]
[CLS] the dose of 145 mg / m 2 onzeald in phase ii clinical trial almost equals the dose of 350 mg / m 2 irinotecan . [SEP]
[CLS] the protracted exposure between continuous dosing cycles and lower c max has improved the therapeutic effects of the treatments and clinical outcomes . [SEP]
[CLS] its open - label , single - arm 2016 phase ii clinical trial had completed the onzeald ' s efficacy in bevacizumab - resistant high - grade gliomas . [SEP]
[CLS] nu - 0129 . nu - 0129 is a class of gene regulation spherical nucleic B-material acid I-material ( sna ) nanostructures having well - orientated sirna oligonucleotides finely designed and synthesized at their surface . such snas are made up of sirna oligonucleotides constructed on gold B-material ( au ) nanoparticle B-nanoparticle centers with oligo ethylene glycol ( oeg ) or peg ( oeg / peg ) on their surface to improve stability and circulatory half - life . [SEP]
[CLS] based on the earlier investigations , using au as the sna heart allows for accurate spatial analysis of au distribution in cells B-material and tumors B-material using inductively coupled plasma mass spectrometry ( icp - ms ) , x - ray fluorescence B-technique microscopy I-technique ( xfm ) , and silver B-material histopathology staining . [SEP]
[CLS] for the treatment of glioblastoma , the sirna gold nanoparticles B-nanoparticle target the bcl - 2 - like protein B-material 12 ( bcl2l12 ) domains . [SEP]
[CLS] the tumor B-material cells B-material die as a result of the inhibition of bcl2l12 expression . [SEP]
[CLS] in glioblastoma , bcl2l12 is hypothesized to be upregulated in a tumorpromoting direction . [SEP]
[CLS] it inhibits the activation of effector caspase - 3 and caspase - 7 . [SEP]
[CLS] it can also bind wild - type p53 and its mutants , destabilizing them and preventing p53 from attaching to target gene promoters . [SEP]
[CLS] in order to test safety , pharmacokinetics , and intratumoral sna nanoconjugate accumulation , the very first phase 0 clinical trial involving the systemic microdose delivery of nu - 0129 in adults with recurrent glioblastoma was done . [SEP]
[CLS] as a consequence , it was discovered to be safe and well tolerated by endothelial , immunological , and tumor B-material cells B-material , and it was linked to lower target protein B-material levels in patients . [SEP]
[CLS] liposomal B-nanoparticle rhenium - 186 ( 186re ) . [SEP]
[CLS] radiation is an essential part of brain cancer treatment , but due to the toxicity B-property of high doses , its usage is limited . [SEP]
[CLS] rhenium - 186 ( 186re ) is a diagnostic imaging I-technique rhenium isotope B-material that is chemically similar to technetium - 99m ( 99mtc ) and is a reactor - produced isotope B-material with great potential for medical therapy only after successful delivery ( table 2 ) . [SEP]
[CLS] it has a 90 - hour half - life with a 2 mm tissue path length . [SEP]
[CLS] its low tissue penetration has provided higher administration doses with the least toxicity B-property . [SEP]
[CLS] localized radiation at the tumor B-material site is achievable through the 100 nm liposomal B-nanoparticle formulations of the 186re . [SEP]
[CLS] its applicability in the failed glioblastoma treatment procedures and accumulation at the tumor B-material site is because of the epr effect [SEP]
[CLS] 2b3 - 101 . [SEP]
[CLS] the phase i / iia clinical study for 2b3 - 101 or glutathione ( gsh ) pegylated liposomal B-nanoparticle dox in patients with glioma and breast cancer brain metastases B-event has concluded . [SEP]
[CLS] the g - technology employed to create this ideal nanostructure is established on the gsh identified transporter on bbb endothelial ( km of 6 mm ) . [SEP]
[CLS] it is generally considered safe and utilizes micromolar glutathione targeting molecules on pegylated nanoliposomal dosage forms . [SEP]
[CLS] the brain - to - blood ratio of doxorubicin B-material was 4 . 8 times greater upon injection of 2b3 - 101 than generic pegylated nanoliposomal dox in discoveries . [SEP]
[CLS] as a result , the brain ' s doxorubicin B-material concentration rises without compromising the bbb ' s integrity . [SEP]
[CLS] it greatly slowed tumor B-material growth when compared to nontargeted pegylated liposomal B-nanoparticle dox . [SEP]
[CLS] pegylated liposomal B-nanoparticle dox ( doxil ® ) . [SEP]
[CLS] dox refers to a wide range of effective chemotherapeutics for the most aggressive malignancies such as glioblastoma . [SEP]
[CLS] however , its effectiveness in vivo is under question due to the poor penetration as the result of the bbb . [SEP]
[CLS] its csf and brain tissue concentrations have dramatically increased in tumor B-material models after being sterically stabilized . [SEP]
[CLS] a pegylated liposomal B-nanoparticle dox formulation with or without another chemotherapeutic agent like temozolomide not only has enhanced drug delivery to the brain but also case series and two - phase ii studies concerning recurrent glioblastoma have demonstrated modest promising results . [SEP]
[CLS] egfr ( v ) - edv - dox . [SEP]
[CLS] the engeneic edvtm technology - based egfr ( v ) - edv - dox is a 400 nm dox - loaded bacterial minicell that utilizes bispecific antibodies B-material to function as a targeted therapy in cancer treatment ( table 2 ) . [SEP]
[CLS] in pigs and dogs , they were well tolerated with modest and temporary toxicity B-property and inflammation , according to earlier investigations . [SEP]
[CLS] the minicells go from the bloodstream to the tumor B-material microenvironment , where they assault the tumor B-material cells B-material ' surface and release dox . [SEP]
[CLS] furthermore , remnant edv bodies in the tumor B-material microenvironment that were unable to infiltrate malignant cells B-material signal the immune system to the tumor B-material site , counteracting the tumor B-material ' s immunosuppression . [SEP]
[CLS] overall , these microcells polarize m1 macrophages and engage nk cells B-material at the same time , resulting in a th1 cytokine response with powerful anticancer B-property activity . [SEP]
[CLS] upon that , dendritic cell B-material maturation and antigen presentation proceeds , leading to tumor B-material - specific cd8 + t cells B-material and durable tumor B-material remission . [SEP]
[CLS] brain drug delivery systems have significantly advanced over the past few years with current research progressing the field every further . [SEP]
[CLS] the latest advanced biological and physicochemical properties of the nanocarriers have taken them to higher levels . [SEP]
[CLS] furthermore , they have enhanced blood circulation and bioavailability B-property . [SEP]
[CLS] not only do they provide a productive functionalized surface for a variety of molecules , but also they can be modified for controlled release over time . [SEP]
[CLS] however , translating brain tumor B-material treatment into clinical trials encounter unique barriers B-property , largely in part due to the cns biological barriers B-property such as the bbb . [SEP]
[CLS] reduced tumor B-material accumulation seems to be another obstacle for such delivery systems to reach the clinic . [SEP]
[CLS] optimizing the physicochemical parameters may overcome the disadvantages of novel nanoformulation . [SEP]
[CLS] shape , size , functionality , and surface charge have been modified in a variety of studies in the field of brain drug delivery research . [SEP]
[CLS] designing a triggered release , as well as stimuli - responsive formulations , are among the most novel fields of drug delivery studies . [SEP]
[CLS] nonetheless , only a handful of studies have attempted for cancer treatment examining stimuli - responsive systems . [SEP]
[CLS] temperature , ph , magnetic B-property field , enzymes , oxidative stress , etc are triggering signals that have been frequently used for cancer treatment . [SEP]
[CLS] the advantages of employing intrinsic environmental features in the tumor B-material site in comparison with normal tissues for improved and efficient stimuli - responsive systems have been comprehensively discussed in the literature . [SEP]
[CLS] furthermore , external stimuli such as light , heat , and magnetic B-property fields are other options for controlled release . [SEP]
[CLS] liu et al have obtained physicochemical sensitive ( ph , temperature , etc ) nanopolymers B-material with both anticancer B-property effects and brain tumor B-material magnetic B-property resonance imaging . [SEP]
[CLS] their application for both diagnosis and treatment procedures has been positively approved . [SEP]
[CLS] the concept of these multifunctional nanosystems demonstrates the future of nanocarriers with vast room for growth and advancements in the field . [SEP]
[CLS] nonetheless , the aforementioned nanoparticles B-nanoparticle ' potential systemic toxicity B-property and neurotoxicity in the clinic should be considered . [SEP]
[CLS] despite numerous efforts to successfully improve the use of nanocarriers in different areas of clinical research , there remain challenges that need to be addressed . [SEP]
[CLS] investigating immortalized brain endothelial cell B-material models for the bbb penetration assessment and testing the nanocarriers ' efficiency to get through the bbb with the minimum cellular damaging effects are considered as examples of challenging aspects of this area . [SEP]
[CLS] the high cost of these cell B-material lines , such as hcmec / d3 , bend3 , and rbe4 , limited availability and accessibility , and susceptibility to media and cultural contaminations have widely affected in vitro studies . [SEP]
[CLS] however , they have been used more frequently in comparison with astrocyte - or pc - derived cell B-material lines in bbb studies . [SEP]
[CLS] preclinical in vivo studies in the field of brain cancer is often hindered by the difficulty of modeling the biological heterogeneity that is observed between humans and mouse models , which are additional challenges for brain cancer . [SEP]
[CLS] through advancements in pathological imaging methods and factoring in higher animal - to - human translational success rates , the in vivo complications may be reduced . [SEP]
[CLS] the design of clinical trials is faced with difficulty in group classifications given the heterogeneity of tumor B-material and the affected cell B-material types . [SEP]
[CLS] furthermore , additional prognostic factors such as the low number of participants in each group may lead to a lack of statistical power to detect significant differences between control and therapeutic arms . [SEP]
[CLS] although more research and development are needed for effective nanocarriers with optimal clinical kinetics , they show promise as a suitable method of delivery for brain cancer treatment based on recent clinical studies . [SEP]
[CLS] nanomedicine provides innovative opportunities in the development of tissue - specific targeted therapeutics , and imaging agents for brain tumor B-material management . [SEP]
[CLS] we have reviewed the challenges and advantages of several development efforts using nanocarriers for the treatment of brain tumors B-material . [SEP]
[CLS] here we highlight the importance of further investigations for the development of effective treatments using nanotechnology for monitoring and treatment . [SEP]
[CLS] this will facilitate the extremely effective development of nanomedicine in brain cancer disease . [SEP]
[CLS] taken together , the main goals of the upcoming brain cancer researches should be not only about having a higher survival rate , but also the patient ' s quality of life and especially the burden of treatment morbidities . [SEP]
[CLS] therefore , other challenges in brain cancer development may take the nanomedicine drug development under , which a few will be discussed . [SEP]
[CLS] first , we found an unmet need in coordinating a multifaceted team of specialists like researchers , neurologists , surgeons , neuropathologists , and other health professionals that will cause less suffering for those who burden the disease . [SEP]
[CLS] moreover , clarifying , organizing , and improving the fund and support in cancer investigations and research is required to make small communities of brain cancer research and investigations more developed . [SEP]
[CLS] second , considering prioritized molecular and genetic tumor B-material detection can lead to precise diagnosis and patient stratification and consequently move us toward specific and efficient drug development that more than likely will promote and facilitate current challenging brain cancer medications . [SEP]
[CLS] this is in line with broadening tailored individualized therapy areas . [SEP]
[CLS] finally , a clinical trial center in this field mostly cannot by itself recruit a considerable statistically powerful number of patients to run the study . [SEP]
[CLS] exploiting and extending more clinical trial centers can be a solution plus donating more grants for the brain cancer research portfolio . [SEP]
[CLS] thus , even though nanomedicine is a crucial milestone in brain cancer treatment but it requires a better understanding of other essential key elements for further reliable advancements . [SEP]
[CLS] the authors declared no potential conflicts of interest with respect to the research , authorship , and / or publication of this article . [SEP]
[CLS] schematic representation of the bbb . [SEP]
[CLS] endothelial cells B-material are made which are tightly attached via tight junctions . [SEP]
[CLS] blood vessels , and mural cells B-material including vascular smooth muscle cells B-material and pcs , are placed on the outer layer . [SEP]
[CLS] the pcs have incompletely covered the abluminal microvascular side that is attached to the vascular basement membrane . [SEP]
[CLS] astrocytes are polarized glial cell B-material types , cover the entire vessel ' s tube . [SEP]
[CLS] abbreviations : bbb , blood - brain barrier B-property ; pcs , pericytes . [SEP]
[CLS] nanocarrier and brain delivery . [SEP]
[CLS] various types of nanocarriers including inorganic B-nanoparticle nanoparticles I-nanoparticle , dendrimers B-nanoparticle , slns , polymeric B-nanoparticle nanoparticles I-nanoparticle , micelles B-material , exosomes , minicells , and liposomes B-nanoparticle encounter 4 types of transport mechanisms including transcellular , receptor B-material - mediated , paracellular , and carrier - mediated transport to pass through bbb . [SEP]
[CLS] abbreviations : bbb , blood - brain barrier B-property ; slns , solid B-nanoparticle lipid I-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] preclinical studies . [SEP]
[CLS] nanocarriers that were used in preclinical studies for brain delivery . [SEP]
[CLS] nanocarrier - based clinical trials . [SEP]
[CLS] the prevalence , distinctive reactivity and biological significance of sulphur - based groups in proteins B-material and nucleic B-material acids I-material means that analysis of sulphur is of prime importance in biochemistry , biotechnology , and medicine . [SEP]
[CLS] we report steps in the development of a method to aid in the detection of these moieties using gold B-nanoparticle nanoparticles I-nanoparticle as adjuncts in polyacrylamide B-technique gel I-technique electrophoresis I-technique ( gold B-material - page ) . [SEP]
[CLS] the chemistry of sulphur is key in biological systems . [SEP]
[CLS] in proteins B-material , the correct formation of disulphide bonds is frequently required for folding ( and hence function ) , and the distinctive nucleophilicity of thiols B-material is routinely exploited within chemical biology to enable labelling of proteins B-material via cysteine B-material . [SEP]
[CLS] in nucleic B-material acids I-material , natural modifications of rnas include sulphurised nucleobases , and the phosphorothioate linkage B-property is an essential tool for increasing the biostability of nucleic B-material acid I-material therapeutics a field boosted recently by the first phase iii success of an rnai therapy . [SEP]
[CLS] phosphorthioate dna can reduce prion infectivity , and thioaptamers have also been shown to combine nuclease resistance with binding of human endothelial growth B-material factor I-material receptor I-material 2 ( her2 ) , associated with many cancers such as lung and breast . [SEP]
[CLS] sulphur groups in dna are also widely used in structural nanotechnology . [SEP]
[CLS] there is therefore an important need for tools to analyse the presence and chemical state of sulphur in proteins B-material and nucleic B-material acids I-material . [SEP]
[CLS] mass spectrometry , usually the first port - of - call , can often be challenging under native conditions ( needed to assess for example protein B-material - protein complexes ) , while other methods for assessing sulphur chemistry are either destructive ( e . g . icp - ms ) or indirect ( e . g . circular dichroism ) . [SEP]
[CLS] gel B-technique electrophoresis I-technique on the other hand is a fast and well - known process which allows for analysis of biomolecule size distributions , with high ( ng ) sensitivity ( determined by the stain ) , but in its standard form does not discriminate between chemistries . [SEP]
[CLS] the inclusion of a mercury B-material layer within polyacrylamide B-technique gel I-technique electrophoresis I-technique ( page ) has been shown to identify sulphurised rnas by diminished mobility B-property mediated by soft - soft hg - s interactions . [SEP]
[CLS] however , the technique utilises highly toxic B-property organomercury building blocks , which are embedded within a crosslinked slab of gel , greatly amplifying waste disposal costs . [SEP]
[CLS] gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) are far less toxic B-property and are well known for their affinity for sulphur ; the replacement of weakly bound ligands by thiols B-material , disulphides , and thioethers B-material [SEP]
[CLS] introductionis widely employed in their functionalisation . [SEP]
[CLS] the integration of aunps B-nanoparticle into page ( fig . 1a ) could therefore be used to provide information on the number and chemistry of sulphur atoms B-material in proteins B-material and nucleic B-material acids I-material ( fig . 1b ) . [SEP]
[CLS] this would mirror the use of boronic B-material acids in page to detect glycation . [SEP]
[CLS] aunps B-nanoparticle have been incorporated into hydrogels previously , but not for electrophoresis B-technique . [SEP]
[CLS] to achieve the integration of page with the sulphur affinity of aunps B-nanoparticle , we initially looked into creation of thiolated acrylamide monomers B-material to ensure that any overall charge on aunps B-nanoparticle ( dependent upon surface capping ) did not result in their own migration under a voltage . [SEP]
[CLS] these monomers B-material proved difficult to isolate due to thiol - ene self - reactivity . [SEP]
[CLS] however , during preliminary screening of conditions it became apparent that concern about nanoparticle B-nanoparticle migration was unwarranted ( fig . 1c ) : citrate - coated aunps B-nanoparticle were retained within their original gel B-technique layer under normal electrophoresis B-technique conditions . [SEP]
[CLS] the addition of b - mercaptoethanol ( a common additive used to reduce protein B-material disulphides ) was required to transport the aunps B-nanoparticle through the gel . [SEP]
[CLS] this finding also verified the displacement of weakly coordinating ligands by thiols B-material within the gel matrix . [SEP]
[CLS] having established both aunp B-nanoparticle entrapment within the polymer B-material gel and ligand exchange , we examined the scope of nanoparticle B-nanoparticle capping systems which could be used . [SEP]
[CLS] citrate - capped aunps B-nanoparticle have an overall negative charge , and are routinely used as precursors to functionalised aunps B-nanoparticle in aqueous conditions . [SEP]
[CLS] they were synthesised using the frens method , and their size assessed by transmission B-technique electron I-technique microscopy I-technique ( tem , fig . s1 and s2 ( esi † ) , d = 17 . 8 ae 3 . 8 nm ) and a citrate - specific uv - visible spectroscopy B-technique method ( fig . s3 ( esi † ) , d = 10 nm ) . [SEP]
[CLS] cetyltrimethylammonium bromide B-material ( ctab ) capped nanoparticles B-nanoparticle are also water B-material soluble , but have a surfactant - type coating B-material , and ligand exchange is known . [SEP]
[CLS] they were synthesised by a modified version of the brust - schriffen synthesis and sized by tem ( fig . s4 and s5 ( esi † ) , d = 7 . 0 ae 3 . 6 nm ) . [SEP]
[CLS] dimethylaminopyridine ( dmap ) - capped aunps B-nanoparticle are stabilised by weak coordinative bonds and have been used as precursors to thiolated aunps B-nanoparticle . [SEP]
[CLS] they were produced by brust reduction followed by phase - transfer ligand exchange , and sized by tem ( fig . s10 and s11 ( esi † ) , d = 4 . 5 ae 2 . 1 nm ) . [SEP]
[CLS] all aunp B-nanoparticle solutions displayed the typical red - purple colouration due to the plasmon resonance band ( prb ) and were stable in solution at 4 1c on the order of months . [SEP]
[CLS] a series of dna samples ( fig . 2a ) was then used to assess the ability of the aunps B-nanoparticle to selectively retain sulphurous functionalities . [SEP]
[CLS] these 54mer strands ( taken from an aptamer had identical nucleoside sequences , but differing degrees and types of sulphur : none ( 0s ) , a single phosphorothioate linkage B-property before the terminal nucleoside ( 1s - term ) , a single phosphorothioate in the centre of the strand ( 1s - cent ) , two and three of the same linkages B-property ( 2s and 3s respectively ) , and a terminal thiol B-material - modifier ( sh ) . [SEP]
[CLS] the aunp B-nanoparticle - dna binding affinity was analysed in pure water B-material by dynamic B-technique light I-technique scattering I-technique ( dls ) to assess changes in apparent diameter as dna bound , and uv - visible spectroscopy B-technique ( uv - vis ) , to assay alterations in surface chemistry ( see esi † for methods ) . [SEP]
[CLS] citrate aunps B-nanoparticle showed negligible differences in their uv spectra in the presence of unmodified , thiolated or phosphorothioated dna ( fig . s3 , esi † ) . [SEP]
[CLS] ctab aunps B-nanoparticle displayed an increase in apparent size by dls ( fig . s6 - s9 , esi † ) in the presence of all dna strands , regardless of their degree of sulphurisation , but minor changes in the prb ( fig . s9 , esi † ) . [SEP]
[CLS] dmap aunps B-nanoparticle displayed changes both in apparent diameter and prb , for the addition of all dna samples , and showed some discrimination between them ( fig . s12 - s15 , esi † ) . [SEP]
[CLS] these findings suggest that the negatively charged citrate aunps B-nanoparticle repel dna which hinders interaction , the positively charged ctab aunps B-nanoparticle bind , but with the electrostatic component dominating , while the dmap aunps B-nanoparticle are able to bind through some interaction with the sulphurous moieties . [SEP]
[CLS] gold - page gels were cast for electrophoresis B-technique consisting of a standard polyacrylamide layer topped with an aunp B-nanoparticle - containing layer , and then tested using the series of dna strands indicated above . [SEP]
[CLS] the citrate aunps B-nanoparticle were found to remain well dispersed throughout the polymerisation process in ph 8 tris - borate - edta ( tbe ) buffer . [SEP]
[CLS] however , there was no visible change in the retention of the strands with respect to their degree or type of sulphur chemistry ( fig . 2b ) . [SEP]
[CLS] this remained the case despite additional variation of aunp B-nanoparticle size ( d = 8 - 40 nm ) and concentration ( 1 - 60 mg au ml a1 ) . [SEP]
[CLS] having already noted that both dna and citrate aunps B-nanoparticle are polyanionic and therefore could experience too much electrostatic repulsion to interact , we switched to a ph 8 tris - acetate - magnesium B-material ( tamg ) buffer in which the dications could act to screen the charge ( akin to use of mg 2 + in native page to observe dna hybridisation ) . [SEP]
[CLS] however , this resulted only in aggregation of the aunps B-nanoparticle ( presumably via neutralisation of the negatively charged citrate shell B-material ) to form large clumps within the gel ; these aggregates still did not affect dna migration . [SEP]
[CLS] ctab - aunps B-nanoparticle were also found to aggregate during the polymerisation process , even in tbe buffer - this may be due to the weakly capped aunps B-nanoparticle having non - innocent interactions with the free radicals . [SEP]
[CLS] however , in this case there was an influence on the migration of the oligonucleotides - they now appeared as diffuse smears , but did not show any discrimination according to sulphur content ( fig . 2c ) . [SEP]
[CLS] this confirms that the oligonucleotides can interact attractively with the aunps B-nanoparticle entrapped within the gel matrix , although in these cases electrostatics appears to be the determining factor rather than sulphur chemistry . [SEP]
[CLS] we then assessed the dmap aunps B-nanoparticle in gold B-material - page . [SEP]
[CLS] the colloids remained well dispersed at the end of the gelation period , although seemingly batch - sensitive changes in rate of gelation occurred . [SEP]
[CLS] this was addressed by increasing the amount of the aps initiator used ( see esi † ) . [SEP]
[CLS] it was found that at a concentration of 10 mg ml a1 of au within the upper gel layer , a progressive retention of the dna strands according to their sulphur content could be observed , albeit in very diffuse bands ( fig . 2d ) . [SEP]
[CLS] importantly , the thiol B-material - modified strand was retained significantly more than the phosphorothioates , indicating sensitivity to both number and type of sulphur functionality . [SEP]
[CLS] with dmap aunps B-nanoparticle , a direct interaction of the gold B-material surface with sulphur functionality can therefore be confirmed . [SEP]
[CLS] we further examined the effect of oligonucleotide length and sequence on the ability of dmap aunps B-nanoparticle to retain phosphorothioates ( fig . 3 ) . [SEP]
[CLS] here we saw a change of migration aligned with the number of sulphurs , independent of dna sequence and length confirming that these properties do not play a part in retention of sulphur containing dna in denaturing conditions . [SEP]
[CLS] we did attempt to analyse the same sequences in non - denaturing conditions however the presence of magnesium B-material ions B-material caused aggregation of the dmap aunps B-nanoparticle . [SEP]
[CLS] preliminary tests with proteins B-material show complete retention of proteins B-material containing cysteine B-material residues , regardless of the sulphur oxidation state ( fig . s16 , esi † ) . [SEP]
[CLS] we endeavoured to examine the gel itself in more detail in order to better understand the influence the aunps B-nanoparticle was having on the gel matrix and properties , and thus optimise the method . [SEP]
[CLS] firstly , the rheological properties of the gel were examined ( fig . 4 and fig . s17 - s30 , esi † ) to assess whether the presence of dmap aunps B-nanoparticle resulted in bulk alteration of the gel matrix which might affect the way in which the dna strands migrate . [SEP]
[CLS] an amplitude sweep was performed on tbe gels at acrylamide concentrations between 5 and 20 % , and with and without 2 . 5 mg au ml a1 dmap aunps B-nanoparticle . [SEP]
[CLS] these studies revealed negligible variation in the storage modulus ( g 0 ) between + au and aau at the same acrylamide concentration , however the loss modulus ( g 00 ) was significantly lower in the presence of gold B-material . [SEP]
[CLS] frequency sweeps were then performed within the linear viscoelastic region as determined by the amplitude sweeps ( see esi † ) . [SEP]
[CLS] again , the difference made by the presence of aunps B-nanoparticle was only seen in the loss modulus , which was both lower and more consistent across the range of frequencies in the presence of aunps B-nanoparticle . [SEP]
[CLS] these results show that the gel retains its elasticity but loses a little of its viscosity B-property when aunps B-nanoparticle are present . [SEP]
[CLS] for the purposes of gold B-material - page , we can therefore conclude that the smearing is due to the chemistry or arrangement of the aunps B-nanoparticle rather than a bulk effect on the physical properties of the gel . [SEP]
[CLS] to further examine the structure of the gel matrix , we used optical B-technique coherence I-technique tomography I-technique ( oct , fig . s31 , esi † ) . [SEP]
[CLS] oct can be used to create 3d models of a transparent sample based upon spatial variations in its refractive B-property index I-property . [SEP]
[CLS] gels consisting of an aunp B-nanoparticle and au - free layer were cast within a glass cuvette ( fig . s32 , esi † ) and volumes were produced using spectral domain oct . [SEP]
[CLS] the interface between the layers could be clearly resolved ( fig . 5a and fig . s33 , s34 , esi † ) - the nongold gel provided no contrast , whereas the gold B-material layer had a strong , speckled appearance . [SEP]
[CLS] this is indicative of variations in gold B-material concentration across the gel , resulting in modulation of refractive B-property index I-property . [SEP]
[CLS] to understand this observation , we undertook transmission B-technique electron I-technique microscopy I-technique of the gels ( fig . 5b and fig . s35 , s36 , esi † ) , which revealed large aggregates of aunps B-nanoparticle within the gel matrix , and in some places , partial agglomeration . [SEP]
[CLS] we believe that this inhomogeneity is responsible for the smeared nature of the bands in gold B-material - page in its current form : the amount of gold B-material which each dna strand ' sees ' as it passes through the gel varies greatly , resulting in elongated bands . [SEP]
[CLS] we are therefore exploring new ways to improve aunp B-nanoparticle dispersion within the gel to apply the method to a wider range of macromolecules and produce cleaner images . [SEP]
[CLS] we have reported three core B-material findings for the development of gold B-material - page as a method for analysis of sulphur in biomolecules . [SEP]
[CLS] firstly , we have demonstrated the entrapment of aunps B-nanoparticle within polyacrylamide gel matrices , and shown that they are capable of binding sulphurous moieties within that matrix . [SEP]
[CLS] secondly , we have identified dmap - aunps B-nanoparticle as being able to distinguish different levels of sulphur modification in oligonucleotides in solution and in electrophoresis B-technique , albeit with suboptimal band quality . [SEP]
[CLS] thirdly , we have identified the cause of smearing within the gel studies . [SEP]
[CLS] we anticipate that the quality of aunp B-nanoparticle dispersal within the gel matrix is critical to reproducible and accurate detection of sulphur by gold B-material - page . [SEP]
[CLS] to make this method more fully applicable will therefore require embarkation on fine tuning of buffer , polymerisation conditions , and aunp B-nanoparticle ligand structure . [SEP]
[CLS] 1 ( a ) polymerisation of acrylamide and bisacrylamide to give crosslinked gels containing aunps B-nanoparticle . [SEP]
[CLS] aps = ammonium persulphate ; temed = tetramethylethylenediamine . [SEP]
[CLS] ( b ) resultant architecture of gel slabs for electrophoresis B-technique and expected alterations in migration according to sulphurous functionality . [SEP]
[CLS] ( c ) image of gel containing citrate aunps B-nanoparticle run in the absence and presence of excess thiol B-material . [SEP]
[CLS] 2 ( a ) sulphur - modified dna used herein . [SEP]
[CLS] ( b ) - ( d ) gold B-material - page using three types of aunp B-nanoparticle . [SEP]
[CLS] lanes : 1 = 0s ; 2 = 1s - term ; 3 = 1s - cent ; 4 = 2s ; 5 = 3s ; 6 = sh ; x = unused . [SEP]
[CLS] all gels run under denaturing conditions ( urea ) in tbe buffer at room temperature . [SEP]
[CLS] ( b ) citrate aunps B-nanoparticle ( 1 mg au ml a1 ) in 8 % page . [SEP]
[CLS] ( c ) ctab aunps B-nanoparticle ( 1 mg au ml a1 ) in 8 % page . [SEP]
[CLS] ( d ) dmap aunps B-nanoparticle ( 10 mg au ml a1 ) in 12 % page . [SEP]
[CLS] 3 ( a ) gold B-material - page using dmap aunps B-nanoparticle ( 10 mg ml a1 ) in 12 % page . [SEP]
[CLS] ( b ) 12 % page without gold B-material . [SEP]
[CLS] 1 = ladder 2 = 0s , 3 = sh , 4 = 0s - trn , 5 = 1s - trn , 6 = 2s - trn , 7 = 3s - trn , 8 = 0s - pri and 9 = 3s - pri . [SEP]
[CLS] all gels were ran in denaturing conditions ( urea ) at room temperature in tbe buffer . [SEP]
[CLS] 4 comparison of rheology of gels with ( + aunp B-nanoparticle ) and without ( aaunp ) gold B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] strain sweep ( top ) and frequency sweep ( bottom ) of 15 % pa gel . [SEP]
[CLS] in the lower graph the two g 0 curves follow nearly exactly the same path . [SEP]
[CLS] 5 ( a ) oct volume illustrating inhomogeneity of + aunp B-nanoparticle layer compared with that of the pure acrylamide ( aaunp ) layer ( scan of 1 . 5 mm a 1 . 5 mm lateral a 1 . 5 mm , measured in air ) . [SEP]
[CLS] ( b ) tem image of dmap aunps B-nanoparticle forming large aggregates within the pa gel matrix . [SEP]
[CLS] further images can be found in fig . s35 and s36 ( esi † ) . [SEP]
[CLS] the potential of oligonucleotides is exceptional in therapeutics because of their high safety , potency , and specificity compared to conventional therapeutic agents . [SEP]
[CLS] however , many obstacles , such as low in vivo stability and poor cellular uptake , have hampered their clinical success . [SEP]
[CLS] use of polymeric carriers can be an effective approach for overcoming the biological barriers B-property and thereby maximizing the therapeutic efficacy of the oligonucleotides due to the availability of highly tunable synthesis and functional modification of various polymers B-material . [SEP]
[CLS] as loaded in the polymeric carriers , the therapeutic oligonucleotides , such as antisense oligonucleotides , small interfering rnas , micrornas , and even messenger rnas , become nuclease - resistant by bypassing renal filtration and can be efficiently internalized into disease cells B-material . [SEP]
[CLS] in this review , we introduced a variety of systematic combinations between the therapeutic oligonucleotides and the synthetic polymers B-material , including the uses of highly functionalized polymers B-material responding to a wide range of endogenous and exogenous stimuli for spatiotemporal control of oligonucleotide release . [SEP]
[CLS] we also presented intriguing characteristics of oligonucleotides suitable for targeted therapy and immunotherapy , which can be fully supported by versatile polymeric carriers . [SEP]
[CLS] health is one of the most important things in our lives . [SEP]
[CLS] currently , we suffer from the global pandemic , covid - 19 caused by severe acute respiratory syndrome coronavirus 2 , 1 and as our lives can be frequently threatened by various types of diseases , including infectious diseases , genetic disorders , and even degenerative diseases , developments of their appropriate treatments have attracted a great deal of attention up to date , and extensive studies are still ongoing . [SEP]
[CLS] in particular , oligonucleotide therapeutics , which employs chemically synthesizable oligonucleotides such as deoxyribonucleic B-material acids I-material ( dnas ) and ribonucleic B-material acids I-material ( rnas ) as potent therapeutic agents , has proven its clinical potential in treating various diseases since the first report in 1978 . [SEP]
[CLS] on the basis of the central dogma of molecular biology , the mechanism of action for this oligonucleotide - based therapy involves in controlling cellular signals by targeting specific messenger rnas ( mrnas ) that are translated into the proteins B-material with pathological effects ; compared to conventional therapies relying on small molecules , peptides B-material , and proteins B-material , the oligonu - cleotide therapy enabling selective regulation of gene expression demonstrates greater safety , stronger potency , and higher specificity , along with an unrestricted choice of drug targets . [SEP]
[CLS] even though various therapeutic oligonucleotides have opened a new era of disease treatments through their novel and efficient mechanisms , several problems , including low in vivo stability and fast renal clearance of the oligonucleotides , still remain . [SEP]
[CLS] to address these problems , there have been many studies on the development of oligonucleotide carriers using a variety of organic and inorganic materials . 9 , 10 among them , lipid - based carriers , including liposomes B-nanoparticle , have been clinically used for oligonucleotide delivery as improving tissue penetration . [SEP]
[CLS] allowing tunable surface modification , inorganic or metal B-nanoparticle nanoparticles I-nanoparticle have shown a long - term stability and a photothermal effect , all of which are highly advantageous for therapeutic applications of oligonucleotides . [SEP]
[CLS] even spherical forms of nucleic B-material acids I-material have been used as nanocarriers of therapeutic oligonucleotides ; as designed in a highly oriented manner and recognize other target nucleic B-material acids I-material , the spherical nucleic acids have been also applied in theranostics . [SEP]
[CLS] by effective collaboration with functional synthetic polymers B-material , the oligonucleotide - based therapy can be more powerful . [SEP]
[CLS] polymer - based nanoparticles B-nanoparticle can secure the loaded oligonucleotides during in vivo circulation , and simultaneously enhance cellular uptake ; for example , polyethylene glycol ( peg ) and polyethylenimine ( pei ) have proven their effectiveness in avoid - moohyun han † , 1 jiyun beon † , 1 ju young lee 2 seung soo oh * , macromolecular research ing renal filtration and interacting with cell B-material membranes for the maximal therapeutic effect of oligonucleotides . [SEP]
[CLS] compared to other material B-material types of carriers , various polymers B-material ( e . g . , poly ( lactic acid ) ( pla ) and poly ( lactic - co - glycolic acid ) ( plga ) ) can be prepared to be biodegradable B-property and biocompatible B-property for in vivo applications , and the availability of highly tunable , chemical synthesis and functional modifications allows the resulting polymers B-material to perform programmed functions capable of significantly improving the therapeutic effects of oligonucleotides . [SEP]
[CLS] elaborate construction of polymeric carriers is also achievable ; surface charge and size of nanocarriers can be controlled for therapeutic purposes , and within dendrimers B-nanoparticle and core - shell structured polymers B-material , inorganic and metal B-material nanomaterials B-material are encapsulated for further employing their diagnostic and therapeutic functions . [SEP]
[CLS] taken together , the versatile polymers B-material could be regarded not as simple drug carriers , but as effective treatment boosters . [SEP]
[CLS] in this review , we presented how oligonucleotides and synthetic polymers B-material can be synergistically combined for therapeutic applications . [SEP]
[CLS] first of all , we discussed the distinct advantages of the oligonucleotide - polymer B-material combination in improving therapeutic efficacy and efficiency ; to guide specific gene - recognizing oligonucleotides to be delivered into disease cells B-material , the polymeric carriers with cell B-material penetration capabilities secure the loaded oligonucleotides against nuclease degradation , simultaneously prolonging their circulation time . [SEP]
[CLS] in terms of unique characteristics of the therapeutic oligonucleotides , we also introduced what kinds of synthetic polymers B-material have been explored for effective oligonucleotide delivery , exemplifying a wide range of stimuliresponsive polymeric systems . [SEP]
[CLS] furthermore , we demonstrated that newly emerging functions of oligonucleotides , such as molecular recognition and immune response induction , can benefit the applications of the polymeric drug carriers . [SEP]
[CLS] finally , we briefly discussed the challenges in clinically using the combination of functional oligonucleotides and polymers B-material toward creation of the next - generation therapeutic applications . [SEP]
[CLS] strategic combination between therapeutic oligonucleotides and various polymers B-material is highly valuable for maximizing the therapeutic efficacy in oligonucleotide therapy ( figure 1 ) . [SEP]
[CLS] based on figure 1 . [SEP]
[CLS] many advantages of combining oligonucleotides with synthetic polymers B-material for therapeutic applications . [SEP]
[CLS] specifically designed oligonucleotides can be used to inhibit disease - related genes or express therapeutic proteins B-material in our bodies . [SEP]
[CLS] various polymeric carriers can be used to improve the therapeutic efficacy and efficiency of the oligonucleotides ; by preventing nuclease degradation and renal filtration , the in vivo stability and blood circulation time of the oligonucleotides can be further enhanced , while the availability of co - drug delivery makes synergistic oligonucleotide therapy achievable . [SEP]
[CLS] moreover , the cellular uptake of the oligonucleotides can be promoted by the polymeric carriers , and in particular , the stimuli - responsive polymers B-material enable the localization of oligonucleotide release into disease sites for minimizing side effects . [SEP]
[CLS] the valuable polymeric carriers can be multifunctionalized to perform not only the oligonucleotide delivery , but also disease diagnosis and in vivo imaging for advanced theranostics . [SEP]
[CLS] the flow of genetic information in a biological system from dna to rna to protein B-material , regulation of the transient rnas can be an effective approach capable of modulating the levels of proteins B-material relevant to various diseases [SEP]
[CLS] to date , there have been several gene regulation mechanisms mediated by therapeutic oligonucleotides , such as antisense oligonucleotides ( asos ) , small interfering rnas ( sirnas ) , and micrornas ( mirnas ) , and their therapeutic effects have been validated by a number of scientific reports and clinical studies . [SEP]
[CLS] in addition to the therapeutic functions , some oligonucleotides , which can form specific 3d conformations at specific ph and ionic conditions , even perform molecular recognition like antibodies B-material ( e . g . , aptamers ) and catalytic reactions like enzymes ( e . g . , dnazymes and ribozymes ) [SEP]
[CLS] based on dynamic dna structures ( e . g . , i - motif and azobenzene - modified dna ) , oligonucleotides themselves serve as smart nanocarriers . [SEP]
[CLS] these oligonucleotides , however , have exposed inherent limitations in therapeutically applying them for in vivo systems , which can be readily addressed by use of polymeric carriers . [SEP]
[CLS] compared to viral carriers , the polymeric oligonucleotide carriers have many different advantages including a large capacity in oligonucleotide delivery , structural flexibility in carrier design , and even biological safety in clinical use . [SEP]
[CLS] furthermore , to respond to unique conditions around abnormal cells B-material , such as a tumor B-material microenvironment , they can be functionalized to selectively release the delivered oligonucleotides at specific sites . [SEP]
[CLS] using watson - crick base - pairing interactions , oligonucleotides can recognize disease - relevant specific genes , thereby exhibiting strong therapeutic effects . [SEP]
[CLS] as comprising a string of nucleotides including adenine , guanine , cytosine , and thymine ( or uracil ) , the therapeutic oligonucleotides can be complementary to target genes as held together by hydrogen B-material bonding . [SEP]
[CLS] for therapeutic applications , the oligonucleotides are typically designed to bind or degrade specific mrnas , which are destined not to interact with ribosomes , thereby inhibiting a subsequent translation process . [SEP]
[CLS] by silencing mrnas , there can be no expression of disease - relevant proteins B-material , alleviating the symptoms of the disease . [SEP]
[CLS] oligonucleotides are unstable in our bodies because of their rapid degradation by ubiquitous nucleases . [SEP]
[CLS] to assess their in vivo stability , the viability of rnas was tested in clinical samples , and as a result , the concentration of endogenous rnas decreased by more than 99 % in blood , serum , and plasma within 15 seconds [SEP]
[CLS] even though dnas are known to be more stable than the rnas , the dna administration into a mouse showed that the degradation time of intravenously injected naked plasmid dnas ( pdnas ) was only 15 minutes . [SEP]
[CLS] in addressing this low in vivo stability of oligonucleotides , the complex formation between the oligonucleotides and biocompatible B-property polymers B-material have been useful to avoid unwanted interactions with various blood components including nucleases . [SEP]
[CLS] in a study , cat - ionic polymers B-material carrying negatively charged oligonucleotides have shown more than 650 % increase in the degradation time of the oligonucleotides by blocking the accessibility of nucleases . [SEP]
[CLS] renal filtration is a size - dependent removal process of metabolic waste products ( e . g . , urea and creatinine ) , but it can also cause therapeutic agents with a small size and low molecular weight , including oligonucleotides , to be eliminated from our bodies , resulting in significant reduction of therapeutic efficiency ( figure 2 ( a ) ) . [SEP]
[CLS] use of polymeric carriers can be a great way to bypass the renal filtration by increasing the total size of the complex ; it can improve the therapeutic effect of the oligonucleotides by prolonging their circulation time in bloodstream . [SEP]
[CLS] previously , polyethylene glycol ( peg ) was conjugated with small interfering locked nucleic B-material acids I-material ( silnas ) to prolong the blood circulation time of the oligonucleotides and prevent their urine excretion . [SEP]
[CLS] to exclude the effect of enzymatic degradation , the silnas ( i . e . , the modified rnas with enzymatic resistance ) were used instead of common sirnas . [SEP]
[CLS] when the naked silnas and the silnas conjugated with 20 kda pegs ( peg20k - silnas ) were compared each other , the naked silnas were degraded over 90 % within 15 minutes , but the peg20k - silnas remained 50 % and 25 % after 1 h and 2 h , respectively . [SEP]
[CLS] this result demonstrated that the pegylated oligonucleotides can effectively avoid the renal filtration , thereby lengthening their circulation time and half - life in the blood stream . [SEP]
[CLS] the polyplex formation between negatively charged oligonucleotides and cationic B-material polymers B-material is highly effective to improve the in vivo stability of the oligonucleotides and simultaneously , their internalization efficiency into cells B-material ( figure 2 ( b ) ) . [SEP]
[CLS] similar with the oligonucleotides , the lipid B-material membranes I-material of the cells B-material are charged negatively , so the repulsive charge interaction makes the cell B-material internalization of the oligonucleotides significantly challenging . [SEP]
[CLS] from this electrostatic point of view , cationic B-material polymers B-material can not only neutralize the negative charge of oligonucleotides , but also exhibit an overall positive zeta B-property potential I-property which can strongly interact with cell B-material membranes ; as non - specific endocytosis B-event is promoted , the cell B-material internalization rate of oligonucleotides can increase . [SEP]
[CLS] moreover , the polymeric carriers can be further protonated within the endosome by the acidic environment , and due to the proton sponge effect that facilitates the influx of counterions and water B-material molecules at the endosomal ph , the membrane of the endosome can be disrupted ; 42 consequently , the endocytosed oligonucleotides can readily escape from the endosome , leading to the improvement of their therapeutic efficiency . [SEP]
[CLS] co - delivery systems are effective for immunotherapy and cancer therapy by promoting a synergistic effect of therapeutic oligonucleotides ; [SEP]
[CLS] macromolecular researchthe resulting polymeric carriers to carry multiple types of drugs , regardless of their hydrophobicity B-property and hydrophilicity B-property [SEP]
[CLS] many studies have reported that chemotherapy drugs have a greater effect on cancer therapy when combined with oligonucleotide therapy . [SEP]
[CLS] similarly , the therapeutic effect of immunotherapy is also known to be magnified by delivering immune - inducing antigens and immune - enhancing cpg oligodeoxynucleotides together . [SEP]
[CLS] in the co - delivery system , block copolymers can be synthesized for well - defined structures suitable for loading all the required therapeutic agents . [SEP]
[CLS] for example , as amphiphilic B-property block copolymers can form micelles B-material , hydrophobic B-property drugs are condensed into the hydrophobic B-property inner core B-material , and negatively charged oligonucleotides are loaded onto the cationic B-material shell B-material . [SEP]
[CLS] 2 . 6 . [SEP]
[CLS] responding to specific stimuli polymeric carriers can be smart enough to perform spatiotemporal control of loaded oligonucleotides in response to various stimuli , such as ph , temperature , and even levels of reactive oxygen B-material species ( ros ) . [SEP]
[CLS] when the oligonucleotide - loaded polymers B-material arrive at disease sites , the therapeutic oligonucleotides should be selectively unloaded to exclude side effects ( e . g . , killing normal cells B-material ) . [SEP]
[CLS] for example , as the ph around tumors B-material ( 6 . 6 ~ 6 . 8 ) is lower than physiological ph ( ~ 7 . 4 ) , ph - responsive polymeric carriers can be highly useful to localize the release of therapeutic oligonucleotides around the tumors B-material . [SEP]
[CLS] moreover , the polyplexes are typically internalized into the cells B-material through nonspecific endocytosis B-event , so their endosomal escape should be followed to prevent lysosomal degradation of the oligonucleotides . [SEP]
[CLS] in this regard , synthetic polymers B-material can be designed to increase the transfection B-property efficiency I-property of delivered oligonucleotides into target cells B-material ; by responding to environment - specific stimuli , they can selectively release the therapeutic oligonucleotides at specific locations , such as tumors B-material and inflammation cells B-material , thereby maximizing the therapeutic efficiency of oligonucleotide therapy . [SEP]
[CLS] some oligonucleotides are capable of recognizing biological molecules , such as proteins B-material and metabolites , and even performing enzymatic reactions , so the capability of polymeric carriers can be further enhanced and multiplexed as coupled with such functional oligonucleotides . [SEP]
[CLS] even though the oligonucleotides can be chemically produced by solid - phase synthesis , the sequencedefined structures can fold into unique conformations at physiological environments ; like monoclonal antibodies B-material or membrane receptors , aptamers selectively bind to target molecules , and like biological enzymes , nucleozymes conduct biocatalytic reactions . [SEP]
[CLS] for instance , some aptamers are capable of specifically recognizing cancer cells B-material and have been used as active targeting B-property agents I-property for various drug delivery systems . [SEP]
[CLS] as the biological functions cannot be achieved by typical polymers B-material , so systematic combination of functional oligonucleotides and polymers B-material can expand the versatility of the polyplex in therapeutic applications . [SEP]
[CLS] even though the potential of oligonucleotide therapeutics has been widely investigated by exploring the unique characteristics of oligonucleotides ( e . g . , specific gene recognition and induction of gene silencing ) , the negatively charged and vulnerable oligonucleotides are susceptible to decomposition by enzymes in our bodies , causing the low efficiency of cellular uptake . [SEP]
[CLS] by employing functionalized polymers B-material , we can compensate for the drawbacks of the therapeutic oligonucleotides ; for example , the polymers B-material with a large hydrodynamic volume ( e . g . , pegs ) protect them from enzymatic digestion and reduce their immunogenicity B-property , while cationic B-material polymers B-material ( e . g . , polyethylenimine ( pei ) ) can improve the cellular uptake efficiency of the oligonucleotides by electrostatically interacting with negatively charged cell B-material membranes ( figure 2 ) . [SEP]
[CLS] in this chapter , we introduced various therapeutic oligonucleotides that can be readily loaded into polymeric carriers and subsequently delivered inside the cells B-material . [SEP]
[CLS] to maximize the therapeutic efficacy of the oligonucleotides , a broad range of smart polymeric systems that can respond to various stimuli have been developed to date , so we also introduced their applications for advanced oligonucleotide drug delivery . [SEP]
[CLS] oligonucleotides can perform various therapeutic functions , such as gene inhibition , gene activation , vaccination , and even gene editing , through modulating gene expression . [SEP]
[CLS] typically , the oligonucleotide therapeutics relies on several forms of oligonucleotides , including aso , sirna , mirna , and mrna ( figure 3 ) . [SEP]
[CLS] whereas aso , sirna , and mirna are delivered into the cytoplasm to target specific mrna for gene expression inhibition , mrna as a therapeutic agent induces gene expression as delivered inside the cells B-material . [SEP]
[CLS] their structural differences are distinct ; for instance , aso is a form of a single strand , but sirna and mirna hold a duplex structure with a short length , while mrna is a much longer single strand . [SEP]
[CLS] in consideration of these differences , a variety of polymeric carriers have been extensively studied . [SEP]
[CLS] the first aso therapy was reported in the late 1970s , and since then , a number of studies on the aso therapy have been demonstrated [SEP]
[CLS] as the aso structure is short and single - stranded , the chemical modification of asos is relatively easy , taking advantage of the advances in nucleotide chemistry . [SEP]
[CLS] for instance , introduction of chemical modifications , such as phosphorothioate backbones , 2 ' - o - methoxyethyl ribose sugars , and n - acetylgalactosamine , into the asos led them to be less vulnerable to nucleases and more probable to be endocytosed by cells B-material . [SEP]
[CLS] however , it cannot be sufficient for the in vivo use of the therapeutic asos ; along with the chemical modifications , the combination of the asos with polymeric carriers has been considered to protect the loaded asos for successfully inhibiting target genes within the cells B-material . [SEP]
[CLS] in particular , polymeric B-nanoparticle nanoparticles I-nanoparticle can be positively charged to enhance the cellular uptake of asos , and by controlling the degradation rate of the polymeric due to the small size , oligonucleotides are rapidly cleared by the renal filtration process , leading to urine secretion . by pegylation , the hydrodynamic volume of the oligonucleotides can be enlarged , thereby prolonging their circulation time in blood . [SEP]
[CLS] ( b ) use of cationic B-material polymers B-material to promote cellular uptake . [SEP]
[CLS] as both oligonucleotides and cell B-material membranes are negatively charged , electrostatically repulsive interactions occur , making the oligonucleotides difficult to be internalized into cells B-material . [SEP]
[CLS] cationic B-material polymers B-material , such as polyethylenimine , can be complexed with the anionic oligonucleotides , making the net charge of the resulting polyplex advantageous for the following cellular uptake . [SEP]
[CLS] once internalized into the endosome , the polymeric carriers begin to be protonated at the endosomal ph , causing the membrane disruption by the influx of counterions and water B-material molecules . [SEP]
[CLS] as resulted by the proton sponge effect , the therapeutic oligonucleotides escape from the endosome and diffuse into the cytoplasm . [SEP]
[CLS] macromolecular researchcarriers [SEP]
[CLS] , sustained release of asos has been achievable [SEP]
[CLS] cationic B-material polymers B-material have been frequently used to decrease the electrostatic repulsive force between asos and cell B-material membranes , thereby improving the asos ' cellular uptake . [SEP]
[CLS] amine - rich pei has a high density of positive charge , so it can be well combined with the negatively charged asos by electrostatic interactions . [SEP]
[CLS] because the net charge of aso / pei complex can become neutral , the electrostatic repulsion against the negatively charged cell B-material membranes can be cancelled . [SEP]
[CLS] moreover , as the pei can load lots of negatively charged oligonucleotides , including the asos , there have been many studies that utilized the cationic B-material pei as an oligonucleotide drug carrier . [SEP]
[CLS] in one study , the pei / aso complex was delivered into the human bladder carcinoma cell B-material ( t24 ) for inhibition of ha - ras mrna , one of the oncogenes . [SEP]
[CLS] compared to the native asos without the pei carrier , the pei / aso complex increased cellular uptakes by 750 % , confirming the usefulness of the pei in neutralizing the negative charge of asos to improve the cellular uptake efficiency . [SEP]
[CLS] importantly , asos are highly susceptible to nucleases , so they can be rapidly degraded in serum . [SEP]
[CLS] this means that the therapeutic asos are necessary to be protected by nuclease - resistant carriers , which is effective to maintain the concentrations of the asos above a certain level for high therapeutic efficacy . [SEP]
[CLS] accordingly , many studies have been conducted to develop polymeric carriers which can protect the asos from nucleases and simultaneously release them in a sustained manner . [SEP]
[CLS] to this end , biodegradable B-property polymers B-material have often been studied , and among them , poly ( lactic - co - glycolic acid ) ( plga ) has been widely used because this polymer B-material is degraded into lactic B-material acids I-material and glycolic B-material acids I-material by esterase in our bodies and eventually decomposed into carbon B-material dioxide and water B-material through the krebs cycle . [SEP]
[CLS] even after phosphodiester modification , the asos were reported to be completely decomposed in serum within an hour , but the asos combined with the plga retained their full length over 21 days , enabling a sustainable release with more than a 500 - fold enhancement in in vivo stability . [SEP]
[CLS] compared to other therapeutic oligonucleotides , sirna would be the most specific for gene silencing . [SEP]
[CLS] it can effectively downregulate the expression of undesired gene by explicitly recognizing target mrna , and its gene silencing effect is more reproducible than that of aso . [SEP]
[CLS] when we know the sequence of the target gene , the gene - specific sirna can be easily designed and synthesized , so the sirna therapy has attracted the strong attention for treating various diseases , ranging from cancers to even chronic and genetic disorders . [SEP]
[CLS] however , similar with asos , poor cellular uptake and nucleolytic degradation by nucleases hampers the efficacy of the therapeutic sirna , and importantly , the sirna is easily cleared by kidney due to its small size . [SEP]
[CLS] to overcome these critical barriers B-property , the sirna inevitably requires a carrier such as a polymeric B-nanoparticle nanoparticle I-nanoparticle . [SEP]
[CLS] pegylation is an effective way to enhance in vivo stability of sirna , which is vulnerable to degradation by nucleases and macrophages in the body . [SEP]
[CLS] the sirna has a well - defined structure as a double - stranded rna , and its length is quite short ( 19 to 23 bp ) , yielding low molecular weight ( 13 ~ 15 kda ) . [SEP]
[CLS] because of the small volume , the sirna is easily removed from the body by kidney filtration . [SEP]
[CLS] considering the pore size of the glomerular filtration barrier B-property is 8 nm , the sirna carrier should be enlarged up to 20 nm in diameter . [SEP]
[CLS] the pegylation can make the sirna resist against nucleases and opsonins by steric hindrance , because highly flexible peg chains can coordinate many water B-material molecules to increase the hydrodynamic volume of the pegylated sirna . [SEP]
[CLS] for example , while naked 25 kda pei protected only 9 % of sirna from rnases , the peis with 2 , 5 , and 20 kda pegs prevented the decomposition of sirna more efficiently , and after 0 . 5 hr , 32 % , 68 % , and 92 % of the loaded sirnas remained , respectively . [SEP]
[CLS] this result showed that pegylation can provide the sterically shielding effect to sirnas and the shielding efficiency depends on the length of peg chains . [SEP]
[CLS] low transfection B-property efficiency I-property of sirnas is one of the biggest problems in sirna delivery , and copolymers with cleavable bonds can enhance endosomal release of the oligonucleotides , thereby promoting their transfection into cells B-material . [SEP]
[CLS] by pegylation , the volume of polymeric B-nanoparticle nanoparticles I-nanoparticle can become sufficiently large to avoid renal filtration and prevent non - specific interactions with opsonin . [SEP]
[CLS] however , the pegylation lowers the specificity of plasma membranes , resulting in a reduction of cellular uptake and endosomal escape . [SEP]
[CLS] even though the cellular uptake can be overcome to some extent with the help of membrane receptor B-material ligands , the use of the ligands is insufficient to address the inefficient endosomal release of sirnas . [SEP]
[CLS] as the gene regulation process occurs in cytoplasm , the sirna should be released from the endosome to form the rna - induced silencing complex ( risc ) . [SEP]
[CLS] to improve the endosomal release of sirnas , reducible poly ( triethylenetetramine / cystamine bisacrylamide ) ( poly ( teta / cba ) ) nanoparticles B-nanoparticle were developed . [SEP]
[CLS] disulfide B-property linkages I-property in cba were cleaved by glutathione ( gsh ) , which is abundant in the cytoplasm , so as liberated from the polymeric carriers , the sir - nas were encouraged to release for the endosome . [SEP]
[CLS] in this study , the use of poly ( teta / cba ) carrying sirnas resulted in twice less expression of vascular endothelial growth factor ( vegf ) than that of linear pei in human prostate cancer cells B-material ( pc - 3 ) . [SEP]
[CLS] this effective suppression of vegf was caused by gsh - responsive cleavage of disulfide bonds within the polymeric carriers ; when buthionine B-material sulfoximine , a gsh inhibitor , was present , there was no difference in the level of vegf expression , regardless of the presence of poly ( teta / cba ) carriers . [SEP]
[CLS] 3 . 1 . 3 [SEP]
[CLS] mirna sirna and mirna share some similarities in that both oligonucleotides possess duplex forms and need to form risc for gene regulation . [SEP]
[CLS] however , whereas the sirna is highly specific to one target gene , the mirna is capable of recognizing multiple mrna targets . [SEP]
[CLS] previous studies reported that more than 60 % of all protein B-material - coding genes have at least one conserved binding site with mirnas . [SEP]
[CLS] moreover , approximately 50 % of mirna - targeting genes are found at cancer - associated genomic regions or fragile sites . [SEP]
[CLS] by taking advantage of the mirna ' s multi - targeting capability and close relationship with cancer diseases , mirna therapy can become a promising treatment for multigenic diseases , including various types of cancers . [SEP]
[CLS] by delivering mirnas complexed with cationic B-material polymers B-material , it was demonstrated that multiple genes associated with colon carcinoma can be regulated simultaneously . [SEP]
[CLS] if only one gene is regulated to treat multigenic diseases , it may have a less therapeutic effect . [SEP]
[CLS] as cancer is involved with highly complicated mechanisms , delivery of multi - targeting mirnas would be highly effective . [SEP]
[CLS] in this study , chemically unmodified mirnas were complexed with cationic B-material peis , which displayed the improved therapeutic effects in a mouse model of colon carcinoma . [SEP]
[CLS] the therapeutic effect of two different mirnas , mir - 145 and mir - 33a , were investigated ; mir - 145 has an important role in reducing tumor B-material proliferation and increasing apoptosis B-event , and mir - 33a is capable of downregulating the oncogenic kinase , pim - [SEP]
[CLS] compared to other oligonucleotides for gene silencing , mrnas are delivered into the cells B-material for protein B-material expression , holding broader therapeutic potential . [SEP]
[CLS] however , as the mrnas are significantly large and hydrophilic B-property , their intracellular delivery is generally more challenging than that of other therapeutic oligonucleotides . [SEP]
[CLS] the mrnas contain open reading frames ( orfs ) , along with 5 ' capping and 3 ' poly - a tailing , to be translated by ribosome . [SEP]
[CLS] their molecular weight roughly ranges from 300 to 5000 kda , which is much larger than that of other oligonucleotides in therapeutics ( aso : 4 ~ 10 kda and sirna : ~ 15 kda ) . [SEP]
[CLS] some early studies have demonstrated the ability of naked mrnas when internalized into cells B-material via scavenger - receptor mediated endocytosis B-event . [SEP]
[CLS] however , it is challenging for the naked mrnas to diffuse across cell B-material membranes , due to their large size and negatively charged backbones . [SEP]
[CLS] nevertheless , the advantage of mrna delivery is clear ; compared to pdnas , mrnas are used for transient , yet rapid expression of therapeutically useful proteins B-material , making the mrnas highly valuable for a safe and fast gene therapy [SEP]
[CLS] currently , several studies of mrna delivery have focused on therapeutic applications , such as vaccination , protein B-material therapy , and gene editing . [SEP]
[CLS] to load the large mrnas , polymeric carriers , which are biocompatible B-property and biodegradable B-property , are inherently necessary to be composed of large - sized polymers B-material . [SEP]
[CLS] the terpolymer of poly ( βamino ester ) - co - poly - ε - caprolactone ( pbae - co - pcl ) was developed as an mrna carrier through ring open polymerization ( rop ) . [SEP]
[CLS] the pbae is suitable for mrna delivery ; this cationic B-material polymer B-material is less toxic B-property than others to cells B-material and can be synthesized from more than 2000 libraries with no byproducts , allowing easy post - modifications . [SEP]
[CLS] the pcl is a commonly used biodegradable B-property and biocompatible B-property polymer B-material for oligonucleotide delivery , due to its high colloidal stability and facile cellular uptake by endocytosis B-event . [SEP]
[CLS] the pbae - co - pcl - based terpolymer was complexed with the mrnas coding for firefly luciferase , and the result - ing complex was given to mice by intravenous ( iv ) injection . [SEP]
[CLS] as a result , it showed a 5 - times higher transfection B-property efficiency I-property in spleen than the mrnas complexed with jetpei , a commercial transfection reagent . [SEP]
[CLS] recently , there have been several studies to treat lung - related diseases using a drug delivery system ( dds ) through inhalation . [SEP]
[CLS] unlike the dds by iv injection , that by inhalation accompanies a nebulization process , making the inhalation delivery challenging . [SEP]
[CLS] polymeric B-nanoparticle nanoparticles I-nanoparticle are more suitable for the inhaled delivery than other nanoparticles B-nanoparticle , such as lipid - based nanoparticles B-nanoparticle , due to their higher resistance to the shear force caused by the nebulization . [SEP]
[CLS] for the inhaled mrna delivery , hyperbranched pbae has been developed and applied to the treatment of lung - related diseases . [SEP]
[CLS] the branched structures of pbae are more stable and have greater positive surface charge than its linear structures , so the use of the hyperbranched structures could be applicable for inhalation . [SEP]
[CLS] when the hyperbranched pbae with the mrnas coding luciferase genes were inhaled every three days , 10 - times larger luciferase proteins B-material were produced compared to the linear pei , and no symptom of local and systemic toxicity B-property was observed . [SEP]
[CLS] to maximize mrna transfection B-property efficiency I-property , one study controlled the end - group composition and molecular weight of poly ( amine - co - ester ) ( pace ) , which is composed of cationic B-material diol monomers B-material , lactone monomers , and diacid monomers B-material . [SEP]
[CLS] cationic B-material diol monomers B-material provide a low cationic B-material charge density , leading to electrostatic interactions with mrnas when lactone monomers B-material stabilize the polyplex using hydrophobic B-property interactions I-property . [SEP]
[CLS] in addition , use of diacid monomers B-material allows the main chain of the polymer B-material to contain biodegradable B-property ester bonds . [SEP]
[CLS] previously , the same group demonstrated the therapeutic efficiency of pace / pdna polyplexes , and at this time , the dependence of mrna transfection B-property efficiency I-property on the molecular weight and end - group composition of pace was thoroughly clarified . [SEP]
[CLS] as the molecular weight of pace decreased from 20 kda to 5 kda , the transfection B-property efficiency I-property of mrnas increased 100 - fold . [SEP]
[CLS] this tendency reached a plateau at 5 kda , but the transfection B-property efficiency I-property at 2 kda dramatically dropped , providing the guideline in choosing the molecular weight of pace for efficient mrna delivery . [SEP]
[CLS] in addition , methylamino ethanol ( - mae ) and carboxyl B-material ( - cooh ) groups as end - groups of pace were investigated in the same work . [SEP]
[CLS] the result showed that 5 kda pace - mae encapsulated 98 % of mrnas , while pace - cooh of the same mw did only 18 % , indicating the end - group dependency in mrna loading . [SEP]
[CLS] even after low in vivo stability and short circulation time of therapeutic oligonucleotides are addressed well by use of polymeric carriers , the carriers can be further evolved to be stimuli - responsive ; the stimuli - responsive carriers can be highly useful for preventing side effects and improving transfection B-property efficiency I-property , thereby enhancing therapeutic effects . [SEP]
[CLS] if specifically loaded molecules are selectively released at a desired location and time , the smart carriers can avoid potential side effects to healthy tissues and organs . [SEP]
[CLS] moreover , their capability to selectively unload the delivered oligonucleotides within endosome is valuable for macromol . res . , 29 ( 10 ) , 665 - 680 ( 2021 ) [SEP]
[CLS] macromolecular researchimproving the transfection B-property efficiency I-property of the oligonucleotides [SEP]
[CLS] for therapeutic applications , the stimuli that polymeric carriers can be responsive to could be divided into two categories : ( 1 ) endogenous stimuli , such as ph , redox levels , and enzyme concentrations , and ( 2 ) exogenous stimuli , such as heat , ultrasound , light , and magnetic B-property field . [SEP]
[CLS] the endogenous stimulus response system can achieve precise spatial control by spontaneously releasing the loaded molecules when the oligonucleotide - carrying carrier arrives at the disease - specific environments . [SEP]
[CLS] on the other hand , the exogenous stimulus response system requires external triggers , but it can exhibit a significantly high transfection B-property efficiency I-property and temporal control accuracy through immediate release at the local area with the external stimuli . [SEP]
[CLS] the environment of abnormal cells B-material , such as tumors B-material , differs from that of normal cells B-material ; for example , most tumor B-material cells B-material exhibit relatively low ph , produce abundant ros , and overexpress specific enzymes , compared to normal cells B-material . [SEP]
[CLS] when polymeric carriers are designed to respond to the different environment in releasing therapeutic oligonucleotides , the therapy can be localized to the targeted cells B-material , leading to higher efficacy and efficiency in therapeutic effects . [SEP]
[CLS] considering the therapeutic mechanisms of various oligonucleotides ( figure 2 ) , the endosomal escape of the delivered oligonucleotides is significantly important , and , there have been the advancement of polymeric carriers in response to the distinct environment within the endosome . [SEP]
[CLS] 3 . 2 . 1 . 1 . [SEP]
[CLS] endogenous stimuliph ph can be a crucially important stimulus in biological systems as it is an intrinsic property of various diseases , pathological conditions , and cellular microenvironments . [SEP]
[CLS] in particular , by endocytosis B-event , a cellular B-event uptake I-event process I-event , drug carriers are typically internalized into the endosome of cells B-material , and the endosome undergoes a maturation process to lysosome , progressively making the internal ph decrease up to ph 4 . 5 . [SEP]
[CLS] considering that the ph of the cytoplasm is 7 . 4 , the endosome is a unique environment to serve as a trigger of ph - responsive system that selectively releases delivered oligonucleotides at low ph . moreover , compared to physiological ph ( ~ 7 . 4 ) , the ph around tumor B-material and inflammatory cells B-material is lower ( 6 . 6 ~ 6 . 8 ) , so the polymeric carriers capable of responding to low ph enable spatial control in the release of therapeutic oligonucleotides . [SEP]
[CLS] given the intrinsic ph of the physiological and pathological microenvironments , the oligonucleotide delivery based on the polymeric carriers with ph - responsive moieties can be an excellent way to improve the therapeutic efficiency of the delivered oligonucleotides without adverse effects . [SEP]
[CLS] when polymeric carriers include ph - responsive linkers , they can enhance cellular uptake by breaking down the size of the carriers near desired locations , such as tumor B-material cells B-material . [SEP]
[CLS] even though pegylation improves the in vivo stability of therapeutic oligonucleotides , it lowers the cellular uptake of the resulting large particles . [SEP]
[CLS] the sirna - loaded polymeric B-nanoparticle nanoparticles I-nanoparticle wherein peg is linked by an acid - cleavable amide B-material bond ( dlink ) were developed , and their behavior near tumor B-material cells B-material was subsequently investigated . [SEP]
[CLS] in response to acidic ph , the dlink - containing nanoparticles B-nanoparticle increased the release rate of sirnas by 60 % compared to the nanoparticles B-nanoparticle permanently linked with peg . [SEP]
[CLS] the ph - responsive displacement of pegs from the dlink - containing nanoparticles B-nanoparticle was effective to improve their gene silencing efficiency ; while the silencing efficiency of plk1 sirnas was 66 . 9 % at ph 7 . 4 , it increased to 80 . 2 % at ph 6 . 5 . [SEP]
[CLS] moreover , when the same dose was administered to mice , the tumor B-material weight decreased from 0 . 54 g to 0 . 29 g , confirming that the acid - cleavable linker system can exhibit high tumor B-material suppression efficacy according to its ph - responsive ability . [SEP]
[CLS] chemically modified cationic B-material polymers B-material can be used to promote oligonucleotide releasing by inducing disruption of endosomal membrane at low ph . positively charged pei has been widely used due to its high loading capacity of oligonucleotides , and its proton sponge effect is attracting the strong attention for ph - responsive oligonucleotide releasing . [SEP]
[CLS] this effect has been observed in certain cationic B-material polymers B-material containing protonatable groups by endosomal ph . [SEP]
[CLS] when endosomal acidification proceeds , the polymeric carriers are further protonated , and counter ions B-material ( e . g . , cl - ) and water B-material enter the endosome to neutralize them . [SEP]
[CLS] for this reason , the endosome becomes swollen and eventually ruptured , making the internalized oligonucleotides diffuse in cytoplasm . [SEP]
[CLS] to exploit the protonation by endosomal acidification , a pei derivative , poly [ n - [ n - ( 2 - aminoethyl ) - 2aminoethyl ] aspartamide ] ( p [ asp ( det ) ] ) has been developed , and its transfection B-property efficiency I-property of delivered oligonucleotides was examined ( figure 4 ( a ) ) [SEP]
[CLS] this polymer B-material exhibited two different pka values , resulting in diprotonation at endosomal ph , so it strongly interacted with vesicular membranes , thereby efficiently releasing the carried oligonucleotides through membrane disruption . [SEP]
[CLS] in an in vivo test to mice , this polymeric carrier achieved nearly 600 % decrease in target protein B-material expression by successful oligonucleotide release into cytoplasm . [SEP]
[CLS] in addition to the p [ asp ( det ) ] , various cationic B-material polymers B-material , including poly ( n , ndimethylamino ethyl methacrylate ) ( pdmaema ) , have been evaluated for their in vitro and in vivo uses . [SEP]
[CLS] there are many different redox agents , such as nicotinamide adenine dinucleotide phosphate , and gsh in the extracellular or intracellular environments of disease - related tissues and cells B-material , and these redox agents play various roles including modulation of cell B-material growth , differentiation , and even death . among them , the most well - known reducing B-property agent I-property would be gsh , of which level is twice higher in the intracellular environment ( i . e . , endosome ) than in the extracellular environment , and importantly , the gsh level increases up to 1000 times in disease - related milieu [SEP]
[CLS] as ros involves in cellular signaling , pathogenic resistance , homeostasis , immunity , cell B-material growth and differentiation , it has been also therapeutically important ; it is known to be overexpressed near several disease cells B-material . [SEP]
[CLS] therefore , there have been many studies to enable spatially controllable release of oligonucleotides by responding to the disease - specific redox level change . [SEP]
[CLS] by incorporating disulfide bonds into polymers B-material , the oligonucleotide carriers can be responsive to intracellular milieu , which can be a great way to enhance the blood circulation time and the cellular uptake of therapeutic oligonucleotides together . [SEP]
[CLS] © the polymer B-material society of korea and springer 2021 while high molecular - weight polymers B-material can be advantageous for avoiding renal filtration , low molecular - weight polymers B-material can be more easily internalized into cells B-material . [SEP]
[CLS] this means that to improve the therapeutic efficacy , the polymeric carriers should not be degraded outside cells B-material , but selectively inside the cells B-material . [SEP]
[CLS] this selective degradation capability can be achieved by the development of disulfide bond - containing polymers B-material , which can be separated into low molecular - weight polymers B-material by gsh - induced reduction at specific locations . [SEP]
[CLS] to assess the therapeutic usefulness of the spatioselective cleavage , three different types of polymeric carriers were synthesized ; while two pei polymers B-material had different molecular weight ( pei25 : 25 kda and pei1 . 8 : 1 . 8 kda ) , the third one ( sspei1 . 8 ) was prepared by linearly linking 1 . 8 kda pei through cleavable disulfide linkers , thereby yielding a molecular weight of 25 kda . [SEP]
[CLS] compared to the cytotoxic B-property pei25 , pei1 . 8 showed the improved cell B-property viability I-property ( up to 250 % ) , and interestingly , the effect of sspei1 . 8 was similar with that of pei1 . 8 . [SEP]
[CLS] despite the similar size , sspei1 . 8 carrying oligonucleotide drugs exhibited 3 times higher transfection B-property efficiency I-property than pei25 . [SEP]
[CLS] similar trends have been observed in various in vitro and in vivo assessments . [SEP]
[CLS] the level of ros , such as superoxides , hydrogen B-material peroxides , peroxyl radicals , and hydroxyl radicals , can be also great to trig - ger the spatially controlled oligonucleotide release ; the ros involves in regulating cell B-material signaling , but abundant ros can induce oxidative stress , by which various diseases ( e . g . , cancer , inflammation , cardiovascular disease , and alzheimer ' s disease ) can occur . [SEP]
[CLS] these diseases have their own pathways to locally produce a high level of ros , so the disease sites have 10 ~ 100 times higher ros concentrations than normal tissues . [SEP]
[CLS] an rosresponsive polymeric carrier using poly ( amine thioketal ) ( patk ) was used for targeted oligonucleotide delivery to cancer cells B-material . [SEP]
[CLS] the half - lives of the polymer B-material were 48 , 20 , and 11 hours in 50 mm , 100 mm , and 200 mm h 2 o 2 solutions , respectively , indicating that the degradation of patk heavily depends on the h 2 o 2 concentrations . [SEP]
[CLS] compared to non - cancerous cells B-material , the patk with oligonucleotide drugs displayed ~ 60 % higher transfection B-property efficiency I-property to cancerous cells B-material , proving the potential of ros - responsive polymers B-material for disease - selective oligonucleotide delivery . [SEP]
[CLS] the exceptional stimulus specificity of ros - responsive polymers B-material enabled their application in oral oligonucleotide delivery ( figure 4 ( b ) ) . [SEP]
[CLS] in this work , sirna - loaded thioketal nanoparticles B-nanoparticle were orally delivered to inhibit tnf - α on intestinal inflammation . [SEP]
[CLS] with a daily oral gavage of the nanoparticles B-nanoparticle , biodistribution of sirnas in the organs of mice was investigated . [SEP]
[CLS] as a result , the orally delivered sirnas were highly localized in the inflamed ) ] has two different protonatable moieties ; one is protonated at ph 7 . 4 , i . e . , the ph of extracellular matrix and cytoplasm , and the other is protonated at ph 5 . 5 , which can be reached during endosomal acidification . [SEP]
[CLS] the diprotonated polymer B-material can induce membrane disruption by strongly interacted with vesicular membranes , thereby making therapeutic oligonucleotides escaped from the endosome . [SEP]
[CLS] ( b ) ros - responsive polymeric carrier system including thioketal groups . [SEP]
[CLS] the thioketal group - including polymeric carriers respond to a high level of ros on inflammation sites . [SEP]
[CLS] due to the specificity of the ros - sensitive linkers , the ros - responsive polymers B-material enable oral oligonucleotide delivery . [SEP]
[CLS] when the polymeric B-nanoparticle nanoparticles I-nanoparticle loaded with tnf - α sirnas were administered to mice by oral gavage , the tnf - α sirnas were exceptionally localized in the inflamed intestinal tissues , leading to effective degradation of tnf - α mrnas . [SEP]
[CLS] ( c ) enzyme - responsive polymeric carrier system that contains cleavable moieties by matrix metalloproteinase 2 ( mmp - 2 ) , one of the proteases . [SEP]
[CLS] as the mmp - 2 is generally overexpressed in disease cells B-material such as cancer cells B-material and inflammatory cells B-material , the polymeric carriers can detach the polyethylene glycol ( peg ) blocks in the presence of mmp - 2 on tumors B-material , promoting the cellular uptake of the smaller and more hydrophobic B-property polyplexes . [SEP]
[CLS] macromol . res . , 29 ( 10 ) , 665 - 680 ( 2021 ) [SEP]
[CLS] macromolecular researchintestinal tissues [SEP]
[CLS] compared to the liver , the intestine had 3 times more populated sirnas , confirming the selective release of the delivered sirnas in the inflamed tissues . [SEP]
[CLS] the transfection B-property efficiency I-property of the thioketal nanoparticles B-nanoparticle was significantly high ; as the transfected tna - α sirna was used to degrade the tnf - α mrna , the amounts of tnf - α mrnas was decreased by l0 times compared to the sirna delivery without the rosresponsive carriers . [SEP]
[CLS] as the high level of ros is a major and unique marker of diseases , a variety of ros - responsive , polymeric oligonucleotide carriers have been studied as safe and efficient delivery systems . [SEP]
[CLS] as biocatalyst , enzymes involve in various reactions in our bodies . [SEP]
[CLS] the biochemical processes are performed at specific conditions due to the environment - dependent activity B-property of I-property the enzymes , and to initiate the reactions , the biological enzymes are necessary to recognize target substrates selectively . [SEP]
[CLS] moreover , dysfunctions of cells B-material can cause some enzymes to be highly overexpressed in tumors B-material and inflammatory tissues . [SEP]
[CLS] such disease - relevant enzyme overexpression is significantly useful for developing stimuli - responsive systems , including smart oligonucleotide drug delivery . [SEP]
[CLS] for example , when polymeric carriers are synthesized to include substrate - mimicking moieties , they can be recognized by the enzymes overexpressed by disease cells B-material . [SEP]
[CLS] the enzyme - responsive polymeric carriers would be valuable for lowering the side effects , but increasing the therapeutic efficiency through releasing oligonucleotide in targeted sites only . [SEP]
[CLS] proteases that involve in cell B-material proliferation , invasion , and apoptosis B-event are one of the most suitable enzymes for enzymeresponsive oligonucleotide delivery as they are upregulated in diseases such as cancer and inflammation . [SEP]
[CLS] among various proteases , matrix metalloproteinase 2 ( mmp - 2 ) was chosen to develop sirna - loaded polymeric carriers ; 138 the mmp - 2 is an enzyme engaged in the degradation of extracellular matrix as overexpressed by almost all tumors B-material . [SEP]
[CLS] to make the polymeric carrier recognizable as the substrate of mmp - 2 , mmp - 2degradable peptides B-material , lags , were used as bridges between pegs and pcls within the synthetic polymer B-material ( figure 4 ( c ) ) . [SEP]
[CLS] in the presence of mmp - 2 , the lag bridges were successfully cleaved , so peg chains were subsequently displaced from the polymeric carrier . [SEP]
[CLS] this peg displacement resulted in reducing the size of sirna - loaded carrier , thereby improving its cellular uptake . [SEP]
[CLS] in in vivo mice model , the mmp - 2 - responsive polymeric carrier selectively delivered sirnas into tumor B-material tissues , reducing the tumor B-material volume by 63 % . [SEP]
[CLS] due to the environment specificity , various enzyme - responsive systems have been recently developed for effective oligonucleotide delivery . [SEP]
[CLS] by artificially generated external stimuli , spatiotemporal control of oligonucleotide - releasing system is achievable to increase the therapeutic efficiency of oligonucleotides . [SEP]
[CLS] for clinical applications , a large dose of oligonucleotides is administered to patients for a long time to result in a practical therapeutic effect . [SEP]
[CLS] moreover , in terms of inter - patient variability , the levels of endoge - nous stimuli can be quite different among individuals , making the same endogenous stimuli - responsive system not generally applicable for all the patients . [SEP]
[CLS] to solve these problems , exogenous stimuli can be exploited in developing the smart oligonucleotide drug carriers ; it is possible to precisely control the release of oligonucleotides by adjusting various parameters , including exposure time of heat , frequency of ultrasound , and wavelength of light . [SEP]
[CLS] moreover , when the oligonucleotide drug carriers are intravenously administered , they can release a high concentration of the therapeutic oligonucleotides at the local area where various external stimuli are applied during blood circulation . [SEP]
[CLS] in pathological environments , such as tumors B-material , the temperature can be slightly higher than that in normal physiological environments ; for example , malignant tumors B-material show the temperature of 40 ~ 42 [UNK] , while the temperature around normal tissues is 36 . 5 ~ 37 . 5 [UNK] for human beings . [SEP]
[CLS] however , this temperature difference may not be large enough for oligonucleotide drug carriers to use as a disease - relevant stimulus , so raising the local temperature by external heating has been extensively considered instead . [SEP]
[CLS] importantly , increasing the local temperature cannot only induce a direct cytotoxic B-property effect in tumors B-material , but also enhances the permeability of the tumors B-material , thereby facilitating oligonucleotide penetration . [SEP]
[CLS] some polymers B-material undergo a phase transition , changing hydrophilicity B-property and hydrophobicity B-property at the lower critical solution temperature ( lcst ) , and their thermos - responsive behaviors are appropriate for selective oligonucleotide release . [SEP]
[CLS] above the lcst , dehydration occurs as the hydrophobicity B-property of the polymeric carriers increases , and the tight complexes are formed between oligonucleotides and the polymers B-material . [SEP]
[CLS] these complexes can prevent degradation of the loaded oligonucleotides and simultaneously improve the cellular uptake due to their compact structures . [SEP]
[CLS] in contrast , under the lcst , hydrogen B-material bonds are newly formed between the polar compartments of the polymers B-material and water B-material molecules , so the carried oligonucleotides can be subsequently dissociated from the polymeric carriers , which is highly advantageous for the following transcription B-event and translation . [SEP]
[CLS] poly ( n - isopropylacrylamide ) ( pnipam ) is a representative lcst - type temperature - responsive polymer B-material , widely known for its lcst at 32 [UNK] . [SEP]
[CLS] however , by adjusting the ratio of hydrophobic B-property and hydrophilic B-property groups , the lcst is controllable for desired therapeutic applications . [SEP]
[CLS] a copolymer of pei - pni - pam was synthesized for thermo - responsive oligonucleotide delivery ( figure 5 ( a ) ) , and to increase the transition temperature up to 42 [UNK] , vinylpyrrolidone was introduced in the copolymer as a hydrophilic B-property monomer B-material . [SEP]
[CLS] when heat was applied , the polymeric carrier with luciferase genes increased the gene expression level by more than 2 orders of magnitude , compared to no heating . [SEP]
[CLS] in an in vivo test of mice , the polymeric carriers with pdnas were injected into the tumor B-material - bearing legs , which were incubated B-technique in a hot water B-material bath at 42 [UNK] . [SEP]
[CLS] as a result , the transfection B-property efficiency I-property was more than 10 times higher than the negative control , the mice without water B-material bath incubation B-technique . [SEP]
[CLS] in a similar way , temperature - responsive polymers B-material consisting of synthetic polymers B-material and polypeptides B-material have been exploited to increase the transfection B-property efficiency I-property of delivered oligonucleotides through heating treatment . [SEP]
[CLS] using ultrasound , spatial and temporal control of oligonucleotide delivery system is also attainable , preventing unnecessary side effects on healthy tissues . [SEP]
[CLS] this non - invasive stimulus is quite attractive due to the easy control of frequency , exposure time and duty cycles , achieving personalized ultrasound intensities . [SEP]
[CLS] ultrasound - responsive oligonucleotide carriers were constructed by the combination between cationic B-material polymers B-material and microbubble - filled liposomes B-nanoparticle ( figure 5 ( b ) ) ; 157 when ultrasound is applied to the carriers , the encapsulated microbubbles rapidly expand , compress , and collapse , and this vigorous movement can cause cavitation and sonoporation , mediating the oligonucleotide carrier destabilization and cell B-material membrane permeabilization . [SEP]
[CLS] using these phenomena , the delivered therapeutic oligonucleotides can be readily liberated from the carriers and enter the cell B-material through the transient pores formed by the cavitation and sonoporation , thereby enhancing the transfection B-property efficiency I-property . [SEP]
[CLS] in a study , to maximize the transfection B-property efficiency I-property of dnas , various parameters such as acoustic pressures , microbubble concentrations , and dna dosages have been tested . [SEP]
[CLS] at an optimal condition , the ultrasound treatment increased the gene expression level by 100 times compared to no treatment . [SEP]
[CLS] along with the ultrasound , various stimuli , such as ph and temperature , have been exploited together to develop dual or multi - responsive oligonucleotide carriers . [SEP]
[CLS] light is also an exogenous and non - invasive stimulus applicable for the oligonucleotide delivery by adjusting its wavelength , polarity , intensity and duration . [SEP]
[CLS] currently , ultraviolet ( uv ) has been frequently used as the external stimulus for selective release of therapeutic oligonucleotides , even though the penetration depth of uv radiation in human skin is quite shallow . [SEP]
[CLS] the uv has more energy than the other types of light , so it can easily induce chemical cleavage and isomerization of various uv - figure 5 . development of polymeric carriers that utilize exogenous stimuli for selective release of oligonucleotides . [SEP]
[CLS] ( a ) heat - responsive polymeric carriers using poly ( n - isopropylacrylamide ) ( pnipam ) - pei to enhance cellular uptake and endosomal escape of oligonucleotides . [SEP]
[CLS] pnipam is one of the representative temperature B-property - I-property sensitive I-property polymers B-material featuring lower critical solution temperature ( lcst ) . [SEP]
[CLS] when the temperature becomes higher than lcst by external heating , the pnipam undergoes hydrophilic - hydrophobic phase transition , resulting in globulization of polymer B-material chains in an aqueous solution . [SEP]
[CLS] due to hydrophobic B-property interactions I-property , the polymeric carriers can be aggregated each other , followed by the endocytosis B-event of the resulting aggregates . [SEP]
[CLS] compared to individual polymeric carriers ( left ) , the aggregated ones ( right ) are eager to make a stronger proton sponge effect ; in this way , by heat stimuli , the heat - responsive polymers B-material enhance the endosomal escape of the delivered oligonucleotides . [SEP]
[CLS] ( b ) ultrasound - responsive polymeric carriers constructed by the combination between cationic B-material polymers B-material and microbubble - filled liposomes B-nanoparticle . [SEP]
[CLS] with the ultrasound , the microbubbles in the polymeric carriers can cause to form small pores at cell B-material membranes through sonoporation and cavitation . [SEP]
[CLS] through the pores , the delivered oligonucleotides easily enter the cells B-material , thereby improving transfection and therapeutic efficiency . [SEP]
[CLS] ( c ) uv - responsive polymeric carriers based on poly ( β - amino ester ) ( pbae ) . [SEP]
[CLS] when non - degradable pbae chains are synthetized to include uv - cleavable nitrobenzyl groups , the resulting polymeric carriers can be rapidly decomposed upon external uv irradiation , which can be synchronized with the release of encapsulated oligonucleotides . [SEP]
[CLS] ( d ) magnetic B-property field - responsive nano - composites for therapeutic oligonucleotide delivery . [SEP]
[CLS] when the surface of superparamagnetic B-nanoparticle iron I-nanoparticle oxide I-nanoparticle nanoparticles I-nanoparticle ( spions ) is modified with pei , therapeutic oligonucleotides , such as mirna - encoding plasmid dnas ( pdnas ) can be well - loaded on the magnetic B-property nano - composites . [SEP]
[CLS] when the spions with pdnas are injected into a body , they can be selectively localized on tumor B-material sites during blood circulation by use of a magnet . [SEP]
[CLS] due to the high concentrations of pdnas around the tumors B-material , their transfection B-property efficiency I-property can be improved with the increase of mirna transcription B-event . [SEP]
[CLS] macromol . res . , 29 ( 10 ) , 665 - 680 ( 2021 ) [SEP]
[CLS] macromolecular researchreactive moieties , which is highly suitable for selective degradation of polymeric carriers and oligonucleotide release . [SEP]
[CLS] for instance , isomeric transformation of azobenzene group by uv can be used as a trigger for the oligonucleotide release of polymeric carriers to increase the efficiency B-property of I-property oligonucleotide transfection I-property . [SEP]
[CLS] when irradiated with 350 nm uv light , the azobenzene group is converted from a hydrophobic B-property trans - isomer to a hydrophilic B-property cis - isomer , and in the presence of visible light at 436 nm , it is converted back to the trans form . [SEP]
[CLS] the hydrophobic B-property transisomers can facilitate dense and stable packing of oligonucleotides within the polymeric carrier , but upon uv exposure , a large number of oligonucleotides can be rapidly released as the formation of hydrophilic B-property cis - isomers causes unpacking of the hydrophilic B-property oligonucleotides . [SEP]
[CLS] for a light - responsive oligonucleotide delivery carrier , the polymer B-material composed of a central azobenzene moiety and two terminal pdmaema blocks ( azo - pdmaema ) was synthesized , and its transfection B-property efficiency I-property was examined by an in vitro cell B-material test . [SEP]
[CLS] with uv irradiation on the azo - pdmaema , the delivered oligonucleotides were transfected into the cells B-material more efficiently than without irradiation , yielding up to 2 . 7 times higher efficiency . [SEP]
[CLS] the cytotoxic B-property effect of the uv irradiation was minimal ; there was no significant change in the cell B-property viability I-property when measured after 15 - minute uv exposure . [SEP]
[CLS] a uv - cleavable nitrobenzyl group can be introduced into a polymeric carrier to accelerate its degradation for the immediate release of therapeutic oligonucleotides . [SEP]
[CLS] pbae is widely used for the material B-material of oligonucleotide delivery carriers due to its synthesis easiness and versatility . [SEP]
[CLS] however , this ester linkage - containing polymer B-material is too slowly degraded by hydrolysis ; the degradation takes several hours to a few days under cellular conditions , which would not be appropriate for the therapeutic application with high concentrations of oligonucleotides . [SEP]
[CLS] the 2 - nitrobenzene moiety was introduced into the backbone of pbae for on - demand oligonucleotide release ( figure 5 ( c ) ) . [SEP]
[CLS] this oligonucleotide carrier improved transfection B-property efficiency I-property about 70 % upon 2 - minute uv irradiation and showed negligible cytotoxicity B-property . [SEP]
[CLS] similarly , other types of light - responsive linkers have been introduced to various polymers B-material , which would be highly useful to rapidly release a high concentration of oligonucleotides without notable toxicity B-property . [SEP]
[CLS] 3 . 2 . 2 . 4 . magnetic B-property field when synthetic polymers B-material are combined with magnetic B-nanoparticle nanoparticles I-nanoparticle , the resulting nano - composites can be the great carriers capable of localizing oligonucleotides in a body as guided by a magnetic B-property field , another non - invasive stimulus . [SEP]
[CLS] for example , by applying the exogenous magnetic B-property field to tumors B-material , the magnetic B-nanoparticle nanoparticles I-nanoparticle during blood circulation can be concentrated near the disease sites . [SEP]
[CLS] however , as unmodified nanoparticles B-nanoparticle cannot readily capture therapeutic oligonucleotides , surface modification with cationic B-material polymers B-material , such as pei , n - acylated chitosan B-material , and polylysine , should be performed to electrostatically load the negatively charged oligonucleotides . [SEP]
[CLS] superparamagnetic B-nanoparticle iron I-nanoparticle oxide I-nanoparticle nanoparticles I-nanoparticle ( spions ) have been chemically coated with chondroitin sulfate - pei copolymers for mirna - encoding pdna delivery ( figure 5 ( d ) ) . [SEP]
[CLS] while the peis hold the pdnas for mirna - 128 transcription B-event on the surface of the spions , the chondroitin sulfates were labeled to the nano - composites for active tumor B-material targeting and cd44 - mediated endocytosis B-event . [SEP]
[CLS] in the presence of the magnetic B-property field , the site - specific accumulation of the nano - composites were successfully induced ; when a static magnet was placed on the xenografted tumors B-material of mice after injection of nanocomposites , the localized biodistribution of the injected nanocomposites was observed within 3 hours , along with the improved transfection of delivered pdnas . [SEP]
[CLS] as the magnetic B-nanoparticle nanoparticles I-nanoparticle allow further in vivo imaging , the oligonucleotide - carrying nano - composites can be also useful for theranostic applications . [SEP]
[CLS] even though synthetic polymers B-material can be prepared to be highly responsive to many different types of endogenous and exogenous stimuli , one of the most deficient abilities as effective oligonucleotide carriers would be disease site targeting . [SEP]
[CLS] to maximize therapeutic efficacy with minimal adverse effects , the therapeutic oligonucleotides should be delivered to the only targeted cells B-material , which are encircled by their own specific surface receptors . [SEP]
[CLS] to empower the polymeric carrier to specifically recognize target cells B-material , many different types of ligands , such as monoclonal antibodies B-material , receptor B-material - specific peptides B-material , and nucleic B-material acid I-material aptamers , 57 have been employed . [SEP]
[CLS] among these ligands , aptamers are the chemically synthesized oligonucleotides , and as they fold into desired conformations at specific conditions , they are capable of recognizing many kinds of targets including cell B-material surface I-material receptors I-material . [SEP]
[CLS] interestingly , the denaturation of their 3d conformations is reversible , so unlike irreversibly denatured , protein - based ligands , further chemical modifications in harsh conditions are allowed , making the aptamers readily conjugated with a variety of highly structured polymers B-material . [SEP]
[CLS] due to the conjugation availability between the aptamers and the polymers B-material , tree - like or star - like dendrimers B-nanoparticle and hyperbranched polymers B-material have been decorated with the synthetic aptamers , and by the multivalency effect of the polymer - decorating aptamers , the resulting hybrid carrier exhibited the enhanced targeting capability , revealing the potential for target - specific oligonucleotide delivery . [SEP]
[CLS] by chemically conjugating cancer cell - targeting aptamers with the peg - based polymers B-material capable of controlled drug release , aptamer - polymer B-material hybrid ( aph ) molecules ( < 10 nm in diameter ) were created for the programmed drug release only into cancer cells B-material . [SEP]
[CLS] using click chemistry , one nucleolin - specific aptamer and one block copolymer , composed of ethylene glycol and ethylene glycol vinyl glycidyl ether B-material , were site - specifically conjugated to produce the aph molecule ; even after the conjugation reaction in organic solvents , the aptamer within the aph maintained the high affinity to target cancer cells B-material . [SEP]
[CLS] moreover , multiple chemotherapy drugs , doxorubicin B-material , were further conjugated to the polymer B-material through enzyme - cleavable linkers with a high yield , so when the aph was internalized in the cell B-material , the delivered drugs could be selectively released due to the linker cleavage by endosomal esterases . [SEP]
[CLS] the aph specifically recognized breast cancer cells B-material that overexpressed nucleolin on their cell B-material surfaces , and successfully released the chemotherapy drugs within the endosomes of the only targeted cells B-material , decreasing the cell viability I-property up to 64 % . [SEP]
[CLS] in contrast , the healthy cells B-material were not targeted at all by the aph , and no internalization and drug release was observed , indicating the minimal side effect by the use of the aph drug carrier . [SEP]
[CLS] rather than a single aptamer , multiple cancer - specific aptamers have been conjugated to a poly ( amidoamine ) ( pamam ) dendrimer B-nanoparticle nanocarrier to improve cell B-material targeting and transfection B-property efficiency I-property . [SEP]
[CLS] the pamam dendrimer B-nanoparticle possessed a large empty inner space , which could load hydrophobic B-property drugs , such as camptothecin ( cpt ) that is commonly used for colorectal cancer treatment . [SEP]
[CLS] at the sphere surface of the dendrimer B-nanoparticle , functional B-material end I-material groups were located , so a number of aptamers could be conjugated for surrounding the dendrimer B-nanoparticle carrier , leading to enhancement of binding interactions with target receptors . [SEP]
[CLS] when the aptamer - decorated dendrimer B-nanoparticle loaded the cpt , the encapsulation B-property efficiency I-property was almost 100 % , and the improved target binding and cytotoxicity B-property to colorectal cancer cells B-material were observed in in vitro and in vivo experiments . [SEP]
[CLS] currently , the treatment paradigm for cancer is rapidly changing from chemotherapy , the treatment by chemical substances , to immunotherapy in which immune cells B-material are activated for killing malignant cells B-material . [SEP]
[CLS] in promoting the ability of t - cells B-material to specifically target cancer cells B-material , chimeric antigen receptor B-material ( car ) genes are necessary to be transfected into the t - cells B-material , and the simultaneous delivery of both antigens and cpg oligodeoxynucleotides to the immune cells B-material would be effective to activate the immune system in our bodies . [SEP]
[CLS] to address this need , there have been different types of polymeric carriers for co - drug delivery , which have great advantages in drug loading capacity compared to viral vectors that have a limit on the size of cargo drug ( e . g . , ~ 10 kb for lentiviral vectors ) . [SEP]
[CLS] moreover , the polymeric carriers can be functionalized to be responsive to various stimuli , such as ph ; for instance , selective endosomal release of delivered oligonucleotides is achievable for increasing their transfection B-property efficiency I-property . [SEP]
[CLS] in this last chapter , we introduced some examples of immunotherapy using copolymer oligonucleotide carriers . [SEP]
[CLS] graft copolymers have been used to efficiently transfer car gene - encoding pdnas or mrnas into t - cells B-material . [SEP]
[CLS] t lymphocytes play a key role in recognition and clearance of antigens . [SEP]
[CLS] however , tumors B-material secrete several immunosuppressive cytokines , so t - cell B-material activation is significantly interfered . [SEP]
[CLS] to avoid the interference of t - cell B-material activation , the car gene is necessary to be transfected into the t - cells B-material , and the genetically modified t - cells B-material can specifically bind to the receptors of cancer cells B-material , recovering the immune response . [SEP]
[CLS] comb - shaped and sunflower - shaped graft copolymers were developed to deliver pdnas and mrnas into t - cells B-material and poly ( 2 - hydroxyethyl methacrylate ) and poly ( 2 - ( dimethylamino ) ethyl methacrylate ) were used to synthesized the graft copolymers with various molecular weights . [SEP]
[CLS] the graft copolymers efficiently transfected pdnas into t - cells B-material ( 25 - 50 % in efficiency ) compared to bpei , of which transfection was negligible in serum - free medium . [SEP]
[CLS] in particular , the comb - shaped graft copolymer showed successful transfection of primary human t - cells B-material with higher efficiency than other types of copolymers , exhibiting 18 % and 25 % transfection by pdnas and mrnas , respectively . [SEP]
[CLS] by systematically designing the polymer B-material to facilitate the endosomal escape of delivered antigens and adjuvants , the polymeric carrier dramatically improved the activation efficiency of immune systems . [SEP]
[CLS] when the adjuvants , such as cpg oligonucleotides , are transferred along with antigens to antigenpresenting cells B-material , the immune response is highly promoted . [SEP]
[CLS] in this work , the di - block copolymers with two multifunctional modules were synthesized to deliver ovalbumin ( ova ) and cpg oligonucleotides . [SEP]
[CLS] in a block , a small percentage of pyridyl disulfide ethyl methacrylate was conjugated with disulfide bonds to bind ova antigens , and the positively charged dmaema containing tertiary amines B-material was included to electrostatically interact with cpg oligodeoxynucleotides . [SEP]
[CLS] in the other block , propylacrylic acids ( paas ) were introduced for ph - responsive endosomal escape of delivered molecules . [SEP]
[CLS] as the carboxylic B-material acid I-material residue was protonated at endosomal ph , the paa experienced a hydrophilic - to - hydrophobic transition , leading to membrane disruption . [SEP]
[CLS] the di - block copolymers with both ovas and cpgs showed 7 times higher t - cell B-material response compared to the co - polymer B-material only with ovas . [SEP]
[CLS] moreover , the use of polymeric carriers was indeed effective , exhibiting 18 times higher response than the free use of ovas and cpgs without carriers . [SEP]
[CLS] to maximize the therapeutic effects of oligonucleotides and minimize their adverse effects simultaneously , the therapeutic oligonucleotides , such as asos , sirnas , mirnas , and mrnas , have been systematically combined with a wide range of functionalized polymers B-material . [SEP]
[CLS] as the physical and chemical properties of the synthetic polymers B-material were finely tuned , the resulting oligonucleotide carriers enabled loading of numerous oligonucleotides and their subsequent delivery to disease sites without renal clearance or nucleolytic degradation . [SEP]
[CLS] moreover , the polymeric carriers have been further engineered to respond to various endogenous and exogenous stimuli ; the release of therapeutic oligonucleotides was highly localized to disease tissues , and the endosomal escape of the large oligonucleotides was significantly promoted , thereby increasing therapeutic efficacy and efficiency dramatically with reduced side effects . [SEP]
[CLS] currently , several clinical trials are ongoing based on the systematic combination of oligonucleotides and polymers B-material , and the medical uses of the synergistic combination ( e . g . , immunotherapy ) are also expanding [SEP]
[CLS] even though oligonucleotides and polymers B-material have complemented each other to actualize highly effective and efficient oligonucleotide therapy , several issues still remain . [SEP]
[CLS] for example , inherent cytotoxicity B-property of the polymer B-material and relevant side effects should be mitigated . [SEP]
[CLS] the cytotoxicity B-property of polymeric carriers is typically attributed to their cationic B-material surface charge and hydrophobicity B-property , which are though essential for them to transfect disease cells B-material with the therapeutic oligonucleotides . [SEP]
[CLS] with a better macromol . res . , 29 ( 10 ) , 665 - 680 ( 2021 ) [SEP]
[CLS] macromolecular researchunderstanding of cellular uptake and disease action mechanisms , various disease - specific stimuli could be further explored to develop the polymeric carriers that can become cationic B-material or hydrophobic B-property at desired locations only . [SEP]
[CLS] the administered dose of the oligonucleotide - loaded polymers B-material is also critical in terms of the side effects ; by reducing the dosage , the side effects of oligonucleotides and polymers B-material would be less influential , and importantly , the reduction of the medical care costs would be expected additionally . [SEP]
[CLS] moreover , as specifically folding oligonucleotides , i . e . , aptamers , can recognize target cells B-material like monoclonal antibodies B-material , the incorporation of the synthetic affinity reagents can strengthen the power of synthetic polymers B-material toward creation of target - specific oligonucleotide carriers . [SEP]
[CLS] furthermore , to solve the off - target effects of simply designed , therapeutic oligonucleotides , it can be considered to deliver the self - regulating oligonucleotides by polymeric carriers . [SEP]
[CLS] by systematically incorporating toehold switches and riboswitches into the therapeutic oligonucleotides , the expression of the resulting oligonucleotides can be regulated by intracellular levels of disease - related biomolecules , which could be highly effective to increase the safety of oligonucleotide therapeutics in clinical use . [SEP]
[CLS] with all efforts , we envision the development of safer and more effective combinations between therapeutic oligonucleotides and polymers B-material , holding the potential for the next generation therapy even applicable for lots of rare or currently untreatable diseases . [SEP]
[CLS] polymeric strategies to overcome the barriers B-property of in vivo oligonucleotide delivery . [SEP]
[CLS] ( a ) oligonucleotide pegylation to avoid renal filtration . [SEP]
[CLS] due to the small size , oligonucleotides are rapidly cleared by the renal filtration process , leading to urine secretion . [SEP]
[CLS] by pegylation , the hydrodynamic volume of the oligonucleotides can be enlarged , thereby prolonging their circulation time in blood . [SEP]
[CLS] ( b ) use of cationic B-material polymers B-material to promote cellular uptake . [SEP]
[CLS] as both oligonucleotides and cell B-material membranes are negatively charged , electrostatically repulsive interactions occur , making the oligonucleotides difficult to be internalized into cells B-material . [SEP]
[CLS] cationic B-material polymers B-material , such as polyethylenimine , can be complexed with the anionic oligonucleotides , making the net charge of the resulting polyplex advantageous for the following cellular uptake . [SEP]
[CLS] once internalized into the endosome , the polymeric carriers begin to be protonated at the endosomal ph , causing the membrane disruption by the influx of counterions and water B-material molecules . [SEP]
[CLS] as resulted by the proton sponge effect , the therapeutic oligonucleotides escape from the endosome and diffuse into the cytoplasm . [SEP]
[CLS] therapeutic mechanisms of several oligonucleotides and the roles of polymeric carriers for successful oligonucleotide therapy . [SEP]
[CLS] polymeric carriers can encapsulate various therapeutic oligonucleotides , such as asos , sirnas , mirnas , and mrnas , preventing the nucleolytic degradation by nucleases and macrophages in blood , while the enlarged size of the polyplexes was effective to avoid renal filtration . [SEP]
[CLS] when the oligonucleotide - loaded polymers B-material successfully reach disease cells B-material by passing through extracellular matrix , the anionic oligonucleotides neutralized by the cationic B-material polymers B-material are well accepted by negatively charged cell B-material membranes , promoting cellular uptakes . [SEP]
[CLS] when the various oligonucleotides are internalized into the cytoplasm of disease cells B-material , their therapeutic mechanisms are quite different . [SEP]
[CLS] asos can directly bind to pre - mrnas in nucleus or to mrnas in cytosol in inhibiting target genes . [SEP]
[CLS] figure 3 . sirnas and mirnas are required to form rna - induced silencing complexes ( riscs ) in cytosol , and the riscs degrade target mrnas to inhibit further translation ; while sirnas are specific to bind one target gene , less specific mir - nas enable recognition of multiple mrnas , leading to multiple gene silencing . [SEP]
[CLS] mrnas are decoded by ribosome in cytoplasm , inducing protein B-material expression for vaccination and therapy . [SEP]
[CLS] development of stimuli - responsive polymers B-material to improve the therapeutic efficacy of delivered oligonucleotides . [SEP]
[CLS] ( a ) ph - responsive polymeric carrier using poly [ n - [ n - ( 2 - aminoethyl ) - 2 - aminoethyl ] aspartamide ] ( p [ asp ( det ) ] ) . [SEP]
[CLS] the p [ asp ( det ) ] has two different protonatable moieties ; one is protonated at ph 7 . 4 , i . e . , the ph of extracellular matrix and cytoplasm , and the other is protonated at ph 5 . 5 , which can be reached during endosomal acidification . [SEP]
[CLS] the diprotonated polymer B-material can induce membrane disruption by strongly interacted with vesicular membranes , thereby making therapeutic oligonucleotides escaped from the endosome . [SEP]
[CLS] ( b ) ros - responsive polymeric carrier system including thioketal groups . [SEP]
[CLS] the thioketal group - including polymeric carriers respond to a high level of ros on inflammation sites . [SEP]
[CLS] due to the specificity of the ros - sensitive linkers , the ros - responsive polymers B-material enable oral oligonucleotide delivery . [SEP]
[CLS] when the polymeric B-nanoparticle nanoparticles I-nanoparticle loaded with tnf - α sirnas were administered to mice by oral gavage , the tnf - α sirnas were exceptionally localized in the inflamed intestinal tissues , leading to effective degradation of tnf - α mrnas . [SEP]
[CLS] ( c ) enzyme - responsive polymeric carrier system that contains cleavable moieties by matrix metalloproteinase 2 ( mmp - 2 ) , one of the proteases . [SEP]
[CLS] as the mmp - 2 is generally overexpressed in disease cells B-material such as cancer cells B-material and inflammatory cells B-material , the polymeric carriers can detach the polyethylene glycol ( peg ) blocks in the presence of mmp - 2 on tumors B-material , promoting the cellular uptake of the smaller and more hydrophobic B-property polyplexes . [SEP]
[CLS] specific design of block copolymers allows [SEP]
[CLS] the delivery of materials for genetic engineering with transitory activity constitutes a promising field in biology and medicine with potential applications in the treatment of disease , from cancer and infectious diseases to inheritable disorders . [SEP]
[CLS] the possibility to restore the expression of a missing protein B-material , the potential correction of the splicing of defective genes , or the silencing or modulation of the expression of other genes constitute powerful tools that will have a great impact in the future of biology and medicine . [SEP]
[CLS] impressive progress has been made in the last decade , with several products reaching the market as novel technologies for gene editing emerge . [SEP]
[CLS] however , the transference of these technologies to functional therapies is hindered by the suboptimal performance of vehicles B-material in capturing , protecting and delivering the corresponding nucleotide cargoes with safety and efficacy . [SEP]
[CLS] chemistry and the chemical sciences will play a key role in the development of the innovative synthetic materials that will overcome the upcoming challenges of the next generation gene delivery therapies and protocols . [SEP]
[CLS] in this review we address the newest chemical advances in the production of materials at the forefront of nucleotide cell B-material delivery and gene therapy . [SEP]
[CLS] chemistry and molecular biology have always been involved in the development of new technologies with potential therapeutic applications . [SEP]
[CLS] dna automated synthesis has been essential for pcr , or site - directed mutagenesis [SEP]
[CLS] these have allowed the generation of optimized fluorescent B-property proteins B-material , phage display technology [SEP]
[CLS] , unnatural amino B-material acids I-material incorporation into proteins B-material , catalytic antibodies B-material and synthetic genomes [SEP]
[CLS] among all of these discoveries and techniques , genetic engineering and gene therapy constitute one of the most promising technologies for the treatment of disease and for the future of human health [SEP]
[CLS] the use of nucleic B-material acids I-material as therapeutic agents to repair a protein B-material deficiency or to modulate gene expression has a great potential not only for the treatment of inherited disorders , but also in the treatment of acquired diseases through their utilization as dna vaccines , antiviral B-property therapies and cancer immunotherapy ( table 1 ) . [SEP]
[CLS] however , the growth of the expectations in gene therapy and medicine have sometimes been dramatically crushed by the delivery problem . [SEP]
[CLS] nucleic B-material acids I-material are negatively charged and are occasionally very unstable molecules and thus their intracellular delivery , at the right place at the right moment , still constitutes a challenge for synthetic chemistry , materials science and biotechnology . [SEP]
[CLS] a gene vector has to overcome different barriers B-property before the successful delivery of its cargo ( fig . 1 ; table 2 ) . [SEP]
[CLS] first , the nucleotide cargo should be protected from degradation , which is usually achieved by complexation or encapsulation in different structures . [SEP]
[CLS] the size and charge of these complexes has to be controlled as it affects uptake efficiency and bio - distribution , as too large or too small particles can be cleared by the mononuclear phagocyte system , the liver or the kidneys [SEP]
[CLS] in systemic delivery , molecules larger than individual oligonucleotides cannot cross the endothelial barrier B-property except for tissues with fenestrated or leaky vasculature [SEP]
[CLS] improving carrier selectivity by conjugation of the vector with targeting molecules can increase selectivity towards targeted cells B-material increasing uptake and decreasing off - target effects [SEP]
[CLS] finally , vector particles have to translocate the plasma membrane , avoid endosomal entrapment and , in some cases , cross the nuclear envelope . [SEP]
[CLS] moreover , the nucleotide cargo has to be released from the transported complexes to be free to interact with its target or be recognized by the cellular elements required for silencing , transcription B-event or translation . [SEP]
[CLS] therefore , the delivery efficiency is not only a question of plasma membrane translocation and cellular internalization . [SEP]
[CLS] the ideal gene carrier should be able to deliver different nucleic B-material acids I-material , protect the cargo from nucleases , avoid fast clearance , toxicity B-property , and immune detection , prevent non - specific interactions with proteins B-material and non - target cells B-material , reach the cells of interest , escape the endosome , release the cargo and , when necessary , transport the cargo into the nucleus ( table 2 ) [SEP]
[CLS] this is obviously not a simple task for synthetically scalable materials but the latest achievements in the field point to a promising future for the wide variety of non - viral vectors . [SEP]
[CLS] viruses function as natural gene carriers as they are able to bind the cell B-material membrane , internalize in the cell B-material , and escape the endosome to reach the cytosol . [SEP]
[CLS] as viruses survival requires the efficient delivery of their own genetic material B-material into cells B-material , they have been artificially manipulated for therapeutic gene transfection . [SEP]
[CLS] therefore , some modified viral vectors have been approved for the treatment of human disease , such as glybera for lipoprotein lipase deficiency , gendicine for cancer treatment , or luxturna for retinal dystrophy . [SEP]
[CLS] however , viruses and derived materials present drawbacks , mainly related with their low cargo capacity and their immunogenicity B-property that can cause fatal adverse reactions or abrogate their activity , or require additional immunosuppresive therapy [SEP]
[CLS] furthermore , this is the author ' s accepted manuscript of the following article : lostale - seijo , i . , & montenegro , j . ( 2018 ) . [SEP]
[CLS] synthetic materials at the forefront of gene delivery . [SEP]
[CLS] nature reviews chemistry . [SEP]
[CLS] doi : 10 . 1038 / s41570 - 018 - 0039 - 1 [SEP]
[CLS] the limitations in the large - scale production of viruses and their potential to induce undesired insertional mutagenesis strongly complicates their real applicability in gene therapy . [SEP]
[CLS] for instance , glybera has been withdrawn from the market due to the high production costs and lack of demand [SEP]
[CLS] the combination of synthetic chemistry and molecular biology has been widely explored for the development of artificial carriers that could accomplish efficient and selective intracellular gene delivery . [SEP]
[CLS] traditional non - viral vectors showed low efficiency due to low cell B-material selectivity as well as endosomal entrapment . [SEP]
[CLS] additionally , most of the delivery studies are performed on monolayers of immortal cell B-material lines that usually contain alterations in dna / rna sensing or survival pathways , which can overestimate their efficiency when transferred to in vivo [SEP]
[CLS] nevertheless , impressive conceptual advances in synthetic gene vehicles B-material have emerged in the last few years and several compounds have shown good activity , not only in vitro but also in vivo . [SEP]
[CLS] nucleotide chemical modifications have delivered a new set of artificial nucleic B-material acid I-material analogues with higher stability , improved activity and lower immunogenicity B-property . [SEP]
[CLS] additionally , the current covalent and supramolecular synthetic tools allow the preparation of an unlimited number of new compounds that can be applied for the delivery of bioactive nucleotides . [SEP]
[CLS] nowadays , several formulations for gene - based therapies are reaching the clinic . [SEP]
[CLS] although viral vectors predominate in the medical applications of gene therapy , synthetic vectors are becoming a real alternative in nucleotide - mediated therapeutics . [SEP]
[CLS] for instance , naked antisense phosphorothioates have been recently approved for human use , non - viral sirna delivery systems are being tested for cancer treatment [SEP]
[CLS] , and [SEP]
[CLS] patisiran , a lipid B-nanoparticle nanoparticle I-nanoparticle for the delivery of sirna for the treatment of hereditary transthyretin - mediated amyloidosis , has recently finished phase 3 studies . [SEP]
[CLS] this review will address the latest advances in non - viral gene delivery and we have sorted these synthetic carriers according to their chemical nature in order to highlight the importance of their molecular structure and connect this structural information to its particular functional application ( table 2 ) . [SEP]
[CLS] nucleotide chemical modifications [SEP]
[CLS] the activity and specificity of bioactive oligonucleotides depends mostly on their base sequence , while the biopolymer B-material backbone usually plays a structural role . [SEP]
[CLS] therefore , nucleotides can tolerate the introduction of certain modifications on their structure that can improve their stability and uptake efficiency without affecting the recognition of its target . [SEP]
[CLS] the chemical structure of oligonucleotide - based drugs has been in a state of permanent evolution to optimize their delivery and clinical applications [SEP]
[CLS] synthetically modified antisense oligonucleotides ( asos ) include , among others , phosphorothioate nucleotides ( ps ) in combination with locked nucleic B-material acids I-material ( lnas ) , tricyclo - dna oligomers ( tcdna ) or 2 ' - deoxy - 2 ' - fluoroarabinonucleotides ( 2 ' - f - ana ) ( fig . 2a ) . [SEP]
[CLS] the substitution of the oxygen B-material for a sulphur atom B-material in phosphorothioate - modified antisense oligonucleotides ( ps - asos ) strongly reduces their nucleolytic degradation . [SEP]
[CLS] the spontaneous uptake , or gymnosis , of these stabilized single stranded nucleotides has been observed both in vitro and in vivo and a number of formulations of phosphorothioate oligonucleotides have been approved for human use , such as fomivirsen for the treatment of cytomegalovirus retinitis , or mipomersen for homozygous familial hypercholesterolemia [SEP]
[CLS] the inclusion of additional hydrophobic B-property carbon B-material atoms I-material , between the c5 ' and c3 ' positions of the nucleotide of a phosphorothioate tricyclo - dna ( tcdna ) , triggered its self - assembly into nanoparticles B-nanoparticle with a suitable size ( 40 - 100 nm ) for in vivo delivery , and therapeutic improvement on a mouse model of duchenne ' s muscular dystrophy [SEP]
[CLS] however , nucleotide chemical modifications can sometimes give rise to adverse effects such as complement activation , trombocytopenia , increased off - target interactions , or intrinsic toxicity B-property of the corresponding metabolites [SEP]
[CLS] particularly , modifications that increase nucleotide hydrophobicity B-property at the 2 ' position such as in 2 ' - fluoro - modified phosphorothioate - asos and phosphorothioate - lnas have been shown to be hepatotoxic in mice . [SEP]
[CLS] intriguingly , less hydrophobic B-property modifications like in 2 ' - o - moe ( 2 ' - o - ( 2 - methoxyethyl ) ) or in constrained 2 ' - o - ethyl nucleotides did not show the same effect . [SEP]
[CLS] however , there is growing evidence that this toxicity B-property , probably due to an increased affinity of these nucleotides towards the hepatic proteins B-material , can be controlled by sequence design optimization [SEP]
[CLS] charge reduction [SEP]
[CLS] the highly negatively charged phosphate backbone of nucleic B-material acids I-material blocks their interaction with the anionic components of the membrane and hinders its crossing of the non - polar membrane , a situation that prevents the uptake of therapeutic oligonucleotides . [SEP]
[CLS] the phosphate charges can be reduced by esterification and phosphotriester formation . [SEP]
[CLS] the modification of the nucleotide phosphate , with the s - acyl - 2 - thioethyl ( sate ) moiety , was optimized for 2 ' modified nucleotides ( 2 ' - f or 2 ' - o - me ) by employing the mild basic - sensitive phenoxyacetyl protecting B-technique group I-technique , which avoids phosphotriester hydrolysis at the nucleobase deprotection step . [SEP]
[CLS] although it cannot be applied to the whole sequence due to duplex destabilization , this esterification strongly increased the nucleotide bioavailability B-property by binding to serum albumin . [SEP]
[CLS] additionally , fusion to cell B-material penetrating peptides B-material ( cpps ) or glycan moieties ( i . e . tris - n - acetylgalactosamine ) enhanced the cellular uptake of the resulting hybrid oligonucleotides . [SEP]
[CLS] this elegant strategy allowed the high yield production of neutral sirna derivatives ( sirnn ) , in which the artificial thioester can be de - caged by cytosolic thioesterases to regenerate the original fully functional sirna inside cells B-material ( fig . 2b ) . [SEP]
[CLS] programmable hybridization . [SEP]
[CLS] the predictable and programmable folding of nucleotides can be employed to control the presentation of functional nucleotides and cell targeting sequences . [SEP]
[CLS] this concept has been applied in gene targeted delivery by generation of active oligonucleotide / targeting aptamer chimeras such as aptamer - sirna or aptamer - mirna chimeras ( fig . 2c ) . [SEP]
[CLS] analogously , a y - shaped rna scaffold containing anti mir - 21 was combined with an egfr aptamer , and the resulting chimera showed promising activity against breast cancer [SEP]
[CLS] the programmable sequential recognition of a nanovehicle by two aptamers that are bound to two different cell B-material receptors was employed for targeted gene delivery . [SEP]
[CLS] the interaction of the nanovehicle with the first aptamer , bound to a cell B-material surface I-material receptor I-material , cleaves a dna loop by reconstitution of a mnazyme . [SEP]
[CLS] after this cleavage , a hybridization sequence of the nanovehicle is exposed and presented to the second aptamer that is bound to a different cell B-material receptor B-material . [SEP]
[CLS] hybridization with this second aptamer anchors the dna nanovehicle to the membrane and enables specific endocytic internalization only in the cells B-material where both receptors are present . [SEP]
[CLS] in addition to aptamers , fusion to other ligands can also be applied to trigger specific receptor - mediated endocytosis B-event . [SEP]
[CLS] the conjugation of antisense oligonucleotides or sirna to tri - antennary - n - acetyl galactosamine is a widely used strategy for the targeted delivery to the hepatic asialoglycoprotein receptor B-material ( asgpr ) ( fig . 2c ) . [SEP]
[CLS] the attachment of this particular glycan has also been confirmed to reduce the toxicity B-property of the oligonucleotide conjugates . [SEP]
[CLS] peg brushes have been also connected to antisense dna ( pacdna ) to increase the stability of the bioactive nucleotide against nucleases and facilitate its cellular uptake by endocytosis B-event ( fig . 2d ) . [SEP]
[CLS] larger single stranded dna or rnas , generated by rolling circle amplification or transcription B-event techniques , can fold into globular structures or " nanoflowers " ( fig . 2d ) . [SEP]
[CLS] these nanoassemblies can include aptamer sequences for tumour targeting and dnazymes to silence specific genes required for cell B-material survival [SEP]
[CLS] similar stimuli - responsive rna nanoparticles B-nanoparticle have been recently developed to release multiples copies of sirna inside the cell B-material . [SEP]
[CLS] these cleavable rna nanoparticles B-nanoparticle consist of a long ssrna containing repetitions of a sirna antisense strand hybridized with chimeric dna - rna sense strands . [SEP]
[CLS] these dna / rna duplexes were coated with a glutathione sensitive chitosan B-material polymer B-material for delivery and they were cleaved inside the cell B-material by the cellular rnaseh . [SEP]
[CLS] attaching large structures to a nucleotide cargo may impair its biological activity . [SEP]
[CLS] covalent dynamic linkers ( i . e . disulfide ) can be used to connect the nucleic B-material acid I-material and the non - viral vehicle B-material . [SEP]
[CLS] these dynamic linkers can be cleaved by external stimuli ( i . e . proteases , ph changes , reducing B-property agents I-property ) and thus release the intact nucleic B-material acid I-material material B-material at the suitable tissue or intracellular location . [SEP]
[CLS] a multifunctional cationic B-material amphipathic polymer B-material can be grafted , via ph or protease sensitive linkages B-property , to peg and n - acetylgalactosamine pendants and connected to a sirna cargo by a disulfide bond . [SEP]
[CLS] in the first step , the protease or ph - mediated cleavage restored ligand targeting and membrane interaction and , once inside the cell B-material , the cleavage of the disulfide bonds triggered cargo release [SEP]
[CLS] in a different conceptual strategy , nucleic B-material acids I-material that are recognised by a polypeptide B-material sequence can function as simple carriers for the delivery of transcription B-event factors for gene expression regulation . [SEP]
[CLS] in dna assembled recombinant transcription B-event factors ( darts ) , the nucleic B-material acid I-material acts as a simple carrier and it can be decorated with glycan residues via a dynamic acetal linker that after hydrolysis unmasked hydrophobic B-property endosomal disrupting domains to facilitate cargo delivery ( fig . 2e ) [SEP]
[CLS] the efficiency and low cost of physical gene transfection has recently triggered a rebirth of this field . [SEP]
[CLS] although with certain limitations for in vivo applications , excluding cutaneous treatment or local hydrodynamic gene delivery to liver or muscle , physical methods can be very useful for ex vivo gene therapy . [SEP]
[CLS] in ex vivo therapies , patient cells B-material are removed , modified ( i . e . transfected ) under in vitro conditions and then reinfused into the patient . [SEP]
[CLS] electroporation , osmotic shocks or microinjection allow direct delivery of the oligonucleotide to the cytoplasm or nucleus , circumventing any endosomal pathway and / or nuclear membrane barrier B-property , because of the transient extensive disruption of the plasma and nuclear membranes . [SEP]
[CLS] however , this aggressive technique can lead to severe cellular damage and new technologies are being developed to allow a better control of the degree of membrane disruption [SEP]
[CLS] indeed , physical gene delivery is currently in the pipeline for therapeutic applications such as cancer immunotherapy by chimeric antigen receptors ( car ) , in where the t - cells B-material of a patient can be extracted , physically transfected ex vivo and the new reprogrammed t - cells B-material re - injected into the same patient to seek and destroy the tumour [SEP]
[CLS] recently , the combination of electroporation and microfluidic cell B-material deformation has been explored for high throughput plasmid , mrna or protein B-material delivery . [SEP]
[CLS] the application of nanoneedles in drug and gene delivery is generally associated with inflammation which can be useful for vaccination but undesirable for other purposes . [SEP]
[CLS] biodegradable B-property porous silicon B-material nanoneedles have recently been developed , which increases their in vivo tolerance and safety , and extends their potential applications . [SEP]
[CLS] the collapse of gas - filled microbubbles , under target - directed ultrasound , was applied in systemic delivery of drugs . [SEP]
[CLS] however , the stabilization of microbubbles for dna delivery usually requires microbubble functionalization with binary lipid B-material mixtures to avoid perturbation of their acoustic properties . [SEP]
[CLS] a recent screening of these stabilizing lipids B-material identified a single dimyristoyl cationic B-material lipid B-material branched with polyspermine and partially decorated with peg . [SEP]
[CLS] this single lipid B-material formulation was able to deliver a miniplasmid to hepatocytes achieving long term gene expression . [SEP]
[CLS] microbubbles can also be used for the targeted delivery of promiscuous cationic B-material liposomes B-nanoparticle . [SEP]
[CLS] these plasmid loaded cationic B-material liposomes B-nanoparticle can be functionalized with peg and heparin to inhibit off - target uptake . [SEP]
[CLS] the precise ultrasonic disruption of co - injected microbubbles triggered the local release of the liposomal B-nanoparticle contents . [SEP]
[CLS] acoustic transfection , directly based on membrane disruption due to cavitation , can also achieve in vitro plasmid , mrna or protein B-material transfection with single cell resolution 71 . [SEP]
[CLS] virus - like particles [SEP]
[CLS] protein B-material vehicles B-material can also be employed to protect and deliver bioactive nucleotides . [SEP]
[CLS] the encapsulation of nucleotidic cargo into a protein B-material container is a ubiquitous strategy in nature in viruses and in higher organisms [SEP]
[CLS] for instance , the arc protein B-material , evolutionary related to transposon ' s gag protein B-material , forms enveloped viruslike particles that are able to deliver its own mrna across neurons or the neuromuscular junction in mammals and insects . [SEP]
[CLS] virus like particles ( vlps ) that are reconstructed from proteins B-material of the natural virus can encapsulate active oligonucleotides , inhibiting tumour growth by intracellular delivery of a suicide gene . [SEP]
[CLS] interestingly , the strong immunogenicity B-property of vlps can be reduced by particle surface modification with polymers B-material such as polynorbornene or polyethylenglycol . [SEP]
[CLS] decoration of vlps with targeting moieties can redirect these artificial capsids to other tissues , as was demonstrated by the attachment of transferrin or aptamers to polyomavirus vlps 75 or phages . [SEP]
[CLS] rna binding B-event proteins I-event [SEP]
[CLS] dsrna - binding domains are common in proteins B-material of viral and non - viral origin and can be used for sirna protection and delivery ( fig . 3a ) . [SEP]
[CLS] a fusion protein B-material composed of dsrna - binding domains , from the protein B-material kinase pkr , fused to a penetrating peptide B-material sequence , was confirmed to deliver sirna in hard - to - transfect cells B-material and in vivo by intranasal inoculation . [SEP]
[CLS] to achieve an efficient intracellular sirna delivery , the binding B-event between I-event the I-event protein I-event and the nucleotide has to be strong enough to protect the cargo , but it also has to be disrupted in the last step of the delivery process . [SEP]
[CLS] a systematic study using high - affinity dsrna binding proteins B-material , derived from p19 of carnation italian ringspot [UNK] showed that , even for dissociation constants in the low pm range , the inhibitory binding - limit was not reached [SEP]
[CLS] however , to achieve cytosolic delivery of the sirna in this study a second pore forming perfringolysin o protein B-material was required to achieve endosomal escape [SEP]
[CLS] bioavailability B-property enhancers [SEP]
[CLS] association with certain proteins B-material can improve cargo pharmacokinetics , as the bigger protein B-material / cargo complexes can circumvent renal clearance and extend their serum half - life by the exocytosis - recycling pathway . [SEP]
[CLS] the non - covalent binding of lipid modified oligonucleotides to albumin has been applied to enhance the bioavailability B-property of sirna and other antisense oligonucleotides . [SEP]
[CLS] conjugation of nucleotides to antibodies B-material can also provide improved pharmacokinetics and targeting capacities . [SEP]
[CLS] a fusion protein B-material composed of hiv specific f105 antibody B-material fragment and protamine , as a nucleic acid binding protein B-material , was designed and confirmed to deliver a sirna specifically to hiv infected cells B-material . [SEP]
[CLS] after attachment of sirna to antibodies B-material , by redox sensitive disulfides in thiomab antibodies B-material , no improvement in activity was found between a dynamic linker and a covalent connector . [SEP]
[CLS] these results indicate that a reversible bond is not a prerequisite for activity , as the linker is at the sense strand and it might not affect the antisense strand loading into the risc complex . [SEP]
[CLS] however , in all these examples of protein mediated delivery , the endosomal escape is usually the limiting factor and the addition of endosomolytic agents as pore forming toxins or cpps [SEP]
[CLS] is sometimes required to achieve efficient cargo delivery . [SEP]
[CLS] cell penetrating peptides B-material ( cpps ) have been extensively applied to the delivery of nucleic B-material acids I-material , as covalent or non - covalent conjugates . [SEP]
[CLS] recent research on cpps has focused on the development of new synthetic peptides B-material for the delivery of artificial nucleotides ( table 3 ) . [SEP]
[CLS] artificial pmo nucleotides have been conjugated to peptides B-material of the pip family and arginine - rich peptides B-material that include non - proteinogenic aminoacids such as 6 - aminohexanoic or βalanine . [SEP]
[CLS] the resulting conjugates achieved therapeutic levels of cargo delivery in different animal models of muscular dystrophies . [SEP]
[CLS] the cationic B-material amphiphilic B-property clip6 cell penetrating peptide B-material presents a d - proline at the middle of its sequence , together with a glutamic B-material acid I-material that disrupt its potential β - sheet secondary structure [SEP]
[CLS] this " intrinsically disordered " peptide B-material was able to reach the cell B-material cytosol by a non - endocytic mechanism for pna efficient delivery . [SEP]
[CLS] amphiphilic B-property peptide B-material nanofibers B-nanoparticle , composed of a short cationic B-material sequence and a β - sheet seed followed by a hydrophobic B-property tail , can also be employed for slow release of antisense oligonucleotides in vitro and for localized sirna delivery in vivo by stereotactic surgery . [SEP]
[CLS] peptide B-material nanofibrils or nanosheets can be implemented as enhancers of viral gene transfer . [SEP]
[CLS] these amyloids have the ability to concentrate and increase infectivity of lentiviruses , a very promising technology with multiple potential ex vivo applications . [SEP]
[CLS] in the rala peptide B-material , a variant of the amphiphilic B-property gala , the protonation of glutamate residues prevents the anionic repulsion between peptide B-material side chains and triggers helical folding . [SEP]
[CLS] this secondary structure fluctuation allows membrane disruption at low ph and rala mediated intracellular delivery of plasmids or mrna [SEP]
[CLS] artificial non - peptidic foldamers have also emerged as biocompatible B-property vehicles B-material for gene delivery . [SEP]
[CLS] disulfide connected dimers of amphiphilic B-property oligourea helical foldamers , equipped with imidazole and isopropyl moieties , were recently shown to deliver plasmid dna in cells B-material with low toxicity B-property . [SEP]
[CLS] dynamic bonds such as oximes or hydrazones B-material are formed in mild aqueous conditions , with good yields , short reaction times and in a fully bio - orthogonal fashion . [SEP]
[CLS] combinations of cationic B-material hydrophilic B-property peptides B-material with hydrophobic B-property tails of different lengths and properties have allowed the straightforward screening and quick identification of new simple formulations for sirna , plasmid or cas9 delivery [SEP]
[CLS] the recently introduced guanidiniocarbonylpyrrole group can form four hydrogen B-material bonds with the phosphate of nucleotides ( fig . 3b ) and thus enhance the plasmid delivery capabilities of short peptides B-material [SEP]
[CLS] cyclic peptides B-material of alternating chirality incorporating guanidiniocarbonylpyrrole can self - assemble into fibers that can also be applied in plasmid transfection . [SEP]
[CLS] alternation of guanidiniocarbonylpyrrole and cyclohexylalanine afforded large amyloid peptide B-material fibers that can be further processed with gold B-nanoparticle nanoparticles I-nanoparticle to obtain nanoparticles B-nanoparticle of the right size for cell B-material transfection . [SEP]
[CLS] another recent and interesting approach is the use of peptides B-material with biological activity as carriers . [SEP]
[CLS] for instance , the peptide B-material pepm , derived from a dengue virus protein B-material , reduces by itself the number of mitotic cells B-material and can also be used for sirna delivery in combined therapy [SEP]
[CLS] sequence notes pip6 [SEP]
[CLS] rxrrbrrxryqflirxrbrxrb x = aminohexanoic acid , b = β - alanine peptide B-material b rxrrbrrxrrbrxb x = aminohexanoic acid , b = β - alanine clip6 kvrvrvrvpptrvrervk p = d - proline cross directly the cell B-material membrane . [SEP]
[CLS] lys - pa lauryl - vvagk nanofibers B-nanoparticle forming peptide B-material . [SEP]
[CLS] pnf palmitoyl - gggaaakrk nanofibers B-nanoparticle forming peptide B-material ef - c + 2 92 qckikqiinmwq derived from hiv gp120 . enhances retroviral infection . [SEP]
[CLS] amyloidforming heptapeptide klvffak derived from the italian familial form of alzheimer ' s aβ . [SEP]
[CLS] enhances retroviral infection rala wearlaralaralarhlaralaralracea derived from gala peptide B-material . table 3 [SEP]
[CLS] selected examples of peptide B-material sequences for gene delivery . [SEP]
[CLS] enhancing membrane fusion . [SEP]
[CLS] lipofection has been one of the most popular methods of transfection since the discovery that cationic B-material lipids B-material spontaneously condense dna and fuse with cell B-material membranes . [SEP]
[CLS] current efforts in this field are focused on enhancing the fusogenic properties of the liposomes B-nanoparticle and minimizing their toxicity B-property . [SEP]
[CLS] in most cases , membrane fusion of liposomes B-nanoparticle is optimized to overcome the endosomal entrapment , while direct fusion with plasma membrane and subsequent direct delivery in the cytosol has received much less attention . [SEP]
[CLS] inspired by snare proteins B-material responsible for vesicle fusion in cells B-material , artificial liposomes B-nanoparticle exposing one of the peptides B-material of a coiled - coil motif specifically delivered sirna and splice correcting oligonucleotides in cells B-material that were previously loaded with the corresponding coil counterpart peptide B-material ( fig . 3c ) . [SEP]
[CLS] the application of this methodology has allowed the preparation of different fusogenic peptide B-material liposomes B-nanoparticle for the direct intracellular release of vesicular contents . [SEP]
[CLS] cell B-material surface engineering by ketone - containing lipids B-material has been recently applied to cell B-material transfection with the corresponding lipoplex containing bio - orthogonal reactive alkoxyamine functionalities ( fig . 3d ) . [SEP]
[CLS] however , the in vivo application of these creative approaches will require the challenging selective ketone - lipid or coil modification of the target cells B-material [SEP]
[CLS] the lipid B-material transfection helper dope bears two bulky oleyl hydrophobic B-property tails giving rise to a conical shape lipid B-material that tends to assemble into the less stable hexagonal phase instead of the lamellar phase adopted by other lipids B-material with higher head to tail surface ratio [SEP]
[CLS] the safinya group recently demonstrated that inverse bicontinuous gyroid cubic nanostructures , confirmed by x - ray scattering , could also enhance fusion with the endosome membrane and efficiently deliver sirna without the requirement of a high cationic B-material charge ( fig . 3e ) . [SEP]
[CLS] although these cuboplexes [SEP]
[CLS] were not able to transport longer dnas , they can be further modified with peg for additional stabilization without activity loss , pointing towards future in vivo applications [SEP]
[CLS] lipid B-nanoparticle nanoparticles I-nanoparticle engineering [SEP] B-nanoparticle
[CLS] lipid B-nanoparticle nanoparticles I-nanoparticle can be modified by using mixtures of different lipids B-material in which not only the lipid B-material composition , but also the synthesis method , can have a great impact on the size , the biodistribution and the delivery efficiency [SEP]
[CLS] typically , they contain a pegylated lipid B-material , cholesterol , a helper lipid B-material and an ionizable B-property lipid B-material . [SEP]
[CLS] the cross - reaction of alkylamines with alkylacrylates with different hydrophobic B-property tails generated a library of ionizable B-property lipidoids that were combined with cholesterol , dspc and peg - dmg to formulate nanoparticles B-nanoparticle for sirna delivery . [SEP]
[CLS] this strategy allowed the extraction of structural insights about ionizable B-property lipid B-nanoparticle nanoparticles I-nanoparticle for endosomal escape and for in vivo delivery . [SEP]
[CLS] the most effective formulations included lipidoids with three or more hydrophobic B-property tails , secondary and tertiary B-material amines B-material and a particle surface pka of around 5 . 5 - 7 . [SEP]
[CLS] recent studies on ionizable B-property lipids B-material also stressed the importance of tertiary amines B-material and the steric constraints for the conformational switch of the lipids B-material that triggers endosomal escape by changing the relative orientation of the hydrocarbon chains ( fig . 3f ) . [SEP]
[CLS] the net surface charge of liposomes B-nanoparticle can alter their biodistribution , as shown by dotma and dope mixed liposomes B-nanoparticle that accumulated in the lung when having a positive charge or in the spleen as the negative charges increased . [SEP]
[CLS] this property was exploited to deliver mrna to dendritic cells B-material for the development of mrna vaccines against cancer . [SEP]
[CLS] in a similar strategy , lipid B-nanoparticle nanoparticles I-nanoparticle containing phospholipids , ionizable B-property and peg - modified lipids B-material , cholesterol , and a lipopolysaccharide adjuvant , were loaded with mrna and the subcutaneous injection of these liposomes B-nanoparticle triggered an immune response that could overcome the critical self - tolerance against auto - antigens required in cancer immunotherapy [SEP]
[CLS] thiol - conjugated lipids B-material / aptamers can be also assembled into nanoparticles B-nanoparticle and employed in targeted delivery of sirna to different tissues such as bone to improve osteogenesis . [SEP]
[CLS] polydiacetylenic derivatives with protonable amine B-material or imidazol groups can be crosslinked with uv light to generate interesting structures , such as nanomicelles or nanofibers B-nanoparticle that are suitable for plasmid and sirna delivery . [SEP]
[CLS] the clinical potential of lipid B-material engineering is exemplified by patisiran , a sirna drug against hereditary transthyretin - mediated amyloidosis , which is delivered by lipid B-nanoparticle nanoparticles I-nanoparticle containing dlin - mc3 - dma [SEP]
[CLS] exosomes are naturally produced vesicles containing lipids B-material , proteins B-material and nucleic B-material acids I-material that can act as natural carriers of proteins B-material and nucleic B-material acids I-material . [SEP]
[CLS] generally , extravesicular engineering has been carried [SEP]
[CLS] out by the loading of exogenous agents and by the genetic engineering of parental cells B-material [SEP]
[CLS] however , until recently the chemical modification of exosomes for gene delivery has been surprisingly overlooked . [SEP]
[CLS] in an interesting biotechnological strategy , fusogenic liposomes B-nanoparticle were used to load the membrane of living cells B-material with azide modified artificial lipids B-material . [SEP]
[CLS] the chemically modified exosomes produced by these cells B-material can then be easily further functionalized by click chemistry , for example by conjugation with targeting peptides B-material . [SEP]
[CLS] in recent examples , the loading of exosomes with synthetic oligonucleotides was simplified by the use of hydrophobic B-property cholesterol - modified sirna . [SEP]
[CLS] despite their promising properties , the cellular origin of exosomes constitutes an important challenge in terms of their large - scale production with standard sizes and compositions . [SEP]
[CLS] sterosomes , cationic B-material liposomes B-nanoparticle enriched in steroles and stearylamine , can provide a more stable alternative to phospholipid liposomes B-nanoparticle , as they are less affected by hydrolysis and oxidation ( fig . 3g ) . [SEP]
[CLS] sterosomes have been embedded in hydrogels for sustained noggin sirna delivery to enhance osteogenic differentiation during bone regeneration [SEP]
[CLS] ( pdpa ) offers an excellent conceptual advance for micelle B-material disruption and cargo release at the tumour microenviroment ( fig . 4a ) . this concept was exploited in pdpa ultrasensitive ph co - polymers B-material containing rgd peptide B-material , peg , and grafted with cationic B-material lipid - like structures [SEP]
[CLS] the resulting grafted nanoparticles B-nanoparticle showed a very narrow ph triggered disassembly at the ph of the early endosome that was employed for in vivo sirna tumour targeted delivery . [SEP]
[CLS] in an analogous approach , the pka of low uptake cationic B-material micelles B-material was tuned by adjusting the ratios of its components ( pdpa and pdmaema ) . [SEP]
[CLS] at the optimal pka ( 6 . 8 - 7 ) , an excellent sirna delivery was observed even at low cellular uptake or in hard - to - transfect cells B-material . [SEP]
[CLS] the ph - dependent aggregation of oligoethyleneimines ( oei ) was optimized by the attachment of different aromatic pendants , such as salicylate ( saoei ) , to maximize the ph - triggered disassembly and cargo release of polyplexes with different nucleic B-material acids I-material ( fig . 4a ) . the ph - triggered dissociation of polyplexes can also be used to release a hidden membrane - lytic peptide B-material to enhance nucleotide release [SEP]
[CLS] this method avoided the membrane disruption until the complex reached the endosome and thus reduced the toxicity B-property of the final formulation [SEP]
[CLS] a different ph - triggered strategy to change polymer B-material properties , from hydrophobic B-property to hydrophilic B-property , to trigger polyplex disassembly and plasmid delivery , explored the ph dependent cleavage of benzoic aromatic imines B-material to generate cationic B-material amines B-material . [SEP]
[CLS] charge altering releasable transporters ( carts ) are composed of oligo ( carbonate - b - alpha - amino ester ) and a lipophilic B-property block . [SEP]
[CLS] carts polymers B-material lose their charge and self - immolate at cytosolic ph , by intramolecular amide B-material formation and piperazines release , to efficiently deliver mrna both in vitro and in vivo ( fig . 4a ) . [SEP]
[CLS] self - replicating alphavirus replicons are large rna molecules that can sustain prolonged protein B-material expression but they do not tolerate structural chemical modifications . [SEP]
[CLS] to protect them from nucleases , these replicons can be formulated with cationic B-material ionizable B-property dendrimeric poly ( amido amine B-material ) and pegylated lipids B-material before intramuscular injection for their in vivo use as rna vaccines . [SEP]
[CLS] redox , enzymes and other strategies for polyplex disassembly [SEP]
[CLS] several other strategies have been studied to trigger nucleotide release from polymeric formulations . [SEP]
[CLS] the disulfide bond can also be exploited in polymers B-material that disassemble in the reducing cytosolic environment . [SEP]
[CLS] the transfection B-property efficiency I-property of branched pei was enhanced by polymer B-material disulfide functionalization with zinc B-material dipicolylamine analogues , which increases vehicle dna affinity , membrane binding and transfection B-property efficiency I-property ( fig . 4b ) . [SEP]
[CLS] pegylated polymers B-material , carrying dithiolane rings for disulfide crosslinking , can be assembled into nanoparticles B-nanoparticle loaded with sirna ( fig . 4b ) . [SEP]
[CLS] these nanoparticles B-nanoparticle were decorated with tumour targeting peptides B-material and showed virus - like cell B-material attachment and " capsid uncoating " behaviour in the cytosol [SEP]
[CLS] in a different enzymatic disassembly strategy , a core - shell artificial virus was prepared by covering a fluorinated cationic B-material polymer B-material and a dna core B-material with an outer shell B-material made of a polymer B-material of hyaluronan with peg and r8 - rgd peptides B-material . [SEP]
[CLS] the degradation of this layered polyplex was mediated by the hyaluronidase overexpressed in the tumour environment , which exposed the particle core B-material and allowed the in vivo delivery of problematic large plasmids . [SEP]
[CLS] polyols , including sirna , can be stabilized by reversible ester formation of the 2 - cis - diols of the terminal ribose or other polymers B-material with phenylboronate . [SEP]
[CLS] after uptake , these boronate - stabilized particles can be disrupted by the high levels of internal atp triggering sirna or plasmid release ( fig . 4b ) . [SEP]
[CLS] in other cases , instead of a stimulus , timed degradation can be used to protect the cargo until cytosolic delivery . [SEP]
[CLS] this is true in the case of poly ( 2 - dimethylaminoethyl acrylate ) ( pdmaea ) , a cationic B-material polymer B-material that slowly self - degrades into a negatively charged compound that repels its cargo for sirna release ( fig . 4b ) . [SEP]
[CLS] fluorinated dendrimers B-nanoparticle and polymers B-material . [SEP]
[CLS] pamam perfluorinated dendrimers B-nanoparticle condensate plasmid dna at very low n / p ratios , reducing its positive charge and thus lowering toxicity B-property and increasing efficiency in the presence of serum . [SEP]
[CLS] their hydrophobic B-property and lipophobic properties reduce their membrane interactions and increase their ability to penetrate tissues and spheroids . [SEP]
[CLS] this intriguing principle was applied in bioreducible pei micelles B-material with a perfluorinated core B-material that showed good activity in dna delivery . [SEP]
[CLS] polymer B-material fluorination B-event has also shown excellent potential for the delivery large plasmids 140 and proteins B-material . [SEP]
[CLS] supramolecular chemistry and dynamic chemistry . weak non - covalent bonds like electrostatic and hydrophobicinteractions control the dynamic processes of nucleotide complexation , protection , membrane translocation and complex disassembly . [SEP]
[CLS] therefore , supramolecular chemistry and supramolecular templates are powerful tools to understand and to develop conceptually new non - viral vectors . [SEP]
[CLS] cyclodextrin - based carriers consist of a linear cationic B-material polymer B-material containing cyclodextrins that can complex with sirna and then incorporate targeting and stabilizing moieties by supramolecular host - guest chemistry with adamantane conjugates . [SEP]
[CLS] this modular strategy originally developed by mark davis has been recently modified by using non - polymeric cyclodextrin with lmw pei pendants that are stabilized with ph sensitive polyketal poly - adamantanes ( fig . 4c ) . [SEP]
[CLS] cyclodextrins polymers B-material have also been recently used in tumour targeted combined therapy with a chemotherapeutic agent and plasmid dna . [SEP]
[CLS] amphiphilic B-property polymers B-material with a hydrophobic B-property core B-material , capped with dendronized dipeptides , enriched in aromatic tryptophan B-material or ph responsive histidine B-material residues , performed as good in vitro delivery vectors for sirna . [SEP]
[CLS] however , cyclodextrins have recently showed toxicity B-property problems in phase one clinical trials . [SEP]
[CLS] the use of thiol B-material click - chemistry allows the fast preparation of libraries of polyesters for sirna delivery that can show selectivity to tumour cells B-material . [SEP]
[CLS] hydrazone - modulated polymers B-material have also been recently introduced as a promising alternative to screen for polymer B-material nucleotide vehicles B-material . [SEP]
[CLS] in this strategy , poly - hydrazides can be modified with different combinations of cationic B-material and hydrophobic B-property aldehydes B-material to generate a library of amphiphilic B-property polymers B-material that can complex and deliver sirna and plasmid in living cells B-material ( fig . 4d ) . [SEP]
[CLS] in several recent examples , the polymer B-material can also play an active role and contribute to an additional therapeutic function instead of acting as a mere carrier . [SEP]
[CLS] polymetformin , derived from the anticancer B-property and antidiabetic drug metformin ( dimethyl biguanide ) , has the ability to complex sirna while preserving its anticancer B-property properties and it can be formulated with lipids B-material and co - delivered into tumour cells B-material ( fig . 4e ) . [SEP]
[CLS] a dendronized dithiophene semiconducting polymer B-material can also deliver a plasmid and trigger the expression of genes regulated by heat shock proteins B-material by near infrared photothermal activation . [SEP]
[CLS] glycan , protein B-material and antibody B-material conjugates . [SEP]
[CLS] n - acetyl - d - galactosamine pendants have been incorporated in guanidinium polymers B-material to enhance targeting and reduce toxicity B-property in the transfection of hepg2 cells B-material . [SEP]
[CLS] similar behaviour has been observed in poly ( glycoamidoamines ) and pei derived polymers B-material that incorporate carbohydrate B-material ( d - glucaric acid ) co - monomers B-material . [SEP]
[CLS] these materials have been further modified by adding alkyl chains to obtain polymer B-material brush materials loaded with sirna , mrna and lipids B-material to produce nanoparticles B-nanoparticle with in vivo activity . [SEP]
[CLS] the introduction of alkyl chains to enable self - assembly with other hydrophobic B-property compounds , such as pegylated lipids B-material , has been applied to poly ( β - amino esters ) ( pbae ) , to generate nanoparticles B-nanoparticle to deliver mrna to the lungs . [SEP]
[CLS] another problem with mrna delivery is that certain cationic B-material carriers can mask the m7g cap at the 5 ' end , blocking its recognition by cellular proteins B-material and inhibiting mrna translation . [SEP]
[CLS] to improve translation initiation , mrna can be pre - assembled with eif4e , the protein B-material involved in cap recognition , a technique that enhanced mrna expression , both in vivo and in vitro [SEP]
[CLS] programmable self - assembly was exploited to prepare dendrimeric sirnas that were efficiently complexed to pbae polymers B-material or branched and linear pei . [SEP]
[CLS] pbae polymers B-material containing peptides B-material with microtubule - associated sequences and nuclear localization signals were employed to overcome the limitation of crossing the nuclear membrane for plasmid delivery in non - dividing cells B-material [SEP]
[CLS] in a recent and extraordinarily promising approach , a plasmid dna packed with this pbae polymer B-material was coated with poly ( glutamic ) acid fused to a targeting antibody B-material and the resulting polyplex allowed in vivo reprogramming of t - cells B-material with cars for the treatment of a leukemia model . [SEP]
[CLS] c ) cyclodextrins can be incorporated into cationic B-material polymers B-material ( left ) and then modified with different compounds ( peg , targeting moieties . . . ) using adamantane or another hydrophobic B-property residue that interact with the cyclodextrin . [SEP]
[CLS] alternatively , individual cyclodextrins can be decorated with shorter cationic B-material polymers B-material for nucleic B-material acid I-material interaction and then assembled into larger structures using poly - adamantanes ( right ) . [SEP]
[CLS] d ) conceptual scheme for dynamic polyhydrazones . [SEP]
[CLS] dynamic hydrazide B-material polymers B-material can be combined with different aldehydes B-material to afford cationic B-material amphiphilic B-property polyhydrazone for nucleotide delivery . [SEP]
[CLS] ( reproduced with permission from ref . 155 ) . [SEP]
[CLS] e ) example of a polymeric nucleotide vehicle B-material , polymetformin , that presents intrinsic biological activity for combined therapy . [SEP]
[CLS] carbon B-material allotropes [SEP]
[CLS] the use of inorganic structures for plasmid delivery can be loosely tracked back to the 70s , with the development of the calcium B-material phosphate co - precipitation method for dna delivery into cells B-material . [SEP]
[CLS] since then , the nanoparticle B-nanoparticle field has evolved tremendously with substantial improvements being made in the characterization of nano - structures and the implementation of novel functionalities . [SEP]
[CLS] carbon B-material nanoforms constitute promising materials for a range of diagnostic tools and biomedical therapies . [SEP]
[CLS] the potential toxicity B-property of carbon B-material nanostructures can be modulated by controlling the size and by modifying the surface of the nanoparticles B-nanoparticle , and thus chemistry plays a key role in turning carbon B-material allotropes into biocompatible B-property scaffolds [SEP]
[CLS] in gene delivery , chemically functionalized carbon B-nanoparticle nanotubes I-nanoparticle ( cns ) exploit their high aspect ratio to rupture or to slip through the lipid B-material bilayer I-material ( fig . 5a ) . [SEP]
[CLS] the 1 , 3dipolar cycloaddition of carbon B-nanoparticle nanotubes I-nanoparticle with azomethine ylides can be employed to equip cns with pendants of oligoethyleneglycol bearing terminal amines B-material for the binding and delivery of dna plasmids . [SEP]
[CLS] the carboxylic B-material groups I-material at the tips of oxidized cns allow the attachment of ammonium and guanidinium dendrons by amide B-material bond formation or click chemistry for sirna complexation and delivery . [SEP]
[CLS] in an alternate approach to covalent modification , supramolecular hydrophobic B-property interactions between cns and lipid / peg amphiphiles B-property can also be employed to stabilize cns in water B-material and functionalize them by disulfide bonds with a sirna cargo . [SEP]
[CLS] as the bio - distribution of cns is influenced by their width or length [SEP]
[CLS] , this property can be exploited in targeted gene delivery [SEP]
[CLS] recent studies have shown that cns have the ability to insert and cross the zona pellucida to deliver dna into embryos without the requirement of individual manipulation [SEP]
[CLS] cationic B-material fullerenes B-nanoparticle , such as the tetra ( piperazino ) fullerene epoxide , can efficiently condense and deliver a plasmid dna with suitable low toxicity B-property when used in vivo . [SEP]
[CLS] polycationic fullerene B-nanoparticle hexakis - adducts have also been employed in plasmid transfection in vitro . [SEP]
[CLS] the ros generation of fullerenes B-nanoparticle when excited by light can also be exploited to enhance the endosomal escape of sirna for cationic B-material dextran decorated fullerene B-nanoparticle vehicles B-material . [SEP]
[CLS] conjugation of cpps to graphene oxide B-material nanosheets reduces cpp toxicity B-property and increases their activity for delivering pdna and aso , but not sirna . [SEP]
[CLS] nanodiamonds , a potentially more biocompatible B-property carbon B-material allotrope , can also be complexed to nucleic B-material acids I-material when decorated with amines B-material or cationic B-material polymers B-material ( fig . 5b ) . [SEP]
[CLS] the polymer B-material pendants can be covalently attached by reaction with the carboxylated diamond surface or non - covalently conjugated by the electrostatic interactions between the cationic B-material polymer B-material and the anionic oxidized nanodiamond . [SEP]
[CLS] metal B-nanoparticle nanoparticles I-nanoparticle . [SEP] B-nanoparticle
[CLS] the precise control of size , shape and the responsiveness to external stimuli are examples of the critical features that metal B-nanoparticle nanoparticles I-nanoparticle offer for biomedical applications . [SEP]
[CLS] additionally , the surface of metal B-material particles can be straightforwardly functionalized with different bioactive and biocompatible B-property targeting molecules . [SEP]
[CLS] the thiol - gold B-material linkage B-property constitutes a simple and reliable method for the functionalization and modification of gold B-nanoparticle nanoparticles I-nanoparticle ( fig . 5c ) . [SEP]
[CLS] tissue - targeting of sirna was achieved by the thiol B-material - gold B-material connection of metal B-nanoparticle nanoparticles I-nanoparticle and a block copolymer shell B-material of poly - l - lysine - poly - ethylenglycol terminated by cyclic rgd peptides B-material ( fig . 5c , left ) . [SEP]
[CLS] the embedding of gold B-nanoparticle nanoparticles I-nanoparticle in hydrogels has been applied for colon cancer treatments by triplecombination therapy ( sirna , drug and phototherapy ) . [SEP]
[CLS] this multifunctional approach involved gold B-material nanorods B-nanoparticle decorated with the avastin antibody B-material drug against vegf and the colorectal cancer targeting peptide B-material tcp - 1 . [SEP]
[CLS] the nanorods B-nanoparticle were combined with gold B-material nanospheres B-nanoparticle that were covered with sirna and further functionalized with endosomolytic peptides B-material for improved release . [SEP]
[CLS] gold B-material nanoclusters , prepared by glutathione and oligoarginine controlled au 3 + reduction , have been recently confirmed as excellent sirna delivery vehicles B-material for silencing the ngf gene against the challenging pancreatic cancer ( fig . 5c , middle ) . [SEP]
[CLS] the tightly packing of nucleic B-material acids I-material over the surface of nanoparticles B-nanoparticle gives rise to spherical shaped nucleic B-material acids I-material conjugates ( snas ) with interesting properties such as increased stability , enhanced affinity and the ability to transfect cells B-material despite their negative surface charge due to potential interaction with scavenger receptors . [SEP]
[CLS] different snas with gold B-material cores B-material have been used for the delivery of sirna ( fig . 5c , right ) or ribozymes into cells B-material and animals . [SEP]
[CLS] topical wound application of snas made of oligoethylene glycol and sirna ( against gm3s ) restores wound healing in diabetic mice . [SEP]
[CLS] snas surface modification with nuclear localization signal peptides B-material ( nls ) strongly enhanced nuclei sirna delivery and induced long term gene silencing by rnadirected dna methylation [SEP]
[CLS] although snas were initially assembled on gold B-nanoparticle nanoparticles I-nanoparticle , further studies confirmed that their emerging properties were independent from the metal B-material core B-material . [SEP]
[CLS] these findings triggered the emergence of more biocompatible B-property cores B-material such as dna , [SEP]
[CLS] spherical nucleic B-material acids I-material . polymers B-material ( hopo ) , liposomes B-nanoparticle [SEP]
[CLS] or other structures ( poss ) . [SEP]
[CLS] these " organic " snas have been successfully tested in sirna delivery . [SEP]
[CLS] porous particles . [SEP]
[CLS] mesoporous silica B-nanoparticle nanoparticles I-nanoparticle can achieve an extremely high sirna loading capacity of up to 380 µg / mg . [SEP]
[CLS] such porous nanoparticles B-nanoparticle can be grafted with silanes ( aptes , teos , mptes ) to modify their surface with amino and mercapto - groups ( fig . 5d ) . [SEP]
[CLS] sirna was then loaded by incubation B-technique in solution at low ph and the particles were finally covered with a block copolymer that was further modified with hydrophobic B-property oleic pendants and terminal cysteines B-material for cross - linking . [SEP]
[CLS] the final silicon based porous ensemble achieved one of the highest sirna delivery efficiencies described for silica B-nanoparticle nanoparticles I-nanoparticle [SEP]
[CLS] a similar porous silicon B-material platform can also be functionalized with aminopropyl - triethoxysilane ( aptes ) , followed by amide B-material connection with l - arginine B-material and pei . [SEP]
[CLS] the pores of the cationic B-material silicon B-material carrier were loaded with sirna , which was released in the form of polyplexes after silicon B-material degradation . [SEP]
[CLS] the prolonged slow release of these polyplexes reduced the toxicity B-property of the formulation after systemic injection . [SEP]
[CLS] aptes grafted silica B-nanoparticle nanoparticles I-nanoparticle can be also functionalized by coordination with ce 3 + for further anchoring of branched pei polymer B-material . [SEP]
[CLS] the resulting nanoparticles B-nanoparticle ( spa - ce - pei ) delivered sirna with higher efficiency than branched pei alone . [SEP]
[CLS] anionic mesoporous silicon B-nanoparticle nanoparticles I-nanoparticle , decorated with icp ( 3isocyanatopropyltriethoxysilane ) , can be covered with cyclodextrin - grafted polyethylenimine . [SEP]
[CLS] doxorubicin B-material was then encapsulated in the porous nanomaterial B-material and the resulting nanoparticles B-nanoparticle were applied to the delivery of a cytotoxic B-property drug and a sirna against pkm2 in combined therapy against orthotopic breast tumours 197 . [SEP]
[CLS] metal organic frameworks ( mofs ) constitute promising scaffolds for nucleotide encapsulation and delivery due to their controlled porosity . [SEP]
[CLS] zr 4 + based mofs were applied in combined therapy for the co - delivery of a cis - platin pro - drug and a sirna . [SEP]
[CLS] in a similar strategy , cysteine grafted mofs were filled with se or ru because of their ability to disrupt microtubules and act as antitumour agents . [SEP]
[CLS] these mofs were also loaded with vegf sirna by the coordination of the vacant fe ( iii ) sites with the nucleotide phosphates . [SEP]
[CLS] the final nanoparticles B-nanoparticle were applied for combined therapy against multidrug resistance breast cancer cells B-material ( fig . 5d ) . [SEP]
[CLS] the increasingly active field of non - viral gene delivery holds great promise for the future of biotechnology and human health . [SEP]
[CLS] the improvement of the synthetic methods has recently granted the access to new encouraging molecular entities and nucleotide modifications such as phosphotriesters or guanidiniocarbonylpyrrole groups [SEP]
[CLS] furthermore , the recent advances in microscopy B-technique and cell B-material biology have allowed a higher level of understanding of the highly dynamic mechanism of uptake and escape of the cargo . [SEP]
[CLS] these and other advances continuously assist and push chemists and material B-material scientists to design and develop the next generation of non - viral carriers that can be sensitive to critical stimuli such as the specific ph required for disassembly or the optimal pka of the lipid B-nanoparticle nanoparticles I-nanoparticle surface for enhanced uptake [SEP]
[CLS] a clear trend for the future of the field will be the combination of new materials with novel formulation techniques . [SEP]
[CLS] by blurring the barriers B-property between different categories , the strength of each material B-material can compensate the weakness of its counterpart and thus improve the performance of the final functional composite . [SEP]
[CLS] endosomolytic peptides B-material buried in ph - sensitive micelles B-material , polymer - coated silica B-nanoparticle nanoparticles I-nanoparticle or peptide B-material - lipid B-material hybrids are just a few recent examples that outline potential directions of the field . [SEP]
[CLS] additionally , combined therapy emerges as an encouraging strategy in where the nucleic B-material acid I-material function is supplemented with other therapeutics such as bioactive polymers B-material and co - delivery of cytotoxic B-property agents . [SEP]
[CLS] furthermore , the still mostly unexplored combination of chemically modified nucleotides and the next generation of synthetic transporters constitute a promising strategy to tackle the future challenges of synthetic materials at the forefront of gene delivery . [SEP]
[CLS] combinatorial libraries and high - throughput screening also constitutes an important and complementary technique compared to rational design , as it can identify structure - activity relationships and lead to unexpected results [SEP]
[CLS] importantly , the synthesis and formulation of the nanocarriers has to be scalable , reproducible and stable and although the field strongly demands new conceptual designs and strategies , the transference of these technologies to therapeutic applications will require a careful consideration of the synthetic scaling up process . [SEP]
[CLS] in lipid B-material particles , film hydration is sensitive to organic contaminants and in batch ethanol dilution homogeneous mixing is hard to achieve . [SEP]
[CLS] therefore , crossflow or microfluidics constitutes an excellent alternative for reproducibility and scalability [SEP]
[CLS] the standard electrostatic complexation of nucleotides with polymers B-material or peptides B-material requires a good control of mixing and thus concentrations , charge ratios and ph and ionic strength have to be carefully considered [SEP]
[CLS] polyplex stability upon dilution is lower than for other formulations , although pegylation might improve this situation . [SEP]
[CLS] in gold B-material nano - conjugates , salt B-material reduction followed by ligand exchange or direct reduction with thiolated ligands can be potentially scaled up . [SEP]
[CLS] however , the election of the method in this case , will depend on the availability of the suitable thiolated ligands . [SEP]
[CLS] silica B-nanoparticle nanoparticles I-nanoparticle seem also promising scalable materials as they can be prepared by condensation of inexpensive silicates under different conditions [SEP]
[CLS] it seems nowadays clear that not all in vitro results will work in vivo . [SEP]
[CLS] most of the in vitro experiments have been done [SEP]
[CLS] in serum free conditions on immortalized cell B-material lines that usually contain alterations that affect their ability to detect nucleic B-material acids I-material and enhance the cells B-material tolerance to stress [SEP]
[CLS] these critical points for in vitro studies could lead to an overestimation of the delivery efficiency and an underestimation of the vehicle B-material toxicity B-property . [SEP]
[CLS] better in vitro models , such as primary cell B-material cultures , or spheroids may predict better the outcome of in vivo experiments , but they still lack the complexity of a living organism . [SEP]
[CLS] in addition , the translation of these results to the clinic will always have to pass regulatory challenges , as all new formulations must be proven safe before further testing , and in this regard , formulations related to well established technologies , such as lipid - based carriers , will be in an advantageous position . [SEP]
[CLS] so far , the most advanced methods in the race for clinical application of non - viral vectors are phosphorothioates , naked plasmid dna , lipid - based carriers and , to a lesser extent , polymeric carriers , which have already some examples approved or nearly approved [SEP]
[CLS] for human use [SEP]
[CLS] the simplicity of the method of delivery of the naked cargo and the strong experience with liposomal - based drug delivery vehicles B-material justify their predominance . [SEP]
[CLS] on the other hand , non - biodegradable B-nanoparticle nanoparticles B-nanoparticle ( i . e . carbon B-nanoparticle nanotubes I-nanoparticle , fullerenes B-nanoparticle , metal B-nanoparticle nanoparticles I-nanoparticle , etc . ) may be problematic in the long - term , especially in cases where repeated administration may be necessary , as they tend to accumulate in liver and kidneys . [SEP]
[CLS] to address this concern , there are several initiatives that try to replace the metal B-material core B-material of snas for biocompatible B-property alternatives . [SEP]
[CLS] however , it is also possible that the therapeutic use of non - biodegradable nanoparticles B-nanoparticle might be restricted to cases in which the nature of the particle contributes to the treatment , as in photothermal therapy [SEP]
[CLS] despite impressive advances in the last decade , there are still considerable challenges that need to be met to help broaden the scope of potential future therapeutic applications of non - viral vectors such as vehicle B-material bioavailability B-property , reduction of immune response , balance of stability and release and endosomal escape [SEP]
[CLS] in this regard , the growing number of synthetic materials for the efficient in vivo transfection of hard - to - transfect cells B-material demonstrates the enormous power of chemistry and biology working together ( table 4 ) . [SEP]
[CLS] all these great advances help inspire new approaches to gene therapy and bring hope for the future of human health . [SEP]
[CLS] potential problems by unwanted protein B-material interactions . [SEP]
[CLS] limited by the synthetic method ; typically short . [SEP]
[CLS] yes , but modified nucleotides increase costs . [SEP]
[CLS] yes , by attaching ligands . [SEP]
[CLS] some approved for human use physical methods several days in serum . [SEP]
[CLS] from low to moderately toxic B-property . [SEP]
[CLS] from aso / sirna to pdna . [SEP]
[CLS] in peptide B-material - oligonucleotide conjugates , length is limited by the synthetic method . [SEP]
[CLS] yes , but in some cases synthesis can be expensive . [SEP]
[CLS] with targeting sequences or ligands . [SEP]
[CLS] i . c . [SEP]
[CLS] , i . v . , and intradermal 95 injection of mice . i . v . , i . c . or i . m . in dogs 87 [SEP]
[CLS] lipid - based 50 - 400 nm [SEP]
[CLS] low toxicity B-property , except for highly cationic B-material particles . [SEP]
[CLS] from aso / sirna to pdna . [SEP]
[CLS] yes , although procedures that require sonication can be hard to scale up . [SEP]
[CLS] by epr or with targeting B-material ligands I-material . [SEP]
[CLS] i . v . [SEP]
[CLS] , s . c . and combined with hydrogels in mice ; lipid B-nanoparticle nanoparticles I-nanoparticle in clinical trials 34 [SEP]
[CLS] from 10 nm ( smallest dendrimers B-nanoparticle ) to almost 1 µm ( fibers and large polyplexes ) [SEP]
[CLS] typically 50 - 300 nm [SEP]
[CLS] most polymers B-material are very biocompatible B-property , but some toxicity B-property issues have been observed ( immune response , cytotoxicity B-property . . . ) [SEP]
[CLS] from aso / sirna to pdna . [SEP]
[CLS] yes , although some of the more complex formulations can be challenging . [SEP]
[CLS] by epr , with targeting B-material ligands I-material or stimuli responsive motifs . [SEP]
[CLS] i . c . [SEP]
[CLS] , i . p . , i . m . , i . t . , i . v . [SEP]
[CLS] in mice [SEP]
[CLS] some reached clinical trial strage , but were retired because of adverse effects [SEP]
[CLS] from 20 nm ( snas ) to 60 µm ( cns ) [SEP]
[CLS] in general , inert and with low toxicity B-property , but hard to degrade in the body . [SEP]
[CLS] some carbon B-material allotropes can be toxic B-property depending on functionalization and contaminants from synthesis . [SEP]
[CLS] from aso / sirna to pdna . [SEP]
[CLS] yes , but influenced by the modifications . [SEP]
[CLS] with targeting B-material ligands I-material , epr , size . . . in some cases light stimulation . [SEP]
[CLS] i . v . [SEP]
[CLS] , s . c . or topically in mice ; bovine embryo transfection [SEP]
[CLS] different synthetic materials and current challenges for efficient intracellular gene delivery . [SEP]
[CLS] the successful delivery of a functional nucleic B-material acid I-material requires overcoming important biological barriers B-property . [SEP]
[CLS] an initial condensation of the nucleotide cargo in particles of the right size and charge for delivery also contributes to the protection of the cargo against ubiquitous nucleases . [SEP]
[CLS] the endothelial barrier B-property , the hepatic and renal clearance , and the accumulation outside the target tissue might hinder the bioavailability B-property of the therapeutic nucleotide in the in vivo systemic delivery . [SEP]
[CLS] to reach its final intracellular destination , nucleotide vehicles B-material have to cross the plasma membrane , escape from the endosome and cross the nuclear membrane , if necessary , for their activity . [SEP]
[CLS] modifications for self - delivering oligonucleotides . [SEP]
[CLS] figure 2 . a ) chemical modifications of nucleotides for gymnotic delivery . [SEP]
[CLS] these modifications usually involve a phosphorothioate backbone ( left ) combined with additional modifications of the ribose , such as those pictured here . [SEP]
[CLS] b ) phosphotriester modification reduces the negative charge of the oligonucleotide and can be reversed by the activity of cellular thioesterases , to get the natural phosphodiester . [SEP]
[CLS] c ) examples of delivery systems that exploit cellular interaction for uptake , such as receptor - binding aptamers or glycan moieties attached to sirna . [SEP]
[CLS] d ) formation of globular structures , by the folding of long ssdna or ssrna strands obtained by rolling circle amplification or transcription B-event ( rca or rct ) that can contain aptamer sequences or multiple repetitions of the active sequence , or by attachment of polymeric brushes that turn antisense oligonucleotides into compact particles . [SEP]
[CLS] e ) an example of the ph - sensitive dynamic linkers found in darts . [SEP]
[CLS] 72 of dengue virus 2c protein B-material [SEP]
[CLS] perturbs bcr - abl1 signalling . [SEP]
[CLS] 3 . peptide B-material and lipid based delivery systems . [SEP]
[CLS] figure 3 . a ) dsrna binding B-event proteins I-event can be used for the recognition and delivery of sirna . [SEP]
[CLS] b ) guanidiniocarbonylpyrrole ( gpc ) , showing the four potential hydrogen B-material bonds that can be established with the phosphate backbone . [SEP]
[CLS] c ) mechanism of fusion enhancement between cellular membranes and liposomes B-nanoparticle , by exploiting the formation of coiled - coils of peptides B-material present in the liposome B-nanoparticle carrier and the cell B-material membrane target . [SEP]
[CLS] according to the cartoon , the helical wheel representation of the coil of the vesicle ( in blue ) and the coil of the cell B-material membrane ( in red ) include the following amino B-material acids I-material : hydrophobic B-property ( a , l , i in black ) , cationic B-material ( k in purple ) and anionic ( e in orange ) . [SEP]
[CLS] d ) bio - orthogonal reaction between the alkoxyamine bearing lipids B-material of the liposome B-nanoparticle vehicle B-material and a cell B-material membrane loaded with lipids B-material exposing ketone groups . [SEP]
[CLS] e ) structure of cuboplexes , whose topology allows membrane fusion without the requirement of a high cationic B-material charge ( reproduced with permission from ref . 112 ; copyright © 2010 , american chemical society ) . [SEP]
[CLS] f ) ionizable B-property lipids B-material contain a head with protonable groups and flexible bonds that allow conformational changes when the head is protonated . [SEP]
[CLS] the conformational change induced by protonation in the lipid B-material library synthesized by viricel et al . is depicted . [SEP]
[CLS] g ) sterosomes are alternatives to phospholipid liposomes B-nanoparticle prepared by mixing stearylamine with esteroles such as cholesterol and its derivatives . [SEP]
[CLS] polymers B-material , dendrimers B-nanoparticle and micellesph B-material disassembly . [SEP]
[CLS] polymers B-material can display , in a multivalent fashion , the functionalities that are required for membrane recognition , interaction and translocation . [SEP]
[CLS] this structural versatility and the potential synthetic scalability turn synthetic polymers B-material into one of the most promising artificial materials for gene delivery . [SEP]
[CLS] the current research efforts in polymeric materials for gene delivery are focused on improving condensation of the cargo , the reduction of the toxicity B-property and the enhancement of the endosomal escape . [SEP]
[CLS] to achieve these challenging goals , new polymers B-material have been designed to disassemble upon the exposure to external stimuli such as reducing B-property agents I-property , enzymes , light , temperature , and above all , ph . [SEP]
[CLS] the cooperative ph triggered disassembly of poly ( 2 - ( diisopropylamino ) ethylmethacrylate ) [SEP]
[CLS] 4 . polymeric approaches to gene delivery . [SEP]
[CLS] figure 4 . a ) structures of ph - sensitive polymers B-material . [SEP]
[CLS] pdpa gets protonated at low ph and electrostatic repulsion disassembles the polymeric structure . [SEP]
[CLS] salicylic acid modification of oligoethyleneimine ( saoei ) reduces its affinity for nucleic B-material acids I-material at low ph and contributes to the disassembly of the polyplexes . [SEP]
[CLS] carts are self - immolative polymers B-material that degrade at ph higher than 7 . [SEP]
[CLS] b ) polymers B-material that respond to other stimuli . [SEP]
[CLS] disulfide bonds can be used for disassembly in the reducing conditions of the cytosol , as in the case of zincdipicolylamine modified pei or dithiolane rings that can be introduced in the polymer B-material sequence for crosslinking and stabilization of the nanoparticles B-nanoparticle . [SEP]
[CLS] cationic B-material pdmaea spontaneously degrades into the negatively charged poly ( acrylic acid ) . [SEP]
[CLS] atp responsive polymers B-material are based on the reversible interaction of phenylboronic acid with diols . [SEP]
[CLS] 5 . nanoparticles B-nanoparticle for gene delivery . [SEP]
[CLS] figure 5 . a ) covalent and non covalent modifications of carbon B-nanoparticle nanotubes I-nanoparticle for the delivery of nucleic B-material acids I-material . [SEP]
[CLS] b ) surface functionalization of nanodiamonds for nucleic B-material acid I-material interactions . c ) delivery systems involving gold B-nanoparticle nanoparticles I-nanoparticle : a gold B-nanoparticle nanoparticle I-nanoparticle modified with a targeting motif ( crgd ) , peg and polylysine for sirna binding 182 ; gold B-material nanoclusters formed by reduction in the presence of glutathione and oligoarginine 184 and an example of spherical nucleic B-material acids I-material 186 , in which sirna is bound to the nanoparticle B-nanoparticle through a thiol B-material group and decorated with peg ; d ) porous nanoparticles B-nanoparticle . [SEP]
[CLS] left : metal - organic framework ( mil - 101 ) incorporating se or ru ions B-material for microtubule disruption and sirna . [SEP]
[CLS] right : surface functionalization of mesoporous silica B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] coordination via cerium B-material of pei ( spa - ce - pei ) , engraftment with aptes , or further modification of aptes with arginine B-material and pei to generate pcps ( polycation - functionalized nanoporous silicon B-material ) to obtain the positive charge necessary for nucleic B-material acids I-material interaction , or introduction of thiol B-material groups with mptes for crosslinking with other polymers B-material . [SEP]
[CLS] oligonucleotides , polymer B-material , physical prodrug , drug delivery intercalation of drugs into assembled dna systems offers versatile new mechanisms for controlled drug delivery . [SEP]
[CLS] however , current systems are becoming increasingly complex , reducing the practicality of large scale production . [SEP]
[CLS] here , we demonstrate a more pragmatic approach where a short dna sequence was modified with poly [ ethylene glycol ] ( peg ) of various lengths at both 5 ′ - termini to provide serum stability and compatibility . [SEP]
[CLS] the anti - cancer drug doxorubicin B-material was physically loaded into two designed binding sites on the dsodn . [SEP]
[CLS] the polymer B-material conjugation improved the stability of the dsodn towards serum nucleases while its doxorubicin B-material binding affinity was unaffected by the presence of the polymers B-material . [SEP]
[CLS] we examined the effects of polymer B-material size on the dsodn carrier characteristics and studied the resulting [SEP]
[CLS] in recent decades dna - based materials have revolutionized the scope and complexity of nanomaterials B-material that can be produced . [SEP]
[CLS] exploiting the specificity of watson - crick base pairing has produced complex dynamic structures such as molecular computers , ( 1 ) motors , ( 2 ) nanoreactors , ( 3 ) as well as sensors and diagnostics . ( 4 - 10 ) more recently , the ability of certain drugs to intercalate within the dna double helix and form strong physical complexes has been utilized to form physical prodrugs for cytotoxics B-property . [SEP]
[CLS] farokhzad et al . first demonstrated that a dna aptamer could be loaded with doxorubicin B-material ( dox ) , allowing targeted uptake into cells B-material presenting the aptamer target . [SEP]
[CLS] this was extended to aptamer - targeted quantum B-nanoparticle dots I-nanoparticle and polymer B-material particles allowing the addition of detection and delivery . [SEP]
[CLS] dabrowiak et al . have demonstrated several systems based upon gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) coated with dna . [SEP]
[CLS] both dox and actinomycin d ( actd ) were intercalated into the helix , and targeting was achieved by introduction of folic B-material acid I-material onto one of the dna strands . [SEP]
[CLS] ) " dna origami " has also been used for intercalated delivery . [SEP]
[CLS] ahn et al . [SEP]
[CLS] introductionhave demonstrated that a dna tetrahedron , formed by annealing 4 strands , can be used to deliver dox to cells B-material , overcoming resistance in a multi - drug resistant cell B-material line . [SEP]
[CLS] leong et al . have used similar tetrahedra , decorated with aunps B-nanoparticle and loaded with actd , for theranostic delivery to bacteria . [SEP]
[CLS] hogberg et al . have taken the origami approach a step further and designed large dna bundles that can twist upon intercalation of dox . [SEP]
[CLS] drug release kinetics , and thus cytotoxicity B-property , can be modulated by varying the induced twist . [SEP]
[CLS] although these systems have demonstrated the feasibility of using dna B-event intercalation I-event as a drug delivery mechanism , problems still remain . [SEP]
[CLS] first , none have the shielding components required to prevent degradation and reduce potential immunogenic B-property interactions in vivo , although some of the nonnatural dna conformations have been shown to be resistant to degradation . [SEP]
[CLS] second , the systems being developed are displaying increasing complexity , in one case requiring over 200 short dna " staples " to hold the drug delivery system together , where each of which has to be synthesized and purified . [SEP]
[CLS] peg polymers B-material have been extensively used to improve the pharmacokinetics of biologics . [SEP]
[CLS] despite recent concerns about potential immunogenicity B-property of peg , this remains controversial , and they are still the polymer B-material of choice for in vivo applications . [SEP]
[CLS] herein we describe a more pragmatic approach to intercalating drug delivery . [SEP]
[CLS] a simple , short , double stranded dna was assembled from complementary oligonucleotides , modified with poly ( ethylene glycol ) ( peg ) at the both 5 ′ positions . [SEP]
[CLS] the effects of peg polymer B-material size on the odn carrier delivery characteristics were examined . [SEP]
[CLS] dox was physically conjugated by intercalation into a pair of binding sites designed into the oligonucleotide sequence forming the dox @ dna - peg delivery system ( fig . 1 ) . [SEP]
[CLS] 1 . schematic representation of intercalating dox @ dna delivery system described herein . [SEP]
[CLS] peg - dna conjugates were synthesized in a similar manner to that described previously . [SEP]
[CLS] polyethylene glycol monomethyl ether B-material ( mpeg , m n 550 , 1900 and 5000 da ) were activated with n , ndisuccinimidyl carbonate B-material to form amino - reactive mpeg - succinimidyl carbonates B-material ( peg - sc ) . [SEP]
[CLS] the peg - scs were subsequently conjugated to 5 ′ - aminohexyl modified single - stranded oligonucleotides to form a small library of peg - ssdna conjugates with 3 different peg lengths , and consisting of two complementary dna sequences ( oligo a : taa cag gat tag cag agc gag g and oligo b : [SEP]
[CLS] cct cgc tct gct aat cct gtt a ) [SEP]
[CLS] conjugates were purified by semi - preparative hplc with typical isolated yields of 50 - 80 % independent of mpeg length . [SEP]
[CLS] hplc and page analysis ( fig . 2a and s1 ) confirmed no residual unmodified oligonucleotides in any of the conjugate samples . [SEP]
[CLS] molecular masses from maldi - tof mass spectrometry were generally in good agreement with theoretical values , however slight deviation from theoretical values was observed . [SEP]
[CLS] this is not unexpected as mpeg is a disperse polymer B-material and some molecular weight enrichment is likely to occur during hplc purification i . e . polymer B-material fractionation . [SEP]
[CLS] lyophilized conjugates were dissolved in dnase - free annealing buffer and hybridized with the appropriate complementary strands ( e . g . peg550 - a and peg550 - b ) to form dsdna conjugates with mpeg chains at both 5 ′ - termini . [SEP]
[CLS] consequently , a series of three peg dna conjugates was produced , dna - peg550 , dna - peg1900 and dna - peg5k , as well the unpegylated , " unmodified " dna . [SEP]
[CLS] after annealing , the conjugates were analyzed by page to ensure that no single stranded odns were remaining after the annealing ( fig . 2a and fig s1a ) . [SEP]
[CLS] page analysis revealed that the peg - odns had successfully formed the double stranded structures . [SEP]
[CLS] weak high molecular weight bands were observed for the single strands in all of the gels which arose from self - association of the unhybridized strands under the non - denaturing conditions . [SEP]
[CLS] to determine the effect of peg on dna stability towards nucleases , dna - peg conjugates and unmodified dna were incubated B-technique in presence of fetal calf serum . [SEP]
[CLS] the conditions were chosen to mimic as closely as possible those used for later in vitro experiments to ensure that any effects that arose from differential degradation could be accounted for . [SEP]
[CLS] the samples were analyzed by native page and individual band intensity estimated using image analysis software . [SEP]
[CLS] the band intensity of each conjugate at the start of the experiment ( 0 hours ) was set as 100 % . [SEP]
[CLS] all dna - peg conjugates were stable for the duration of the experiment with 80 - 90 % remaining after 72 h . [SEP]
[CLS] conversely , the unmodified dna had completely degraded after 72 h and was reduced to approximately 40 % after 48 h ( fig . 2b and s2 ) . [SEP]
[CLS] the conjugation of the peg polymers B-material to each terminus of the dna therefore improved its stability significantly and provided protection against the nucleases . [SEP]
[CLS] however , no effect of polymer B-material molecular weight on stability was observed under these conditions . [SEP]
[CLS] the affinity of doxorubicin B-material ( dox ) to the dna carriers was examined in order to determine the binding of the drug and if this was affected by pegylation . [SEP]
[CLS] doxorubicin B-material binds preferentially to 5 ′ - gc - 3 ′ and 5 ′ - cg - 3 ′ double stranded sequences , thus it is expected that the sequence used here would have two preferential binding sites . [SEP]
[CLS] affinity measurements were performed by a previously established fluorescence B-property quenching protocol . [SEP]
[CLS] the fluorescence B-property of doxorubicin B-material was plotted as a function of the oligonucleotide concentration to produce a hill plot from which dissociation constants ( k d ) were calculated ( fig . 2c and s3 ) . [SEP]
[CLS] comparison of the calculated k d values showed no difference beyond experimental error between the pegylated and non - pegylated dna , with all being 200 ± 60 nm . [SEP]
[CLS] in vitro cytotoxicity B-property studies of dna - peg and dox @ dna - peg conjugates dna conjugates promote cell B-material proliferation : to determine if the dna - peg conjugates were capable of delivering dox they were assessed in vitro in a549 cells B-material ( lung adenocarcinoma epithelial cells ) . [SEP]
[CLS] carriers were loaded at a 1 / 10 ( w / w ) with dox based on their oligonucleotide content ( i . e . 1 mg dox per 10 mg of odn ) forming the dox @ dna - peg conjugates . [SEP]
[CLS] cells B-material were incubated B-technique with free dox , dox @ dna - peg and empty dna - peg carriers for 72 h . [SEP]
[CLS] dox concentrations ranged from 0 . 15 nm - 10 μm and were maintained across groups . [SEP]
[CLS] for the unloaded conjugates the concentrations were matched to the oligonucleotide concentrations used in the dox @ dna - peg group . [SEP]
[CLS] metabolic activity was assessed by mtt B-technique assay I-technique , untreated cells B-material were normalized as 100 % metabolic activity . [SEP]
[CLS] as expected , free dox resulted in complete suppression of metabolic activity at higher concentrations ( [UNK] μm ) , with a calculated ic50 of 89 nm ( 95 % ci 57 - 131 nm , fig . 3a ) . [SEP]
[CLS] however , for dox @ dna - peg conjugates complete suppression was not seen even at very high concentrations of dox ( 10 μm ) , with approximately 30 % metabolic activity remaining compared to untreated cells B-material ( fig . 3b and s3 ) . [SEP]
[CLS] although intercalation of the dox within a carrier is expected to affect cell B-material entry , and thus cytotoxicity B-property , this should be compensated for at the higher concentrations . [SEP]
[CLS] when considered with the effect of the unloaded dna - peg ( fig . 3c ) it is clear that the carriers alone promote metabolic activity / cell B-material growth , and this promotion is also dose dependent . [SEP]
[CLS] visual inspection during the course of the experiment revealed that the cell B-material density increased as a function of dna concentration which was in an agreement with the mtt results . [SEP]
[CLS] it has previously been shown that pyrimidine nucleosides are growth promoting when they are present in media at a concentration of 1µg / ml with endothelial cells B-material . [SEP]
[CLS] cytotoxicity B-property of dox @ dna - peg and dna - peg . [SEP]
[CLS] to counteract enhanced cell B-material proliferation in the presence of oligonucleotide carriers further in vitro studies were performed in media supplemented with free nucleosides ( a , c , g and t ; 10 µg / ml each ) for both cell B-material lines . [SEP]
[CLS] cytotoxicity B-property experiments were performed with a549 cells B-material as before . [SEP]
[CLS] with the addition of free nucleosides to the media no enhancement in cell B-material proliferation was observed in the presence of unloaded dsodn carriers when compared to untreated cells B-material ( fig . s4 ) . [SEP]
[CLS] for free dox complete suppression of metabolic activity was observed at high concentrations as before ( fig 4 ) . [SEP]
[CLS] however , the calculated ic50 value increased to 124 nm ( 95 % ci 102 - 151 nm ) indicating that a higher dose of dox is required to inhibit cell B-material growth in the presence of nucleosides , further supporting our previous conclusions . [SEP]
[CLS] the half maximal inhibitory concentration ( ic50 ) is derived as the mid - point between the maximum and minimum of the dose response curve . [SEP]
[CLS] however , since the maximal inhibition response varies between dox @ dna carriers and to that of the free drug direct ic50 comparison between the groups was difficult . [SEP]
[CLS] consequently , ic50 values were also calculated by interpolation of the fitted curve at 50 % viability . [SEP]
[CLS] in the case of free dox , where the maximum inhibition nears 100 % , this has little effect on the calculated ic50 ( 126 nm c . f . 124 nm ) however it provides a more realistic value for the dox @ dna samples . [SEP]
[CLS] values calculated by both methods are contained with table 1 . [SEP]
[CLS] for non - pegylated dox @ dna , an ic50 of 132 nm ( 95 % ci 104 - 165 nm ) is comparable to that of free dox . [SEP]
[CLS] this is not unexpected considering that the unshielded dna is readily degraded in the presence of serum and this is likely to be exacerbated in the presence of cells B-material or if internalized . [SEP]
[CLS] however , even at the highest non - pegylated dox @ dna concentration ( 1 μm ) cell B-property viability I-property remains at ~ 20 % compared to the untreated control . [SEP]
[CLS] for the dox @ dna - peg samples ic50 values are strongly dependent on the molecular weight of the shielding polymer B-material . [SEP]
[CLS] dox @ dna - peg5k and dox @ dna - peg550 , with ic50s of 232 nm ( 95 % ci 189 - 282 ) and 541 nm ( 95 % ci 411 - 718 ) respectively , are approximately 2 - and 4 - fold less toxic B-property than dox @ dna . [SEP]
[CLS] dox @ dna - peg1900 maintains an ic50 similar to that of dox @ dna and free drug with an ic50 of 122 nm ( 95 % ci 98 - 151 ) . [SEP]
[CLS] the dox @ dna - peg conjugates all exhibited dose saturation at highest dox @ dna concentrations and inhibition was limited to approximately 70 % in comparison to 95 % inhibition for the free drug . [SEP]
[CLS] in vitro studies were extended to a mcf7 cell B-material line ( breast adenocarcinoma epithelial cells B-material ) to determine if the limiting toxicity B-property was cell B-material line specific . [SEP]
[CLS] mcf7 cells B-material were more sensitive to dox with an ic50 of 27 nm ( 95 % ci 20 - 37 ) for free drug and all systems were capable of ~ 90 - 95 % inhibition at the highest concentrations . [SEP]
[CLS] the greater sensitivity results in less pronounced effects seen with polymer B-material molecular weight , with little variation between treatments , particularly when the large confidence intervals are considered . [SEP]
[CLS] lines are calculated four - parameter logistic fits . [SEP]
[CLS] confocal microscopy B-technique analysis of cells B-material treated with dox @ dna dox @ dna systems display cell line dependent localization . [SEP]
[CLS] to further understand the cytotoxicity B-property data dox @ dna systems were studied by confocal microscopy B-technique in a549 and mcf7 cells B-material . [SEP]
[CLS] cells B-material were incubated B-technique with dox @ dna carriers and free dox and the localization studied at various time points using the inherent fluorescence B-property of the drug for detection . [SEP]
[CLS] due to sensitivity requirements the drug concentration was fixed at 3 μm in all cases . [SEP]
[CLS] after 3 hours ( fig . 5 ) , free dox showed intense staining in the nucleus in both cell B-material lines indicating rapid uptake , while in all dox @ dna nuclear staining was considerably less intense . [SEP]
[CLS] dox @ dna samples contain punctate regions within the cytoplasm and in the perinuclear region ; this was particularly pronounced in a549 cells B-material . [SEP]
[CLS] this demonstrates that dsodn are readily internalized into both a549 and mcf7 cells B-material , albeit at lower levels than free drug , and allow trafficking into the target organelle . [SEP]
[CLS] by 18 hours ( fig . 6 ) nuclear staining in mcf7 cells B-material remains intense for both free drug and dsodn - dna while other conjugates have lower levels . [SEP]
[CLS] in contrast a549 cells B-material have lower levels of nuclear staining compared to mcf7 cells B-material but retain staining in cytoplasmic regions for all dox @ dna samples and free dox . [SEP]
[CLS] in order to investigate the intracellular localization of internalized dox , cells B-material were incubated B-technique for 18 hours with free dox or dox @ dna and the lysosomal compartments were counterstained ( fig . 7 and s6 for non - merged images ) . [SEP]
[CLS] in mcf7 cells B-material intense doxorubicin B-material staining was observed in the nucleus for all dsodn and free dox . [SEP]
[CLS] furthermore , punctate doxorubicin B-material positive regions co - localized with the lysosomal marker . [SEP]
[CLS] in contrast , a549 cells B-material showed no detectable nuclear staining at 18 hours , even for free dox , while retaining cytoplasmic doxorubicin positive lysosomal staining . [SEP]
[CLS] this contrasts with the findings of the previous experiment ( fig . 6 ) where some low level signal was still detected in nucleus of a549 cells B-material at 18 hours . [SEP]
[CLS] however , due to the broad excitation / emission spectra of both doxorubicin B-material and the lysosomal marker , the collection range and signal gain for doxorubicin B-material had to be narrowed to prevent signal bleed through . [SEP]
[CLS] the co - localization assay ( fig . 7 ) therefore only visualized the most intense regions of doxorubicin B-material staining i . e . the punctate cytoplasmic regions . [SEP]
[CLS] lower levels of doxorubicin B-material fluorescence B-property may reflect increased efflux of doxorubicin B-material from a549 cell B-material nuclei in comparison to mcf7 cells B-material which could explain the cell B-material lines elevated resistance to the drug . [SEP]
[CLS] both cell B-material lines have been shown to express multidrug resistance associated - proteins B-material ( mrp ) which can limit the efficiency of chemotherapy . [SEP]
[CLS] ( 25 ) encapsulation of doxorubicin B-material in dsodn allows the drug to traffic into the nucleus and lysosome however it does not alter the final destination of the doxorubicin B-material nor prevent drug efflux . [SEP]
[CLS] however , replacement of the oligonucleotide segment with one that exerts its own therapeutic effect , e . g . sirna or t - oligos , may provide a synergistic effect . [SEP]
[CLS] initial in vitro assays with a549 cells B-material revealed that the carriers promoted cell B-material growth compared to untreated control , with later experiments requiring supplementation with free nucleosides to enable comparison . [SEP]
[CLS] comparisons of the metabolic activity , uptake and localization of dox @ dna systems in a549 and mcf7 cells B-material demonstrated several differences . [SEP]
[CLS] in mcf7 cells B-material , metabolic activity and uptake were comparable to that of free dox . [SEP]
[CLS] conversely , in a549 cell B-material line dox @ dna systems had a lesser effect on metabolic activity than free dox with no system able to achieve more than 70 % inhibition . [SEP]
[CLS] uptake studies determined that dox @ dna systems were taken up to a lower extent than free dox , and that dox levels in nuclei reduced with time in a549 cells B-material compared to mcf7 cells B-material our findings highlight the issues that need to be addressed when designing and evaluating oligonucleotide based drug delivery systems . [SEP]
[CLS] the vast difference observed between the two cell B-material lines underlines the importance of adopting appropriate in vitro models early on and supplementing tissue culture medium with free nucleosides . [SEP]
[CLS] our results suggest that our pegbased system could be used to treat doxorubicin sensitive cancer cells B-material in instances where adverse drug effects are limiting . [SEP]
[CLS] the data derived from a549 cell B-material line also accentuates the fact that pegylated - dsodn system would be not suitable to treat drug resistance cells B-material , in those circumstances amphiphilic B-property polymers B-material and known inhibitors of multidrug resistance protein B-material ( mrp ) should be considered instead . [SEP]
[CLS] we are currently evaluating alternative dsodn - derived systems to address this issue of drug resistance . [SEP]
[CLS] all other solvents and reagents were of analytical or hplc grade and purchased from sigma aldrich unless otherwise specified . [SEP]
[CLS] nmr spectra were recorded on a bruker avance 400 spectrometer at 400 . 13 mhz ( 1 h ) and 100 . 62 mhz ( 13 c , 1 h decoupled at 400 . 13 mhz ) in chloroform - d . [SEP]
[CLS] all chemical shifts are reported in ppm relative to the signal for tetramethylsilane . [SEP]
[CLS] reverse phase high performance liquid B-technique chromatography I-technique ( rp - hplc ) was performed on a shimadzu prominence uplc system fitted with a dgu - 20a5 degasser , lc - 20ad low - pressure gradient pump , cbm - 20a lite system controller , sil - 20a autosampler and an spd - m20a diode array detector . [SEP]
[CLS] analytical separations were performed on a phenomenex clarity 3 μm oligo - rp c18 column ( 4 . 6×50 mm ) with a gradient of meoh ( 10 - 70 % for peg5k and peg1900 odns and 10 - 50 % for peg550 odns over 20 min ) in 0 . 1 m triethylammonium acetate ( teaa , ph 7 . 5 ) / mecn ( 95 / 5 ) as the mobile B-property phase at a flow rate of 1 . 0 ml / min . [SEP]
[CLS] semipreparative separations were performed on a phenomenex clarity 3 μm oligo - rp c18 column ( 10×50 mm ) under the same conditions at a flow rate of 5 . 0 ml / min . [SEP]
[CLS] matrix - assisted laser desorption / ionization B-property time - of flight ( maldi - tof ) mass spectrometry was performed on a bruker maldi - tof ultra flex iii spectrometer operated in linear , positive ion B-material mode . [SEP]
[CLS] 3 - hpa containing dahc was used as the matrix for oligonucleotide analysis . [SEP]
[CLS] briefly , a saturated solution of 3 - hpa ( 50 mg / ml ) was prepared by adding 25 µg of 3 - hpa to 500 µl of 50 % mecn / water B-material . [SEP]
[CLS] 25 µl of dahc solution ( 100 mg / ml ) was added to 225 µl of the 3 - hpa solution , to give a final dahc concentration of 10 mg / ml . odn solutions were desalted prior to mixing with matrix using c18 ziptips . [SEP]
[CLS] equal volumes of matrix solution and odn solution ( 0 . 2 mm ) were mixed and 2 µl of the mixture was spotted onto the maldi plate and allowed to dry . [SEP]
[CLS] polyacrylamide B-technique gel I-technique electrophoresis I-technique ( page ) analysis was performed at 160 mv using a 15 % acrylamide running gel . [SEP]
[CLS] native gels were prepared using acrylamide - bis - acrylamide ( 29 : 1 ) and [SEP]
[CLS] strands were hybridized in annealing buffer which consisted of 10 mm tris , 50 mm nacl and 1 mm edta ph 7 . 5 . [SEP]
[CLS] for the hybridisation the strands were mixed in equimolar quantities to give a final concentration of 75 µm of each strand and placed in a water B-material bath at 95 °c for 5 minutes . [SEP]
[CLS] the water B-material bath was then turned off and allowed to cool slowly to room temperature . [SEP]
[CLS] annealed strands were desalted using a pd spintrap g - 25 column ( ge healthcare ) and oligonucleotide concentration determined by optical density @ 260 nm ( 1 od = 30 ug / ml ) . [SEP]
[CLS] annealing was verified using page gel under non - denaturing conditions . [SEP]
[CLS] solutions were aliquoted , lyophilized and stored at - 20 °c until needed . [SEP]
[CLS] doxorubicin B-material hcl ( dox hcl ) ( 1 mg ) was dissolved in uhq water B-material ( 1 ml ) . [SEP]
[CLS] the solution was filtered through a 0 . 22 µm pes syringe filter and stored at 4 °c until use . [SEP]
[CLS] dna carriers were dissolved in sterile dpbs . [SEP]
[CLS] dox hcl was added to the dna solution in order to achieve a 10 % ( w / w ) dox hcl to dna drug loading . [SEP]
[CLS] the dox @ dna complexes were allowed to form for 30 min . and used without further purification . [SEP]
[CLS] for in vitro experiments the dox @ dna complexes were diluted 25 - fold with media to a dox concentration of 10 µm ( 5 . 8 µg / ml of dox , 58 µg / ml odn ) . [SEP]
[CLS] dox @ dna systems were incubated B-technique at an oligonucleotide concentration of 0 . 63 µg / ml in dpbs containing 10 % fetal calf serum ( fcs ) . [SEP]
[CLS] solutions were incubated B-technique at 37 c with mild agitation . [SEP]
[CLS] at each time point ( 0 , 24 , 48 , 72 h ) a sample ( 10 μl ) was removed , flash frozen in liq . n 2 , and stored at - 20 °c until analysis by page . [SEP]
[CLS] the samples were diluted with 10 µl of loading buffer and 10 µl of the diluted sample loaded per well . [SEP]
[CLS] page was carried out at 160 mv using a 15 % acrylamide gel . [SEP]
[CLS] the oligonucleotide / polymer B-material bands were visualized using methylene blue . [SEP]
[CLS] intensity of gel bands was estimated by image analysis using scion image ( macro : gelplot2 ) . [SEP]
[CLS] a fluorescence B-technique spectroscopy I-technique method [SEP]
[CLS] affinity of doxorubicin B-material hcl to hybridized oligonucleotide strandswas used to assess the binding affinity of dox for dna systems . [SEP]
[CLS] the assay was performed with a constant dox concentration ( 1 . 5 µm ) while dna carrier was titrated from 0 to 15 molar ( dna concentrations : 15 , 10 , 7 . 5 , 4 . 5 , 1 . 5 , 0 . 75 , 0 . 45 , 0 . 15 , 0 . 045 , 0 . 015 , 0 . 005 and 0 µm ) . [SEP]
[CLS] a hill plot was used to determine the binding affinity of the double stranded oligonucleotide sequence to doxorubicin B-material hcl ( y : ( f0 - f ) , x : concentration odn µm ) . [SEP]
[CLS] all analyses were performed in graphpad prism 6 . 0 . [SEP]
[CLS] general [SEP]
[CLS] the mcf - 7 cells B-material were grown in standard t75 flasks . [SEP]
[CLS] a549 cells B-material were grown in t75 flasks treated for optimal attachment ( corning t75 cat no 430641 ) . [SEP]
[CLS] both cell B-material lines were grown in minimum essential medium eagle ( mem ) media containing 4 mm l - glutamine B-material , penicillin / streptomycin and 10 % fcs . [SEP]
[CLS] the media was supplemented with nucleosides ( g , c , t , a ) at the later stages of the cell B-material work , each nucleoside was added at a final concentration of 10 µg / l . [SEP]
[CLS] cytotoxicity B-property assays . [SEP]
[CLS] cells B-material were allowed to reach confluence in the t75 flasks before being seeded onto 96 - well plates . [SEP]
[CLS] cells B-material were seeded at 5×10 3 cells B-material / well for both cell B-material lines ( 100 µl , 5×10 4 cells B-material / ml ) . [SEP]
[CLS] cells B-material were allowed to attach for 24 hours before the media was removed and replaced with 100 µl of media containing the appropriate dox concentration . [SEP]
[CLS] for experiments with carrier alone , an equivalent concentration of dna ( without drug ) was used . [SEP]
[CLS] 6 wells were used for each concentration . [SEP]
[CLS] cells B-material were treated for 72 hours before metabolic activity was determined using an mtt B-technique assay I-technique . [SEP]
[CLS] mtt solution ( 10 µl of 5 mg / ml ) in media was added to each well and allowed to develop for 75 minutes . [SEP]
[CLS] the media was then carefully aspirated and wells washed with pbs ( 100 μl ) . [SEP]
[CLS] the formazan crystals were finally dissolved by adding dmso ( 100 µl ) to each well . [SEP]
[CLS] the plate was read using a tecan m200 platereader at 562 nm and the mtt response compared to untreated cells B-material . [SEP]
[CLS] untreated cells B-material were normalized as 100 % metabolic activity . [SEP]
[CLS] toxicity B-property curves were fitted with graphpad prism 6 . 0 using the 4 - parameter variable slope log ( inhibitor ) vs response model . [SEP]
[CLS] due to incomplete inhibition ic50 calculated are considered as relative . [SEP]
[CLS] real ic50 values were calculated by interpolation of the fitted curve within the same software . [SEP]
[CLS] cells B-material were seeded on nunc labtekii coverglass 8 well slides at 20 , 000 cells B-material / well and incubated B-technique overnight . [SEP]
[CLS] conjugates were loaded with drug as described above and added to cells B-material at a final doxorubicin B-material concentration of 3µm . [SEP]
[CLS] after the required incubation B-technique time cells B-material were fixed in cold 4 % paraformalydehyde in pbs for 10 minutes . [SEP]
[CLS] samples were stored at 4 °c and imaged by laser scanning confocal microscopy B-technique ( lscm ) within 24 hours . [SEP]
[CLS] settings were excitation 532 nm laser , emission 552 - 654 nm . [SEP]
[CLS] lysosomal staining was conducted using cytopainter lysosomal staining kit - blue ( abcam , ab112135 ) following the manufacturer ' s instructions with an incubation B-technique time of 20 minutes . [SEP]
[CLS] cells B-material were imaged immediately by lscm using settings excitation 405 nm , emission 430 - 480 nm and excitation 532 nm , emission 553 - 672 . [SEP]
[CLS] 2 . a . page analysis of peg5k - oligos and dna - peg5k conjugates . [SEP]
[CLS] lanes l - r : ( 1 ) idt [SEP]
[CLS] cells B-material which are proliferating rapidly require a high rate of dna synthesis . [SEP]
[CLS] for those cells B-material the rate of dna synthesis can become the growth limiting factor . [SEP]
[CLS] to administer 10 µm dox , 58 µg / ml of the dsodn carrier was required . [SEP]
[CLS] the proliferation of the a549 cell B-material line was thus promoted by degradation of the dsodn carrier and subsequent release of free nucleosides . [SEP]
[CLS] the dsodn carrier was not shown to promote the proliferation of a mcf - 7 breast cancer cell B-material line . [SEP]
[CLS] 3 . cytotoxicity B-property of a . doxorubicin B-material ; b . dox @ dna - peg1900 ; and c . dna - peg1900 dsodn [SEP]
[CLS] 4 . cytotoxicity B-property of dox @ dna - peg in a549 and mcf - 7 cell B-material lines . [SEP]
[CLS] cells B-material were cultured in the [SEP]
[CLS] 5 . cellular uptake dox @ dna samples . [SEP]
[CLS] cells B-material were incubated B-technique with doxorubicin loaded [SEP]
[CLS] cellular uptake dox @ dna samples . [SEP]
[CLS] cells B-material were incubated B-technique with doxorubicin loaded [SEP]
[CLS] doxorubicin B-material co - localizes with a lysosomal marker . [SEP]
[CLS] mcf - 7 and a549 cells B-material were incubated B-technique [SEP]
[CLS] all oligonucleotides ( hplc purified ) were purchased from biomers . net gmbh ( ulm , germany ) and used without further purification . [SEP]
[CLS] mcf - 7 and a549 cell B-material lines were received from crn nci - 60 cell B-material bank . [SEP]
[CLS] polyethylene glycol monomethyl ether B-material ( mpeg , m n 5000 , 1900 and 550 da ) were purchased from polysciences inc . cytopainter lysosomal staining kit blue fluorescence B-property was purchased from abcam . [SEP]
[CLS] n , n ′ - disuccinimidyl carbonate B-material ( dsc , ≥95 . 0 % ) , triethylamine ( et 3 n , ≥99 % ) , thiazolyl blue tetrazolium bromide B-material ( mtt , 98 % ) , water B-material ( bpc grade ) , diammonium hydrogen B-material citrate ( dahc , ≥99 % ) , stains - all ( 95 % ) , 3 - hydroxypicolinic acid ( 3 - hpa , ( ≥99 % ) ) , methylene blue hydrate ( > 97 % ) , tris - borate - edta buffer ( tbe , 10× concentrate ) , ammonium persulfate ( ≥98 % ) , n , n , n ′ , n ′ - tetramethylethylenediamine ( temed , 99 % ) , ethylenediaminetetraacetic acid disodium salt B-material ( edta , > 99 % ) , glycerol anhydrous ( fluka ) , bromophenol blue solution ( 0 . 04 wt % h 2 o ) , acrylamide : n , n ′ - methylenebisacrylamide ( 29 : 1 ) 40 % solution and dulbecco ' s pbs ( modified , without calcium B-material chloride B-material and magnesium B-material chloride ) were purchased from sigma aldrich . [SEP]
[CLS] cytotoxicity B-property data for doxorubicin B-material and dox @ dna complexes in a549 and mcf7 cells B-material [SEP]
[CLS] the residue was dissolved in toluene and any insoluble material B-material removed by filtration . [SEP]
[CLS] toluene was removed under reduced pressure and the pegylated oligonucleotides were synthesized as previously described . ( 10 ) specific reaction conditions are detailed in table 2 . [SEP]
[CLS] briefly , 5 ′ - aminohexyl oligonucleotide ( 1 eq . ) was dissolved in dpbs to a concentration of 2 . 5 mg / ml . a solution of mpeg - sc ( 5 eq . ) was added with gentle agitation and the reaction allowed to proceed for up to 72 h . [SEP]
[CLS] conversion was monitored by hplc and additional mpeg - sc added if required . [SEP]
[CLS] pegylated oligonucleotides were purified by semi - preparative hplc and lyophilized . [SEP]
[CLS] lyophilized materials were redissolved in dnase free water B-material and concentration determined by measurement of optical density at 260 nm ( 1 od = 20 μg / ml ) . [SEP]
[CLS] solutions were then aliquoted and lyophilized before storage at - 20 °c . [SEP]
[CLS] conditions and analytical data for the pegylation of oligonucleotides sequences . [SEP]
[CLS] oligo a : taa cag gat tag cag agc gag g ; oligo b : cct cgc tct gct aat cct gtt a . b for dissolution of mpeg - sc . c calculated from measured od values . [SEP]
[CLS] table 2 . d theoretical / found . [SEP]
[CLS] doi : 10 . 1002 / advs . 201802095and indeed the current front - runner mp in translational studies is based on poly ( glutamic acid ) . [SEP]
[CLS] herein , we propose that superior mp can be engineered on the nucleic B-material acid I-material scaffold , specifically for nucleoside analogues ( figure 1 ) . [SEP]
[CLS] the latter comprise a highly important class of drugs with approved applications in anticancer B-property and antiviral B-property therapies . [SEP]
[CLS] nucleic B-material acid I-material scaffolds offer a fully natural mechanism for degradation and drug release . [SEP]
[CLS] they can be engineered to have the highest possible deliverable payload ( each monomer B-material unit can contain a therapeutic nucleoside ) , possibly comprised of different nucleoside analogues to make up a combination therapy . [SEP]
[CLS] existing prior art is limited to the nucleic B-material acids I-material containing 5 B-material - I-material fluorouracil I-material and the registered antiproliferative effects thereof . [SEP]
[CLS] beyond these reports , the landscape of possibilities associated with the nucleic acid scaffolded mp ( termed herein " therapeutic nucleic B-material acids I-material , " tna ) is undefined and remains highly appealing . [SEP]
[CLS] in this work , we designed tna using idoxuridine , an approved drug against herpes simplex virus ( hsv - 1 / 2 ) , and trifluridine , an anticancer B-property agent ( lonsurf ) also used in antiviral B-property therapy ( figure 1a ) . [SEP]
[CLS] synthesis of tna was developed such as to capitalize on the existing methodology for automated syntheses of nucleic B-material acids I-material . [SEP]
[CLS] toward this end , trifluridine and idoxuridine were first converted to the 4 , 4 ′ - dimethoxytrityl ( dmt ) protected phosphoramidite derivatives ( figure 1b , for details on synthesis see supporting information ) . [SEP]
[CLS] resulting tnas were either 7 - mers or 14 - mers with sequences containing only the antiviral B-property drug , or the antiviral B-property drug and thymidine in an alternating sequence . [SEP]
[CLS] the latter were designed for a broader understanding of the structure - activity relationship of tnas . [SEP]
[CLS] products were characterized by mass spectrometry , hplc , and maldi to confirm purity and composition ( figure 1c - e ) . [SEP]
[CLS] these analyses demonstrate that the synthesized mp were essentially monodisperse as chemical entities with both the composition and the degree of polymerization precisely controlled by the synthesis . [SEP]
[CLS] this comes in stark contrast to the conventional mp obtained through , radical polymerization techniques in which case dispersity of chains is inevitable and the resulting polymers B-material are an ensemble of products rather than a molecularly defined structure . [SEP]
[CLS] tnas are therefore advantageous from the perspective of translational potential since fda imposes increasingly tighter regulations on polymers B-material for drug delivery . [SEP]
[CLS] finally , to illustrate the natural degradation mechanism for drug release , the tnas were exhaustively digested using a nuclease / phosphodiesterase enzyme . [SEP]
[CLS] lc - ms macromolecular prodrugs ( mp ) built on the natural phosphodiester and deoxyribose backbone are developed using marketed antiviral B-property nucleoside analogues . [SEP]
[CLS] these mp are synthesized using automated synthesis , have defined molecular composition , and have a natural mechanism for drug release . [SEP]
[CLS] these unique attributes , coupled to the efficient cell B-material entry and potent antiviral B-property effects , position the prodrugs scaffolded on nucleic B-material acids I-material favorably for translational studies . [SEP]
[CLS] nucleic B-material acid I-material scaffolds based on ( deoxy ) ribose and phosphodiester linkage B-property have evolved as the most reliable tool to store , read , and copy information . [SEP]
[CLS] in modern days , nucleic B-material acids I-material are also highly warranted in biomedicine , biotechnology , and nanotechnology . [SEP]
[CLS] the defining characteristics of these scaffolds are their stability and the availability of methods to precisely control their sequence and therefore the molecular structure and function . [SEP]
[CLS] herein , we offer a new prospect on nucleic B-material acids I-material and hypothesize that these form a unique scaffold for macromolecular prodrugs ( mp ) . [SEP]
[CLS] the latter hold immense promise for targeted drug delivery . [SEP]
[CLS] however , carbon - chain mp thus far have failed in clinical trials and are disadvantaged due to the nondegradable nature of the polymer B-material backbone and the inevitable dispersity of chains by molar mass meaning inhomogeneity of the formulation . [SEP]
[CLS] the main - chain biodegradable B-property peptide B-material or polysaccharide B-material scaffolds present viable alternatives analyses revealed that degradation of tna affords the expected nucleosides ( thymidine , trifluridine , or idoxuridine ) as the main product of natural oligomer decomposition ( figure 1f ) , thus providing the final element of validation of the composition for tna . [SEP]
[CLS] tna engineered as macromolecular prodrugs , unlike antisense oligonucleotides , sirna , and other nucleic acid based therapeutic molecules , do not have to stay intact for cytosolic intracellular delivery but in fact must be degraded to elicit their therapeutic effect , figure 2a . [SEP]
[CLS] nuclease - mediated processing of tna can occur intracellularly and / or extracellularly ( either way contributing to the overall natural drug release from the prodrug ) . [SEP]
[CLS] to investigate this , we used hplc quantification to monitor the intact tna and the released idoxuridine . [SEP]
[CLS] we observed that tna - i 14 is stable in serum - free cell B-material culture medium over at least 24 h but undergoes a natural degradation in the presence of serum , figure 2b . [SEP]
[CLS] we observed disappearance of the full - length oligonucleotide already within 2 h of incubation B-technique of tna in serum ( data not shown ) . [SEP]
[CLS] however , during this time , we documented only a minor concentration of the released idoxuridine , figure 2c , which suggests preprocessing of tna into shorter oligomer species during this time . [SEP]
[CLS] idoxuridine release became prominent with incubation B-technique times between 2 and 6 h . [SEP]
[CLS] independently , we monitored the kinetics of tna cell B-material entry using time - lapse confocal B-technique laser I-technique scanning I-technique microscopy I-technique . [SEP]
[CLS] cell B-material entry was fast and clearly observable already within 20 - 30 min of tna incubation B-technique with the human foreskin fibroblast cells B-material , and within 50 - 60 min for a549 lung cancer cells B-material , figure 2d . [SEP]
[CLS] furthermore , rate of cell B-material entry for the tna was independent of the presence of serum , that is , independent of tna degradation . [SEP]
[CLS] together , data in figure 2 strongly suggest that tna may undergo initial scission in the extracellular space in the presence of serum , but this process is not a strict requirement for cell B-material entry , and tna exhibit fast translocation into cells B-material irrespective of this preprocessing . [SEP]
[CLS] this conclusion agrees well with prior data on the subject illustrating that oligonucleotides of this length exhibit efficient cell B-material entry via endocytosis B-event , possibly aided by specific membrane - bound proteins B-material . [SEP]
[CLS] to determine the antiviral B-property effects of the synthesized tna , as well as idoxuridine and trifluridine as controls , we used a luminescence - based cell B-technique viability I-technique assay I-technique that allows quantification of the hsv - 2 induced cytopathic effects and its inhibition by antivirals B-property ( figure 3a ) . [SEP]
[CLS] tna based on thymidine , tna - t 14 , elicited a surprising antiviral B-property effect , which was nevertheless characterized by low potency and moderate efficacy of treatment . [SEP]
[CLS] in turn , the trifluridine - based tna - f 14 proved to have cell B-material growth inhibitory effects , which is not surprising given that trifluridine is a marketed anticancer B-property agent . [SEP]
[CLS] interestingly , the tna of alternating t and f , ( t / f ) 7 , did not affect hsv - 2 infection , and also did not show any cytotoxic B-property effects . [SEP]
[CLS] most importantly , tnas based on idoxuridine proved to be potent , efficacious inhibitors of hsv - 2 , with virtually no toxicity B-property . [SEP]
[CLS] strikingly , activity - related ic 50 for tna based on idoxuridine ( 75 nm for tna - i 14 , 110 nm for tna - i 7 , and 263 nm for tna - ( t / i ) 7 expressed in molarity of the nucleoside ) was superior to that of the parent drug ( 26 µm ) . [SEP]
[CLS] in other words , tna - i 14 was ≈35 - fold more potent than idoxuridine when expressed in molarity of the incorporated drug , and more potent than idoxuridine even if expressed in molarity of tna chains . [SEP]
[CLS] this phenomenon cannot be explained by the extracellular drug release , in which case the best outcome would be a matched potency of the prodrug and the drug . [SEP]
[CLS] we believe that enhanced potency of the tna is therefore due to facilitated cell B-material entry for tna ( figure 2d ) , with ensuing exhaustive intracellular processing of the prodrugs for drug release [SEP]
[CLS] encouraged by the above data , we tested tna in an advanced setting using clinical isolates of hsv - 1 ( from bronchoalveolar lavage , bal ) and hsv - 2 ( from vaginal swab ) . [SEP]
[CLS] tna based on trifluridine ( tna - f 14 ) revealed virtually no antiviral B-property activity against hsv - 1 and minor , variable antiviral B-property activity against hsv - 2 ( figure 3b ) . [SEP]
[CLS] the counterpart based on thymidine ( tna - t 14 ) exhibited modest antiviral B-property effect at highest concentrations . [SEP]
[CLS] idoxuridine - based tna ( tna - i 14 and i 7 ) proved to be the lead formulations with potent inhibitory effects against hsv - 1 from bal and for hsv - 2 from vaginal swab , with ic 50 values close to those against hsv - 2 shown in figure 3a . [SEP]
[CLS] confocal B-technique laser I-technique scanning I-technique microscopy I-technique imaging provided visual illustration of the tna cell B-material entry and concurrent antiviral B-property effects mediated by these prodrugs ( best viewed on human foreskin fibroblast cells B-material , hff ) , figure 3c . [SEP]
[CLS] while hsv infection led to the loss of the typical fibroblast cell B-material phenotype , exposure to tna - i 14 ( but not the inactive and cytotoxic B-property tna - f 14 ) restored the cell B-material phenotype . [SEP]
[CLS] to gain further insights into the antiviral B-property mechanism , we used acyclovir ( acv ) - resistant strains of the viruses ( hsv - 1 from throat flushing and hsv - 2 from anus swab ) , figure 3b and figure s3 ( supporting information ) . [SEP]
[CLS] in these strains , acv resistance is mediated through a frameshift mutation in the viral thymidine kinase , which renders phosphorylation of acv and other nucleosides inefficient , whereas the virus surface remains unaltered . [SEP]
[CLS] general polyanion - mediated inhibition of viral infectivity is achieved by diverse negatively charged polymers B-material through a direct contact with the viral particles and for dna / tna should be largely sequence - independent and similar for the hsv - 1 / 2 , resistant to acv or not . [SEP]
[CLS] our experiments revealed that tna were largely inactive against the acv - resistant virus strains , figure 3b and figure s3 ( supporting information ) . [SEP]
[CLS] this observation strongly suggests that antiviral B-property effects elicited by the tna are not due to a polyanion - mediated inhibition of virus cell B-material entry , but caused by tna processing and the released drug . [SEP]
[CLS] furthermore , tna - i 14 and tna - i 7 were potent and efficacious antiviral B-property agents in serum - free cell B-material culture conditions that exclude extracellular processing of tna ( figure s4 , [SEP]
[CLS] supporting information ) , strongly suggesting that tna processing for drug release is intracellular . [SEP]
[CLS] drug release from tna may afford nucleosides ( that require subsequent kinase activity to have an antiviral B-property effect ) or nucleotides ( with ensuing kinase - independent antiviral B-property activity ) . [SEP]
[CLS] since tna - i 14 or i 7 were not active against the acv - resistant strains of hsv ( figure 3b and figure s3 in the supporting information ) , the activity of the viral kinase likely remained a limiting factor . [SEP]
[CLS] in other words , our data demonstrate that the intracellular drug release from tna affords the dephosphorylated nucleoside analogue , idoxuridine . [SEP]
[CLS] taken together , our results indicate that in the presence of serum tna undergo preprocessing in the extracellular space followed by cell B-material entry and an exhaustive intracellular processing to release the nucleoside antiviral B-property drug . [SEP]
[CLS] this process results in a remarkable potency of tna as prodrugs . [SEP]
[CLS] for in vivo use , stabilization of nucleic B-material acids I-material in circulation can be achieved using diverse methodologies currently in ( pre ) clinical development for the delivery of small interfering rna or antisense nucleic B-material acids I-material . [SEP]
[CLS] however , our preliminary work suggests that tools of gene transfer such as lipofectamine and the " spherical nucleic B-material acids I-material , " that is , tna immobilized on gold B-nanoparticle nanoparticles I-nanoparticle , do not aid but impeded activity of tna ( see figures s5 - s7 , supporting information ) , likely due to restricting the tna processing . [SEP]
[CLS] we currently investigate alternative methodologies such as albumin protraction . [SEP]
[CLS] in this study , we present the first - in - class macromolecular prodrugs built on the natural nucleic B-material acid I-material scaffold using marketed nucleoside analogues . [SEP]
[CLS] tna have molecularly defined composition , which is a highly favorable attribute concerning the regulatory approval , and release the drug via a natural , nuclease - mediated mechanism . [SEP]
[CLS] tna exhibited fast , unaided cell B-material entry and exerted antiviral B-property effects with high potency superior to that of the parent nucleoside analogues . [SEP]
[CLS] together , the natural mechanism of drug release , the perfectly controlled composition , and the high antiviral B-property activity position tna is highly favorably for translational studies [SEP]
[CLS] a ) chemical formula of trifluridine and idoxuridine ; b ) schematic illustration for the conversion of antiviral B-property drugs into monomers B-material for automated synthesis of nucleic B-material acid I-material ( r = i for idoxuridine or r = cf 3 for trifluridine ) . [SEP]
[CLS] c ) mass spectrometry characterization of tna with regards to the molar mass . d ) hplc characterization of tna - i 7 and tna - i 14 . sequences elute at distinctly different times , as individual peaks , with minimal content of shorter oligomers thus illustrating that tna are essentially monodisperse reaction products . [SEP]
[CLS] e ) maldi characterization of the tna - i 7 and tna - i 14 supporting the notion that tna are obtained as monodisperse reaction products . [SEP]
[CLS] f ) lc - ms analyses illustrating that upon exhaustive digestion ( phosphodiesterase i from crotalus adamanteus venom ) , tna release the expected idoxuridine , trifluridine , or thymidine . [SEP]
[CLS] a ) schematic illustration of the nuclease - mediated degradation of the tna resulting in the release of idoxuridine . [SEP]
[CLS] b ) hplc elution profiles for tna - i 14 after a 24 h incubation B-technique in cell B-material culture medium with or without fcs . [SEP]
[CLS] c ) hplc - based quantification of the idoxuridine release from tna - i 14 upon its incubation B-technique in serum - containing cell B-material culture medium . [SEP]
[CLS] presented results are mean of three independent experiments ± s . d . d ) time - lapse confocal B-technique laser I-technique scanning I-technique microscopy I-technique images illustrating cell B-material entry for tna - i 14 in the presence of fcs ( dmem containing 10 % fcs ) or absence of fcs ( x - vivo medium ) in human foreskin fibroblasts ( hff ) and lung carcinoma cell B-material line ( a549 ) . [SEP]
[CLS] nuclei were stained with hoechst 33 342 ; tna - i 14 - cy5 is false - colored in yellow . [SEP]
[CLS] scale bar ( same for all panels ) : 20 µm . [SEP]
[CLS] adv . sci . 2019 , 6 , 1802095 [SEP]
[CLS] a ) dose response curves for tna in preventing hsv - 2 infection of vero e6 cells B-material and corresponding cell viability I-property data . [SEP]
[CLS] cells B-material were incubated B-technique with tnas for 2 h prior infection , and 72 h afterward infection was determined by using celltiter - glo ( r ) ( ctg ) . [SEP]
[CLS] to determine the cell B-property viability I-property , vero e6 was incubated B-technique with tnas for 74 h without infection . [SEP]
[CLS] figure 3 . n = 3 in triplicates , ±sem . [SEP]
[CLS] * p < 0 . 1 , * * p < 0 . 01 , and * * * p < 0 . 001 , by one - way anova with bonferroni post - test . [SEP]
[CLS] b ) dose response curves illustrating activity of tna against hsv - 1 and hsv - 2 strains sensitive or resistant to acyclovir . [SEP]
[CLS] vero e6 cells B-material were incubated B-technique with tnas for 2 h prior infection , infected and 72 h later infection rates were determined by using a cell B-technique viability I-technique assay I-technique . [SEP]
[CLS] n = 3 in triplicates , ±sem . [SEP]
[CLS] c ) confocal B-technique laser I-technique scanning I-technique microscopy I-technique images illustrating infection of a549 cells B-material with hsv - 1 and hff cells B-material with hsv - 2 in the presence of tna - cy5 - i 14 and tna - cy5 - f 14 . [SEP]
[CLS] after fixation , cells B-material infected with hsv - 1 were stained with an antibody B-material against the glycoprotein e ( ge ) of hsv - 1 and a secondary antibody B-material labeled with alexa fluor 488 . [SEP]
[CLS] the same was done for cells B-material infected with hsv - 2 by using an antibody B-material against the gd of hsv - 2 . [SEP]
[CLS] nuclei were stained with hoechst 33 342 , cellular membranes with cell B-material mask deep orange . [SEP]
[CLS] the nucleic B-material acids I-material are coupled to the fluorophore cy5 . [SEP]
[CLS] tna - cy5 - i 14 and tna - cy5 - f 14 did not show any cytotoxic B-property effects in used cell B-material lines determined with cell B-technique viability I-technique assay I-technique ( data not shown ) . [SEP]
[CLS] scale bar ( same for all panels ) : 20 µm . [SEP]
[CLS] embedded in a unique language , deoxyribonucleic B-material acid I-material ( dna ) carries the lion ' s share of the hereditary information in living cells B-material . [SEP]
[CLS] ever since friedrich miescher isolated dna in 1869 , the scientific community extensively investigated its properties and possible applications . [SEP]
[CLS] james watson and francis crick identified the molecular structure of dna in 1953 , starting the age of genetics and modern molecular biology . [SEP]
[CLS] the watson - crick base pairing rules provide dna with unique self - recognition and sequence programmability , which enabled dna and dna - based materials to find their applications in biomedicine , which includes drug delivery , gene dna is more than a carrier of genetic information : it is a highly versatile structural motif for the assembly of nanostructures , giving rise to a wide range of functionalities . [SEP]
[CLS] in this regard , the structure programmability is the main advantage of dna over peptides B-material , proteins B-material , and small molecules . [SEP]
[CLS] dna amphiphiles B-property , in which dna is covalently bound to synthetic hydrophobic B-property moieties , allow interactions of dna nanostructures with artificial lipid B-material bilayers I-material and cell B-material membranes . [SEP]
[CLS] these structures have seen rapid growth with great potential for medical applications . [SEP]
[CLS] in this review , the current state of the art of the synthesis of dna amphiphiles B-property and their assembly into nanostructures are first summarized . [SEP]
[CLS] next , an overview on the interaction of these dna amphiphiles B-property with membranes is provided , detailing on the driving forces and the stability of the interaction . [SEP]
[CLS] moreover , the interaction with cell B-material surfaces in respect to therapeutics , biological sensing , and cell B-material membrane engineering is highlighted . [SEP]
[CLS] finally , the challenges and an outlook on this promising class of dna hybrid materials are discussed . [SEP]
[CLS] 5th anniversary article silencing , and diagnostics . [SEP]
[CLS] apart from that technologies have been developed to evolve dna molecules , which strongly bind a wide variety of target molecules ( aptamers ) or exhibit catalytic activity ( dnazymes ) . [SEP]
[CLS] as therapeutics , nucleic B-material acids I-material inhibit either dna or rna expression , thereby blocking the production of proteins B-material related to a disease . [SEP]
[CLS] however , the clinical application of therapeutic nucleic B-material acids I-material ( tnas ) is still facing limitations due to unsolved challenges regarding delivery . [SEP]
[CLS] for instance , negatively charged cellular membranes act as a natural barrier B-property to prevent entry of foreign polyanionic nucleic B-material acids I-material . [SEP]
[CLS] once inside the cell B-material , dnases or rnases degrade foreign nucleic B-material acids I-material to prevent their integration into the genome . [SEP]
[CLS] tnas further have to be delivered to the correct cells B-material with minimal side effects to other cells B-material . [SEP]
[CLS] when using tnas as artificial receptors , the failed anchoring or insertion of the dna in the cell B-material membrane restricts its excellent recognition properties . [SEP]
[CLS] these challenges potentially decrease the applicability of dna reporting signals from the cell B-material or tissue . [SEP]
[CLS] the unique programmability gives dna an edge over other molecules that interact with membranes , such as peptides B-material , proteins B-material , and small molecules . [SEP]
[CLS] in order to realize successful insertion of dna in the cell B-material membrane and efficient delivery of tnas both in vitro and in vivo , one of the most commonly used strategies is increasing the hydrophobicity B-property of nucleic B-material acids I-material . [SEP]
[CLS] to this end , dna is chemically conjugated with hydrophobic B-property moieties , resulting in dna amphiphiles B-property . [SEP]
[CLS] efficient and stable insertion into live cell B-material membranes allows amphiphilic B-property dna conjugates to cross the cell B-material membrane . [SEP]
[CLS] importantly , these dna amphiphiles B-property can be modified with additional functional groups that enable specific targeting and biocompatibility B-property in vivo , providing them with a tremendous potential for biomedicine . [SEP]
[CLS] to date , the synthesis and application of amphiphilic B-property dna conjugates have been well demonstrated and reviewed . [SEP]
[CLS] a dna amphiphile B-property is based on hydrophilic B-property dna that contains a covalently connected hydrophobic B-property segment . [SEP]
[CLS] usually , the hydrophobic B-property moiety is a polymer B-material or a small molecule . [SEP]
[CLS] the lipophilic B-property modifications B-event of I-event dna I-event can be achieved by conjugation at either the 3 ′ - or 5 ′ - terminal , or within the dna sequence , allowing the construction of complex structures . [SEP]
[CLS] these hydrophobic B-property moieties can be conjugated to dna , either on a solid support during dna synthesis or by coupling to already synthesized dna units in solution . [SEP]
[CLS] the first successful chemical synthesis of a dinucleotide was achieved in 1955 . [SEP]
[CLS] stable deoxynucleoside phosphoramidites were introduced as synthons in 1985 , opening up the field . [SEP]
[CLS] nowadays , solid phase synthesis ( sps ) allows generating dna fragments of up to 200 nucleotides . [SEP]
[CLS] this technology allows functionalization or introduction of non - natural nucleotides . [SEP]
[CLS] the fully automated synthesis can be precisely controlled , monitored , and is characterized by a high reproducibility . [SEP]
[CLS] to broaden the scope of synthesis robots by introducing special solvents , catalysts B-property , extreme reaction conditions or long reaction times , the automated process can be replaced by the syringe synthesis technique or in - flask reactions to realize various modifications of the dna with hydrophobic B-property units . [SEP]
[CLS] coupling of dna with specific motifs in solution phase has been demonstrated as another highly versatile strategy , which was reviewed by our group before . [SEP]
[CLS] solution phase synthesis is used for covalent bond formation between functional groups such as amines B-material or thiols B-material , with groups such as carboxylic B-material acids I-material or maleimides [SEP]
[CLS] however , aqueous solution coupling of dna with hydrophobic B-property molecules often results in low yields due to the solvent incompatibility of starting materials . [SEP]
[CLS] to overcome this limitation , we reported a conjugation protocol for coupling of hydrophobic B-property molecules to dna with high efficiency . [SEP]
[CLS] by complexing dna with positively charged quaternary ammonium surfactants B-property , we neutralized the charge on the dna , making it soluble B-property in organic solvent . [SEP]
[CLS] the organic phase coupling technique expands the number of possibilities to generate amphiphilic B-property dna hybrids . [SEP]
[CLS] one of the most commonly used lipids B-material in dna amphiphiles B-property is cholesterol . [SEP]
[CLS] in addition to cholesterol or one of its derivatives , other synthetic single - chain fatty acids , steroid molecules , [ 34 ] α - tocopherol , hydrophobic B-property polymers B-material , such as poly ( propylene oxide B-material ) ( ppo ) , or the π - conjugated system porphyrin have been successfully introduced to dna ( figure 1 ) . [SEP]
[CLS] hence , synthetic protocols to introduce a wide range of hydrophobic B-property moieties into dna at various positions are available , allowing for the exploration of new functionalities in nanotechnology . [SEP]
[CLS] dna amphiphiles B-property can be designed to assemble into a variety of nanoscale structures . [SEP]
[CLS] in general , nanoscale structures can be constructed " top - down " or " bottom - up " : the bottom - up approach makes use of assembling single molecules into nanostructures by intermolecular B-property interactions I-property , yielding a level of molecular control that is out of reach to a top - down strategy . [SEP]
[CLS] dna amphiphiles B-property that contain both hydrophobic B-property moieties and nucleic B-material acids I-material possess advantageous features derived from the dna part as well as from the hydrophobic B-property moieties combined in one molecule . [SEP]
[CLS] the watson - crick base pairing rules that govern dna nanotechnology allow the rational design of complex nanostructures which result in novel functions . [SEP]
[CLS] this molecular technology is based on bottom - up self - assembly , which was initiated by nadrian seeman in the early 1980s and has been growing rapidly ever since . [SEP]
[CLS] depending on the design , the structures can be 1d , 2d , or 3d . in addition , single - stranded overhanging sequences in the final structure enable further functionalization by hybridization with complementary sequences . [SEP]
[CLS] more detail on the assembly of dna nanostructures and their emerging applications in areas such as biophysics , drug delivery , synthetic biology , can be found in ref . [ 41 , 46 ] . [SEP]
[CLS] on the other hand , hydrophobic B-property units in amphiphiles B-property tend to microphase separate due to hydrophobic B-property interactions I-property . [SEP]
[CLS] this structural concept can be further combined with assembly mechanisms relying on electrostatic forces , π - π stacking interactions , hydrogen B-material bonding and van der waals interactions . [SEP]
[CLS] hence , dna amphiphiles B-property have the ability to self - assemble into predictable morphologies ( figure 2 ) , such as spherical micelles B-material , rods , vesicles , and bilayers . [SEP]
[CLS] an inspiring example of engineering such morphologies was reported by baglioni and co - workers in 2007 : they synthesized nucleolipids in which the choline headgroup of phosphatidylcholines was replaced by a nucleoside , either uridine or adenosine . [SEP]
[CLS] the resulting molecules had a negatively charged nucleotide group as polar head . [SEP]
[CLS] depending on the length of the alkyl chains , globular micelles B-material , flexible cylindrical aggregates , or bilayers were obtained from these nucleolipids . [SEP]
[CLS] the shape of the amphiphile B-property dictates the obtained structures : a short hydrophobic B-property chain provides an amphiphile B-property with a conical shape , resulting in globular micellar aggregates , while a long alkyl chain gives a cylindrical shape that results in wormlike micellar aggregates . [SEP]
[CLS] the latter morphology is further modulated by improved orientation of the bases that interact with each other . [SEP]
[CLS] when above its critical micelle B-material concentration , dna amphiphiles B-property self - assemble into micellar systems with nanometer dimensions . [SEP]
[CLS] this occurs spontaneously because the amphiphiles B-property phase separate in aqueous media . [SEP]
[CLS] micellar structures are composed of a hydrophobic B-property core B-material and a hydrophilic B-property dna shell B-material . [SEP]
[CLS] dna amphiphiles B-property form spherical micelles B-material with a diameter from 6 . 7 to 36 . 4 nm , as measured by atomic B-technique force I-technique microscopy I-technique ( afm ) and dynamic B-technique light I-technique scattering I-technique ( dls ) . [SEP]
[CLS] similar to inorganic B-nanoparticle nanoparticles I-nanoparticle , the size of the spherical micelles B-material can be regulated by adjusting the dna or hydrophobic B-property segments . [SEP]
[CLS] afm revealed that such micelles B-material deform , depending on the hydrophobic B-property segments attached to the dna molecules . [SEP]
[CLS] amphiphiles B-property with different dna lengths or different lipids B-material form micelles B-material with tunable size , indicating a relationship between micelle B-material size and length of the constituent segments . [SEP]
[CLS] in this context , dna polymerase can be utilized to control the size of micelles B-material : treatment of micelles B-material consisting of dna - b - ppo ( ppo block covalently connected to the 5 - end of a 22 nt single - stranded dna ) with the enzyme terminal deoxynucleotidyl transferase ( tdt ) increases the size from 10 to 23 nm , depending on the incubation B-technique time ( figure 3a ) . [SEP]
[CLS] similarly , the use of enzymes to digest and ligate nucleic B-material acids I-material resulted in dna amphiphiles B-property containing dsdna with molecular weights of up to three million daltons . [SEP]
[CLS] these strategies offer post - synthetic control over the growth of dna nanostructures in aqueous medium . [SEP]
[CLS] furthermore , the size and stability of dna amphiphile B-property micelles B-material is determined by the number of hydrophobic B-property moieties : increasing the number of nucleotides containing dodec - 1 - ynyl chains attached to the nucleobases resulted in smaller micelles B-material with increased stability . [SEP]
[CLS] the position of the hydrophobic B-property nucleotide units in the short sequences proved to have little influence on micelle B-material structure and stability . [SEP]
[CLS] hybridization allows precise post - synthetic control over the shape of a dna micelle B-material ( figure 3b ) . [SEP]
[CLS] the shape of micelles B-material can be changed from spheres to rods by addition of complementary single - stranded dna to the dna amphiphiles B-property , forming double - stranded dna . [SEP]
[CLS] morphology can be controlled reversibly with for example dna - brush amphiphiles B-property that assemble into spherical micelles B-material ( ≈25 nm ) and contain a rna nucleotide as an enzymatic cleavage site ( figure 3c ) . [SEP]
[CLS] mixing spherical micelles B-material with a dna - based phosphodiesterase that is specific for the dna sequence and cuts at the rna site , resulted in a long cylindrical structure ( > 1000 nm in length ) . [SEP]
[CLS] to facilitate a subsequent cylinder - to - sphere transformation , a 19 - base ssdna sequence was added , which forms a 9 nt duplex with the truncated dna of the cylinder shell B-material . [SEP]
[CLS] the reverse sphereto - cylinder transition was achieved again by the addition of a complementary 19 - base ssdna designed to invade into the shorter nine - base duplex in the micelle B-material shell B-material . [SEP]
[CLS] thus , dna is a superb tool for encoding supramolecular structure information allowing exquisite control over morphology of dna amphiphiles B-property . [SEP]
[CLS] our group synthesized an additional type of structure , based on a mixed hybrid micellar architecture ( figure 3d ) . [SEP]
[CLS] here , dna - b - ppo and pluronic f127 ( a triblock copolymer with a peg ( polyethylene glycol ) - b - ppo - b - peg architecture ) were combined . [SEP]
[CLS] in this construct , the ppo from both dna amphiphile B-property and pluronic copolymer formed the core B-material of the micelles B-material , while dna from dna - b - ppo and peg from pluronic were located in the corona . [SEP]
[CLS] the resulting self - assembled structures were finally cross - linked by forming a semi interpenetrating polymer B-material network in the micelle B-material core B-material . [SEP]
[CLS] the peg domain did not undermine the hybridization of dna and the hydrophobic B-property core B-material could be loaded with hydrophobic B-property drugs . [SEP]
[CLS] the resulting aggregates exhibit the potential for combining block copolymers of different nature , facile functionalization of dna amphiphiles B-property by hybridization and the possibility for stabilization of such aggregates by polymer B-material network formation within the micelle B-material core B-material . [SEP]
[CLS] as a result , micelles B-material were obtained that are stable in regard to dilution , temperature increase and the possibility for attaching conveniently targeting units . [SEP]
[CLS] likely , such a peg corona shields the dna backbone and improves the biocompatibility B-property and immune compatibility of the mixed hybrid micelles B-material , vide infra . [SEP]
[CLS] dna amphiphile B-property micelles B-material can be functionalized to introduce new properties . [SEP]
[CLS] micellar aggregation of dna amphiphiles B-property aligns the single - stranded dna in its corona , which allows dna - templated organic reactions to proceed in 3d space . [SEP]
[CLS] therefore , the ssdna of the corona needs to be hybridized with sequences , which are equipped with reactants . [SEP]
[CLS] ( figure 3e ) moreover , dna amphiphiles B-property were functionalized to a high degree for combined mrna detection and gene therapy in molecular beacon micelle B-material flares ( mbmfs ) , which are selfassembled diacyllipid - molecular - beacon dna conjugates . [SEP]
[CLS] the mbmfs showed efficient cell B-material uptake , enhanced enzymatic stability , excellent target selectivity , and superior biocompatibility B-property compared to pristine dna . [SEP]
[CLS] diperfluorodecyl - dna conjugates allow further improvement of target binding affinity and enzymatic resistance by virtue of the physicochemical properties of fluorination B-event . [SEP]
[CLS] however , loss of integrity of micelles B-material compromised the recognition ability of the aptamer when interacting with cells B-material . [SEP]
[CLS] therefore , the same group developed a more stable cross - linked dna - methacrylamide - lipid B-material micelle B-material ( x - dlm ) system ( figure 3f ) , which incorporates a methacrylamide functionality between the hydrophilic B-property and hydrophobic B-property portions of dna - lipid amphiphiles B-property that can be cross - linked after self - assembly in aqueous solution . [SEP]
[CLS] this x - dlm system offers further improved stability in the cellular environment and better specificity regarding cell B-material recognition . [SEP]
[CLS] besides cross - linking of dna amphiphiles B-property , these nanoobjects can be encapsulated via a facile self - assembly process . [SEP]
[CLS] therefore , the nucleic B-material acid I-material micelles B-material were incubated B-technique with virus capsid ( vc ) proteins B-material ( figure 3g ) . [SEP]
[CLS] in this approach , the negatively charged dna particles induced capsid formation , allowing the entrapment of oligonucleotides as a constituent selected lipid - oligonucleotide conjugates , exemplifying the variety of lipophilic B-property residues that can be appended to dna . [SEP]
[CLS] structures of dna conjugated with , from top to bottom , cholesterol obtained via a 1 , 3 - dipolar huisgen ' s cycloaddition between alkyne modified cholesterol and 5 ′ - azido - 5 ′ - deoxythymidine ; a single hydrocarbon chain obtained via a 1 , 3 - dipolar huisgen ' s reaction between alkyne modified c18 chain and 5 ′ - azido - 5 ′ deoxythymidine ; tocopherol obtained by covalent attachment to the 5 ′ end of the strand ; a single fluorocarbon chain obtained via a huisgen ' s reaction between 5 ′ - azide deoxythymidine and propargylated fluorocarbon chain ; a ppo chain obtained via a ppo phosphoramidite during sps [SEP]
[CLS] ; double hydrocarbon chains obtained via a reaction of stearoyl chloride B-material with 1 , 3 - diamino - 2 - dydroxypropane [SEP]
[CLS] and [SEP]
[CLS] double fluorocarbon chains obtained by a diperfluorodecyl phosphoramidite during sps . [SEP]
[CLS] www . advancedscience . com part of the micellar template . [SEP]
[CLS] the preloading of entities in the core B-material or by hybridization of micelles B-material enables encapsulation of various small molecules inside vcs , which marked a significant step forward in chemical virology due to the flexibility of loading these protein B-material nanocontainers with various payloads . [SEP]
[CLS] thus , dna amphiphiles B-property form micelles B-material that are tunable , versatile , and allow realization of functions . [SEP]
[CLS] next to micelles B-material , amphiphilic B-property dna molecules can be aligned to form liposomes B-nanoparticle or bilayers , similar as indicated for conventional adv . sci . 2019 , 6 , 1900043 figure 2 . schematic models of self - assembled lipids B-material . a ) micelles B-material are preferentially formed by lipids B-material with a conical shape . [SEP]
[CLS] b ) vesicles are composed of spherical lipid B-material bilayers I-material with a water B-material core B-material . [SEP]
[CLS] c ) planar lipid B-material bilayers I-material are formed by lipids B-material with a cylindrical shape . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2014 , american chemical society . [ 61 ] [SEP]
[CLS] c ) dnazyme induced reversible transformation of the aggregate shape of a dna - brush block copolymer . [SEP]
[CLS] reproduced from ref . [ 62 ] . [SEP]
[CLS] d ) schematic of the mixed micelle B-material architecture . [SEP]
[CLS] two amphiphilic B-property block copolymers , dna - b - ppo and peo - b - ppo - b - peo with the trade name pluronic f127 , form mixed micellar structure and this micelle B-material can be stabilized by formation of a semi - interpenetrating network in its core B-material . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2010 , royal society of chemistry . [SEP]
[CLS] e ) schematic representation of the drug delivery system based on dna amphiphiles B-property . [SEP]
[CLS] liposomes B-nanoparticle from dna amphiphilesa ) targeting units ( red dots ) that are connected to the complementary sequence of the micelles B-material are hybridized to equip the nanoparticle B-nanoparticle surface with folic B-material acid I-material units . [SEP]
[CLS] b ) the anticancer B-property drug ( green dots ) is loaded into the core B-material of the micelles B-material . [SEP]
[CLS] reproduced from ref . [ 55 ] . [SEP]
[CLS] f ) schematic of dna micelle - templated vc formation . [SEP]
[CLS] loading hydrophobic B-property molecules ( top , green ) into micelle B-material core B-material and hybridization of a complementary dna connected to functional moieties ( bottom , red ) to the dna micelle B-material . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2010 , american chemical society . g ) photoinduced cross - linking of selfassembled dna - methacrylamide - lipid B-material micelles B-material . [SEP]
[CLS] green dot between dna and lipid B-material represents methacrylamide molecules which can be crosslinked . [SEP]
[CLS] reproduced from ref . [ 65 ] . [SEP]
[CLS] www . advancedscience . com surfactant B-property molecules in figure 2 : liposomes B-nanoparticle are flat bilayer sheets folded to form closed spherical objects , with the structure of the assembly determined by the conical shape of the dna amphiphiles B-property . [SEP]
[CLS] nucleic B-material acid I-material functionalization of lipids B-material allows additional control over lipid B-material self - assembly through specific interactions among the polar heads . [SEP]
[CLS] as in micelles B-material , the hydrophobic B-property lipid B-material tail and hydrophilic B-property dna head combined determine the phase behavior and aggregate microstructure . [SEP]
[CLS] dna amphiphiles B-property that form vesicular structures can be made for example by linking poly ( butadiene ) covalently to poly - cytidine during solid phase synthesis . [SEP]
[CLS] the resulting amphiphilic B-property copolymer selfassembled into 80 nm vesicles as demonstrated by tem and confocal microscopy B-technique . [SEP]
[CLS] by using a functional dna moiety as head group , one can induce more complex behavior : conjugation of the lipid B-material tail with a dna sequence that forms an i - motif renders the liposome B-nanoparticle structure ph sensitive upon acidification ( figure 4a ) . [SEP]
[CLS] the c - rich dna segment undergoes a structural change from random coil ssdna to an i - motif structure upon acidification ( ph = 5 ) , triggering the transformation of the vesicles into an entangled 3d network . [SEP]
[CLS] this process was reversed when the ph was increased to 7 . 3 . [SEP]
[CLS] this structure allowed the encapsulation of a hydrophobic B-property molecule and a ph - triggered release , showing that these dna amphiphile systems can be engineered to be sensitive to external stimuli . [SEP]
[CLS] moreover , vesicles can be prepared with programmed geometry and dimensions using ssdna - modified gold B-nanoparticle nanoparticles I-nanoparticle or dna origami as scaffolds . [SEP]
[CLS] the ssdna on the scaffold hybridizes with corresponding dna amphiphiles B-property and the resulting frame allows generation of the desired bilayer upon mixing with additional dna amphiphiles B-property ( figure 4b ) . [SEP]
[CLS] strikingly , a variety of vesicle shapes was obtained by templating the dna amphiphile assembly , i . e . , cuboids and dumbbells . [SEP]
[CLS] in a similar way , dna origami can be used to template vesicle formation in the interior of the origami structure . [SEP]
[CLS] this allows size - controlled liposome B-nanoparticle formation with the added feature that the origami can be removed . [SEP]
[CLS] in this case , the inner surface of the dna origami ring is decorated with ssdna extensions , which can hybridize with lipid - dna conjugates , thus acting as an exoskeleton for liposome B-nanoparticle formation ( figure 4c ) . [SEP]
[CLS] using this approach , a series of highly monodisperse sub - 100 nm ( 29 , 46 , 60 , and 94 nm ) liposomes B-nanoparticle with a variety of different lipid B-material compositions were produced . [SEP]
[CLS] thus , dna amphiphile B-property vesicles with desired sizes or shapes can be synthesized using templated vesicle formation . [SEP]
[CLS] besides exclusively preparing vesicles from dna amphiphiles B-property , liposomes B-nanoparticle formed from other lipids B-material can be functionalized by nucleic B-material acids I-material with the help of amphiphilic B-property dna conjugates . [SEP]
[CLS] thereby , the hydrophobic B-property unit of the dna amphiphile B-property pierces into the lipid B-material membrane I-material . [SEP]
[CLS] in this context , dna amphiphiles B-property are excellent tools for controlled vesicle fusion and formation of multivesicle assemblies . [SEP]
[CLS] for vesicle fusion , bilayers are brought into close proximity after which the lipid B-material head - groups from one vesicle insert into the other , creating the basis for the fusion pore . [SEP]
[CLS] dna hybridization connects vesicles and brings them together to initiate fusion . [SEP]
[CLS] using vesicles modified with double cholesterol terminated dna strands that were complementary to each other , hook and co - workers reported for the first time amphiphilic B-property dna induced fusion of lipid B-material vesicles . [SEP]
[CLS] the hybridization occurs in a zipper - like fashion by forcing the vesicles into close contact , enabling opening of the fusion pore between the two vesicles . [SEP]
[CLS] dna - induced fusion was more efficient with liposomes B-nanoparticle that consisted of cone shaped lipids B-material such as dope ( 1 , 2 - dioleyl - sn - glycero - 3 - phospho - ethanolamine ) and cholesterol , showing the importance of the geometry of those lipids B-material for efficient fusion . [SEP]
[CLS] in a separate study involving dna conjugated to 1 , 2 - o - dioctadecyl - rac - glycerol at either the 3 ′ or 5 ′ end , it was shown that both lipid B-material and content mixing of the vesicles took place , indicating vesicle fusion . [SEP]
[CLS] the fusion kinetics depended on the dna sequence and the average number of lipid - dna per vesicle . [SEP]
[CLS] notably , vesicles without lipid - dna or ones presenting noncomplementary sequences underwent lipid B-material mixing or exchange of membrane molecules , but no content mixing . [SEP]
[CLS] to test the effect of membrane - membrane spacing on fusion , a series of amphiphilic B-property conjugates was synthesized by adding 2 - 24 noncomplementary nucleotides at the membrane - proximal ends of the two complementary sequences . [SEP]
[CLS] it was found that increasing the lengths of the linkers reduced lipid B-material and content mixing , but increased vesicle docking rates . [SEP]
[CLS] to further improve vesicle fusion , we employed dna modified with four hydrophobic B-property chains , which resulted in stable incorporation of dna into the liposomal B-nanoparticle bilayer with limited dissociation , which allowed for an efficient full fusion of the two liposome B-nanoparticle populations with complementary sequences [SEP]
[CLS] increased affinity of the hydrophobic B-property domain of the dna amphiphiles B-property or stronger mechanical coupling between the anchor and the oligonucleotides may improve fusion further . [SEP]
[CLS] in a striking example of the application of vesicle fusion between an artificial pathogen and a protocell , as shown in figure 4d , dna templated docking and subsequent fusion induced by the oppositely charged membranes resulted in gene delivery . [SEP]
[CLS] another excellent example of dna - programmed membrane fusion deals with efficient intracellular protein B-material delivery on both suspended and adherent cells B-material . [SEP]
[CLS] thereby , dna hybridization provides targeting and spatiotemporal control of the fusion between protein - loaded liposomes B-nanoparticle and cell B-material membranes , resulting in fast release of proteins B-material into the cytoplasm . [SEP]
[CLS] docking of vesicles in the absence of fusion may lead to vesicle assemblies , which can be controlled by the design of the amphiphilic B-property oligonucleotides . [SEP]
[CLS] this assembly process , to some extent , is similar to the assembly of dna - inorganic B-nanoparticle nanoparticle I-nanoparticle conjugates , which was initiated in the 1990s by mirkin et al . in contrast to dna - covered inorganic B-nanoparticle nanoparticles I-nanoparticle , [ 68 ] . [SEP]
[CLS] b ) schematic of the frame - guided assembly process with a dna origami scaffold . [SEP]
[CLS] dna origami cuboid with a20 sequences protruding from the surface is folded by a template and corresponding staple strands . [SEP]
[CLS] then , d t doeg dendron is anchored on dna origami by hybridization . [SEP]
[CLS] when g 2 cl - 18 is added , hydrophobic B-property groups on the dna origami guide g 2 cl - 18 dendrons to form hetero - vesicles around the dna frame . [SEP]
[CLS] reproduced from ref . [ 70 ] . [SEP]
[CLS] c ) size - controlled liposome B-nanoparticle formation through a dna scaffold . [SEP]
[CLS] a dna - origami ring ( red ) with multiple single - stranded empty handles is constructed first . [SEP]
[CLS] then dna antihandles ( oligonucleotides with complementary sequence to handle sequence that are chemically conjugated to dope , shown as green curl with orange head ) are hybridized to the dna ring . [SEP]
[CLS] afterward , this lipid - modified ring is mixed with extra lipid B-material and detergent , and dialysed to allow vesicle formation . [SEP]
[CLS] after purification and release , uniform liposomes B-nanoparticle with sizes being determined by the dna template are generated . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2016 , nature publishing group . d ) schematic figure of pathogen dna delivery to protocell by dna - mediated fusion . [SEP]
[CLS] when anchoring a set of complementary dna on a protocell and an artificial pathogen membrane , dna hybridization brings the two membranes in close proximity to enable fusion . [SEP]
[CLS] thereby , pathogen dna is released into the protocell . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , american chemical society . [SEP]
[CLS] e ) illustration of reversible control over the assembly of liposomes B-nanoparticle . [SEP]
[CLS] when the liposome B-nanoparticle surface is equipped with self - complementary dna bearing a terminal azobenzene moiety , the vesicles undergo reversible assembly and disassembly in response to multiple stimuli including uv light , salt B-material addition and temperature . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2016 , american chemical society . [SEP]
[CLS] the assembly of multiple vesicles received much less attention . [SEP]
[CLS] dna - controlled assembly of vesicles in solution and on solid supported membranes has been reported however , using for example a lipid - dna conjugate in which ssdna is coupled to two lipid B-material membrane I-material anchors at either end , with both ends inserting into the lipid B-material membrane I-material while the ssdna protrudes into the solution . [SEP]
[CLS] upon treatment with a complementary dna strand , the increased stiffness of the double - stranded dna releases one of the anchors into the solution , which allows binding to another liposome B-nanoparticle . [SEP]
[CLS] further inter - liposomal B-nanoparticle membrane anchoring occurs , which leads to aggregation of the vesicles . [SEP]
[CLS] this process provides sharp and reproducible thermal aggregation - disaggregation transitions . [SEP]
[CLS] the authors proposed that this system might be used to detect biologically relevant polynucleotides B-material . [SEP]
[CLS] further optimization of the oligonucleotides and hydrophobic B-property anchor parts allowed detection of dna sequences at nanomolar concentrations and enabled sensitive mismatch discrimination of target sequences . [SEP]
[CLS] next to thermal disaggregation , liposome B-nanoparticle assemblies were disconnected into the single vesicle state by means of light ( figure 4e ) : a self - complementary ssdna bearing a terminal switchable azobenzene moiety was anchored on vesicles and hybridization of the dna induced vesicle aggregation . [SEP]
[CLS] upon irradiation with uv light , the azobenzene isomerizes from the trans to a less hydrophobic B-property cis isomer , decreasing its anchoring efficacy in the lipid B-material membrane I-material . [SEP]
[CLS] as a result , the assembly of vesicles was destabilized . [SEP]
[CLS] hence , several means of control are present to reversibly assemble and disassemble multivesicle architectures aided by dna . [SEP]
[CLS] the cell B-material membrane , or plasma membrane , plays an essential role in separating the cytoplasm from the extracellular environment , and consequently determines the size of a cell B-material and is involved in cell signaling . [SEP]
[CLS] the most common components of the plasma membrane are phospholipids . [SEP]
[CLS] another major component is cholesterol , which localizes between the phospholipid molecules and regulates membrane stiffness and stability . [SEP]
[CLS] other types of lipids B-material such as glycolipids take up a minor fraction , while membrane proteins B-material occupy a significant portion of the surface . [SEP]
[CLS] the individual phospholipid molecules are in a dynamic state in which they rotate freely around their long axes and diffuse laterally within each leaflet , thus providing cell B-material membrane fluidity . [SEP]
[CLS] the cell B-material membrane is not a homogeneously mixed lipid B-material bilayer I-material but displays heterogeneity of the spatial arrangement of lipids B-material and proteins B-material . [SEP]
[CLS] in some cases , even lipid B-material rafts may be formed . [SEP]
[CLS] they consist of cholesterol , sphingomyelin and tightly packed saturated phospholipids forming a liquid ordered phase , which is more stable and less fluid than the liquid disordered phase constituting the rest of the membrane . [SEP]
[CLS] here we discuss the interaction of dna amphiphiles B-property with cell B-material membranes , which provide biological applications from diagnostics to biomedicine . [SEP]
[CLS] dna amphiphiles B-property interact with cell B-material membranes by hydrophobic B-property interactions I-property . [SEP]
[CLS] in model membranes , dna amphiphiles B-property dissociate from their micellar aggregates and integrate in model membranes spontaneously . [SEP]
[CLS] next to model membranes , dna micelles B-material have a strong affinity toward the cell B-material membrane . [SEP]
[CLS] the hydrophobicity B-property of the dna amphiphile B-property influences the anchoring on cell B-material membranes , as illustrated by a series of oligonucleotides conjugated to alkyl chains with either 12 , 18 , or 26 carbons B-material , tested in a range of mammalian cell B-material types . [SEP]
[CLS] a strong correlation exists between lipid B-material length and the efficiency with which the amphiphiles B-property are incorporated : nonfunctionalized dna shows negligible incorporation , while for dna with c12 and c18 tails an intermediate B-property insertion efficiency is observed and best piercing into cell B-material membranes is detected for c26 . [SEP]
[CLS] thereby , ssdna strands conjugated with fatty acid tails are in a dynamic equilibrium with the culture medium , but when hybridized with its complementary ssdna that is conjugated with a fatty acid as well , the construct remains in the cell B-material membrane . due to double anchoring of the duplex , its interaction with membrane B-material lipids I-material is enhanced , hence the construct remained incorporated into the lipid B-material bilayer I-material . [SEP]
[CLS] an alternative mechanism for interaction of amphiphilic B-property dna with cell B-material membranes is through receptor - mediated ligand binding . [SEP]
[CLS] in general , two types of receptor - mediated ligand interactions are involved : direct and indirect ones . [SEP]
[CLS] the dna segment of the amphiphile B-property can be an aptamer , which selectively targets a cell B-material membrane receptor B-material . [SEP]
[CLS] thus , the amphiphilic B-property dna attaches to the cell B-material membrane directly . [SEP]
[CLS] another receptor - mediated ligand interaction occurs through an indirect pathway : the lipophilic B-property tail of amphiphilic B-property dna binds to lipoproteins or other proteins B-material , which are subsequently recognized by the corresponding receptors on the cell B-material membrane . [SEP]
[CLS] for instance , cholesterol conjugated sirna can be bound to lipoprotein after intravenous injection into mice . [SEP]
[CLS] the high binding affinity of lipoprotein to cellular scavenger receptor B-material sr - bi facilitates the interaction of cholesterol - sirna amphiphiles B-property with the cell B-material membrane . [SEP]
[CLS] similarly , octadecyl tails of amphiphilic B-property dna bound with albumin and the resulting aggregate was recognized by cell surface albumin receptors gp18 and gp30 . [SEP]
[CLS] when investigating the interaction efficiency of dna amphiphiles B-property with cell B-material membranes , one key factor is the structure of the dna in the amphiphile B-property . [SEP]
[CLS] amphiphilic B-property dna with long dna sequences incorporates slower into the cell B-material membrane than ones with short nucleic B-material acid I-material chains , because longer dna forms large micelles B-material with a more densely charged corona , which reduces the availability of the hydrophobic B-property domain . [SEP]
[CLS] another possibility is that longer oligonucleotides contain more anionic phosphate groups , which are repelled by the anionic glycocalyx on cell B-material surfaces . [SEP]
[CLS] next to this , the hydrophobic B-property tail of dna amphiphiles B-property influences the interaction : diacyllipids dna have a high affinity for insertion into the cell B-material membrane , single chain c18 lipid B-material dna shows modest incorporation , while cholesterol modified dna exhibits the lowest affinity . [SEP]
[CLS] related to these experiments , a single acyl chain dna - mediated membrane anchoring is insufficient to mediate cell B-event - I-event cell I-event adhesion I-event , but the cell - cell interaction is achieved when diacyllipids are used . [SEP]
[CLS] moreover , different lipid B-material tails show preference for various lipid B-material domains : in liposome B-nanoparticle membranes , diacyllipids mainly anchor to liquid or liquid - ordered domains , while tocopherols anchor exclusively to liquid - disordered domains . [SEP]
[CLS] cholesterols incorporate into membranes depending on the lipid B-material composition of the membrane . [SEP]
[CLS] thus , dna amphiphiles B-property show preference for lipid B-material domains on cell B-material membranes . [SEP]
[CLS] due to the fact that the lipid B-material composition varies with cell B-material type , different lipid B-material tails can direct amphiphilic B-property dna to different cell B-material types . [SEP]
[CLS] the interaction between amphiphile B-property and cell B-material membrane also depends on the amount of amphiphilic B-property dna . [SEP]
[CLS] the number of amphiphilic B-property dna molecules that can be anchored to the cell B-material membrane depends on the initial concentration of dna amphiphile B-property in the culture medium . [SEP]
[CLS] a higher starting concentration leads to a higher density of dna tethering . [SEP]
[CLS] for interactions driven by aptamer recognition , densely packed aptamers on an amphiphilic B-property micelle B-material induce a multivalent effect , which leads to higher affinity for the cellular membranes . [SEP]
[CLS] moreover , the cell B-material culture medium influences the interaction of dna amphiphiles B-property with the membrane . [SEP]
[CLS] the culture medium affects the anchoring efficiency in the decreasing order : pbs > dmem > pbs with 10 % fbs ( fetal bovine serum ) > dmem with 10 % fbs . [SEP]
[CLS] the components in the cell B-material culture medium alter the interaction between amphiphilic B-property dna and cell B-material membranes . [SEP]
[CLS] for instance , albumin in albuminrich culture medium binds the lipid B-material domain and forms a complex that prevents amphiphilic B-property dna inserting into the cell B-material membrane . [SEP]
[CLS] hence , cell B-material membranes display additional features that influence their interaction with dna amphiphiles B-property . [SEP]
[CLS] it is important to consider their more complex structure compared to model membranes when applying dna amphiphiles B-property in living systems . [SEP]
[CLS] after binding to the cell B-material membrane , dna amphiphiles B-property or their assemblies are not static in space and time . [SEP]
[CLS] instead , they are in a dynamic exchange with the medium , they can be degraded and they can be subjected to endocytosis B-event . [SEP]
[CLS] all dna amphiphiles B-property are in equilibrium between the aqueous medium and the cell B-material surface . [SEP]
[CLS] even though a dna sequence is connected to a long lipid B-material tail , like c26 , it still displays characteristic re - equilibration . [SEP]
[CLS] a gradual loss of lipid B-material dna on the membrane occurs when replacing the cell B-material culture medium . [SEP]
[CLS] this loss is a result of adjusting a new equilibrium between dna amphiphile B-property on the cell B-material membrane and the culture medium . [SEP]
[CLS] dna conjugated to an alkyl chain showed a gradual decay on the cell B-material surface . [SEP]
[CLS] after the first hour of incubation B-technique , only < 20 % loss was observed . [SEP]
[CLS] however , after 2 . 5 h only 50 % of the initial amount of dna was present on the cell B-material surface . [SEP]
[CLS] when incubated B-technique for 24 h , only a very weak signal originating from the dna remained . [SEP]
[CLS] this gradual decay is temperature - dependent : surface anchored dna decayed to 86 % of its initial concentration after 160 min at 25 °c , while 67 % of its initial concentration was left after the same time period at 37 °c . [SEP]
[CLS] amphiphilic B-property dna anchors to the outer leaflet of the cell B-material membrane and is subjected to slow endocytosis B-event . [SEP]
[CLS] c18 and cholesterol modified oligonucleotides are taken up by cells B-material via an energy - dependent mechanism rather than by passive diffusion . [SEP]
[CLS] indeed , some of the dna amphiphiles B-property enter cells B-material via endocytosis B-event , while the majority possibly flips and translocates from the cell B-material surface to the organelles during membrane recycling . [SEP]
[CLS] as micelles B-material , amphiphilic B-property dna locates initially close to the cell B-material membrane , then disassembles and fuses with the cell B-material membrane . [SEP]
[CLS] this cellular uptake of a dna amphiphile B-property micelle B-material represents a similar uptake mechanism compared to other amphiphilic B-property molecules . [SEP]
[CLS] for interactions driven by receptor B-event binding I-event , endocytosis B-event is suggested as the subsequent step after binding of the amphiphilic B-property dna or amphiphilic B-property dna embedded lipoprotein with the receptors . [SEP]
[CLS] these complexes are recognized by corresponding receptors on the cell B-material membrane and subsequently enter cells B-material via receptor - mediated endocytosis B-event . [SEP]
[CLS] amphiphilic B-property dna and its assemblies interact efficiently with cell B-material membranes and hence offer a facile strategy for further manipulating the cell B-material surface . [SEP]
[CLS] a major characteristic is that amphiphilic B-property dna allows convenient cell B-material surface modification . [SEP]
[CLS] other common strategies for presenting dna at cell B-material surfaces , such as expression of a dna B-event binding I-event domain of a protein B-material at the cell B-material surface , covalent attachment of dna to functional groups at the membrane , or a dna aptamer that binds membrane target sites , either involve complicated stepwise processes or can only be applied to very limited membrane targets . [SEP]
[CLS] instead , employing amphiphilic B-property dna to modify a cell B-material surface is simple and quick . [SEP]
[CLS] coincubating amphiphilic B-property dna with cells B-material allows spontaneous insertion of the amphiphiles B-property into the cell B-material membrane . [SEP]
[CLS] this process is fast and can be performed within only 3 min . [SEP]
[CLS] moreover , amphiphilic B-property dna can be anchored to different cell B-material types , including natural killer cells B-material , t cells , and cancerous cells B-material . [SEP]
[CLS] this quick modification procedure results in stable anchoring of the dna in the membrane : the majority of diacyllipid - dna locates on the outer leaflet and remains even after 2 h incubation B-technique with cells B-material at 37° . [SEP]
[CLS] the easily accessible dna on the membrane is a highly versatile technology platform in vitro and in vivo . [SEP]
[CLS] to target cell B-material membranes in vivo dna amphiphiles B-property can be administered locally . [SEP]
[CLS] dna amphiphiles B-property injected into mice remained 72 h at the injection site , which reduced to 4 h with dna that does not contain a hydrophobic B-property tail . [SEP]
[CLS] more important , compared with nucleic B-material acids I-material coated I-material on nanoparticles B-nanoparticle , modifying cell B-material membrane by amphiphilic B-property dna insertion is noninvasive and does not involve inorganic components . [SEP]
[CLS] www . advancedscience . com [SEP]
[CLS] hydrophobic B-property domains within nucleic B-material acids I-material allow their easy incorporation into lipid B-material bilayers I-material and facilitate their uptake by living cells B-material . [SEP]
[CLS] here we will discuss the biomedical functions of amphiphilic B-property dna structures , which can be derived from this behavior . [SEP]
[CLS] both amphiphilic B-property micelle B-material and liposome B-nanoparticle nanostructures can be exploited for drug delivery . [SEP]
[CLS] until now , a number of examples have been reported demonstrating highly efficient drug delivery with dna amphiphiles B-property and their assemblies in vitro and in vivo . [SEP]
[CLS] our group loaded the hydrophobic B-property anticancer B-property drug ( doxorubicin ) into the interior of dna - b - ppo micelles B-material , which were taken up through receptor - mediated endocytosis B-event and significantly inhibited growth of caco - 2 cancer cells B-material . [SEP]
[CLS] the cellular uptake of the micelles B-material strongly depended on the density of the recognition elements , i . e . , folic B-material acid I-material , on the micellar surface . [SEP]
[CLS] moreover , dna amphiphile B-property micelles B-material were very well suited for loading another hydrophobic B-property anticancer B-property drug : paclitaxel B-material . [SEP]
[CLS] recently , we tackled in vivo functionality of the dna amphiphiles B-property micelle B-material even with human tissue in the context of ophthalmology for treating eye infections . [SEP]
[CLS] therefore , different antibiotics were loaded into the dna amphiphile B-property micelles B-material ( figure 5a ) . [SEP]
[CLS] aptamers were complexed with an aminoglycoside , i . e . , dna aptamer for kanamycin b or rna aptamer for neomycin b , and subsequently conjugated at the 3 ′ end of the dna amphiphiles B-property through hybridization . [SEP]
[CLS] compared with pristine drugs , the dna amphiphile B-property micelles B-material showed extended residence time on the ocular surface and improved efficiency on the cornea in vitro and in vivo . [SEP]
[CLS] this study highlights the potential applicability of amphiphilic B-property dna - based materials in the clinic . [SEP]
[CLS] most recently , a lipid - conjugated drug - incorporated oligonucleotide was developed for hitchhiking with endogenous serum albumin for cancer chemotherapy . [SEP]
[CLS] by incorporating a hydrophobic B-property lipid B-material tail , floxuridine homomeric oligonucleotides inserted into the hydrophobic B-property pocket of albumin to form complexes which accumulate at the tumor B-material site by the enhanced permeability and retention ( epr ) effect and internalize into the lysosomes of cancer cells B-material after intravenous injection . [SEP]
[CLS] upon enzymatic degradation , the cytotoxic B-property floxuridine monophosphate is released and inhibited cancer cell B-material proliferation . [SEP]
[CLS] furthermore , dna amphiphiles B-property and their self - assembled structures find application in immunotherapy . [SEP]
[CLS] surface anchoring of dna amphiphiles B-property directed immune cells B-material to their target cells B-material : modification of natural killer ( nk ) cells B-material with an aptamer named kk1b10 ( figure 5b ) resulted in specific targeting of cancer cells B-material , i . e . , chronic myelogenous leukemia cell line k562 . [SEP]
[CLS] this adv . sci . 2019 , 6 , 1900043 figure 5 . a ) schematic of dna micelles B-material for treating eye infections . [SEP]
[CLS] lipid - modified dna strands form micelles B-material and were then hybridized with antibiotics loaded aptamers . [SEP]
[CLS] upon administration to the eye , dna micelles B-material adhere to the cornea and release antibiotics to treat infections . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , elsevier . [SEP]
[CLS] b ) illustration of targeting cancer cells B-material ( green ) with aptamer - modified immune cells B-material ( red ) . [SEP]
[CLS] immune cells B-material were equipped with lipid modified dna aptamer , which targets the cancer cell B-material surface . [SEP]
[CLS] when cancer cells B-material are mixed with normal cells B-material , immune cells B-material only recognize cancer cells B-material by their surface anchored aptamer and then kill cancer cells B-material . [SEP]
[CLS] reproduced from ref . [ 12 ] . [SEP]
[CLS] c ) schematic of lipid dna micelles B-material as cpg carrier . [SEP]
[CLS] lipid conjugated dna forms micelle B-material and was then hybridized with different amounts of cpg sequences to form immunostimulatory nanoparticles B-nanoparticle ( inps ) with different degree of cpg functionalization . [SEP]
[CLS] these inps activated immune responses both in vitro and in vivo . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , elsevier . [SEP]
[CLS] d ) assembly of liposomal B-nanoparticle spherical nucleic B-material acids I-material by anchoring tocopherol modified dna to dopc small B-material unilamellar I-material vesicles I-material . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2014 , american chemical society . [SEP]
[CLS] resulted in 50 % increased killing efficiency of nk cells B-material toward k562 cancer cells B-material , compared with unmodified nk cells B-material . [SEP]
[CLS] the higher killing efficiency was likely due to the better targeting efficiency of nk cells B-material when the dna aptamer amphiphile B-property is attached . [SEP]
[CLS] moreover , the selectivity of the aptamer modified nk cells B-material was demonstrated when the target k562 cells B-material are mixed with an excess of nontargeted cells B-material . [SEP]
[CLS] in a different approach , the immunological effects of dna amphiphile B-property micelles B-material decorated with the immune adjuvant ( cpg ) were studied in vivo recently . [SEP]
[CLS] different amounts of immunostimulatory adjuvants were established on the surface of spherical micelles B-material through simple stoichiometric incorporation ( figure 5c ) . [SEP]
[CLS] after that , a full immunological assay , including phagocytosis B-event , the expression of costimulatory molecules , and the production of proinflammatory cytokines in spleen dendritic cells B-material ( dcs ) was evaluated and analyzed . [SEP]
[CLS] as a result , dose - dependent activation of spleen dcs by cpgconjugated micelles B-material was observed , which was accompanied by the pronounced up - regulation of costimulatory molecule and cytokine production . [SEP]
[CLS] in addition , labeling 50 % of the dna amphiphile B-property micelles B-material with the cpg segment can fully induce the activation of spleen dc . [SEP]
[CLS] the straightforward functionalization by dna duplex formation makes the dna amphiphile B-property micelles B-material a biocompatible B-property and scalable delivery platform for immunostimulation and immunotherapy . [SEP]
[CLS] since such dna micelles B-material still exhibit single - stranded dna on the surface ready for hybridization , these sites could be easily exploited for the incorporation of antigens to boost the generation of humoral and cellular vaccine - specific immune responses . [SEP]
[CLS] gene silencing offers the potential to cure certain diseases by down - regulating the disease - causing gene expression and protein B-material production . [SEP]
[CLS] one of the most widely used gene silencing strategies is exogenously derived single - stranded antisense oligonucleotides ( asos ) . [SEP]
[CLS] as discussed in the introduction part , the intrinsic physicochemical properties of asos , such as negative charges , high hydrophilicity B-property , and high molecular weight , prevent their efficient delivery to the intracellular target site . [SEP]
[CLS] to this end , conjugation of hydrophobic B-property moieties to asos has been used as a safer and straightforward strategy to assist their cellular uptake . [SEP]
[CLS] early studies from the 1980s used cholesteryl conjugated oligonucleotides to inhibit hiv infections or targeted the intercellular adhesion molecule - 1 gene . [SEP]
[CLS] later , hydrocarbon lipids B-material were conjugated to oligonucleotides to assist antisense efficiency : barthelemy et al . proposed an example involving lipid B-material moieties that were connected to oligonucleotides via click chemistry , which promoted cellular uptake . [SEP]
[CLS] as a result , the hepatitis c virus ( hcv ) internal ribosome entry site ( ires ) - mediated translation was effectively suppressed . [SEP]
[CLS] interestingly , when the aso was conjugated to a c18 lipid B-material or cholesterol unit , a dose - dependent reduction of the translation was measured in the huh7 cell B-material line . [SEP]
[CLS] more importantly , the biological activity of the oligonucleotide was not affected by the lipid B-material conjugation and toxicity B-property was negligible at relevant concentrations . [SEP]
[CLS] in another notable example , mirkin and coworkers synthesized a spherical nucleic B-material acid I-material nanostructure , which consists of a liposomal B-nanoparticle core B-material ( 30 nm ) stabilized with a dense shell B-material of tocopherol - modified dna that intercalates between the phospholipids and defines the liposomal B-nanoparticle structure ( figure 5d ) . [SEP]
[CLS] by using commercially available and fda - approved building blocks , they demonstrated that such monodisperse dna - functionalized vesicles remain stable with no change in dispersity for at least 4 days at 37 °c . [SEP]
[CLS] this behavior is contrary to native nonfunctionalized vesicles , which tend to fuse and form large poly - disperse structures under such conditions . [SEP]
[CLS] the obtained spherical nucleic B-material acid I-material architecture did not only stabilize the liposomal B-nanoparticle constructs but rapidly entered multiple cell B-material lines and resulted in effective gene knockdown of her2 in skov - 3 cells B-material . [SEP]
[CLS] tracking cell B-material functions , metabolism , and cell - cell signaling in their native cellular environment has enormous implications for cell B-material biology and regenerative medicine . [SEP]
[CLS] for the past few decades , molecular sensors or nanoparticles B-nanoparticle tethered on the membrane surface have been utilized to monitor such cell B-material activities . [SEP]
[CLS] however , these sensors exhibit several drawbacks , such as limited targets , a need for complicated chemical modification , allowing measurements only under model conditions , or they do not monitor in real - time . [SEP]
[CLS] fortunately , as a relatively new cell B-material surface biosensor , amphiphilic B-property dna outperforms other methods in several aspects . [SEP]
[CLS] first , aptamers can be selected via a process called systematic evolution of ligands by exponential enrichment ( selex ) to specifically bind to certain target molecules , such as metal B-material ions B-material , small organic molecules or proteins B-material with high affinity . [SEP]
[CLS] second , the straightforward functionalization of dna with fluorophores facilitates signal readout by means of photoluminescence B-property . [SEP]
[CLS] furthermore , hydrophobic B-property tags permit anchoring of the biosensor to the cell B-material membrane . [SEP]
[CLS] finally and importantly , dna hybridization or the fast response of dna aptamers for their targets render monitoring in real time and in situ with high spatiotemporal resolution feasible . [SEP]
[CLS] however , sometimes the action of aptamers is compromised by nuclease degradation , variability of pharmacokinetics or rapid renal filtration in native environments . [SEP]
[CLS] to overcome these limitations , their activity or persistence under physiological conditions were optimized during selection . [SEP]
[CLS] another means of stabilization represents the introduction of chemical modifications to decrease enzymatic digestion , and pegylation to prolong circulation times . [SEP]
[CLS] so far , amphiphilic B-property dna has been used to monitor metal B-material ions B-material , ph and chemical transmitters in cellular environment . [SEP]
[CLS] another notable example is the measurement of formation of lipid B-material membrane I-material domains to monitor and understand the dynamic signaling interactions on the cell B-material surface ( figure 6a ) . [SEP]
[CLS] to achieve this , a ssdna strand named s1 was anchored to the cell B-material membrane via a hydrophobic B-property lipid B-material unit and was partially hybridized with a blocking strand b . [SEP]
[CLS] similarly , a s2 strand was anchored at a second anchor site and was partially hybridized with a walker strand w . [SEP]
[CLS] an initiator strand can completely remove the blocking strand from the s1 strand by a strand displacement reaction , leaving s1 free for hybridization . [SEP]
[CLS] because strand w from the s2 site hybridizes preferentially with the free www . advancedscience . com s1 strand , it will translocate once both sequences are in close proximity . [SEP]
[CLS] to observe this displacement , strand s1 and strand w were labeled with a fluorescence B-property resonance energy transfer ( fret ) pair , leading to quenched fluorescence B-property . [SEP]
[CLS] the fret efficiency becomes a measure of the lipid B-material domain encounter rate since the dna amphiphiles B-property were anchored in different lipid B-material domains . [SEP]
[CLS] three lipid B-material tails were attached to the nucleic B-material acid I-material moieties , i . e . , diacyllipid , cholesterol , and tocopherol , to specifically adv . sci . 2019 , 6 , 1900043 figure 6 . [SEP]
[CLS] a ) schematic illustration of using a dna probe to measure the encounter rate of lipid B-material domains on live cell B-material membranes . [SEP]
[CLS] the s1 strand is anchored to the cell B-material membrane and is partially hybridized with a blocking strand b . [SEP]
[CLS] similarly , the s2 strand is anchored at a second anchor site and partially hybridized with a walker strand w . when an initiator stand i is introduced to the system , it removes strand b from s1 , leaving s1 free for hybridization . [SEP]
[CLS] since the walker strand from the s2 site has priority to hybridize with this free s1 strand over its own s2 strand , it will translocate from the s2 site to the s1 site once both strands meet each other . [SEP]
[CLS] since the s1 and s2 stands are labeled with fret dyes , once they encounter each other , the fluorescence B-property is quenched . [SEP]
[CLS] different hydrophobic B-property moieties attached the nucleic B-material acid I-material units introduce selectivity of the dna strands for certain lipid B-material domains . [SEP]
[CLS] thus the quenching rate is a measure to evaluate the encounter rate of different lipid B-material domains . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2017 , nature publishing group . b ) working principle of switchable aptamer micelle flares for atp imaging inside living cells B-material . [SEP]
[CLS] on the left , aptamers are folded , while upon binding the target molecule , the aptamer unfolds leading to a dequenching of fluorescence B-property ( right side ) . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2013 , american chemical society . [SEP]
[CLS] c ) two populations of cells B-material exhibit anchored dna on their membranes . [SEP]
[CLS] when mixed together , hybridization of membrane - embedded dna induces cell - cell contact . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2009 , national academy of sciences . [SEP]
[CLS] d ) illustration of microtissues constructed by dna hybridization . [SEP]
[CLS] sensing the extra and intracellular environmenta ) illustration of cell B-material adherence by dna hybridization . [SEP]
[CLS] one type of cell B-material is anchored with a particular dna strand on the membrane . [SEP]
[CLS] the other cell B-material type is functionalized with the complementary dna strand . [SEP]
[CLS] b ) when the two cell B-material types are mixed , hybridization induces cell - to - cell aggregation . [SEP]
[CLS] c ) formation of microtissue by dna hybridization . [SEP]
[CLS] iteration of this process allows assembling microtissues into the third dimension . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2015 , nature publishing group . [SEP]
[CLS] locate dna strands in different cellular lipid B-material domains . [SEP]
[CLS] this method transduces transient encounters of nanodomains into a cumulative cell B-material surface fluorescence B-property signal and thus allows to detect signaling events on live cell B-material membranes . [SEP]
[CLS] apart from probing the cell B-material surface , amphiphilic B-property dna was utilized for imaging and detecting intracellular parameters such as the level of atp . [SEP]
[CLS] a switchable aptamer - containing micelle B-material flare allowed detection of atp within cells B-material ( figure 6b ) . [SEP]
[CLS] this design implicated three segments with a dna layer that folds into an aptamer loop against atp . [SEP]
[CLS] the hydrophobic B-property segment was a diacyllipid tail , with a peg unit as spacer B-material between the dna and the hydrophobic B-property tail . [SEP]
[CLS] a fluorophore and a quencher were covalently attached to 3 ′ and 5 ′ ends . [SEP]
[CLS] once atp is binding , the dna loop opens , leading to an increase of fluorescence B-property . [SEP]
[CLS] due to the fact that the micelles B-material interacted with the cell B-material membrane and were internalized into cells B-material , atp in both membrane and cytosolic environment could be detected . [SEP]
[CLS] similar as the dna amphiphile mediated liposome B-nanoparticle assembly discussed in section 3 . 2 . 2 , when tethered onto the cell B-material membrane , the amphiphilic B-property dna facilitates cell B-material capture and assembly through the specific and fast recognition properties of the nucleic B-material acids I-material . [SEP]
[CLS] the length of the dna strands is of crucial importance for the successful cell to cell contact by hybridization . [SEP]
[CLS] a 20 - mer dna strand on the cell B-material surface cannot hybridize with its complementary sequence due to steric hindrance provided by the dense glycocalyx layer . [SEP]
[CLS] however , a 60 - and 80 - mer poly ( dt ) spacer B-material inserted between the lipid B-material anchor and the dna recognition element that will hybridize , significantly increases the cell B-event adhesion I-event to other surfaces . [SEP]
[CLS] eventually , dna - anchored on cell B-material surfaces can be linked to surfaceanchored complementary dna . [SEP]
[CLS] the accessibility of cell B-material membranes with anchored dna amphiphiles B-property also facilitates cell B-material assembly and microtissue formation . [SEP]
[CLS] in one example , bertozzi et al . linked nonadherent jurkat cells B-material together by employing dna anchored on their surfaces ( figure 6c ) . [SEP]
[CLS] this group found that the most important parameters for cell B-material assembly are the cell B-material concentration , dna density on the cell B-material surface , and dna sequence complexity . [SEP]
[CLS] since the cells B-material are attached to each other through dna hybridization , this process can be reversed by dnase addition or thermal melting . [SEP]
[CLS] this allows the construction of microtissues with defined cell B-material composition and stoichiometry . [SEP]
[CLS] this approach can be extended in a bottom - up strategy that uses a dna - patterned substrate as a template and temporary dna - based cellular adhesions as synthetic linkages B-property between cellular building blocks for tissue engineering in 3d ( figure 6d ) . [SEP]
[CLS] in this way , the construction of arrays of 3d cell B-material cultures with many tunable parameters was feasible . [SEP]
[CLS] in the same study , template dna was linked to a glass slide to form dna patterns . [SEP]
[CLS] then , a pdms flow channel was placed on top of the dna pattern . [SEP]
[CLS] a cell B-material population functionalized on the surface with complementary dna to the template dna was added to the flow channel , which directed the cells B-material to the designed 2d pattern . [SEP]
[CLS] the formed cell B-material pattern could be released by enzymatic cleavage of the dna . [SEP]
[CLS] embedding such microtissues constructed from dna in gels allows to study the influence of tissue size , shape and composition on cell B-material behaviors in 3d . [SEP]
[CLS] the extraordinary self - recognition and hybridization properties of dna can be applied for creating various programmable nanostructures . [SEP]
[CLS] an exceptional form of dna amphiphiles B-property are dna - based nanopores . [SEP]
[CLS] dna - based nanopores open exciting opportunities in the field of bio - nanotechnology , as shown by their protein - based counterparts . [SEP]
[CLS] single - stranded nucleic B-material acid I-material scaffolds together with staple strands or short oligonucleotides can fold into dna - based nanopores . [SEP]
[CLS] when conjugated to hydrophobic B-property units , the otherwise hydrophilic B-property nanopores insert into synthetic lipid B-material membranes I-material . [SEP]
[CLS] moreover , these nanopores interact with biological membranes ( figure 7a ) . [SEP]
[CLS] a notable example is a dna nanopore with a 2 nm opening and an outer diameter of 5 . 5 nm and a height of 14 nm , which contained a hydrophobic B-property belt with 72 ethyl phosphorothioates at the bottom of the pore to direct insertion into the cell B-material membrane . [SEP]
[CLS] after incubating B-technique these nanostructures with cervical cancer cells B-material , the dna nanopores mainly located at the membrane and caused cell B-event death I-event . [SEP]
[CLS] nanopores that did not contain a hydrophobic B-property belt were mostly internalized by the cancer cells B-material . [SEP]
[CLS] the cytotoxic B-property effect of dna - based nanopores could allow for anticancer B-property activity , albeit for true applications the selectivity needs to be improved . [SEP]
[CLS] apart from a cytotoxic B-property effect , dna nanostructures on cell B-material membranes enable the transport of membrane B-material lipids I-material . [SEP]
[CLS] a lipidscrambling dna nanostructure , consisting of only eight dna strands , which were modified by tetraethylenglycol ( teg ) cholesterol ( figure 7b ) , spontaneously inserts into biological membranes by forming a toroidal pore that connects the inner and outer leaflets of the membrane . [SEP]
[CLS] the inserted nanostructure facilitates the exchange of lipid B-material molecules between the inner and outer bilayer leaflets rapidly equilibrating the lipid B-material composition . [SEP]
[CLS] the rate of lipid B-material transport catalyzed by the dna nanostructure is three orders of magnitude higher than that reported for lipid B-material transport catalyzed by natural enzymes . [SEP]
[CLS] the stable dna - induced toroidal lipid B-material pore likely induces this exceptional transport behavior . [SEP]
[CLS] the dna - based artificial scramblase also showed translocation of phosphatidylserine lipids B-material from the inner membrane leaflet to the outer leaflet of human cancer cells B-material . [SEP]
[CLS] besides insertion , dna - origami nanodevices B-nanoparticle can be placed onto the surface of living cells B-material ( figure 7c ) . [SEP]
[CLS] the membrane can be functionalized by anchoring dna to the cell B-material surface via cholesterol insertion into the membrane , followed by binding of a bridge - oligonucleotide that partially hybridizes with this surface dna . [SEP]
[CLS] the bridging oligo allows binding of the membrane - bound breadboard ( mbb ) binding sites , but also offers the possibility of removal of this mbb from another surface via a strand displacement reaction . [SEP]
[CLS] several cell B-material types can be functionalized with mbbs , including primary , endothelial , and lymphoma cells B-material . [SEP]
[CLS] furthermore , the mbb can be released from cell B-material surfaces when a detachment strand is added . [SEP]
[CLS] by using dna origami nanodevices B-nanoparticle as engineering tools , mbb acts as a mediator www . advancedscience . com for either homotypic or heterotypic cell - cell interactions , which mimic complex biological processes on I-event the I-event cell B-material membrane . [SEP]
[CLS] dna - based materials have exceptional properties in regard to structural design . [SEP]
[CLS] compared to other building blocks like peptides B-material , proteins B-material , and synthetic macromolecules , dna allows the bottom - up construction of complex architectures and tuning the interaction energy between complementary dna strands . [SEP]
[CLS] recent progress in the design and functionalities of dna amphiphiles B-property builds on these remarkable properties to implement dna hybrid materials into the application areas of diagnostics and biomedicine . [SEP]
[CLS] these efforts are enabled by well - established protocols to synthesize amphiphilic B-property dna molecules and their commercial availability . [SEP]
[CLS] moreover , the topology and interactions of amphiphilic B-property dna is highly controllable , and their aggregation behavior into superstructures such as micelles B-material or vesicles , but also many other geometries can be precisely adjusted . [SEP]
[CLS] it is possible to tune their size , switch their assembly state , and modify their surfaces at will through duplex formation . [SEP]
[CLS] with their hydrophobic B-property units , amphiphilic B-property dna hybrids further provide a simple and efficient strategy for membrane modification of living cells B-material . [SEP]
[CLS] this simple functionalization procedure allows further cell B-material surface engineering , cell B-material assembly , and facilitates potential sensing applications . [SEP]
[CLS] despite these many favorable properties of dna amphiphiles B-property , certain challenges need to be overcome before translating them further toward the clinic . [SEP]
[CLS] one of the most critical issues is the biological stability . [SEP]
[CLS] although enhanced enzymatic stability was reported for dna amphiphile B-property micelles B-material , it remains a challenge to minimize nuclease degradation , especially in vivo . [SEP]
[CLS] next to this , upon exposure to biological medium , amphiphilic B-property dna structures are encapsulated by a protein B-material corona I-material , which possibly shields recognition elements on the surface and compromises its targeting efficiency . [SEP]
[CLS] another big challenge represents maintaining the solubility B-property of amphiphilic B-property dna in biological media and its activity on membranes . [SEP]
[CLS] proteins B-material from serum , like albumin or lipoproteins , are well known to form stable complexes with amphiphilic B-property dna , thus preventing their desired functions . [SEP]
[CLS] approaches to prevent such interactions of amphiphilic B-property dna with serum proteins B-material are urgently needed for extending biomedical applications . [SEP]
[CLS] furthermore , amphiphilic B-property dna molecules in micelle B-material assemblies are always in a dynamic equilibrium within their environment : strong [ 122 ] [SEP]
[CLS] b ) design of the lipid - scrambling dna nanostructure . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , nature publishing group . [SEP]
[CLS] c ) illustration of the sequential mbb functionalization steps . [SEP]
[CLS] oligos ( mio ) are first integrated into the cell B-material membrane , then bridge oligos hybridize with mio strands followed by bridge fortifier oligo hybridization . [SEP]
[CLS] lastly , membrane bound breadboard ( mbb ) binds to the cell B-material membrane by hybridizing with bridge oligos . [SEP]
[CLS] reproduced from ref . [ 124 ] . [SEP]
[CLS] www . advancedscience . com dilution after intravenous injection might result in concentrations below the cmc , which leads to disassembly of micelles B-material and drug release before reaching the target . [SEP]
[CLS] to prevent this , the biological stability of amphiphilic B-property dna micelle B-material needs to be adjusted to the desired delivery function . [SEP]
[CLS] it has been shown that covalent cross - linking of the lipid dna molecules can for example increase the stability of the assembled nanostructures , and therefore we foresee that this challenge will be overcome in the near future . [SEP]
[CLS] although the introduction of a hydrophobic B-property segment into the nucleic B-material acid I-material amphiphiles B-property is essential for their function , the biocompatibility B-property and biosafety of these hybrids should be taken into consideration , especially toward clinical translation . [SEP]
[CLS] for example , too many lipid - dna insertions in the membrane will lead to cell B-material membrane disturbance , damage , and cell B-event death I-event . [SEP]
[CLS] since the insertion mechanism into membranes , which is mediated by hydrophobic B-property interactions I-property , is not specific for a given cell B-material type , it is essential that additional features for selective incorporation are introduced . [SEP]
[CLS] a notable example of such an effort is labeling the dna amphiphiles B-property with folic B-material acid I-material to target cancer cells B-material . [SEP]
[CLS] at this stage , most studies on the biocompatibility B-property of dna amphiphiles B-property were conducted using cell B-material cytotoxicity B-property evaluation , while only a few studies investigated their local or systematic toxicity B-property in vivo . [SEP]
[CLS] as more and more dna amphiphiles B-property are developed for biomedical applications , these activities need to be extended for more comprehensive toxicological evaluations , such as cell B-material membrane damage , cell B-material signaling interference , oxidative stress , genotoxicity , etc . , which are required for predicting long - term biosafety . [SEP]
[CLS] since a lot of knowledge and control over synthesis and assembly mechanisms of dna amphiphiles B-property have been gained , we are in an excellent position to explore the unique properties of dna amphiphiles B-property when combined with hydrophobic B-property molecules . [SEP]
[CLS] similar as native protein B-material clusters on cell B-material membranes , dna nanostructures ( not limited to nanopores ) might act as artificial gate for intracellular / extracellular transportation , as means for cellular environment regulation and as tool to regulate cellular signaling . [SEP]
[CLS] from the perspective of synthetic biology , the exciting examples of interfacing dna amphiphiles B-property with membranes will fuel further activities regarding artificial cell B-material engineering , cell assembly and novel tissue formation . [SEP]
[CLS] moreover , dna amphiphiles B-property might find potential applications in cell - based therapy . [SEP]
[CLS] in addition to immune cells B-material , amphiphilic B-property dna nanostructure hitchhiked on other circulatory cells B-material merits more investigations . [SEP]
[CLS] taken together , dna amphiphiles B-property are at a stage where a large variety of nucleic B-material acid I-material materials is readily available , hence several structural designs were investigated in combination with living sytsems , especially addressing potential biomedical applications . [SEP]
[CLS] we predict a further growth in this area addressing more complex functions including the fields of oncology , vaccination and theranostics . [SEP]
[CLS] huo obtained his ph . d . degree from the national center for nanoscience and technology , chinese academy of sciences ( cas ) , in 2016 under the supervision of prof . xing - jie liang . [SEP]
[CLS] currently , he is a post - doctoral researcher working with prof . herrmann at the dwi - leibniz institute for interactive materials , germany . [SEP]
[CLS] his research focuses on engineered biomacromolecules and inorganic hybrid nanostructures for various medical and technological applications . [SEP]
[CLS] hongyan li received her m . sc . degree in materials science and engineering from xi ' an jiaotong university in 2014 . [SEP]
[CLS] she is presently a ph . d . student under the supervision of prof . herrmann at the university of groningen . [SEP]
[CLS] her research interests are focused on dna - based nanomedicine and other soft materials . [SEP]
[CLS] andreas herrmann studied chemistry at the university of mainz in germany . [SEP]
[CLS] from 1997 to 2000 he pursued his graduate studies at the max planck institute for polymer B-material research in the group of professor k . mullen . [SEP]
[CLS] in 2010 , he became a full professor at the zernike institute for advanced materials at the university of groningen in the netherlands . [SEP]
[CLS] since 2017 prof . herrmann is a scientific board member of the dwi - leibniz - institute for interactive materials in aachen , germany , and chair of macromolecular materials and systems at the institute of technical and macromolecular chemistry , rwth aachen university , germany . [SEP]
[CLS] 1 . selected lipid - oligonucleotide conjugates , exemplifying the variety of lipophilic B-property residues that can be appended to dna . [SEP]
[CLS] structures of dna conjugated with , from top to bottom , cholesterol obtained via a 1 , 3 - dipolar huisgen ' s cycloaddition between alkyne modified cholesterol and 5 ′ - azido - 5 ′ - deoxythymidine ; a single hydrocarbon chain obtained via a 1 , 3 - dipolar huisgen ' s reaction between alkyne modified c18 chain and 5 ′ - azido - 5 ′ deoxythymidine ; tocopherol obtained by covalent attachment to the 5 ′ end of the strand ; a single fluorocarbon chain obtained via a huisgen ' s reaction between 5 ′ - azide deoxythymidine and propargylated fluorocarbon chain ; a ppo chain obtained via a ppo phosphoramidite during sps ; double hydrocarbon chains obtained via a reaction of stearoyl chloride B-material with 1 , 3 - diamino - 2 - dydroxypropane ; and double fluorocarbon chains obtained by a diperfluorodecyl phosphoramidite during sps . [SEP]
[CLS] a ) enzymatic growth of dna - b - ppo micelles B-material . [SEP]
[CLS] reproduced from ref . [ 59 ] . [SEP]
[CLS] b ) schematic representation of hybridization of dna - b - ppo micelles B-material with different dna molecules . [SEP]
[CLS] figure 3 . a ) base pairing with a short complementary sequence yields micelles B-material and maintains the overall shape of the aggregates . [SEP]
[CLS] b ) hybridization with long dna templates results in rod - like micelles B-material . [SEP]
[CLS] reproduced from ref . [ 61 ] . [SEP]
[CLS] c ) dnazyme induced reversible transformation of the aggregate shape of a dna - brush block copolymer . [SEP]
[CLS] reproduced from ref . [ 62 ] . [SEP]
[CLS] d ) schematic of the mixed micelle B-material architecture . [SEP]
[CLS] two amphiphilic B-property block copolymers , dna - b - ppo and peo - b - ppo - b - peo with the trade name pluronic f127 , form mixed micellar structure and this micelle B-material can be stabilized by formation of a semi - interpenetrating network in its core B-material . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2010 , royal society of chemistry . [SEP]
[CLS] e ) schematic representation of the drug delivery system based on dna amphiphiles B-property . [SEP]
[CLS] a ) targeting units ( red dots ) that are connected to the complementary sequence of the micelles B-material are hybridized to equip the nanoparticle B-nanoparticle surface with folic B-material acid I-material units . [SEP]
[CLS] b ) the anticancer B-property drug ( green dots ) is loaded into the core B-material of the micelles B-material . [SEP]
[CLS] reproduced from ref . [ 55 ] . [SEP]
[CLS] f ) schematic of dna micelle - templated vc formation . [SEP]
[CLS] loading hydrophobic B-property molecules ( top , green ) into micelle B-material core B-material and hybridization of a complementary dna connected to functional moieties ( bottom , red ) to the dna micelle B-material . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2010 , american chemical society . [SEP]
[CLS] g ) photoinduced cross - linking of selfassembled dna - methacrylamide - lipid B-material micelles B-material . [SEP]
[CLS] green dot between dna and lipid B-material represents methacrylamide molecules which can be crosslinked . [SEP]
[CLS] reproduced from ref . [ 65 ] . [SEP]
[CLS] www . advancedscience . comadv . sci . 2019 , 6 , 1900043 [SEP]
[CLS] a ) illustration of working principle of reversible ph - responsive dnasome . [SEP]
[CLS] at ph 7 . 3 , c rich dna - pe spontaneously forms a dnasome . [SEP]
[CLS] when ph is lowered to 5 , the i - motif structure forms and the morphology of the dnasome transforms to entangled 3d networks . [SEP]
[CLS] reproduced from ref . [ 68 ] . [SEP]
[CLS] b ) schematic of the frame - guided assembly process with a dna origami scaffold . [SEP]
[CLS] dna origami cuboid with a20 sequences protruding from the surface is folded by a template and corresponding staple strands . [SEP]
[CLS] then , d t doeg dendron is anchored on dna origami by hybridization . [SEP]
[CLS] when g 2 cl - 18 is added , hydrophobic B-property groups on the dna origami guide g 2 cl - 18 dendrons to form hetero - vesicles around the dna frame . [SEP]
[CLS] reproduced from ref . [ 70 ] . [SEP]
[CLS] c ) size - controlled liposome B-nanoparticle formation through a dna scaffold . [SEP]
[CLS] a dna - origami ring ( red ) with multiple single - stranded empty handles is constructed first . [SEP]
[CLS] then dna antihandles ( oligonucleotides with complementary sequence to handle sequence that are chemically conjugated to dope , shown as green curl with orange head ) are hybridized to the dna ring . [SEP]
[CLS] afterward , this lipid - modified ring is mixed with extra lipid B-material and detergent , and dialysed to allow vesicle formation . [SEP]
[CLS] after purification and release , uniform liposomes B-nanoparticle with sizes being determined by the dna template are generated . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2016 , nature publishing group . [SEP]
[CLS] d ) schematic figure of pathogen dna delivery to protocell by dna - mediated fusion . [SEP]
[CLS] when anchoring a set of complementary dna on a protocell and an artificial pathogen membrane , dna hybridization brings the two membranes in close proximity to enable fusion . [SEP]
[CLS] thereby , pathogen dna is released into the protocell . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , american chemical society . [SEP]
[CLS] e ) illustration of reversible control over the assembly of liposomes B-nanoparticle . [SEP]
[CLS] when the liposome B-nanoparticle surface is equipped with self - complementary dna bearing a terminal azobenzene moiety , the vesicles undergo reversible assembly and disassembly in response to multiple stimuli including uv light , salt B-material addition and temperature . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2016 , american chemical society . [SEP]
[CLS] adv . sci . 2019 , 6 , 1900043 [SEP]
[CLS] a ) a membrane - spanning dna nanopore ( np B-nanoparticle ) with cytotoxic B-property activity and three negative control nanostructures . [SEP]
[CLS] figure 7 . a ) the np B-nanoparticle - ep pore is composed of a six - duplex bundle ( blue ) and a hydrophobic B-property belt ( purple ) made up of 72 ethyl phosphorothioate ( ep ) groups . [SEP]
[CLS] b ) inserting of np B-nanoparticle - ep pores into cellular membrane induces cell B-event death I-event . [SEP]
[CLS] c ) np B-nanoparticle - p features phosphorothioate groups but no hydrophobic B-property ethyl modification . d ) np B-nanoparticle contains native phosphate groups . e ) nnp contains ep groups but lacks three of the six strands required to generate the six - duplex bundle nanopore . [SEP]
[CLS] reproduced from ref . [ 122 ] . [SEP]
[CLS] b ) design of the lipid - scrambling dna nanostructure . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , nature publishing group . [SEP]
[CLS] c ) illustration of the sequential mbb functionalization steps . [SEP]
[CLS] oligos ( mio ) are first integrated into the cell B-material membrane , then bridge oligos hybridize with mio strands followed by bridge fortifier oligo hybridization . [SEP]
[CLS] lastly , membrane bound breadboard ( mbb ) binds to the cell B-material membrane by hybridizing with bridge oligos . [SEP]
[CLS] reproduced from ref . [ 124 ] . [SEP]
[CLS] the comprehensive understanding and proper use of supramolecular interactions have become critical for the development of functional materials , and so is the biomedical application of nucleic B-material acids I-material ( nas ) . [SEP]
[CLS] relatively rare attention has been paid to hydrophobic B-property interaction I-property compared with hydrogen B-material bonding and electrostatic interaction of nas . [SEP]
[CLS] however , hydrophobic B-property interaction I-property shows some unique properties , such as high tunability for application interest , minimal effect on na functionality , and sensitivity to external stimuli . [SEP]
[CLS] therefore , the widespread use of hydrophobic B-property interaction I-property has promoted the evolution of na - based biomaterials in higher - order self - assembly , drug / gene - delivery systems , and stimuli - responsive systems . [SEP]
[CLS] herein , the recent progress of na - based biomaterials whose fabrications or properties are highly determined by hydrophobic B-property interactions I-property is summarized . [SEP]
[CLS] 1 ) the hydrophobic B-property interaction I-property of na itself comes from the accumulation of base - stacking forces , by which the nas with certain base compositions and chain lengths show properties similar to thermal - responsive polymers B-material . [SEP]
[CLS] 2 ) in conjugation with hydrophobic B-property molecules , na amphiphiles B-property show interesting self - assembly structures with unique properties in many new biosensing and therapeutic strategies . [SEP]
[CLS] 3 ) the working - mechanisms of some na - based complex materials are also dependent on hydrophobic B-property interactions I-property . [SEP]
[CLS] moreover , in recent attempts , na amphiphiles B-property have been applied in organizing macroscopic self - assembly of dna origami and controlling the cell - cell interactions . [SEP]
[CLS] nucleic B-material acids I-material ( nas ) are macromolecules with well - defined molecular structures ; the combination of the four nucleotides ( a , t , g , c ) can precisely encode unlimited information , structures with certain bases . [SEP]
[CLS] therefore , the conformational change of na will drive signal transduction for the detection of these metal B-material ions B-material . [SEP]
[CLS] much more than all of these , the recognition capability of na can be extended to no limitations on targets upon the development of aptamers by systematic evolution of ligands by exponential enrichment ( selex ) technique . [SEP]
[CLS] aptamers are single - stranded dna or rna strands that can bind to targets with high affinity and specificity by folding into certain conformations . [SEP]
[CLS] to date , dna and rna aptamers have been widely used to construct biosensors and diagnostics through identifying various targets , up to bacteria and cell B-material , protein B-material , peptide B-material , amino B-material acid I-material , and down to small molecule and metal B-material ion B-material . [SEP]
[CLS] 2 ) many nas also show therapeutic functions , which can specifically inhibit the function of a particular gene involved in disease , mainly including antisense oligonucleotides ( asos ) and small interfering rnas ( sir - nas ) . [SEP]
[CLS] asos are single - strand dnas ( ssdnas ) in 8 - 50 nt lengths , which bind with mrna to block function or initiate degradation by endogenous ribonuclease h ( rnase h ) . [SEP]
[CLS] on the other side , sirna is a 20 - 28 nt long double - strand dna ( dsdna ) , which suppress gene expression through activating the rna - induced silencing complex ( risc ) and result in target mrna degradation . [SEP]
[CLS] in addition to gene regulation and therapy , unmethylated cytosine - phosphate - guanine ( cpg ) motif - containing dna ( cpg dna ) is also used as immunostimulants . [SEP]
[CLS] as a toll - like receptor B-material 9 ( tlr9 ) agonist , cpg dna can generate immune responses in cancer therapy . [SEP]
[CLS] although na shows many interesting biomedical functions , its effective pharmacological use is still facing challenges . [SEP]
[CLS] na is unstable in the bloodstream and rapidly cleared by the body , can induce immunogenicity B-property , and lack cell B-material membrane permeability . [SEP]
[CLS] therefore , different strategies have been developed to make nas an applicable material B-material . [SEP]
[CLS] all the strategies are based on the idea of densely packing nas to increase biostability and cell B-material uptake capability , which can be realized by covalent and noncovalent approaches . [SEP]
[CLS] an ssdna comprises hydrophobic B-property bases and negatively charged phosphate - sugar backbone . [SEP]
[CLS] thus the available interactions of dna include the hydrogen B-material bonding between nucleotides that can drive complementary strands together , the electrostatic attraction between the negatively charged dna and cations B-material or positively charged molecules , and the coordination interactions between dna bases and metal B-material ions B-material . [SEP]
[CLS] moreover , by the matured synthesis method , dna can be easily modified with a lot of functional groups for further conjugation reactions . [SEP]
[CLS] 1 ) electrostatic interactions are the electric force between any two charged molecules . [SEP]
[CLS] as dna is a negatively charged polyelectrolyte , multivalent cations B-material become the most - used nonviral transfection vectors for gene therapy , including cationic B-material lipids B-material , short - chain polyamides ( e . g . , spermine and spermidine ) , natural and synthetic poly ( amino acids ) , cationic B-material water - I-property soluble polymers B-material ( e . g . , linear and branched polyethyleneimine ) , and cationic B-material amphiphilic B-property polymers B-material . [SEP]
[CLS] this method is efficient and straightforward , but it remains some limitations . [SEP]
[CLS] for instance , generally , cationic B-material polymers B-material show low condensation efficiencies to short nas , such as sirna and aso . [SEP]
[CLS] also , to construct a stable polyplex , the charge ratio between the cationic B-material polymer B-material and dna should be larger than 10 , and the excessive amount of cationic B-material polymer B-material will leilei tian is an associate professor at southern university of science and technology . [SEP]
[CLS] she obtained her ph . d . degree in polymer B-material chemistry and physics from jilin university , where she specialized in synthesis and supramolecular chemistry of organic functional materials . [SEP]
[CLS] she worked as a postdoctoral researcher at the university of chicago , working on developing dna nanobiotechnology for the sensitive detection of diseaserelated biomolecules . [SEP]
[CLS] her research interests focus on designing smart , biologically active , functional materials from the viewpoint of supramolecular chemistry . [SEP]
[CLS] her research group recently engages in developing dna conjugated / complexed materials , which will have interesting applications in biosensors and biomedicines . [SEP]
[CLS] result in high cytotoxicity B-property . [SEP]
[CLS] the strong interaction between the two charged polymers B-material will ensure the biostability of dna during the blood circulation . [SEP]
[CLS] however , the resultant polyplex generally lacks the stimuli - responsive capability , resulting in difficulties in the proper release of therapeutic nas at the cytoplasm or nucleus . [SEP]
[CLS] 2 ) the hydrogen B-material - bonding interactions following the elegant watson - crick base - pairing rule have been widely applied in the fabrication of nanostructures with precise dimensions and smart properties , which makes dna become the most useful building material B-material in nanoscience and technology . [SEP]
[CLS] the most representative technique is dna origami , which fabricates nanostructures by utilizing hundreds of short single strands ( staple strands ) to fold an m13 phage dna into the desired structures . [SEP]
[CLS] recently , ding and co - workers utilized origami technique to construct an autonomous dna robot , which could deliver thrombin specifically to tumor B-material - associated blood vessels and induce intravascular thrombosis , resulting in tumor B-material necrosis and inhibition of tumor B-material growth . [SEP]
[CLS] this work proved that it is potential to apply the dna origami technique in precise and smart drug delivery for cancer therapy . [SEP]
[CLS] however , the disadvantages , such as the complicated fabrication process and the high cost , will also limit its practical applications . [SEP]
[CLS] 3 ) the coordinative or electrostatic interactions between the nucleobases / phosphate backbones of dnas and metal B-material ions B-material provide another approach to fabricate functional dna - metal hybrid nanomaterials B-material . [SEP]
[CLS] on the one hand , the dna sequences act as nucleation sites and the stabilizing ligands for metal B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] on the other hand , the metal B-material components will endow more functionalities to the dna - metal B-material hybrid nanomaterials B-material , promoting their applications in the biological / chemical detecting , cellular and in vivo imaging , and therapeutics . [SEP]
[CLS] recently , li and co - workers utilized the coordination - driven self - assembly of fe 2 + ions B-material and dnas to produce dna nanostructures with well - controlled morphologies and functionalities . [SEP]
[CLS] the dna - fe hybrid nanomaterials B-material showed a high cellular permeability , which could efficiently deliver functional dnas to cells B-material and showed a high in vivo therapeutic efficacy . [SEP]
[CLS] 4 ) dna can be conjugated to the surface of other nanomaterials B-material through the functional groups that are chemically modified at the ends of dna strands , which can also efficiently protect dna from enzymatic cleavage and enhance the cell B-material permeability . [SEP]
[CLS] the most famous example is the conjugation between the thiol - modified dnas and gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) , which is a very reactive and efficient reaction . [SEP]
[CLS] on the one hand , aunp B-nanoparticle is a very efficient fluorescence B-property quencher I-property , which can combine with the molecular recognition properties of dna to develop " nanoflares " for intramolecular imaging and sensing applications . [SEP]
[CLS] nowadays , aunp B-nanoparticle - dna conjugates have become a very ideal platform for the design of biosensors . [SEP]
[CLS] on the other hand , dna strands are densely conjugated on the surface of aunps B-nanoparticle to form a closely packed and orientated dna shell B-material . [SEP]
[CLS] the " cluster effect " of dna makes the aunp B-nanoparticle - dna structures ( which are called spherical nucleic B-material acids I-material in some studies ) show some distinctive properties , such as an enhanced nuclease resistance , a higher cellular uptake capability , and which even show brain - blood barrier B-property crossing capability . [SEP]
[CLS] therefore , these properties also make aunp B-nanoparticle - dna structures perfect systems for gene delivery . [SEP]
[CLS] except for the above - mentioned methods , hydrophobic B-property interactions I-property may provide another possible force to construct dnabased nanomaterials B-material for biomedical applications . [SEP]
[CLS] hydrophobic B-property interaction I-property , also known as hydrophobic B-property effect , is a kind of property of nonpolar molecules ( or hydrophobic B-property moieties of amphiphiles B-property ) , which can drive these molecules to assemble to form anhydrous domains in aqueous solution . [SEP]
[CLS] essentially , the source of the hydrophobic B-property effect is the entropy effect caused by nonpolar solutes destroying hydrogen B-material bonds between water B-material molecules . [SEP]
[CLS] in biophysics , hydrophobic B-property interactions I-property play an essential role in the 3d structure of proteins B-material . [SEP]
[CLS] for a globular protein B-material , its surface is usually surrounded by a layer of hydrophilic B-property residues in aqueous solution , and residues with hydrophobic B-property side chains are usually inside the protein B-material . [SEP]
[CLS] as for the synthetic amphiphilic B-property block - copolymers , hydrophobic B-property interaction I-property is also very important in its assembly ; as the controlled " living " polymerization technique has been well developed , the molecular weights of the hydrophobic B-property and hydrophilic B-property blocks can be precisely tuned to control the degree of hydrophobic B-property interactions I-property , by which the block copolymers can self - assemble into nanoarchitectures with various sizes , morphologies , and smart stimuliresponsive properties , making them distinctive in drug / gene delivery applications . [SEP]
[CLS] regarding constructing dna - based biomaterials through hydrophobic B-property interactions I-property shows several advantages . [SEP]
[CLS] 1 ) hydrophobic B-property interaction I-property can stabilize dna to enhance its biostability . [SEP]
[CLS] 2 ) hydrophobic B-property interactions I-property can enhance the interaction between dna and other nanomaterials B-material to fabricate functional nanocomposites . [SEP]
[CLS] 3 ) hydrophobic B-property interaction I-property is dynamic and highly adjustable , which will allow the desired materials to respond more sensitively to environmental stimuli , enabling the design of smarter biomaterials . [SEP]
[CLS] 4 ) higherorder self - assembly structures will be constructed by introducing hydrophobic B-property interaction I-property into the structural dna assembly . [SEP]
[CLS] intrinsically , dna is a polymer B-material whose molecular weight and conformation can be well - defined by the sequence . [SEP]
[CLS] therefore , dna shows overwhelming advantages in the accurate control of hydrophobic B-property interactions I-property for further self - assembly . [SEP]
[CLS] for one thing , dna shows some amphiphilic B-property nature as its base is hydrophobic B-property , and its phosphate backbone is hydrophilic B-property . [SEP]
[CLS] hence the length change , hybridization / dehybridization , and conformation change ( e . g . , i - motif and g - quadruplex ) of dna can influence the hydrophobicity B-property of dna and eventually affect the assembly behavior of dna - based materials . [SEP]
[CLS] the hydrophobic B-property interaction I-property will not only affect the condensation state of dna and also play an important role in determining the properties of its complex with other nanomaterials B-material . [SEP]
[CLS] for another , dna can be used as the hydrophilic B-property block to synthesize dna - based amphiphilic B-property block copolymer , and the resultant self - assemblies can well reserve the functionality of dna and generate many new properties owing to the hydrophobic B-property interactions I-property . [SEP]
[CLS] in this regard , the hydrophobicity B-property of the hydrophobic B-property block can also be tuned to control the properties of dna - based amphiphilic B-property block copolymer . [SEP]
[CLS] due to the interesting and important role of hydrophobic B-property interactions I-property in dna - based materials , in this review , we will summary the recently developed strategies that mainly use hydrophobic B-property interactions I-property to construct dna - based biomedical materials ( figure 1 ) , including 1 ) hydrophobic - hydrophilic phase separation for intrinsic dna condensation ; 2 ) biomedical materials based on the selfassembly of nucleic B-material acid I-material amphiphiles B-property ; and 3 ) biomedical materials based on the hydrophobic - interaction - stabilized complexes between dna and other nanomaterials B-material . [SEP]
[CLS] many investigations have demonstrated that the dna double helix is mainly stabilized by coin - pile stacking of base pairs and less by the hydrogen B-material bonding between matched bases that most textbooks still refer to . [SEP]
[CLS] stacked bases attract to one another through van der waals forces ; the energy associated with a single interaction has a little significance to the overall dna structure , however , the net effect summed over the numerous ones , results in substantial stability . [SEP]
[CLS] therefore , the hydrophobic B-property base stacking has been considered as the primary contributor to dna doublehelix stability . [SEP]
[CLS] a recent study has revealed that the hydrophobic B-property interaction I-property of dna plays an important role in biological activity , which may have catalytic roles to activate dna polymerase . [SEP]
[CLS] as a result , instead of utilizing hydrogen B-material - bonding base - pairing , the hydrophobic B-property interaction I-property is also necessary to construct dna nanostructures for biomedical applications . [SEP]
[CLS] recently , an interesting phenomenon has been observed from rolling circle amplification ( rca ) , in which a part of the rca product is directly converted to flower - like microsized particles after the reaction . [SEP]
[CLS] motivated by this observation , low - cost functional dna materials fabricated by rca was put forward for its superior biostability endowed by its densely compacted flowerlike dna nano / microstructure . [SEP]
[CLS] rca is a classic isothermal amplification technique , which can efficiently produce a high quantity of long ssdna ( lssdna ) ; the sequence of the lssdna is well defined by and repeatedly copied from the circular template . [SEP]
[CLS] as a result , multiple functional sites are polymerized together into an rca product . [SEP]
[CLS] it has been investigated that the flower - like dna structure was formed by the twine of long flexible rca products , the process of which was induced by the high concentration of magnesium B-material pyrophosphate ( mgppi ) , a byproduct of rca reaction . [SEP]
[CLS] according to our investigations , other than mgppi , the addition of an appropriate concentration of mg 2 + could also condense the rca product and induce the formation of a similar dna structure with biostability against enzymatic degradation . [SEP]
[CLS] the rca nanoparticles B-nanoparticle condensed by excessive mg 2 + showed a smaller size of ≈100 nm , which can still keep intact and stable after going through the desalting column . [SEP]
[CLS] there are three critical factors for the condensation of the rca products and the subsequent biomedical applications . [SEP]
[CLS] 1 ) the aromatic structure of the dna base is hydrophobic B-property , and the phosphate backbone is hydrophilic B-property . [SEP]
[CLS] a dna strand can be well dissolved in aqueous solution without phase separation because the charge of its phosphate backbone allows the dna strands to repel each other and achieves a good dispersion . [SEP]
[CLS] therefore , for an efficient condensation , a high concentration of counter - ions B-material is required to screen the surface charge of dna . [SEP]
[CLS] 2 ) oligonucleotides with low molecular weights cannot be condensed by mg , which suggested that the polymer B-material nature of the enzymatically synthesized lssdna is essential for the condensation process . [SEP]
[CLS] generally , compared with small molecules , polymers B-material show stronger intramolecular B-property interactions I-property and more chain flexibility . [SEP]
[CLS] therefore , only a long dna strand with significant hydrophobic B-property base stacking can go through hydrophobic - hydrophilic phase separation . [SEP]
[CLS] 3 ) poor biostability is the biggest obstacle for the practical application of therapeutic nas . [SEP]
[CLS] for biomedical applications , the rca technique can polymerize multiple strands of functional nas to yield lssdna with super - high molecular weight . [SEP]
[CLS] also , the produced lssdna shows a highly increased tendency to form condensed structures with high biostability . [SEP]
[CLS] as a result , the lssdnas produced by rca can act as both the polymer B-material carrier and therapeutic nas to construct self - delivered all - dna nanomedicines for chemo - / gene therapy ( figure 2a ) . [SEP]
[CLS] in another research , walther et al . demonstrated that the rca products show physical properties similar to thermoresponsive copyright 2018 , nature publishing group . [SEP]
[CLS] polymers B-material , which would go through hydrophobic - hydrophilic phase separation under a certain temperature [SEP]
[CLS] also , they studied the key factors that will affect the phase separation process . [SEP]
[CLS] first , they found that only purine - rich lssdna ( poly a and poly g ) exhibited heat - induced phase separation in the presence of 50 × 10 −3 m mg 2 + ( figure 2b ) . [SEP]
[CLS] in contrast , pyrimidine - rich lss - dna did not show phase separation . [SEP]
[CLS] second , the phase separation due to hydrophobic B-property interaction I-property depends on the degree of polymerization of the dna . [SEP]
[CLS] the authors observed that as the length of the lssdna increases , the cloud point temperature ( i . e . , the temperature at which phase separation occurs ) decreases ( figure 2c ) , which may be due to the increased length of ss - dna , resulting in a stronger hydrophobic B-property interaction I-property that makes phase separation easier . [SEP]
[CLS] it is worth noting that when the length of dna is shorter than ≈100 nucleobases , the dna remains in a dissolved state regardless of the temperature . [SEP]
[CLS] finally , the authors studied the effect of counter - ions B-material on the phase separation of lssdna . [SEP]
[CLS] they found no phase separation observed in the te buffer ( 10 × 10 −3 m tris , 1 × 10 −3 m ethylenediaminetetraacetic acid ( edta ) , ph = 8 ) without mg 2 + , which is because the charge on the phosphate skeleton is not shielded , and the dna exhibits a state of electrostatic stability . [SEP]
[CLS] for lssdna containing poly a of ≈1000 bases in length , the phase transition caused by hydrophobic B-property interaction I-property occurs at ≈75 °c , 17 . 5 × 10 −3 m mg 2 + . [SEP]
[CLS] when the mg 2 + concentration is increased to 100 × 10 −3 m , the cloud point temperature reduces to 40 °c . [SEP]
[CLS] the counter - ion B-material was changed from mg 2 + to ca 2 + ( calcium B-material chloride B-material , cacl 2 ) , and a similar phase tran - sition was observed ( figure 2d ) . [SEP]
[CLS] note that large alkaline earth elements ( such as barium chloride B-material , barium chloride B-material , ≤100 × 10 −3 m ) do not cause heat - induced phase separation behavior of lss - dna , while the transition metal B-material ( zinc B-material chloride B-material , manganese B-material chloride ) can cause phase separation of lssdna at room temperature ( ≥20 × 10 −3 m ) . [SEP]
[CLS] we can conclude from the above work that , when the surface charge of dna is screened by the presence of counter - ions B-material , the rca - produced lssdna shows physical properties similar to thermoresponsive polymers B-material . [SEP]
[CLS] a certain temperature would induce hydrophilic B-property and hydrophobic B-property phase separation , distinct from the properties of oligonucleotides . [SEP]
[CLS] such phase separation behavior is related to the composition and the length of the nas , the temperature , and the concentration of counter - ions B-material . [SEP]
[CLS] indeed , besides the hydrophobic B-property interaction I-property , some other factors also contribute to the self - driving condensation of lssdna , such as the nonspecific hydrogen B-material - bonding and purine - purine [UNK] - [UNK] stacking . [SEP]
[CLS] the formation process of nanoparticles B-nanoparticle by lssdna can be inferred as the following . [SEP]
[CLS] the lssdna can be well dispersed in water B-material due to the electrostatic repulsion of phosphate backbone . [SEP]
[CLS] a certain concentration of counterions , especially mg 2 + , can screen the surface charge of lssdna . [SEP]
[CLS] the aromatic structures of lssdna are hydrophobic B-property , showing the tendency of aggregation and resulting in two situations . [SEP]
[CLS] if lssdna is super long , the phase separation of lssdna will be observed without heat treatment ; or , if lssdna is of medium length ( ≈1000 base ) , it needs to be heated to cause phase separation . [SEP]
[CLS] chemotherapy has become the indispensable cancer - treatment method nowadays ; however , this method shows serious drawbacks as anticancer B-property drugs would nonspecifically attack both cancer cells B-material and nonlesional normal cells . [SEP]
[CLS] therefore , tumor - targeted and microenvironment - responsive chemotherapeutics have attracted a lot of research interest . [SEP]
[CLS] functional nucleic B-material acids I-material ( fnas ) have been widely applied to realize targeted drug delivery and release by its specific recognition ability . recently , the pure dna nanostructures , like dna origami , become famous as the delivery system for its precise and smart controls . [SEP]
[CLS] at the same time , they still face many challenges hindering its biomedical application , such as the unsatisfactory biostability for in vivo application , the tedious fabrication strategy , and the high expense of a large number of oligonucleotides . [SEP]
[CLS] the low - stability in the serum - containing medium is the foremost problem , the primary solutions to which includes reducing the number of nick sites , improving compaction density of dna , and external encapsulation protection . [SEP]
[CLS] our group employed the rca method as mentioned above to fabricate targeted chemotherapy - based nanostructures . [SEP]
[CLS] first , a carefully designed template was replicated through an rca reaction to yield a large quantity of lssdnas with periodic sequences , and each repeat is comprised of an aptamer sequence with the ability to target cancer cells B-material and a hairpin sequence for doxorubicin B-material ( dox ) - loading and ph - responsive release ( figure 3a , b ) . [SEP]
[CLS] after that , a certain concentration of mg 2 + could effectively cause the firm condensation of the lssdna due to the hydrophobic B-property interaction I-property , forming nanoparticles B-nanoparticle with a small size of ≈100 nm ( mg - rnc ) . [SEP]
[CLS] besides , the mg - rnc could remain intact and stable after removing the excess mg 2 + through desalting . [SEP]
[CLS] the mg / p ratios ( molar ratio of mg 2 + to dna phosphate groups ) play an important role in the formation of mg - rnc . [SEP]
[CLS] as the mg / p ratio increased from 0 to 200 , the r g / r h ( r g : radius of gyration , r h : hydrodynamic radius ) ratio decreased from about 1 . 5 to 0 . 77 , which were monitored by laser light scattering ( lls ) . [SEP]
[CLS] this phenomenon indicates that lssdna has changed from a random coil conformation to a solid spherical structure . [SEP]
[CLS] however , hardly any obvious conformation change could be observed if short oligonucleotides were mixed with excessive mg 2 + , which indicates that the high molecular weight is of great significance to the mg - rnc formation . [SEP]
[CLS] transmission B-technique electron I-technique microscopy I-technique ( tem ) images of mg - rnc nanoparticles B-nanoparticle showed that the nanostructures are like " fried eggs " with relatively dense internal and irregular perimeters ( figure 3c ) . [SEP]
[CLS] the stability of the drug delivery system in serum is of great significance for in vivo experiment . [SEP]
[CLS] compared with the pure rca product , mg - rnc nanoparticles B-nanoparticle showed no significant degradation after a 12 h incubation B-technique in serum medium ( figure 3d ) . [SEP]
[CLS] the aptamer ( sgc8 ) targeting the receptor B-material protein B-material tyrosine B-material kinase 7 ( ptk7 ) on the cell B-material membrane was integrated into the rca product . [SEP]
[CLS] these receptors are overexpressed in the human leukemic cell B-material line ( cem ) while low expressed in human lymphoma cell B-material line ( ramos ) . [SEP]
[CLS] the dox - loaded , targetingcapable nanoparticles B-nanoparticle ( mg - rcn1 @ dox ) were incubated B-technique with cem and ramos cells B-material , respectively . [SEP]
[CLS] the results showed that mg - rcn1 @ dox only exhibited significant cytotoxicity B-property to cem , indicating that the prepared nanoparticles B-nanoparticle did show the selectively targeted cytotoxic B-property effects ( figure 3e ) . [SEP]
[CLS] due to the high stability of mg - rcn nanoparticle B-nanoparticle , it can be used for targeted drug delivery in vivo . [SEP]
[CLS] different groups of samples were injected intravenously into tumor B-material - bearing mice . [SEP]
[CLS] after 24 h of in vivo distribution , the mice were sacrificed , and the heart , liver , spleen , lung , and kidney were collected ; and the fluorescence B-property distribution of dox was observed . [SEP]
[CLS] the results showed that mg - rcn nanoparticles B-nanoparticle accumulate more concentrated in the tumor B-material than free dox , and the aptamer sequence improved the in vivo accumulation of nanoparticles B-nanoparticle at the tumor B-material site ( figure 3f ) . [SEP]
[CLS] in a word , the counter - ion B-material mg 2 + can drive lssdnas rather than short oligonucleotides from the random coil conformation to the compact solid spherical structure . [SEP]
[CLS] dna is a polyelectrolyte , the compaction of which could be restricted by charge - repulsions . [SEP]
[CLS] when the surface charge of dna is fully screened by mg 2 + , the intramolecular B-property interactions I-property ( hydrophobic B-property interaction I-property in the majority ) were enhanced due to the high - molecular - weight of lss - dna , and phase separation will take place to condense dnas to form nanostructures , which are stable enough for in vivo chemotherapeutic applications . [SEP]
[CLS] in summary , multifunctional lss - dna for targeted chemotherapy can be produced by rca reaction , and the stable self - delivery nanomedicines with superior biocompatibility B-property could be further fabricated by the simple addition of mg 2 + . [SEP]
[CLS] overall , this new strategy of exploiting the rca technique and mg 2 + condensation , can fabricate nanoparticles B-nanoparticle with a nontoxic composition through a simple fabrication process and provides an efficient way to preserve and promote dna functions , which shows the great potential for broad applications in the biomedical field . [SEP]
[CLS] rna interference ( rnai ) has proven to be an effective treatment strategy . [SEP]
[CLS] however , the biggest hurdle that hinders rnai therapy is the inefficient delivery . [SEP]
[CLS] short - chain length with a rigid structure makes double - stranded sirna more difficult to form stable complexes with cationic B-material transfection agents compared with the long plasmid dna . [SEP]
[CLS] moreover , these unstable complexes are more enzyme sensitive , giving rise to the early release of sirna , which results in low cellular uptake , low serum stability , and even nonspecific immune responses . [SEP]
[CLS] in addition , excessive dosage of cationic B-material transfection agents can also pose many biosafety problems . [SEP]
[CLS] dna nanomaterials B-material stabilized by hydrophobic B-property interaction I-property for gene - therapysirna polymerization has been proven to be a very promising strategy in enhancing the efficiency of sirna delivery by changing the low - charge and rigid properties of single sirnas . [SEP]
[CLS] comparing with the methods of direct sirna linkage B-property , herein , we developed a novel method exploiting the rca product to facilitate the high degree polymerization of sirna and the subsequent formation of the robust complex with transfection agent poly ( ethylenimine ) ( pei ) . [SEP]
[CLS] in this work , we carefully investigated and elucidated the role of the rca lssdna as a cocarrier material B-material for sirna delivery . [SEP]
[CLS] e ) the cytotoxicity B-property of mg - rnc1 @ dox nanoparticles B-nanoparticle on targeted cem cells B-material and nontargeted ramos cells B-material . [SEP]
[CLS] f ) the distribution of mg - rnc1 @ dox , mg - rnc2 @ dox ( without targeted aptamer sequences ) , and free dox in the main organs after intravenous injection . [SEP]
[CLS] from left to right are the hearts , lungs , livers , spleens , kidneys , and tumors B-material . [SEP]
[CLS] the pseudocolor indicates the fluorescence B-property intensity of dox . [SEP]
[CLS] all panels are reproduced with permission . [SEP]
[CLS] copyright 2018 . american chemical society . [SEP]
[CLS] the enzymatically synthesized lssdna shows polymer - like properties ; therefore , its interaction with pei is highly dependent on its molecular weight ( mw ) . [SEP]
[CLS] since the mw and functionality of the dna can be easily tailored according to the application of interest , which makes it surpass the other synthetic polymers B-material . [SEP]
[CLS] through a simple sequence optimization , sirnas could be efficiently hybridized to the rca cocarrier , and the hybrid could be more efficiently complexed by pei ( figure 4a ) . [SEP]
[CLS] we revealed that the length of the binding site was vital to efficient hybridization . [SEP]
[CLS] the sirna with an 18 - base dna tail ( dna 18 - sirna ) was optimized to show a 100 % graft efficiency , while neither dna 12 - sirna nor dna 6 - sirna showed satisfactory hybridization efficiency . [SEP]
[CLS] therefore , with the efficient hybridization , hundreds of sirnas could be grafted to a single rca chain , which will be the most efficient way among all the methods based on the sirna " polymerization " strategy . [SEP]
[CLS] the hybrid of sirnas and the rca cocarrier could be complexed by pei more efficiently . [SEP]
[CLS] different from sirna , the rca product with a large spatial charge density and high molecular flexibility was proved to show stronger affinity to pei , in which the sufficient complexation all panels are reproduced with permission . [SEP]
[CLS] copyright 2019 . american chemical society . [SEP]
[CLS] with pei was achieved when the charge was completely neutralized . [SEP]
[CLS] we found that the interaction between dna and pei is mw - dependent , and the higher mw of rca product brought the better stability of the polyplexes . [SEP]
[CLS] the chain flexibility was decreased due to the introduction of more double - stranded sites to rca cocarriers during hybridization . [SEP]
[CLS] however , the rca - sirna hybrid system exhibited similar pei complexation performance as rca products . [SEP]
[CLS] the loose complexation of pei / sirna is challenged by circulating nuclease degradation , renal clearance , and the reticuloendothelial system uptake . [SEP]
[CLS] although the excessive use of pei can improve the stability of pei / sirna polyplexes , it will induce severe cytotoxicity B-property at the same time . [SEP]
[CLS] starting from the n pei / p sirna ratio of 2 ( approximately at the charge neutralization point ) , the complexation structure of pei / rca - sirna could be well preserved in the biological environment , which ensured the further success of the polyplexes in cellular and in vivo applications ( figure 4b ) . [SEP]
[CLS] also , pei / rca - sirna polyplexes showed efficient cell B-material uptake and proper sirna release . [SEP]
[CLS] finally , based on all these good properties , the best rnai efficiency was determined to be 80 % produced by pei / rca - sirna , which was better than the commercially available lipofectamine under the optimized conditions ( ≈60 % ) . [SEP]
[CLS] pei / rca - sirna polyplex was selected for the in vivo transfection investigation , which exhibited an in vivo gene suppression efficiency of 50 % ( figure 4c ) . [SEP]
[CLS] on the contrary , the pei / sirna complexes did not cause a significant decrease in the in vivo luciferase expression . [SEP]
[CLS] in summary , free of any chemical processes , the very biocompatible B-property rca cocarrier could hybridize with hundreds of sirnas , which could be sufficiently complexed by a reduced amount of pei , resulting in a polyplex with low cytotoxicity B-property and improved rnai efficiency both in vitro and in vivo . [SEP]
[CLS] therefore , the potentials of the rca cocarrier on improving sirna delivery have been carefully proved , making this strategy promising for rnai applications in the future . [SEP]
[CLS] with the excellent physical properties of good water B-material solubility B-property , convenient preparation , and high fluorescence B-property , oligonucleotidestabilized silver B-material clusters ( agncs ) have gained increasing attention in the biomedical field . [SEP]
[CLS] despite their rapid development , the low photo - stability is still the major shortcoming of most oligonucleotide - stabilized agncs . [SEP]
[CLS] especially for the agncs with the ability to emit red light , their emitting light would turn from red to green gradually within 24 h . [SEP]
[CLS] therefore , our group tried to protect agncs with long and condensed rca products to improve their stability for biological applications . [SEP]
[CLS] first , cytosine - rich rca products were synthesized , which were crosslinked by ag + to form dna hydrogels . [SEP]
[CLS] after reduction , the prepared fluorescent B-property rca - stabilized agncs were carefully characterized ( figure 5a ) . [SEP]
[CLS] the ( dynamic B-technique light I-technique scattering I-technique ) dls analysis showed that the diameter of the rca - agnc complex was about 230 nm , and the diameters of agncs were determined to be ≈2 nm characterized by tem . [SEP]
[CLS] therefore , the nanocomposites of rca - agnc complexes are agncs encapsulated within the condensed rca sequences , in which the hydrophobic B-property interaction I-property of rca products played a considerable role to enhance the stability of agncs . [SEP]
[CLS] in comparison with agncs stabilized by oligonucleotide , rca - stabilized agncs showed greatly enhanced photo - and thermostability and declined toxicity B-property . [SEP]
[CLS] as for photostability , the red emission of oligonucleotide - agncs decreased 80 % in the ambient environment after 5 days preservation from light , while the rca - agncs showed superior photostability that there was only a little decline in red emission intensity even after 30 days under the same condition . [SEP]
[CLS] upon 10 h exposure to sunlight , the intensity of the red emissions of the oligonucleotide - agncs and rca - agncs decreased by 70 % and 15 % , respectively . [SEP]
[CLS] for the thermostability , when incubated B-technique at ) illustration of rca - agncs with higher photostability for detecting ros / rns . [SEP]
[CLS] c ) titration curves of the rca - agncs with an increase in • oh concentration ; the green emissions were excited at 440 nm and the red emissions were excited at 560 nm . [SEP]
[CLS] d ) using the rca - agncs as a ratiometric fluorescent B-property probe to monitor the dynamic levels of ros / rns in lipopolysaccharide - treated a549 cells B-material at various incubation B-technique times . [SEP]
[CLS] all panels are reproduced with permission . [SEP]
[CLS] copyright 2018 , american chemical society . [SEP]
[CLS] 37 °c for 60 min , the rca - agnc remained ≈90 % of its original fluorescence B-property , while the oligonucleotide - agncs lost ≈80 % of its fluorescence B-property . [SEP]
[CLS] the selective detection of reactive oxygen B-material or nitrogen B-material species ( ros / rnss ) would be benefited from the superior photostability of rca - agncs , as the common physical factors that cause emission quench could be ignored except for the ros / rnss ( figure 5b ) . [SEP]
[CLS] the reversible response of agncs to redox was improved due to the protection by rca products . [SEP]
[CLS] therefore , the rca - agncs with comparable green or red emission were fabricated by rational dna sequences design to realize multicolor cell B-material imaging and ros / rnss ratiometric detection . [SEP]
[CLS] the ratios of emission integrations ( r = i green / i red ) between the ranges of 500 - 560 nm ( from the green - emission of agncs ) and 610 - 700 nm ( from the red - emission of agncs ) in the absence and the presence of ros / rns , were calculated to evaluate the sensitivity of rca - agncs in response to certain ros / rns species ( figure 5c ) . [SEP]
[CLS] the ratio significantly increased over 10 times for the presence of • oh , slightly increased by ≈3 times for single oxygen B-material ( 1 o 2 ) , and negligibly increased for the other ros / rns , such as alkylperoxyl radical ( roo • ) , hypochlorite ( clo − ) , peroxynitrite ( onoo − ) , and h 2 o 2 . [SEP]
[CLS] all the results certified that rca - agncs showed the best selectivity for the detection of • oh ( the most reactive form of oxygen B-material ) , with a low limit of detection ( lod ) reaching 58 × 10 −9 m . [SEP]
[CLS] in the meantime , rca - agncs could be successfully applied to monitor the dynamic levels of ros / rns in lipopolysaccharides treated human lung cancer ( a549 ) cells B-material ( figure 5d ) . [SEP]
[CLS] these results demonstrated that the rca - agnc with outstanding fluorescence B-property property and application - oriented advantages is a promising nanomaterial B-material for biomedical applications in multicolor cell B-material imaging and intracellular sensing . [SEP]
[CLS] another strategy to stabilize dnas by hydrophobic B-property interactions I-property is to conjugate dna with hydrophobic B-property organic molecules , like dyes , drugs , polymers B-material , or dendrimers B-nanoparticle , to produce dna - organic hybrid amphiphiles B-property , which will self - assemble into many higherorder structures in aqueous solutions , such as micelles B-material , tubes , and vesicles of various shapes . [SEP]
[CLS] these novel dna amphiphilic B-property materials show many distinctive properties due to the incorporation of the hydrophobic B-property block . [SEP]
[CLS] 1 ) as a result of the self - assembly driven by hydrophobic - hydrophilic B-property phase separation , the conjugated dnas are densely packed at the outer shell B-material of the resultant nanostructures , which will enhance the biological stability and cellular penetration capability of dnas , and finally make them more suitable for intracellular and in vivo applications . [SEP]
[CLS] 2 ) meanwhile , the hydrophobic B-property domains of the self - assembled nanostructures can load hydrophobic B-property drugs or organic dyes . [SEP]
[CLS] except for increasing the their solubility B-property in I-property water I-property , the dnas packed at the outer shell B-material will enhance drug capacity in targeted and stimuli - responsive delivery . [SEP]
[CLS] 3 ) besides , the lengths and sizes of the hydrophilic B-property dna and hydrophobic B-property part will substantially affect the self - assembly structures , as dnas can go through conformational switches in response to environmental stimulus , dna - organic hybrid amphiphiles B-property can behave more intelligently in biomedical applications . [SEP]
[CLS] on the basis of the above features , dna - organic hybrid amphiphiles B-property have been applied in a variety of fields , including biosensors , gene regulation , and drug delivery . [SEP]
[CLS] in the following sections , we will discuss the synthesis of dna amphiphiles B-property and summarize their recent applications . [SEP]
[CLS] dna - organic hybrid amphiphiles B-property are synthesized by the conjugation of hydrophilic B-property short - strand dna and hydrophobic B-property blocks through the covalent bonds . [SEP]
[CLS] since dna and most hydrophobic B-property molecules are highly immiscible , the corresponding conjugation reactions become relatively difficult . [SEP]
[CLS] the interesting properties and application potentials of dna amphiphiles B-property have driven researchers to develop different methods to promote conjugation reactions . [SEP]
[CLS] 1 ) hydrophobic B-property molecules are directly conjugated to the terminal of dnas through a common coupling reaction using either solid - phase or liquid - phase reaction . 2 ) a hydrophobic B-property polymer B-material is directly polymerized from the initiator which is attached to the dna end . [SEP]
[CLS] ( 3 ) a polymerizable monomer B-material is attached to a dna unit and dna - grafted amphiphilic B-property polymer B-material is synthesized as a result of the polymerization . [SEP]
[CLS] several kinds of coupling reactions are commonly used to synthesize dna - organic hybrid amphiphiles B-property . for example , amidation B-event between amine B-material and carboxyl B-material group I-material , disulfide bond , michael addition reaction , and copper B-material - catalyzed ( or copper - free ) cycloaddition reaction . [SEP]
[CLS] amide B-material bonds are unstable to the alkaline environment , and the disulfide bonds can be broken when encountering reducing substances . [SEP]
[CLS] the chemical bonds formed by the latter two reactions are relatively stable to the environment and therefore are more broadly used . [SEP]
[CLS] some strategies have been developed to improve the yields of the heterogeneous reactions between hydrophobic B-property molecules and dnas . [SEP]
[CLS] for instance , herrmann ' s group reported a method for obtaining efficient dna amphiphiles B-property in liquid - phase reaction , where the positively charged surfactant B-property can bring dnas to the organic solvent , thereby greatly improving the reaction yield . [SEP]
[CLS] alternatively , the coupling reactions were conducted in solvents that can dissolve both dna and hydrophobic B-property molecules to a comparable extent , such as dimethylformamide ( dmf ) , dimethylsulfoxide ( dmso ) , or a mixed solution of the first two with water B-material . [SEP]
[CLS] besides , the solid - phase reaction can also improve the yields of dna amphiphiles B-property , where the solid support can bring dnas into the organic solvent system under vigorous stirring ; also , there are some attempts to prepare dna amphiphiles B-property by the polymerization of monomer - conjugated dna or directly run polymerization from the initiator - conjugated dna . [SEP]
[CLS] in addition , the purification of dna amphiphiles B-property is also a key issue , and several purification methods have been developed according to the characteristics of the dna amphiphiles B-property . [SEP]
[CLS] these methods include 1 ) the separation is proceeded by the differences in molecular weights , such as dialysis , ultrafiltration , and electrophoresis B-technique gel I-technique , 2 ) reversed - phase chromatography B-technique exerts separation by the polar differences , 3 ) size exclusion I-technique chromatography B-technique exerts separation by the differences in molecular sizes , and 4 ) anion B-material exchange chromatography B-technique exerts separation by the charge density differences . [SEP]
[CLS] the most commonly used hydrophobic B-property moieties ( figure 6a - g ) of dna amphiphiles B-property can be roughly divided into the following categories : 1 ) lipids B-material ( such as fatty chains , cholesterol , and their analogs ) , 2 ) [UNK] - conjugated molecules ( including hydrophobic B-property fluorescent B-property dyes and conjugated polymers B-material ) , 3 ) strongly hydrophobic B-property polymers B-material ( polystyrene , polynorbornene ( pnb ) derivatives grafted with aromatic rings ) , 4 ) biodegradable B-property polymers B-material ( ( polylactic acid ( pla ) , polycaprolactone ( pcl ) , and polylactic acid - glycolic acid ( plga ) ) , 5 ) stimulus - responsive polymers ( ( temperature - responsive poly ( nisopropylacrylamide ) ( pnimap ) , ph - responsive polyacrylic acid ( paa ) ) , 6 ) some other hydrophobic B-property molecules ( ( poly phosphorylated hexaethylene ( he n ) , polypropylene oxide B-material ( ppo ) ) , and 7 ) aggregation - induced emission ( aie ) - based molecules . these hydrophobic B-property moieties are chemically conjugated to dnas ( with different sequence lengths ) through covalent bonds , producing various dna amphiphiles B-property . [SEP]
[CLS] dna - based biosensors play a significant role in many fields , especially in disease diagnosis , environmental monitoring , and food analysis . [SEP]
[CLS] sensitivity is crucial for the development of biosensors , which relies on the sensitive and specific recognition of targets and the subsequently triggered signal transduction . [SEP]
[CLS] the specific watson - crick base - pairing between dna strands guarantees that dna - based biosensors can detect the complementary dna with high sensitivity and fidelity . [SEP]
[CLS] besides , developed by se - lex , aptamers are short , single - stranded dna or rna that can selectively bind to specific targets , which broadens the targets of dna - based biosensors from dna / rna to micromolecules ( such as atp and ions B-material ) , large biomolecules ( proteins B-material ) , and even whole organisms ( cells B-material and tissues ) . [SEP]
[CLS] in pursuit of higher sensitivity , numerous signal transduction approaches have been developed for dna - based biosensors , including fluorescence B-property , electrochemistry , colorimetry , and surface plasmon resonance , and so forth . [SEP]
[CLS] among them , the fluorescence - based detection method is mostly used . [SEP]
[CLS] molecular beacon is a dna hairpin structure that is widely used as fluorescent B-property probes . [SEP]
[CLS] initially , the fluorescence B-property is quenched by the dual - modification dna ends with fluorophore and quencher , which will be restored when the molecular beacon hybridizes to a complementary target sequence . [SEP]
[CLS] similarly , many dna - based fluorescence B-property probes are developed by dna - conformational - switch controlled fluorescence B-property changes . [SEP]
[CLS] in addition to this design strategy , dna amphiphiles B-property bring new insights into the design of dna - based fluorescence B-property probes . [SEP]
[CLS] on one side , dna amphiphiles B-property generally selfassemble into nanostructures through hydrophobic B-property interactions I-property . [SEP]
[CLS] when the dna block recognizes targets by chain hybridizations or conformational changes , the amphiphilicity B-property of the selfassembly system will be altered to result in further aggregation or dissociation of the previous nanostructures . [SEP]
[CLS] on the other side , in addition to the dependence on fluorophore / quencher pair , many hydrophobic B-property dyes showing aggregation - state - sensitive fluorescence B-property properties can be applied to the design of dna amphiphile based biosensors . [SEP]
[CLS] for instance , when the hydrophobic B-property dyes with aggregation - induced emission ( aie ) characteristic , the target - induced aggregation will turn on the dna amphiphile B-property biosensor ( figure 7a ) ; while for the hydrophobic B-property dyes showing the property of aggregation - caused quenching ( acq ) , the target - induced aggregation will result in fluorescence B-property turn off ( figure 7b ) . [SEP]
[CLS] moreover , hydrophobic B-property dyes can be encapsulated into the hydrophobic B-property core B-material of the dna amphiphile self - assembly to realize biosensing and detection ( figure 7c ) . [SEP]
[CLS] in this section , we focus on the discussions of fluorescence B-property biosensors based on dna amphiphiles B-property , for which the signal transduction is through the target - triggered amphiphilicity B-property changes . [SEP]
[CLS] most organic fluorescent B-property materials exhibit acq effect that their fluorescence B-property will be quenched at high concentrations or in the aggregated state , which limits their application as probes since they cannot be used at high concentrations . [SEP]
[CLS] however , these materials with the acq characteristics can be used for the construction of signal - on or signal - off biosensors that based on the change of amphiphilicity B-property of the conjugates . [SEP]
[CLS] since tang and co - workers discovered the hexaphenylsiloles ( hps ) with phenyl rotors structure that emits strong fluorescence B-property in the aggregated state in the year 2001 , this kind of aggregation - induced emitters ( aiegens ) with the characteristics of aggregation - induced emission has provided an alternative candidate for the fluorescence B-property transducer . [SEP]
[CLS] we classify these biosensors into signal - on and signal - off by the photophysical property of the coupled organic material B-material . [SEP]
[CLS] signal - on : conjugated polymer B-material ( cp ) is a typical fluorescent B-property material B-material that has been widely used for the design of biosensors due to its light - harvesting and energy transfer capability . [SEP]
[CLS] for example , xia ' s group designed a conjugated polymer - dna ( cpc - dna ) bipolar beacon . [SEP]
[CLS] the amphiphilic B-property nature of cp - c - dna drives the formation of a micelle B-material , and the fluorescence B-property of cps is quenched due to the acq effect . [SEP]
[CLS] the elongation of the dna length will impair the micelle B-material stability and induces the collapse of micelles B-material , resulting in a signal - on sensing system . [SEP]
[CLS] therefore , this system was applied to the detection of telomerase activity by using a telomerase substrate sequence as the dna block ( fig - ure 8a ) . [SEP]
[CLS] telomerase is a common biomarker B-property for most immortal cell B-material lines and primary human tumors B-material , which adds multiple ttaggg strands to the end of telomere . [SEP]
[CLS] in the presence of telomerase , the telomerase substrate sequences conjugated to cp - c - dna micelle B-material were catalyzed by telomerase to extend to longer chains , as a consequence , the hydrophilicity B-property of the dna part increased and led to the collapse of the micelles B-material , resulting in the recovery of cp fluorescence B-property . [SEP]
[CLS] the lod of this signal - on biosensor was estimated to be 4 bladder cancer cells B-material per microliter ( µl ) , which is comparable to the pcr method . [SEP]
[CLS] in comparison with pcr and other dna amplification methods , this method is accurate and simple by avoiding numerous artifacts and sophisticated optimizations , which was successfully applied in both mimic systems and real urine samples , offering a costeffective , simple , and noninvasive method for bladder cancer diagnosis . [SEP]
[CLS] the aiegens found by tang ' s group show high brightness and photobleaching resistance in the water B-material , which are more suitable for the design of signal - on biosensors . [SEP]
[CLS] several dna - aiegen conjugates have been developed for applications in optical sensing and imaging . [SEP]
[CLS] by coupling the hydrophilic B-property dna segment , the dna - aie conjugates display satisfactory water - solubility B-property that ensures the low fluorescence B-property background in bioassays B-technique . [SEP]
[CLS] liu and tang et al . reported the first " turn - on " probe based on a dna - aie conjugate for specific na detection in 2013 . [SEP]
[CLS] they created a dna - aie probe ( tpe - dna ) consisting of a typical aiegen ( tetraphenylethene , tpe ) and a 20 - mer ssdna sequence . [SEP]
[CLS] principally , the aie effect is mainly caused by the restriction of intramolecular motions ( rim ) . [SEP]
[CLS] therefore , when the dna - aie probe hybridized with its complementary dna strand , the rigid conformation and hydrophobic B-property stacking I-property interactions I-property of the double helix structure caused the rim process of tpe , resulting in a 3 - fold enhancement in the fluorescence B-property intensity . [SEP]
[CLS] the lod of this dna - tpe probe was calculated to be 0 . 3 × 10 −6 m of copyright 2015 , american chemical society . b ) schematic representation of tpe - dna probe to detect mir - 21 with the assistance of exo iii . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2015 , american chemical society . c ) upper panel : synthetic route of tpe - r - dna . lower panel : schematic representation of tpe - r - dna probe to detect mnsod mrna . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , american chemical society . d ) schematic illustration of tpe - dna probe for k + detection . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2017 , elsevier . e ) the working scheme of ifs - tpe conjugates in response to ph changes , and the fluorescence B-property intensity of aie increased when the ph was switched from 7 . 0 to 5 . 0 . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , royal society of chemistry . [SEP]
[CLS] complementary dna . [SEP]
[CLS] to pursue higher signal - to - noise ratio ( snr ) and sensitivity , liu and tang et al . developed a twoarmed tpe - dna conjugate ( tpe - 2dna ) , in which two oligonucleotides were conjugated to one tpe moiety . [SEP]
[CLS] tpe - 2dna probe showed a lower fluorescence B-property background in the absence of target due to its enhanced solubility B-property in I-property water I-property . [SEP]
[CLS] meanwhile , as a result of the presence of two arms , it displayed a stronger fluorescence B-property after target recognition . [SEP]
[CLS] micrornas ( mirnas ) are a kind of noncoding ssrnas , and the dysregulations of mirnas expressions are closely allied to many diseases . [SEP]
[CLS] xia ' s group devised a water - soluble B-property amphiphilic B-property tpe - dna probe to detect mir - 21 . [SEP]
[CLS] the tpe - dna probe contained a tpe group and a particular dna sequence that can hybridize with mir - 21 , as shown in figure 8b . [SEP]
[CLS] the resultant dna - rna duplex would be recognized by exonuclease iii ( exo iii ) , and therefore the dna part was digested to release the target mir - 21 and the residual tpe units . [SEP]
[CLS] the released mir - 21 target could be recognized by another tpe - dna probe , resulting in a cyclic process and amplified detection signal . [SEP]
[CLS] the residual tpe units aggregated in water B-material and lit up due to the aie effect . [SEP]
[CLS] later , xia and co - workers designed a tpe - r - dna probe based on the same exo iii - assisted strategy for the detection of manganese B-material superoxide dismutase ( mnsod ) mrna ( figure 8c ) . [SEP]
[CLS] they demonstrated the hydrophobic B-property residue tpe - r - at aggregated in water B-material and emitted strong fluorescence B-property . [SEP]
[CLS] the lod of the tpe - r - dna was as low as 0 . 6 × 10 −12 m in vitro . [SEP]
[CLS] what is more , tpe - r - dna could detect mnsod mrna in cancer tissues , proving its application capability in bioimaging . [SEP]
[CLS] functional ssdnas can not only hybridize with their complementary strands but also can bind to diverse target molecules ( such as metal B-material ions B-material , organic dyes , amino B-material acids I-material , and enzymes ) by folding into distinct secondary or tertiary structures . [SEP]
[CLS] for instance , dnas can form double - or triple - stranded structures , inter - or intramolecular g - quadruplexes or i - motif structures , which have been widely utilized in the conformation - switchbased dna biosensors . [SEP]
[CLS] the combination of the aiegens with the specific recognition ability of the functional ssd - nas will develop many new capable biosensors . [SEP]
[CLS] guanine - rich dna sequences can fold into a quadruplex structure known as g - quadruplex by the stacking of four hoogsteen - paired guanines . [SEP]
[CLS] dna quadruplexes can be further stabilized by the addition of alkali B-material - I-material metal I-material cations B-material ( such as na + and k + ) through electrostatic interactions . [SEP]
[CLS] the monitoring of k + is vital and urgent because of their unique relationship with various diseases . [SEP]
[CLS] tan and zhang et al . designed an aie - based dna probe ( tpe - dna ) for k + detection , in which a water - I-property soluble tpe derivative was chemically conjugated with a guanine - rich short dna via the amide B-material bond . [SEP]
[CLS] as seen in figure 8d , in the presence of k + , the k + - induced parallel g - quadruplex structure brought four tpe molecules into proximity , and hindered the intramolecular motions of tpe , resulting in a fluorescence B-property - signal enhancement . [SEP]
[CLS] both of the in vitro and cellular results proved that this aie probe showed a [UNK] - fold higher sensitivity over other g - quadruplex probes . [SEP]
[CLS] as illustrated in figure 8e , our group synthesized a new dnabased hybrid fluorescent B-property probe containing an i - motif forming sequence ( ifs ) and monofunctionalized tpe , which showed a distinct ph - responsive aie effect . [SEP]
[CLS] we demonstrated that the conformational switch of the ifs block triggered by a change of external ph could well control the hydrophobicity B-property of the ifs - tpe hybrid , which was applied in ph - responsive biosensing . cytosine - rich dna sequences can undergo a conformational transition from a random coil to a folded i - motif structure under acidic conditions . [SEP]
[CLS] in a neutral environment , the ifs - tpe probe existed as a random - coil conformation , while the ifs block folded into the i - motif structure at acidic ph and induced the aie effect . [SEP]
[CLS] a 10 - time aie enhancement was observed by turning the ph from 7 . 0 to 5 . 0 . [SEP]
[CLS] there were two reasons for the outstanding ph - responsive aie effect . [SEP]
[CLS] one was due to the hydrophobic B-property stacking interaction . [SEP]
[CLS] tpe molecules were stacked on the i - motif quadruplex structure when the ifs sequence folded in response to a decrease in ph , which restricted the internal rotations of tpe and initiated the aie effect . [SEP]
[CLS] the other reason is that the solubility B-property of the hybrid molecule in water B-material will be reduced due to ifs folding , which facilitated the aggregation of the tpe part and initiated the aie effect . [SEP]
[CLS] the tem image indicated that , at an acidic condition , ifs - tpe would form spherical aggregates with diameters of 150 - 200 nm . [SEP]
[CLS] moreover , this turn - on fluorescent B-property probe owned great resistance to oxidation and held the potential for ph monitoring in the complex intracellular environment . [SEP]
[CLS] as a result , the signal - on strategy based on the ph - induced aie effect described in this work will be significant for the development of novel dna - based probes in the future . [SEP]
[CLS] signal - off : besides the signal - on fluorescent B-property probe , a collection of the signal - off biosensor was designed for particular biosensing . [SEP]
[CLS] the above - mentioned conjugated polymer B-material ( cp ) - dna conjugates developed by xia ' s group can also be used as a signal - off biosensor for hg 2 + detecting . [SEP]
[CLS] the cp - dna conjugates were in the dispersed state with the initial fluorescence B-property of cps , while in the presence of hg 2 + , the hydrophobic B-property parts of cp aggregated together by the formation of t - hg 2 + - t complexes . [SEP]
[CLS] also , this sensing strategy can be extended to detect various other analytes ( such as other metal B-material ions B-material or proteins B-material ) , by just replacing the recognition elements of the cp - dna with other functional dna sequences such as aptamers . [SEP]
[CLS] the predictable and programmable features make dnas become the ideal scaffolds for the design of dna - based biosensors . [SEP]
[CLS] recently , quite a few dna biosensors based on the self - assembly of dna amphiphiles B-property are developed . [SEP]
[CLS] roelfes et al . proposed a novel design of dna - lipid B-material conjugates , and the folding of the dna parts into g - quadruplex structures drives the formation of micelle B-material aggregates . [SEP]
[CLS] upon the addition of the complementary dna strand , the hybrid double helix structure unfolded g - quadruplexes that made the micelle B-material disassemble , resulting in cargo release . [SEP]
[CLS] a well - known forster resonance energy transfer ( fret ) pair ( two dyes ) was simultaneously encapsulated in this micelle B-material , which showed the donor emission as a result of the efficient fret . [SEP]
[CLS] when the micelles B-material disassembled in the presence of complementary dna , the two dyes were released , resulting in decreased fret efficiency . [SEP]
[CLS] furthermore , this system was applied to the detection of adenosine 5 ' - triphosphate ( atp ) . [SEP]
[CLS] a dna hairpin containing the atp - binding dna aptamer was designed , which was comprised of an atp - binding domain and a responsive domain . [SEP]
[CLS] in the absence of atp , the responsive domain was locked in the stem region of the hairpin ; and upon binding of atp , the hairpin structure was rearranged to expose the responsive domain , which could hybridize with the dnalipid micelles B-material and resulted in cargo release . [SEP]
[CLS] the presented design convinced the applicable versatility of this strategy for the detection of different kinds of targets or stimuli . [SEP]
[CLS] as shown in figure 9a , tan and yang et al . designed a switchable aptamer micelle flare ( samf ) formed by the self - assembly of dna - diacyllipid conjugates ( the dna part is a hairpin structure with the sequence of atp aptamer locked in the stem region ) to detect and monitor atp molecules in live cells B-material . [SEP]
[CLS] the strong hydrophobic B-property interaction I-property made the dna - diacyllipid conjugates form a uniform spherical nanostructure as clarified by the tem image ( with a diameter of 28 nm as shown in figure 9b ) , displaying quenched fluorescence B-property due to the acq effect . [SEP]
[CLS] upon the binding of target atp , the hairpin dna rearranged its conformation and destabilized the self - assembled nanostructure to make fluorescence B-property restored ( figure 9c ) . [SEP]
[CLS] as the diacyllipids in samf copyright 2013 , american chemical society . [SEP]
[CLS] d ) schematic illustration of the molecular structure of o1 - dtpe , e ) using the g - quadruplex structure as the molecular scaffold for aiegen self - assembly , and f ) the accurate control of aie effect by the g - quadruplex structure . [SEP]
[CLS] the panels ( d ) - ( f ) are adapted with permission . [SEP]
[CLS] copyright 2018 , royal society of chemistry . [SEP]
[CLS] are similar to the cell B-material membranes , samfs could be efficiently uptake by live cells B-material compared with other dna probes . [SEP]
[CLS] therefore , samfs with properties of cell B-material permeability and the controllability at nanoscale show application potentials in bioanalysis , disease diagnosis , and drug delivery . [SEP]
[CLS] lei et al . made a molecular scaffold based on a tetrapod dna quadruplex ( tp - g4 ) for self - assembly and precise control of the aie - effect - based fluorescence B-property signal transduction . [SEP]
[CLS] as shown in figure 9d , the scaffold tp - g4 was formed by four [UNK] - 6g strands that can provide a confined space for the regulation of the aie effect . [SEP]
[CLS] a dna - aiegen conjugate , o1 - dtpe , was synthesized by " copper - free " click coupling , which was water B-property - I-property soluble B-property and exhibited low fluorescence B-property in I-property water I-property . [SEP]
[CLS] when o1 - dtpe was self - assembled with tp - g4 , the fluorescence B-property of dtpe became a 10 . 4 - fold higher than the background . [SEP]
[CLS] the significant aie effect was induced by the aggregation of aiegens refined by the g - quadruplex structure ( figure 9e ) . [SEP]
[CLS] besides , as depicted in figure 9f , the aie effect could be precisely regulated by the distance between the g - quadruplex core B-material and the aiegens and by altering the quartet number of the g - quadruplex . [SEP]
[CLS] furthermore , similar self - assembly and aie regulation system was developed by using i - motif as the clawed scaffold , showed a structure - dependent light response to both tetra - and bimolecular i - motif quadruplex structures . [SEP]
[CLS] these results demonstrated that dna scaffolds could efficiently regulate the aie effect , which could form a universal molecular tool for the design of new biosensing strategies . [SEP]
[CLS] in medicine , agents with pharmacological activity are called drugs . [SEP]
[CLS] the efficacy of drugs is not only related to their chemical formula but also depends on their dosage form and routes of administration , which can be evaluated by pharmacokinetics , the study of the time course of drug absorption , distribution , metabolism , and excretion . [SEP]
[CLS] chemotherapeutic drugs and na drugs play a pivotal role in cancer treatment . [SEP]
[CLS] commonly used chemotherapy drugs include dox , paclitaxel B-material ( ptx ) , and camptothecin ( cpt ) , and na drugs include asos , sirnas , dnazymes , aptamers , and immune adjuvant cpg . [SEP]
[CLS] generally , both drugs cannot be used directly due to their disadvantages in biomedical applications , which will affect their in vivo pharmacological activities . [SEP]
[CLS] therefore , materials science and engineering has been more and more involved in drug design and formulation . [SEP]
[CLS] nanodrug delivery system ( ndds ) based on drugs and other materials was designed to solve the problems of chemotherapy drugs , such as poor water B-material solubility B-property , severe toxicity B-property and side effects , rapid renal excretion , weak biological stability , low cell B-material absorption efficiency , and nonspecific immune inflammation . [SEP]
[CLS] in addition , nddss could facilitate the accumulation of drugs in solid tumor B-material sites due to the well - known enhanced permeability and retention ( epr ) effect , resulting in targeted drug delivery . [SEP]
[CLS] na drugs also suffer from instability in biological environments and poor cellular uptake due to their high negative charge . [SEP]
[CLS] self - assembly and conjugation strategies have been developed to enhance the biostability and cellular penetration properties of na drugs . [SEP]
[CLS] recently , dna amphiphiles B-property attract great research attention due to its capability in improving the pharmacokinetics and pharmacodynamic properties of both chemotherapy drugs and na drugs . [SEP]
[CLS] driving by hydrophobic B-property interactions I-property , dna amphiphilic B-property molecules can self - assemble into nanostructures , such as micelles B-material or vesicles in water B-material . [SEP]
[CLS] for micellar nanostructure , the hydrophobic B-property core B-material of micelles B-material can carry insoluble drugs , such as dox , ptx , and cpt [SEP]
[CLS] vesicle shows a relatively complicated nanostructure , in which the hydrophilic B-property core B-material can be used to transport some hydrophilic B-property drugs , and the hydrophobic B-property bilayer can carry hydrophobic B-property drugs . [SEP]
[CLS] as for the dna part , the densely packed dnas in the aggregate of dna amphiphiles B-property show a " cluster " effect , which become more resistant to nuclease degradation and more capable in cell B-material and tissue penetration compared with the free dnas . [SEP]
[CLS] meanwhile , the dna functions can be well reserved and even enhanced under the dense - pack - state . [SEP]
[CLS] therefore , the na drugs can be used as the dna block of the amphiphile B-property or introduced through chain hybridization with the dna block . [SEP]
[CLS] more than the dual enhancements to the two kinds of drugs , [SEP]
[CLS] dna amphiphile B-property - based biomedicinethe size , morphology , and surface properties of the nanostructures of dna amphiphiles B-property can be well - tuned by adjusting the chemical structures of the hydrophobic B-property block and the hydrophilic B-property dna block , which will significantly affect the pharmacological activity and kinetics of drugs . [SEP]
[CLS] in this section , we summary the design principle of nddss based on dna amphiphiles B-property and exemplify their applications . [SEP]
[CLS] the following points need to be considered when designing ndds based on dna amphiphiles B-property for practical applications : 1 ) the ndds must well protect drugs from early leakage and efficiently deliver drugs to the target site ; 2 ) as for the design of cancer drugs , the drugs also need to be taken up by target cells B-material and reach subcellular organelles to work ; 3 ) in addition , it is better that the nddss can respond to tumor B-material microenvironments and specifically release drugs . [SEP]
[CLS] the recently developed dna amphiphile B-property - based nddss can be roughly classified into the following categories ( figure 10 ) . [SEP]
[CLS] 3d structure improving the performance of nucleic B-material acid I-material : na drugs , such as aso , aptamer , sirna , dnazyme , and cpg dna , have been proven to possess good pharmacological activity in preclinical and clinical applications . [SEP]
[CLS] however , the inherent properties of na drugs , such as low stability in biological environments , poor cell B-material penetration , and undesired immune response , limit their direct application in clinical practice . [SEP]
[CLS] many gene delivery vectors ( such as cationic B-material liposomes B-nanoparticle , viral vectors , and inorganic materials ) have been developed to improve the biological properties of na drugs . [SEP]
[CLS] moreover , recently , the development of na nanotechnology has opened up new directions for the delivery of nas . [SEP]
[CLS] nucleic B-material acid I-material nanostructures , such as dna origami , framework na , and spherical nucleic B-material acid I-material ( sna ) , have made important contributions to improving the bioavailability B-property of na drugs in in vivo applications . [SEP]
[CLS] among them , nanostructures , such as micelles B-material or vesicles , self - assembled from dna amphiphiles B-property , which are chemical conjugation of pharmacologically active nas and hydrophobic B-property molecules , have attracted more and more attentions in na drug delivery ( figure 10a ) . [SEP]
[CLS] these micelles B-material and vesicles have densely packed nas on their surfaces , exhibiting " spherical nucleic B-material acid I-material " properties , which show extraordinary biomedical activity compared with free nucleic B-material acids I-material . [SEP]
[CLS] 1 ) the densely packed nas are resistant to nucleases by preventing the accessibility ; 2 ) the self - assembled nanostructures become more permeable to cells B-material through the interactions with the receptors on the cell B-material surface , which can mediate polyanion transcytosis ; 3 ) it is also reported that nanostructures with densely packed nas on the surface can cross the blood - brain barrier B-property ( bbb ) through receptormediated transcytosis to achieve diagnosis and therapy of central system diseases . [SEP]
[CLS] drug - loaded dna amphiphile B-property micelle B-material : only when the drug reaches the target tissue or cell B-material can it play an ideal pharmacological effect , especially for chemotherapy drugs . [SEP]
[CLS] naked chemotherapeutic drugs reach around the body only through concentrationdependent diffusion and cannot distinguish diseased cells B-material from normal cells . [SEP]
[CLS] therefore , direct intravenous injection of naked chemotherapeutics shows risks in producing great harms such as undesired side effects and systemic toxicity B-property . [SEP]
[CLS] the use of ndds can alleviate this problem to some extent . [SEP]
[CLS] many drug carriers have been applied for targeted delivery of chemotherapeutics , such as liposomes B-nanoparticle , polymeric micelles B-material , inorganic materials , and metalorganic complexes . [SEP]
[CLS] in particular , amphiphiles B-property can self - assemble into 3d nanostructures through hydrophobic B-property interaction I-property in aqueous solutions , and these nanostructures include hydrophobic B-property domains and hydrophilic B-property domains . [SEP]
[CLS] these two domains can simultaneously accommodate drugs with different properties to achieve synergetic diagnosis and treatment ( figure 10b ) . [SEP]
[CLS] the inner core B-material provides a hydrophobic B-property environment for hydrophobic B-property drugs , including but not limited to dox , ptx , and cpt . [SEP]
[CLS] for ndds fabrication , these lipophilic B-property drugs and dna amphiphiles B-property are first dissolved in good solvents ; later , by solvent exchange , the two will form drug - loaded dna amphiphile B-property micelles B-material , which can significantly enhance the bioavailability B-property of naked drugs due to the better targeted - cell B-material - delivery capabilities . [SEP]
[CLS] dna - amphiphile B-property - drug conjugates : instead of direct encapsulating drugs into the dna amphiphile B-property micelles B-material , the conjugation of drugs to dna amphiphiles B-property provides another approach to realize efficient drug delivery , which shows the following characteristics ( figure 10b ) : 1 ) drug can be loaded in a well - defined stoichiometric ratio ; 2 ) it shows better stability than physical encapsulation , as the conjugation through covalent bonds will prevent premature drug release . [SEP]
[CLS] for fabrication , drug molecules are conjugated to the hydrophobic B-property moieties of dna amphiphiles B-property , which can form drug - conjugated micelles B-material due to hydrophobic B-property interactions I-property in aqueous solutions for eventual application in targeted delivery of chemotherapeutics . [SEP]
[CLS] floxuridine - embedded dna amphiphiles B-property : floxuridine is a nucleoside analog , which is used as an antitumor drug . due to its structural similarity to thymine ( t ) nucleoside , fluorouridine can replace t in the na sequence without affecting the base - pairing capability . [SEP]
[CLS] therefore , floxuridine can be embedded in the na sequence by replacing t during dna solid - phase synthesis ( figure 10c ) . [SEP]
[CLS] the resulting floxuridine - embedded dna is then chemically conjugated with a hydrophobic B-property moiety to form a floxuridine - embedded dna amphiphile B-property . [SEP]
[CLS] this special amphiphile B-property can form 3d micelles B-material through hydrophobic B-property interactions I-property in aqueous solution , which helps it efficiently penetrate cells B-material to kill cancer cells . [SEP]
[CLS] dna - drug amphiphiles B-property : despite dna amphiphiles B-property have proven to be safe and nontoxic to a large extent . [SEP]
[CLS] however , the safety of hydrophobic B-property moieties introduced into dna amphiphiles B-property remains to be further confirmed . [SEP]
[CLS] taking all - dna carriers can avoid this problem because dna is an endogenous material B-material showing good biocompatibility B-property . [SEP]
[CLS] during the solidphase synthesis , phosphorothioate groups are used to replace phosphodiester groups , which can react with a benzyl bromide B-material group to form a chemical conjugation . [SEP]
[CLS] therefore , benzyl bromide - modified hydrophobic B-property drugs can be grafted into the phosphodiester sites on the dna backbone ( figure 10c ) . [SEP]
[CLS] the resulting dna ps - drug conjugates show obvious amphiphilicity B-property and can self - assemble into 3d micelle B-material structure through hydrophobic B-property interaction I-property in aqueous solution . [SEP]
[CLS] this drug delivery strategy by the conjugating drugs to the na backbone can well control the drug - na ratio and show low safety risks . [SEP]
[CLS] intelligent design based on dna amphiphiles B-property : to sum up , the dna amphiphile - based nddss show the following advantages : 1 ) dna amphiphile - based ndds shows precise and consistent drug - loading capability , which can be achieved through chemical conjugation between chemotherapy drugs and dna amphiphiles B-property . [SEP]
[CLS] 2 ) the densely packed structure of nas on the surface of ndds micelles B-material formed by the self - assembly of dna amphiphiles B-property can delay the nuclease degradation ; an additional shell B-material of polyethylene glycol ( peg ) can also be designed to enhance the stability of dna micelles B-material during blood circulation and avoid rapid clearance by the liver . [SEP]
[CLS] 3 ) taking advantage of dna nanotechnology , intelligent nddss can be designed to improve the pharmacological activity of drugs . [SEP]
[CLS] for instance , large self - assembly structures can be fabricated to conceal the targeting B-material ligands I-material , which will accumulate at the tumor B-material site due to the epr effect . [SEP]
[CLS] at the tumor B-material site , in response to tumor B-material microenvironments , the large structure can be disassembled into small nanoparticles B-nanoparticle , exposing the active ligands on the surface to mediate the internalization of the nanoparticles B-nanoparticle . [SEP]
[CLS] since the design principle of nddss based on dna amphiphiles B-property has been elucidated in the above part . [SEP]
[CLS] in this section , we will introduce the instances of their applications in the field of biomedicine . [SEP]
[CLS] improving the performance of functional nas through hydrophobic B-property interaction I-property : [SEP]
[CLS] 1 ) asos : asos are short ssdna , of which the length is 8 - 50 nucleotides . [SEP]
[CLS] the function of asos was first observed by zamecnik , and he proved that virus replication could be restrained through downregulating protein B-material expression with the existence of the specific oligonucleotides . [SEP]
[CLS] after that , the mechanism beneath the function of asos has been revealed . [SEP]
[CLS] after the hybridization of aso with its corresponding mrna through watson - crick base pairing , the formation of the dna - rna complex would induce rnase h assisted degradation . [SEP]
[CLS] the degradation of the targeted mrna interrupts rna processing , resulting in the inhibition of protein B-material expression . [SEP]
[CLS] there are some alternative mechanisms for the modulation of gene expression via asos , such as the steric blockade of rna binding proteins B-material , and splicing modulation . [SEP]
[CLS] although asos have great clinic application potentials and several asos drugs have already been approved by food and drug administration ( fda ) such as fomivirsen for cytomegalovirus retinitis , mipomersen for homozygous familial hypercholesterolemia ( hofh ) , eteplirsen for duchenne muscular dystrophy ( dmd ) , and nusinersen for spinal muscular atrophy ( cma ) , the existing challenges including autoimmunity , off - target effect , sensitive to nuclease , and low cell B-material membrane across efficacy , still limit its further development . [SEP]
[CLS] some methods based on dna - hydrophobic B-property conjugates have been proposed by researchers to overcome these challenges . [SEP]
[CLS] asos can hardly diffuse across the cell B-material membrane freely because of the intrinsic negative charged characteristics , which severely limits its gene regulation function . [SEP]
[CLS] therefore , vectors are needed to realize gene delivery . [SEP]
[CLS] although modified virus vectors are widely applied to enhance the delivery efficiency , the unsolved safety problem of immunogenicity B-property and high cost cannot be ignored . [SEP]
[CLS] some synthetic cationic B-material nonviral vectors have been investigated to decline immunogenicity B-property and cost for improving transfection performance . [SEP]
[CLS] among these vector compounds , pei holds excellent performance of combining dna with its positively charged amino moieties . [SEP]
[CLS] however , the cytotoxicity B-property of pei becomes the fundamental concern for its clinical viability . [SEP]
[CLS] sleiman and co - workers have designed and constructed a novel aso - polymer B-material conjugate that maintains high gene knockdown efficiency while less pei vectors needed , resulting in low cytotoxicity B-property . [SEP]
[CLS] they fabricated three samples to investigate their cytotoxicity B-property and gene knockdown capability . [SEP]
[CLS] the first one was phosphorothioate aso against firefly luciferase ( luc - aso ) as the control . [SEP]
[CLS] the other two were the same asos conjugated to the twelve dodecane units ( he 12 - luc - aso ) and the polymer B-material ( with alternating dodecane units and hexamethylene glycol , ( he - heg ) 6 - luc - aso ) , respectively . [SEP]
[CLS] owing to the hydrophobicity B-property of dodecane units , he 12 - luc - aso could self - assemble to form spherical micelles B-material via a simple annealing process . [SEP]
[CLS] by contrast , the presence of hydrophilic B-property hexamethylene glycol in ( he - heg ) 6 - luc - aso decreased the hydrophobicity B-property of the polymer B-material chain and led to the inhibition of spherical micelles B-material formation ( figure 11a ) . [SEP]
[CLS] for the subsequent application , pei was introduced to form asoconjugate : pei complexes . [SEP]
[CLS] they observed that luc - aso : pei and ( he - heg ) 6 - luc - aso : pei complexes showed poor gene silencing capability . [SEP]
[CLS] the gene silencing activity could be significantly enhanced and only observed for he 12 - luc - aso : pei , even with the pei concentration as low as 1 µg ml −1 . [SEP]
[CLS] the compact micelles B-material structure driven by hydrophobic B-property interaction I-property could efficiently reduce the usage of pei for transfection and gene silencing , which provides a safe and efficient strategy for gene therapy . [SEP]
[CLS] by scavenger receptor B-material ( sr ) - mediated internalization , spherical nucleic B-material acids I-material ( snas ) that show the high efficiency in gene regulation without the need for auxiliary transfection reagents have turned to be more and more attractive . [SEP]
[CLS] zhang and co - workers proposed a novel approach to graft asos onto the hydrophobic B-property pcl block , an fda approved clinical application material B-material , to form a biodegradable B-property amphiphilic B-property dna brush block copolymer ( dbbc ) with the ability to self - assemble into snas . [SEP]
[CLS] dbbcs could form uniform monodisperse micelles B-material due to the strong hydrophobic B-property interaction I-property . [SEP]
[CLS] compared with conventional linear dna block copolymer ( ldbc ) , the micelles B-material of dbbc could be functionalized with more nas on the surface . [SEP]
[CLS] the high density of surface nas endowed it with a high melting temperature , about 3 . 5 °c higher than that of ldbc - snas . [SEP]
[CLS] therefore , when the aso against enhanced green fluorescent B-property protein B-material ( egfp ) was introduced to sna structures by the chain hybridization , the dbbc - snas showed better cell B-material uptake efficiency and more effective gene knockdown efficiency than the ldbc - sna ( 54 % vs 37 % knockdown efficiency ) . [SEP]
[CLS] the biodegradable B-property perspective of dna - pcl in the physiology environment reduced adverse effects caused by the inappropriate accumulation , and this dbbc design could be broadened to other biodegradable B-property hydrophobic B-property materials , such as pla and plga . [SEP]
[CLS] the superiorities of dbbc - snas , such as transfection - reagent - free internalization and effective gene regulation efficiency , promise this kind of dna nanomaterials B-material the great potentials in clinical application . [SEP]
[CLS] as discussed above , the formation of snas has been considered as an efficient strategy for gene delivery . [SEP]
[CLS] generally , inorganic B-nanoparticle nanoparticles I-nanoparticle , like aunps B-nanoparticle , are widely used to construct the sna structures , which , however , will result in the risk of cytotoxicity B-property caused by inorganic B-nanoparticle nanoparticles I-nanoparticle . [SEP] B-nanoparticle
[CLS] to solve this problem , gianneschi and co - workers fabricated organic sna material B-material for mrna regulation , which showed comparable cell B-material uptake efficiency to au - sna [SEP]
[CLS] this novel material B-material was constructed by conjugating hydrophobic B-property polynorbonyl and hydrophilic B-property na through the amidation B-event coupling of carboxylic B-material acid I-material and amine B-material on each segment . [SEP]
[CLS] the novel dna amphiphile B-property self - assembled into micelles B-material driven by hydrophobic B-property interaction I-property . [SEP]
[CLS] the highly dense na corona at the outside of micelles B-material still maintained its biofunctions . [SEP]
[CLS] the micelles B-material were characterized by tem and dls with a uniform size of about 10 nm in radius . [SEP]
[CLS] for practical application , the locked nucleic B-material acid I-material ( lna ) , showing enhanced biostability and hybridization stability than the normal nas , was conjugated with the polymer B-material to synthesize the lnapolymer amphiphile B-property ( lpa ) . [SEP]
[CLS] the cellular uptake was verified by fluorescence - activated cell B-material sorting ( facs ) , which revealed that lpa showed cell B-material uptake efficiency 10 times higher than that of single - stranded lna . [SEP]
[CLS] consequently , by introducing the antisurvivin lna , the resultant lpa materials showed a very efficient mrna regulation capability . [SEP]
[CLS] the mechanism of the uptake of copyright 2015 , royal society of chemistry . b ) schematic representation of the rna - polymer B-material amphiphile B-property for sirna delivery ; some chemical modifications , [UNK] - dabcyl , [UNK] - stilbene , [UNK] - dmab , [UNK] - phosphorylated , and [UNK] - hydroxyl , on the outer sirnas were investigated , and only dabcyl and stibenen could help to enhance the transfection B-property efficiency I-property of sirna . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , american chemical society . [SEP]
[CLS] lpa within the cell B-material was investigated ; as methyl - [UNK] - cyclodextrin could significantly decrease the internalization of lpa , class a sr - mediated endocytosis B-event pathway was confirmed [SEP]
[CLS] rather than utilizing inorganic B-nanoparticle nanoparticles I-nanoparticle , organic snas exploited synthetic polymer B-material and hydrophobic B-property interaction I-property to construct sna structures , which made the gene - delivery system more biocompatible B-property for clinical applications . [SEP]
[CLS] moreover , the diversified structures and the tunable properties of synthetic polymers B-material can provide broad possibilities for the dna amphiphiles B-property . [SEP]
[CLS] although some traditional delivery systems for therapeutic nas have been explored , there still exist hurdles , such as the off - target problem , hampering its clinical translation . [SEP]
[CLS] to solve this limitation , sleiman ' s group proposed an idea to design a stimuli - responsive sna that could precisely inhibit a specific gene in target cells B-material . [SEP]
[CLS] the sna was assembled by two " pillar " like dna - polymer B-material conjugates , partially hybridized by a " bridge " like dna , on which the aso " cargo " against luciferase could be further hybridized . [SEP]
[CLS] the responsive process could be initiated by the overexpressed mirna in target cells B-material , which subsequently triggered the release of the aso for mrna regula - tion . [SEP]
[CLS] this sna system delivered genes only in response to the presence of mirna biomarkers B-property , which could improve the performance of aso therapy . [SEP]
[CLS] 2 ) sirna : as a new type of gene therapy , rna interference ( rnai ) has attracted broad attention . [SEP]
[CLS] numerous laboratory and clinical studies have been carried out and showed great application promise for diseases caused by gene mutation as well as the abnormal gene expression . [SEP]
[CLS] sirnas are short dsrna , with 21 - 23 base pairs in length , which are delivered in duplex for stability . [SEP]
[CLS] after entering the cell B-material , sirna is recognized and loaded onto argonaut - 2 protein B-material ( ago2 ) to form the ago2 - risc , the rna - induced silencing complex . [SEP]
[CLS] the sense strand of a sirna is hydrolyzed , and the residual antisense strand can guide the cleavage of specific mrna sequences to inhibit the expression of the corresponding protein B-material . [SEP]
[CLS] the key challenges for sirna therapy come from the delivery and offtarget issues , so the design and construction of stable , nontoxic , and in vivo effective delivery system for sirna are urgently needed . [SEP]
[CLS] 12 . [SEP]
[CLS] a ) schematic representation of the micelles B-material self - assembled from cpg - diacyl lipid B-material amphiphiles B-property . [SEP]
[CLS] b ) fluorescence B-technique imaging I-technique of draining lymph nodes from c57bl / 6 mice which were injected with different samples . [SEP]
[CLS] c ) the quantification of cpg accumulations . [SEP]
[CLS] all panels are reproduced with permission . [SEP]
[CLS] copyright 2014 , nature publishing group [SEP]
[CLS] although some sirna delivery applications have been carried out by inorganic snas , there still exist drawbacks , such as the nanoparticle - determined chemical functionalization efficiency and the accumulation - induced cytotoxicity B-property . [SEP]
[CLS] gianneschi ' s group synthesized an rna - polymer B-material amphiphile B-property , which self - assembled into organic snas with about 100 rna strands on the surface . [SEP]
[CLS] moreover , the surface - conjugated rnas could be further hybridized with the complementary sequences to form sirnas for gene silencing via rnai pathway . [SEP]
[CLS] to enhance its stability against rnase mediated degradation , the 2 ' - fluoropyrimidine ( 2 ' - f ) moiety , a proven nuclease - resistance reagent , was modified to the rna strands ; as a result , only little hydrolysis was observed even in the presence of a high concentration of rnase a ( 100 ng µl −1 ) . [SEP]
[CLS] for intracellular uptake , they found that the internalization of this rna - polymer sna was not as efficient as that of the previously reported dnapolymer sna , which was probably because the secondary structures of rna induced unfavorable receptor B-material recognition . [SEP]
[CLS] however , they also found that some chemical moieties , such as dabcyl and stilbene , can enhance the transfection B-property efficiency I-property of the rnapolymer snas ( figure 11b ) , resulting in a silence efficiency up to 90 % for the survivin mrna . [SEP]
[CLS] this platform provides a convenient approach for sirna loading via chain hybridization , and the introduction of certain chemical moieties on sna surfaces offers a simple and straightforward way to modulate cellular uptake capability of snas . [SEP]
[CLS] 3 ) cpg : the phenomenon that gene could trigger immune responses was first found in 1984 . [SEP]
[CLS] the specific dna extracted from mycobacterium tuberculosis could activate nature killer ( nk ) cells B-material and inhibit the proliferation of cancer cells B-material . [SEP]
[CLS] the mechanism of tumor B-material regression caused by specific bacterial dna later was proved owing to the existing unmethylated cpg sites within the sequences . [SEP]
[CLS] the accumulation of cpg dnas in antigen - presenting cells B-material ( apcs ) in lymphatics will bind with toll - like receptor B-material 9 ( tlr 9 ) and initiate the immunity pathway , thereafter , promote the proliferation of cytotoxic B-property cd 8 + cells B-material . [SEP]
[CLS] now , the synthetic oligonucleotides with cpg sites have been widely investigated and utilized as efficient vaccine adjuvants . [SEP]
[CLS] nevertheless , many challenges remain to be overcome , such as safety , stability , and efficient delivery to the lymph node ( ln ) where the immune process takes place . [SEP]
[CLS] to break the barriers B-property of systemic toxicity B-property and the sophisticated design , irvine and co - workers synthesized a dna amphiphile B-property comprised of the albumin - binding lipid B-material and the cpg dna to enhance the delivery to ln via " albumin hitchhiking " process ( figure 12a ) . [SEP]
[CLS] different amphiphiles B-property with different lipophilic B-property structures , such as cholesterol - cpgs , monoacyl lipid B-material - cpgs and diacyl lipid B-material - cpgs , were synthesized to optimize the albuminbinding capability . [SEP]
[CLS] only the cpg conjugates comprised of diacyl lipids B-material could form liposomes B-nanoparticle driven by hydrophobic B-property inter - adv . sci . 2020 , 7 , 2001048 2001048 ( 20 of 34 ) action and showed efficient albumin binding , which led to 30fold increases in t - cell priming and enhanced antitumor efficacy while greatly reducing systemic toxicity B-property ( figure 12b ) . [SEP]
[CLS] further , they proved that although the formation of micelle B-material structure was critical for the effect of the cpg - lipid B-material amphiphiles B-property , the micelles B-material also had to disassemble into free amphiphile B-property accompanied by interacting with albumin for taking effect . [SEP]
[CLS] when poly - g sequences were introduced between the diacyl lipid B-material and cpg sequence to lock the amphiphiles B-property in the micellar state by forming g - quadruplex hydrogen B-material bonding and block disassembly , the accumulation at ln was found decreased significantly . [SEP]
[CLS] therefore , moderate hydrophobic B-property interaction I-property plays an essential role in the effectiveness of this amphiphile B-property system , which could ensure the dissociation of the micelle B-material structure upon binding with albumin ( figure 12c ) . [SEP]
[CLS] when combined with antigen , these cpglipid adjuvants showed obvious tumor B-material regression ability . [SEP]
[CLS] liu and co - workers utilized this delivery system to examine the immune effects of different cpg sequences and proved the importance of ln - targeting for its vaccine adjuvant clinic application . [SEP]
[CLS] this novel delivery design via " albumin hitchhiking " strategy could also be applied to other gene immunity therapeutics . [SEP]
[CLS] dna conjugates work as chemotherapeutic drugs carrier : because of the cytotoxicity B-property of chemotherapeutic drugs to healthy cells B-material , the development of selective delivery to target cells B-material becomes increasingly important . [SEP]
[CLS] dna - b - ppo block copolymer was synthesized by herrmann as carriers to encapsulate dox within the particle core B-material by hydrophobic B-property interaction I-property . [SEP]
[CLS] through hybridization with folic B-material acid linked complementary dna , the targeting ability was realized owing to the high expression of the folate B-material receptors I-material on several kinds of cancer cells B-material . [SEP]
[CLS] the monodisperse particle of 10 nm with more folic B-material acid I-material ligands showed higher uptake efficiency and a better chemotherapeutic effect . [SEP]
[CLS] precise delivery and release of drugs gain extensive attention nowadays . [SEP]
[CLS] zhang ' s group proposed a novel approach that could precisely control drug release via uv light activation by using the delivery system of dna amphiphiles B-property . [SEP]
[CLS] they synthesized dna amphiphiles B-property by conjugating camptothecin ( cpt ) to dna sequences that were modified with the photo - cleavable linker , 2 - nitrobenzyl ether B-material . [SEP]
[CLS] micelles B-material of different morphologies , such as sphere and rod , could be easily formed in aqueous solution . [SEP]
[CLS] the highly dense dna - corona could protect surface dna from nuclease degradation , and only under the uv trigger , could the interior hydrophobic B-property core B-material release cpt and fulfill the chemotherapeutic function . [SEP]
[CLS] if incorporated with two - photon or nir dye , this platform would gain the potential of precise spatiotemporal drug release . [SEP]
[CLS] not only dna amphiphiles B-property can form micelles B-material and work as drug - delivery vehicles B-material , but some nucleobase analog drugs can also conjugate to hydrophobic B-property molecules and enhance their biomedical function . [SEP]
[CLS] tan and co - workers developed a lipidconjugated floxuridine homomeric oligonucleotide ( fu20 ) , a widely used therapeutic nucleobase analogue , the systemic delivery of which could be enhanced by " hitchhike " with albumin ( figure 13a ) . [SEP]
[CLS] owing to the modification of diacyl lipid B-material , the fu20 - lipid B-material amphiphile B-property self - assembled into micelles B-material and could noncovalently attach with albumin through the interaction of lipid B-material and albumin hydrophobic B-property core B-material during blood circulation , which resulted in a sufficient tumor B-material accumulation . [SEP]
[CLS] after the endocytosis B-event of cancer cells B-material , fu20 - lipid / albumin complexes would be hydrolyzed by lysosome to release fu20 and inhibited cancer cell B-material proliferation . [SEP]
[CLS] this platform provides an idea of designing drug - delivery systems for nucleoside / nucleobase analogue drugs . [SEP]
[CLS] therapeutic dna conjugates integrated with chemotherapeutic drugs for synergistic therapy : one of the main challenges that hinder the development of chemotherapeutic drugs in experimental and clinical practice is drug resistance [SEP]
[CLS] the degree of drug resistance mainly correlates with the expression level of the responsible proteins B-material , so the incorporation with gene therapy provides a potential way to address the drug - resistance issue of chemotherapy . [SEP]
[CLS] zhang ' s group constructed a chemogene sna to realize the codelivery of therapeutic nas as well as the chemotherapeutic drugs . [SEP]
[CLS] one of the most widely used chemotherapy drugs , ptx , was attached to norbornenyl group via reductively cleavable disulfide linker and prepolymerized by ringopening metathesis polymerization . [SEP]
[CLS] through the " click " chemistry , the ptx grafted polymer B-material was subsequently conjugated to a dna strand , which was an aso ( g3139 ) targeting the antiapoptotic b - cell lymphoma 2 ( bcl - 2 ) family proteins B-material . [SEP]
[CLS] with a degree of polymerization of 10 , ptx 10 provided enough hydrophobicity B-property for the amphiphiles B-property to form sna micelles B-material . [SEP]
[CLS] the micelles B-material with 15 nm diameter presented superior stability against dnase i and the high cell B-material uptake efficiency . [SEP]
[CLS] with the ability to load and release both ptx and aso at the same time , this dual delivery design showed the inspiration for the clinic treatment of drug - resistant cancers . [SEP]
[CLS] other than using exogenous hydrophobic B-property polymer B-material to graft with ptx , zhang and co - workers designed an sna material B-material that was self - assembled from the dna strands modified with hydrophobic B-property ptx ( figure 13b ) . [SEP]
[CLS] the amphiphilic B-property dna molecule was composed of therapeutic bcl - 2 aso and ptxgrafted phosphorothiolate dna ( po dna - b - ( ps dna - g - ptx ) ) conjugated together through the disulfide linkers . [SEP]
[CLS] the snas were self - assembled owing to the amphiphilicity B-property , which could be subsequently functionalized with targeting aptamers and imaging dyes via hybridizations . [SEP]
[CLS] after the aptamer - assisted internalization , the structure of sna would be broken through the cleavage of disulfide linkers by gsh and released free aso and ptx . [SEP]
[CLS] treated by this sna system , the expression of bcl - 2 was downregulated by nearly 77 % for drug - resistant models both in vitro and in vivo , and the growth of tumor B-material was substantially hindered by this synergistic treatment . [SEP]
[CLS] besides the drugs attaching with dna via a disulfide linker , zhang ' s group proposed a novel drug conjugating approach to directly replace the thymine ( t ) on the dna strand by nucleoside analog floxuridine ( f ) . [SEP]
[CLS] the f - embedded bcl - 2 aso was conjugated to peg - b - pcl through copper - free click reaction . [SEP]
[CLS] the forming micelles B-material with controllable sizes regulated by pcl length would preferentially accumulate in tumors B-material owing to the epr effect . [SEP]
[CLS] though t was replaced by f , the knockdown efficiency of the aso was still retained , which could efficiently reverse the drug - resistance caused by bcl - 2 protein B-material . [SEP]
[CLS] meanwhile , the chemotherapeutic f would be released after the dna strand was degraded by dnase . [SEP]
[CLS] this synergistic chemo - gene therapeutic system shows the obvious effect of reversing drug - resistance , which could efficiently inhibit the growth of both orthotopic and subcutaneous drug - resistant bel - 7402 ( human liver carcinoma cell B-material line ) tumors B-material . [SEP]
[CLS] 13 . [SEP]
[CLS] a ) schematic illustration of the synthesis of fu20 - lipid B-material amphiphile B-property and its enhanced systemic delivery by the " hitchhike " strategy . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , wiley - vch . [SEP]
[CLS] b ) illustration of the synthesis of po dna - b - ( ps dna - g - ptx ) and its self - assembled sna for multifunctional biomedical applications . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , wiley - vch . [SEP]
[CLS] nir - ii emitting organic sna for brain tumor B-technique imaging B-technique : I-technique brain tumor B-material is one of the diseases that highly affect human health , whose visualization is critical for early diagnosis and imagingguided surgery . [SEP]
[CLS] compared with commonly used x - ray computed B-technique tomography I-technique and magnetic B-property resonance ( mri ) , fluorescence B-technique imaging I-technique is easier to operate and allows real - time imaging during the operation . [SEP]
[CLS] among them , the second near - infrared region ( nir - ii , 1000 - 1700 nm ) fluorescence B-property can provide a more promising method with deeper penetration depth and better resolution than nir - i fluorescence B-property . [SEP]
[CLS] however , the bbb hinders the imaging and diagnosis of brain tumors B-material by using nir - ii nanofluorophore . [SEP]
[CLS] therefore , our group developed a kind of organic sna whose hydrophobic B-property core B-material can accommodate nir - ii emitting fluorescent B-property dyes . [SEP]
[CLS] the resultant nir - ii emitting organic sna can effectively cross bbb and target brain tumors B-material , thereby enhancing the diagnostic B-technique imaging B-technique of I-technique brain I-technique tumors I-technique ( figure 14a ) . [SEP]
[CLS] in order to prevent the early leakage of organic dyes in in vivo applications , polystyrene ( ps ) with high hydrophobicity B-property was selected as the hydrophobic B-property block to synthesize dna block copolymer , ps - b - dna . [SEP]
[CLS] as shown in figure 14b , ps - b - dna was successfully synthesized by a solid - phase " click " reaction . [SEP]
[CLS] after cleaved from beads , ps - b - dna directly assembled into spherical micelles B-material with the size of ≈20 nm in aqueous solution , which was determined by tem and dls . [SEP]
[CLS] the densely packed dnas on the surface of ps - b - dna sna showed hybridization ability comparable to free dna . [SEP]
[CLS] as for biostability , the in vivo half - life of micellar dna was determined to be ≈65 min , which was much larger than the that of free dna ( ≈19 min ) , because dense dna arrangements hinder the accessibility of nucleases . [SEP]
[CLS] compared [SEP]
[CLS] all panels are reproduced with permission . [SEP]
[CLS] copyright 2020 , wiley - vch . [SEP]
[CLS] the nir - ii emitting organic dye ( fe ) was encapsulated into the ps - b - dna polymer B-material matrix by using nanoprecipitation method to obtain fe @ ps - b - dna . [SEP]
[CLS] the sna with 25 wt % fe showed the brightest brightness and a fluorescence B-property quantum yield of 9 . 6 % . [SEP]
[CLS] fe @ ps - b - peg and fe @ ps - b - dna / apt were also prepared under the same conditions . [SEP]
[CLS] subsequently , in vivo brain tumor B-technique imaging I-technique was performed . [SEP]
[CLS] at 24 h administration of different samples , fe @ ps - b - dna and fe @ ps - b - dna / apt showed strong fluorescence B-property signal at brain tumor B-material sites compared with fe @ ps - b - peg . [SEP]
[CLS] after imaging experiment , the isolated brains were collected . [SEP]
[CLS] the enhanced accumulation of fe @ ps - b - dna was observed due to the ability of ps - b - dna in bbb crossing ; the targeted aptamer further enhanced the tumor penetrating ability of fe @ ps - b - dna / apt . [SEP]
[CLS] therefore , compared with fe @ psb - dna , fe @ ps - b - dna / apt showed a 3 . 8 - fold signal enhancement ( figure 14d ) . [SEP]
[CLS] finally , the biocompatibility B-property of fe @ ps - b - dna has also been demonstrated , showing excellent biosafety . [SEP]
[CLS] these results indicate that as a versatile polymer B-material matrix , ps - b - dna can facilitate functional organic dyes to cross bbb for the diagnosis and imaging of brain diseases . [SEP]
[CLS] in the above sections , the self - assemblies of individual dna and dna amphiphiles B-property driven by hydrophobic B-property interactions I-property for biomedical applications have been discussed . [SEP]
[CLS] in addition to behaving as the driving force for self - assembly , the hydrophobic B-property interaction I-property can also be applied to fabricate the composite materials between dna and inorganic B-nanoparticle nanoparticles I-nanoparticle / organic molecules , which is also an important approach in the design of dna - based biomedical materials . [SEP]
[CLS] therefore , in this section , we will summarize the dna - containing composite materials due to the hydrophobic B-property interactions I-property . [SEP]
[CLS] in the field of materials science , the superior properties of a variety of inorganic nanomaterials B-material have been extensively investigated , for example , the superparamagnetism B-property of magnetic B-nanoparticle nanoparticles I-nanoparticle , the optical properties of quantum B-nanoparticle dots I-nanoparticle , the surface plasmon resonance of aunps B-nanoparticle , and the upconversion emission of rare - earth - based nanoparticles B-nanoparticle . [SEP]
[CLS] in addition to these interesting properties , many inorganic nanomaterials B-material show high cellular - uptake capability and high tumor B-material - accumulation by the epr effect . [SEP]
[CLS] they are widely applied in biomedical applications , such as theranostics and biological detection . [SEP]
[CLS] as nanomaterials B-material have large surface - area - to - volume ratios , the complexation with functional biomolecules , including phospholipids , proteins B-material , and nucleic B-material acids I-material , through surface adsorption has become a practical strategy to construct the biomedical materials . [SEP]
[CLS] in this section , we focus on discussing the use of hydrophobic B-property interactions I-property to form complexes between inorganic nanomaterials B-material and nas . [SEP]
[CLS] good water B-material solubility B-property , large surface area , and convenient chemical modification make graphene oxide B-material ( go ) an interesting and unique nanomaterial B-material . [SEP]
[CLS] recently , go has shown great potential in bioanalysis applications , which benefits from its outstanding biocompatibility B-property , as well as its strong but dynamic interactions with biomolecules . [SEP]
[CLS] dna forms complicated binding interactions with go , including the stacking between the dna bases and the hydrophobic B-property domains of go as well as the hydrogen B-material bonding and the electrostatic repulsion with the oxygen - rich domains . [SEP]
[CLS] the following factors affect the interaction between dna and go : 1 ) the interaction will be greatly affected by the dna sequence , i . e . , purine bases ( a and g ) show stronger binding affinity compared with the pyrimidines ( t and c ) . [SEP]
[CLS] 2 ) furthermore , a shorter dna strand can be more rapidly and tightly adsorbed by go . [SEP]
[CLS] 3 ) there is also electrostatic repulsion between dna and go ; thus , higher salt B-material concentrations can screen charges and promote dna adsorption . [SEP]
[CLS] 4 ) likewise , in the acidic buffers , the surface charge of go is neutralized , and consequently , its repulsion toward dna will be reduced , resulting in an enhanced attractive interaction . for instance , liu et al . reported that go absorbed less dna at high ph value . [SEP]
[CLS] 5 ) temperature is also an important factor that will affect the interaction between go and dna , and liu et al . observed that the faster dna adsorption will happen at relatively higher temperatures [SEP]
[CLS] unpaired bases in ssdna strongly interact with go surface through hydrophobic B-property interaction I-property and [UNK] - [UNK] stacking , on the contrary , go shows relatively low affinity to dsdna due to the electrostatic repulsion . [SEP]
[CLS] therefore , go can more efficiently adsorb ssdna compared with dsdna . [SEP]
[CLS] considering its capability to sensitively discriminate between ssdna and dsdna , as well as its super and universal fluorescence B-property quenching ability , go has been extensively used in dna - based fluorescence B-property biosensors for the detection of biomolecules , metal B-material ions B-material , and small molecules . [SEP]
[CLS] the working principle of go - dna nanosensors B-nanoparticle based on physisorption is as follows ( figure 15 ) . [SEP]
[CLS] the fluorophore - labeled ssdna is adsorbed on the surface of go , and the fluorescence B-property is quenched by go at this time . [SEP]
[CLS] when the complementary target dna is treated , the ssdna forms double helix structures and dissociate from the go surface ; thus , the fluorophore will emit fluorescence B-property to realize the detection of the target dna . [SEP]
[CLS] although simple to be constructed , the go - dna nanosensors B-nanoparticle based on physisorption remained some drawbacks . [SEP]
[CLS] first , as the interaction between go and dna is sensitive to ph , it is hard to develop ph sensors based on this method . [SEP]
[CLS] second , there will be a severe specificity problem when the physisorbed sensor is applied to intracellular detection . [SEP]
[CLS] as the intracellular environment contains a high concentration of nucleic B-material acids I-material and proteins B-material , the physically adsorbed dna might be displaced by the nontarget molecules . [SEP]
[CLS] therefore , go - dna nanosystems constructed via chemical conjugation was demonstrated to show a higher specificity for extra - and intracellular applications . [SEP]
[CLS] however , both the conjugation process and the subsequent sensing process will be affected by the high affinity of the go surface to dna . [SEP]
[CLS] to solve this problem , we developed a treatment method using herring sperm dna ( hsd ) , by which the physical adsorption from go was weakened . [SEP]
[CLS] thus , the conjugation yield of dna and the sensing specificity of the resultant go - dna nanosystem were significantly improved . [SEP]
[CLS] ph - sensitive go - dna nanosensors B-nanoparticle have been successfully fabricated through combining the chemical conjugation and hsd passivation method , which showed excellent sensing ability not only in vitro but also in intracellular ph detection . [SEP]
[CLS] also , by the same strategy , go - dna nanosensors B-nanoparticle for the detection of intracellular micrornas were fabricated , which can simultaneously discriminate among three mirnas in live cells B-material as well as monitor the dynamic expression of these mirnas . [SEP]
[CLS] aunp B-nanoparticle is one of the most important nanomaterials B-material in the biomedical field due to its low cytotoxicity B-property , high biostability , and distinct optical properties . [SEP]
[CLS] aunps B-nanoparticle can be easily prepared by the reduction reaction of chloroauric acid ( haucl 4 ) , and the particle size can be controlled by adjusting the concentration of citrate . [SEP]
[CLS] due to the localized surface plasmon resonance ( lspr ) effect , aunps B-nanoparticle show colorimetric changes as varying from the dispersed state to the aggregated state . [SEP]
[CLS] also , aunps B-nanoparticle are efficient quenchers , which show distance - dependent fluorescence B-property quenching . [SEP]
[CLS] based on these two exciting properties , aunps B-nanoparticle have been extensively applied in biological analysis and detections ; and aunps B-nanoparticle are often accompanied by dnas in the design of sensing systems . [SEP]
[CLS] thiolmodified dna can be chemically conjugated to the surface of aunps B-nanoparticle through covalent bonds ( au - s chemical bonds ) to form stable aunp B-nanoparticle - dna probes . [SEP]
[CLS] nonetheless , the interaction of unmodified dnas with aunps B-nanoparticle also attracted a lot of research attention , as this will result in a simpler , more convenient , faster , and cost - friendly method for the preparation of aunp B-nanoparticle / dna - based biomaterials . [SEP]
[CLS] there is still controversy about the driving forces of the physical adsorptions between dnas and aunps B-nanoparticle . li et al . proposed that the adsorption of ssdna onto aunps B-nanoparticle was mainly maintained by electrostatic force through derjaguin - landau - verwey - overbeek ( dlvo ) theory . [SEP]
[CLS] however , nelson et al . believed that the hydrophobic B-property interaction I-property was the main interaction between dnas and aunps B-nanoparticle , and they deduced the conclusion from the following phenomenon . [SEP]
[CLS] first , the affinity of aunp B-nanoparticle to ss - dna is much larger than that to dsdna , which cannot be explained by the debye screening as there is only a linear difference in charge density from ssdna to dsdna . [SEP]
[CLS] also , the interaction between dna and aunp B-nanoparticle is determined by the sequence of dna and the types of salts B-material in the buffer , which cannot be ex - plained by dlvo theory but can be well described by hydrophobic B-property interactions I-property . [SEP]
[CLS] because aunps B-nanoparticle can sensitively and specifically discriminate ssdna from dsdna , biosensors have been developed based on this principle . [SEP]
[CLS] owing to the high absorption coefficients , the color of aunps B-nanoparticle can be visually observed when the concentration is down to the nm level , which is also very sensitive to the salt B-material concentrations . [SEP]
[CLS] under a high concentration of salts B-material , aunps B-nanoparticle can be stabilized by ssdna against aggregation ( figure 16a ) . [SEP]
[CLS] in the presence of cdna , the surface - absorbed ssdna can be recognized to form dsdna , which will dissociate from the surface of aunps B-nanoparticle due to the significantly decreased hydrophobic B-property interaction I-property ( all the hydrophobic B-property bases are locked in the duplex structure ) , resulting in the aggregation of aunps B-nanoparticle and the subsequent colorimetric variations ( figure 16b ) . [SEP]
[CLS] for example , the colorimetric detection of lead ions B-material was carried out by this principle ( figure 16c ) . [SEP]
[CLS] dnazyme with a doublestranded structure cannot prevent the aggregation of aunps B-nanoparticle at first ; when it went through the catalyzed cleavage reaction in the presence of pb 2 + , ssdna as the products of cleavage could prevent salt - induced aggregation of aunps B-nanoparticle , inducing colorimetric changes . [SEP]
[CLS] in another example , in the presence of k + , the grich ssdna underwent conformational changes to form a folded g - quadruplex structure . [SEP]
[CLS] as a result , the salt - induced aunps B-nanoparticle aggregation occurred , enabling the label - free detection of k + ( figure 16d ) . [SEP]
[CLS] noncovalent interactions between cationic B-material polymers B-material and nas have been widely applied in biomedical fields , such as gene delivery and na detection . [SEP]
[CLS] however , the physical interaction of cationic B-material polymers B-material with nas still needs further clarification . [SEP]
[CLS] although electrostatic forces can be intuitively determined between cationic B-material polymers B-material and negatively charged nucleic B-material acids I-material , recent studies have shown that hydrophobic B-property forces also play an important role . [SEP]
[CLS] in the following part , we will discuss the role of hydrophobic B-property forces in the cationic B-material - polymer / dna complexes and their biomedical applications by using two cationic B-material polymers B-material as examples . [SEP]
[CLS] gene - delivery is always a big challenge for gene therapy . [SEP]
[CLS] naked dna or rna generally shows poor cellular uptake capability and low biological stability ; therefore , it is difficult to exert their biological effects and requires a suitable delivery system to overcome these physiological obstacles . [SEP]
[CLS] cationic B-material polymers B-material , as the most commonly used nonviral vector delivery systems , especially pei , have been widely used for intracellular delivery of therapeutic nas . however , the efficiency of pei - based gene delivery is not ideal due to its low efficiency in complexing with dna and the undesired cytotoxicity B-property . [SEP]
[CLS] in order to solve this problem , chemical modifications to pei have been performed to enhance the gene delivery efficiency through enhancing the hydrophobic B-property interactions I-property . [SEP]
[CLS] the introduction of the hydrophobic B-property moiety can be achieved through the following strategies . [SEP]
[CLS] modifying the hydrophobic B-property moieties on the pei ; or modifying the hydrophobic B-property moieties on the nas . [SEP]
[CLS] the introduction of hydrophobic B-property moieties provides the additional driving force , hydrophobic B-property interaction I-property , to the complexation , which makes the pei / dna complex easier to form and more stable ( figure 17 ) . [SEP]
[CLS] the pei / dna complex with the enhanced hydrophobic B-property interactions I-property exhibits many excellent properties , for example , the higher dna condensation efficiency , less cytotoxicity B-property due to the less pei usage , stronger biological stability , and better cell B-material uptake capability due to the hydrophobicitymediated cell B-material endocytosis B-event . [SEP]
[CLS] [UNK] - conjugated polymers B-material have long - range delocalized electrons on their backbone , thereby showing many excellent properties . [SEP]
[CLS] compared with small molecule analogs , [UNK] - conjugated polymers B-material exhibit stronger light - harvesting capabilities . [SEP]
[CLS] besides , the exciton can rapidly migrate along the backbone of the [UNK] - conjugated polymer B-material , achieving an efficient energy transfer to the low - energy receptors , which is also known as the " molecular wire " effect . [SEP]
[CLS] these two advantages make [UNK] - conjugated polymers B-material a promising candidate for the development of biosensors . [SEP]
[CLS] however , if the [UNK] - conjugated polymer B-material is to be applied in the biological environment , it needs to be chemically modified to achieve proper water B-material solubility B-property . [SEP]
[CLS] cationic B-material groups are generally introduced into the side chains to prepare water - soluble B-property cationic B-material [UNK] - conjugated polyelectrolytes . [SEP]
[CLS] related studies have revealed that , except the electrostatic attraction , hydrophobic B-property interaction I-property between ssdna and the backbones of the cationic B-material [UNK] - conjugated polyelectrolytes is also essential . [SEP]
[CLS] xia et al . found that the cationic B-material [UNK] - conjugated polyelectrolyte showed different affinities to ssdna and dsdna ( figure 18a ) . [SEP]
[CLS] the exposed bases of ssdna can be complexed with the hydrophobic B-property backbone of the [UNK] - conjugated polyelectrolyte through hydrophobic B-property interactions I-property . [SEP]
[CLS] while the bases of ds - dna are shielded , so the interaction between dsdna and the polyelectrolyte becomes weaker , which was determined by fret between the fluorophore on the dna and the [UNK] - conjugated polyelectrolyte ( figure 18b , c ) . [SEP]
[CLS] biosensors can be designed based on this principle . [SEP]
[CLS] as shown in figure 18d , in the absence of the target ( for example , cocaine ) , the [UNK] - conjugated polyelectrolyte strongly binds to the ssdna ; therefore , there is an efficient fret from the polyelectrolyte to the fluorophore - labeled on the ssdna . [SEP]
[CLS] when the target is present , the ssdna is recognized to form dsdna , which shows a relatively weaker affinity to the [UNK] - conjugated polyelectrolyte , resulting in an inhibited fret . [SEP]
[CLS] base pairing of nucleic B-material acid I-material is the central principle of dna self - assembly . [SEP]
[CLS] since seeman used dna as a building block to the illustration of a sensor for cocaine detection based on [UNK] - conjugated polyelectrolytes / dna complex . [SEP]
[CLS] the panels ( c ) and ( d ) are reproduced with permission . [SEP]
[CLS] copyright 2010 , american chemical society . [SEP]
[CLS] construct nanomaterials B-material since the 1980s , a variety of dna selfassemblies have been developed , such as dna origami , framework nucleic B-material acid I-material , and dna cage . [SEP]
[CLS] moreover , due to biocompatibility B-property and diverse biological functionalities , these dna selfassemblies have been widely applied in biomedical fields , including but not limited to drug delivery , gene regulation , immune regulation , and biosensors . [SEP]
[CLS] however , nanostructures formed solely by the hydrogen B-material - bonding of base - pairing typically require hundreds of dna strands , which results in the high cost and the complexity in operation . [SEP]
[CLS] therefore , it is necessary to introduce other self - assembly driving forces to enrich the diversity of nanostructures and simplify the preparation of dna - based nanomaterials B-material . [SEP]
[CLS] introducing hydrophobic B-property interactions I-property will increase the diversity and functionality of dna nanomaterials B-material . [SEP]
[CLS] dna amphiphiles B-property show distinct interface properties , i . e . , the dna block can be used for the programmable molecular - hybridization , and the hydrophobic B-property moiety provides the hydrophobic B-property interaction I-property . [SEP]
[CLS] therefore , dna amphiphiles B-property can be considered as the best candidates to introduce hydrophobic B-property interactions I-property into the design of novel dna nanomaterials B-material . [SEP]
[CLS] dna origami is a technique that folds long ssdna into the desired structure with the help of hundreds of short - stranded dnas , in which the long single strand is called a scaffold , and the short strands are called staples . [SEP]
[CLS] nanostructures with exact dimensions can be prepared by the dna origami technique , including smiley faces , vases , and pentagrams . [SEP]
[CLS] however , it is hard to construct macroscopic structures only by the hydrogenbonding of base pairing in dna origami technique . [SEP]
[CLS] introducing additional hydrophobic B-property interactions I-property is a practical approach to solve this problem , and by which more functionalities are possible to be incorporated . [SEP]
[CLS] simmel et al . used dna origami to prepare a hollow stem , on which lipid B-material membranes I-material were inserted to fabricate the columnar channels through the cholesterol anchors . [SEP]
[CLS] the as - prepared nanostructure behaved as the artificial nanoscale transmembrane channel , which showed a response similar to that of natural ion B-material channels ( figure 19a ) . [SEP]
[CLS] in another work , simmel et al . introduced cholesterol - modified dna into the monolayered dna origami structures by chain hybridization . [SEP]
[CLS] because the red is the stem inserted into the cell B-material membrane . [SEP]
[CLS] the orange is cholesterol - modified dna used to anchor this artificial ion B-material channel into the lipid B-material bilayer I-material . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2012 , aaas . [SEP]
[CLS] b ) driven by hydrophobic B-property forces , a single layer of dna origami is folded into a double - layered structure . [SEP]
[CLS] the tem images show the dna origami sheets before and after folding . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2014 , wiley - vch . [SEP]
[CLS] c ) hydrophobic B-property molecules are hybridized to dna origami through dna tail to form a hydrophobic B-property framework , which can self - assemble into higher - order structures . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2016 , wiley - vch . [SEP]
[CLS] d ) dna origami was used as a template to mediate the formation of vesicles with the controlled size and shape . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2016 , nature publishing group . [SEP]
[CLS] e ) different quantized cage assemblies controlled by the length of the hydrophobic B-property polymer B-material ; dna cage - loop structures can be prepared by adjusting the hydrophobicity B-property of the dna amphiphiles B-property . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2016 , american chemical society . [SEP]
[CLS] f ) the hydrophobic B-property polymer B-material is packed into a dna cage by molecular hybridization . [SEP]
[CLS] the hydrophobic B-property environment in the cage can contain the guest molecule and release the guest molecule in the presence of specific dna sequence . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2013 , nature publishing group . [SEP]
[CLS] the hydrophobic B-property part of the dna amphiphile B-property has a strong tendency to aggregate in an aqueous solution , the single - layer dna origami structure with cholesterol folded into a sandwich - like double - layer structure due to the hydrophobic B-property interactions I-property ( figure 19b ) . [SEP]
[CLS] this bilayer structure could also be expanded to mul - tiple layers by adding a surfactant B-property or a phospholipid bilayer membrane . [SEP]
[CLS] generally , the amphiphilic B-property molecules tend to self - assemble into 3d spherical structures rather than the 2d nanostructures in aqueous solution . [SEP]
[CLS] liu et al . used the dna origami structure as a 2d template to rivet the dna amphiphiles B-property by dna hybridization . [SEP]
[CLS] through hydrophobic B-property interactions I-property , a continuous nanosheet was assembled from dna amphiphiles B-property in an aqueous solution ( figure 19c ) . [SEP]
[CLS] similarly , controlling the shape and size of vesicles at the nanoscale is challenging , and achieving this goal may require careful optimization of lipid B-material composition and manufacturing processes . [SEP]
[CLS] lin et al . used dna origami to prepare a circular dna template , and then hybridized the lipid - modified dna to the origami template , which could further mediate the formation of vesicles with the expected sizes and shapes ( figure 19d ) . [SEP]
[CLS] 4 . 3 . 2 . dna cage - dna amphiphiles dna cages can be assembled from only a few dna strands through complementary base pairing . [SEP]
[CLS] however , it is challenging to prepare a large number of complex nanostructures with only a few dna strands from only one self - assembly language ( base pairing ) . [SEP]
[CLS] sleiman et al . hybridized dna amphiphiles B-property to dna cages . [SEP]
[CLS] the hydrophobic B-property block of the amphiphile B-property on the side of the cage can induce aggregation of the dna cages , and the degree of aggregation can be well controlled by the length of the hydrophobic B-property block of the dna amphiphile B-property . [SEP]
[CLS] also , dna cage - loop structures can be prepared by adjusting the hydrophilic B-property / hydrophobic B-property ratios of dna amphiphiles B-property ( figure 19e ) . [SEP]
[CLS] besides , the hybridization of hydrophobic B-property polymers B-material on both sides of a dna cage results in a " handshake " of the polymers B-material within the cage , resulting in a dna micelle B-material cage . [SEP]
[CLS] therefore , they further prepared monodisperse crosslinked polymer B-material nanoparticles B-nanoparticle in dna cages , the hydrophobic B-property environment of which can be used to load guest molecules ; and the guest molecules can be released by the presence of specific dna sequences ( figure 19f ) . [SEP]
[CLS] the self - assemblies of common block copolymers have been widely studied , such as spherical micelles B-material , vesicles , sheets , tubes , which show a large number of applications in the field of materials science and biomedicine . [SEP]
[CLS] unlike the base - pairing language of dna , the self - assembly of classic block copolymers is governed by the interactions including , electrostatic forces , hydrophobic B-property interactions I-property , and [UNK] - [UNK] stacking . [SEP]
[CLS] the incorporation of the specific hybridization properties of dna into the self - assembly of the typical block copolymers will produce incredible assembly structures and satisfy some specific applications . [SEP]
[CLS] classical amphiphilic B-property block copolymers form small - sized micelles B-material in aqueous solutions at specific hydrophilic B-property - hydrophobic B-property ratios . [SEP]
[CLS] its inner core B-material is hydrophobic B-property , and its shell B-material is hydrophilic B-property , which has been widely used in drug and gene delivery . [SEP]
[CLS] through the coassembly method , the peg - based amphiphile B-property and the dna - based amphiphile B-property can form a mixed micelle B-material together , and the dna in this mixed micelle B-material still retains the ability of hybridization . [SEP]
[CLS] zhang et al . coassembled the peg - b - pcl amphiphilic B-property block copolymer with dna - b - pcl to prepare mixed micelles B-material and studied in detail the effect of the ratios of peg / dna on dna properties , including the hybridization dynamics , cellular uptake , biostability , and gene regulation capability ( figure 20a ) . [SEP]
[CLS] park et al . reported that ps - b - dna and psb - pnipam were coassembled into a mixed micelle B-material with a ps core B-material and a dna / pnipam mixed shell B-material ( figure 20b ) . [SEP]
[CLS] due to the temperature - responsive properties of pnipam , when the temperature is higher than the lower critical solution temperature ( lcst ) , pnipam would change from a hydrated coil to a dehydrated collapsed state . [SEP]
[CLS] as a result , the dna strands in the shell B-material of mixed micelle B-material would be exposed , showing faster dna degradation rates and better cell B-material uptake through receptor - mediated endocytosis B-event . [SEP]
[CLS] this dynamically switchable dna recognition properties of dna based nanostructure offers new opportunities for the design of intelligent drug delivery systems in the future . [SEP]
[CLS] amphiphilic B-property molecules can also self - assemble into a vesicle structure in an aqueous solution under a specific hydrophilic B-property / hydrophobic B-property ratio , which has a hollow aqueous cavity and a lipophilic B-property bilayer . [SEP]
[CLS] in one of the examples , dna amphiphiles B-property conjugated with tocopherol tails were anchored into the phospholipid layer of the liposomes B-nanoparticle , which were self - assembled from 1 , 2 - dioleoyl - sn - glycerol - 3 - phosphate choline ( dopc ) , resulting in liposome B-nanoparticle snas ( figure 20c ) . [SEP]
[CLS] this liposome B-nanoparticle sna has a high - density of dnas on the surface and displays typical " sna " properties , such as the robust biological stability and the highly efficient cell B-material internalization . [SEP]
[CLS] also , the properties of the vesicles are related to the sequence of dna , e . g . , the fusion between vesicles can be promoted by the presence of dna [SEP]
[CLS] alternatively , dna amphiphiles B-property can be used to regulate cell - to - cell interactions through the anchoring of hydrophobic B-property moieties and molecular hybridization of dna . [SEP]
[CLS] in another work , park et al . reported that two amphiphilic B-property block copolymers , poly ( butadiene ) - block - poly ( ethylene oxide B-material ) ( pbd - b - peo ) and polymethyl acrylate - block - dna ( pma - b - dna ) , could coassemble to form giant polymersomes ( figure 20d ) ; when two of this kind of polymersomes interacted via strand - hybridization , the dna that originally uniformly distributed on the polymersome would gather to one side and form a dna island . [SEP]
[CLS] in addition to common micelle B-material and vesicle structures , amphiphilic B-property block copolymers can also self - assemble into other unusual architectures , such as cylinders and sheets . [SEP]
[CLS] in particular , amphiphilic B-property conjugated polymers B-material will exhibit different selfassembly properties , due to their special characteristics , such as [UNK] - [UNK] stacking and the rigid molecular - structures . [SEP]
[CLS] park et al . prepared an amphiphilic B-property conjugated polymer B-material , peg - b - pt ( polythiophene ) . [SEP]
[CLS] peg - b - pt can self - assemble into a variety of morphologies in aqueous solutions , including flakes , vesicles , and copyright 2017 , american chemical society . [SEP]
[CLS] b ) thermal - responsive cell B-material - uptake of pnipam / dna hybrid micelles B-material . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , royal society of chemistry . [SEP]
[CLS] c ) the dna - tocopherol amphiphiles B-property can be inserted into the phospholipid bilayer of liposome B-nanoparticle through hydrophobic B-property interactions I-property to form liposomal B-nanoparticle snas , which show higher cellular uptake , more efficient gene knockdown , and higher biostability . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2014 , american chemical society . [SEP]
[CLS] d ) binary coassembly of pbd - b - peo and pma - b - dna into giant mixed polymersomes ; and the imaging of the formation of dna islands at the junction site of two polymersomes . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2016 , american chemical society . [SEP]
[CLS] copyright 2014 , american chemical society . [SEP]
[CLS] nanoribbons [SEP]
[CLS] thereafter , they used dna - b - pt to coassemble with peg - b - pt at a molar ratio of 1 : 200 to form dnafunctionalized nanoribbons , and the dna hybridization ability on the nanoribbons was verified by the presence of cdnamodified aunps B-nanoparticle , which can be observed by tem imaging ( figure 21 ) . [SEP]
[CLS] dna - based biomaterials exhibit unparalleled potential in structural design and functional applications . [SEP]
[CLS] compared with other building blocks , like peptides B-material or synthetic polymers B-material , dna shows many sequence - dependent functionalities and can realize the precise nanostructure construction through the exact basepairing interactions . [SEP]
[CLS] besides , as one of the most basic driving forces in nature , the introduction of hydrophobic B-property interactions I-property has expanded the scope of dna - based biomaterials and reduced their preparation costs . [SEP]
[CLS] moreover , dna self - assemblies driven by hydrophobic B-property interactions I-property exhibit stronger biological stability and contain hydrophobic B-property domains for the loading of functional molecules . [SEP]
[CLS] therefore , hydrophobic B-property interactions I-property play a more and more important role in the development of dna - based biomaterials . [SEP]
[CLS] according to the discussions in this review , utilizing hydrophobic B-property interaction I-property to fabricate dna - based biomaterials shows several advantages over the other strategies , and the application prospects are also discussed . [SEP]
[CLS] 1 ) the phase separation of long strand dna provides an efficient and low - cost method for the fabrication of dna - based biomaterials . [SEP]
[CLS] in this system , dna can be used not only as delivery carriers but also as functional cargoes ( e . g . , sirna , antisense dna , and dnazyme ) . [SEP]
[CLS] additionally , traditional chemotherapeutics ( e . g . , doxorubicin B-material ) can be loaded into the dna nanoparticles . [SEP]
[CLS] hence , this strategy can be applied to drug delivery and gene regulation . [SEP]
[CLS] besides , stimuli - responsive dna sequences ( e . g . , ph and ions B-material ) can also be integrated into the dna nanoparticles , achieving smart drug delivery . [SEP]
[CLS] other materials can also be introduced into this system for increasing functionalities , such as silver B-material nanoclusters for optical properties and gold B-nanoparticle nanoparticles I-nanoparticle for heat effect . [SEP]
[CLS] 2 ) as for dna amphiphiles B-property , many organic functional molecules can be introduced to enrich the functionalities of the resultant selfassemblies , such as fluorescence B-property dyes , organic semiconducting molecules with photothermal or photodynamic capabilities , or stimuli - responsive polymers B-material . [SEP]
[CLS] the assembly formed by dna amphiphiles B-property contains a hydrophobic B-property domain that can accommodate some functional materials for special applications , such as nir - ii dye and magnetic B-property resonance contrast B-technique agents I-technique . [SEP]
[CLS] 3 ) the dynamic and highly tunable hydrophobic B-property interactions I-property will make the resultant materials more sensitively respond to the environmental stimuli , providing a new avenue to develop smart biomaterials . [SEP]
[CLS] for instance , as the aggregation - state of the dna amphiphiles B-property can be well controlled by the hydrophilic B-property dna block , organic dyes are used as the hydrophobic B-property blocks , and some innovative dna - based biosensors have been developed . [SEP]
[CLS] when the dna block recognizes targets by chain hybridizations or conformational changes , the amphiphilicity B-property of the self - assembly system will be altered to result in further aggregation of the dna amphiphiles B-property or dissociation of the self - assembled micelles B-material . [SEP]
[CLS] therefore , the aggregation states of the organic dyes will be changed to show significantly different optical properties . [SEP]
[CLS] in another example , due to the hydrophobic B-property interactions I-property , dna amphiphiles B-property showed more efficient albumin binding . [SEP]
[CLS] thus , by the conjugation with the hydrophobic B-property lipid B-material , the cpg - lipid amphiphiles B-property showed a 30 - fold increase in t - cell B-material priming and the significantly enhanced immunotherapy efficacy . [SEP]
[CLS] 4 ) hydrophobic B-property interaction I-property offers an efficient way to construct higher - order self - assembly structures . [SEP]
[CLS] dna origami is the most potent nanotechnology , which can create almost any arbitrary shapes with precisely defined dimensions at the nanoscales . [SEP]
[CLS] however , one of the major challenges of the dna origami technique is the construction of macroscopic structures only by the hydrogenbonding of base pairing . [SEP]
[CLS] introducing additional hydrophobic B-property interactions I-property is an effective approach to solve this problem . [SEP]
[CLS] the nanosized building blocks created by the dna origami technique are possible to be further organized by dna amphiphiles B-property to self - assemble into higher - order bulky structures . [SEP]
[CLS] on one hand , as the hydrophobicity B-property of the hydrophobic B-property block can be easily tuned , a variety of higher - level self - assembled structures are possible to be realized . [SEP]
[CLS] on the other hand , the hydrophobic B-property blocks can bring in more functionalities to the final materials . [SEP]
[CLS] 5 ) clarifying the rules of controlling and utilizing hydrophobic B-property interactions I-property will promote the development of new dna - based functional materials . [SEP]
[CLS] as discussed in the second part , dna itself shows thermal - responsive properties , which will change from the dispersed state to the aggregated state at a certain temperature driven by the hydrophobic B-property interaction I-property . [SEP]
[CLS] walther et al . revealed that this thermal - controlled phase separation properties of dna are dependent on the composition and the polymerization degree of the dna chains . [SEP]
[CLS] based on their observations , they could construct hierarchical self - assembled architectures by the sophisticated design of the hydrophobic B-property interactions I-property , which also showed temperature - controlled cargo release capability . [SEP]
[CLS] for the dna / inorganic B-nanoparticle nanoparticle I-nanoparticle complexes formed by hydrophobic B-property interactions I-property , it also provides a nonlabeled way to fabricate multifunctional materials . [SEP]
[CLS] 6 ) the application field of the dna - based materials has been significantly extended by the comprehensive understanding and widespread use of hydrophobic B-property interactions I-property . [SEP]
[CLS] for example , amphiphilic B-property dna molecules can be used to regulate the interaction between different cells B-material , which provides great possibilities for cell B-material engineering research , such as cell B-material signal transduction , cell B-material microenvironment regulation , and cell B-material presentation . [SEP]
[CLS] also , amphiphilic B-property dna may show excellent potential in cell - based therapies , such as regulating immune cells B-material or other circulating cells . [SEP]
[CLS] in terms of synthetic biology , dna - based biomaterials combined with hydrophobic B-property interactions I-property may facilitate the preparation of artificial organelles and even artificial cells B-material . [SEP]
[CLS] although dna - based biomaterials combined with hydrophobic B-property interactions I-property exhibit many excellent properties , there are still some challenges that need to be overcome , both in the design of functional materials and in practical clinical transformation . [SEP]
[CLS] first , the precise control of the degree of hydrophobic B-property interactions I-property in pure dnas , dna amphiphiles B-property , and dna complexes is still an unresolved issue . [SEP]
[CLS] more systematic studies on the dna - based materials are still required to give the structurehydrophobic interaction - property relationship . [SEP]
[CLS] also , although nanoscale dna - based biomaterials exhibit improved biological stability compared with free dna , minimizing the degradation of ribozymes is still a challenge , especially in vivo . [SEP]
[CLS] due to the immunogenicity B-property of dna itself , the safety of dna - based biological materials in vivo still needs to be carefully evaluated before the clinical transformation . [SEP]
[CLS] hydrophobic B-property interaction I-property is a weak interaction , which will be affected by many factors , such as solution dilution and interference by other amphiphilic B-property molecules ; therefore , how to protect this dna - based biomaterial based on hydrophobic B-property interaction I-property is also a challenge . [SEP]
[CLS] moreover , amphiphilic B-property dna will tend to be inserted into the cell B-material membrane through hydrophobic B-property interactions I-property , which may cause unpredictable cell B-material damage . [SEP]
[CLS] as for the strategy where dna and other materials complex through hydrophobic B-property interaction I-property , although these composite materials have exhibited great potential in various biological applications , their properties still need to be further explored . [SEP]
[CLS] in summary , dna - based biomaterials combined with hydrophobic B-property interaction I-property are a very comprehensive and extensive platform . [SEP]
[CLS] in this platform , various functional or structural dna can be easily introduced ; and the hydrophobic B-property interaction I-property provides the driving force for self - assembly , which allows these dna - based materials to show diversified functionalities , more sensitive stimuli - responsiveness , hierarchical self - assembly capability , and more complicated interactions with biological molecules or cells B-material . [SEP]
[CLS] therefore , we believe that the integration of dna properties and hydrophobic B-property interactions I-property will become a trend to address the practical problems that need to be solved in the biomedical field , and hydrophobic B-property interaction I-property will become a necessary tool in the design of dna - based biomaterials in the future . [SEP]
[CLS] xiao obtained his bachelor ' s degree in biopharmaceutical engineering from huazhong university of science and technology in 2017 . [SEP]
[CLS] currently , he is a ph . d . student under the supervision of prof . leilei tian at southern university of science and technology . [SEP]
[CLS] his research focuses on dnahybrid conjugates and their assembly . [SEP]
[CLS] zhe chen received his m . sc . degree in pharmaceutical analysis from soochow university in 2016 . [SEP]
[CLS] he is presently a joint ph . d . student under the cosupervision of prof . leilei tian at southern university of science and technology and prof . xuanjun zhang at university of macau . [SEP]
[CLS] his research interests are focused on aie - based dna nanotechnology for biomedical applications . [SEP]
[CLS] 1 . schematic illustration of hydrophobic B-property interaction I-property as a promising driving force for the biomedical applications of nucleic acid - based biomaterials . [SEP]
[CLS] a ) illustration for hydrophobic - hydrophilic phase separation of lssdna produced by rolling circle amplification . [SEP]
[CLS] b ) illustration for heat - induced phase separation of purine - rich lssdna . [SEP]
[CLS] c ) a phase diagram of the cloud point temperature corresponding to the length of dna at [ mg 2 + ] = 50 × 10 −3 m . d ) a phase diagram of the cloud point temperature of poly a ( ≈1000 - base long ) corresponding to the concentration of the counter - ions . [SEP]
[CLS] the panels ( b ) - ( d ) are reproduced with permission . [SEP]
[CLS] copyright 2018 , nature publishing group [SEP]
[CLS] a ) illustration of the phase separation of mg 2 + - stabilized lssdna nanoparticles B-nanoparticle for targeted dox - delivery . [SEP]
[CLS] b ) illustration of the design of mg - rnc . [SEP]
[CLS] the blue part will fold up at ph 5 to release dox in the green part . [SEP]
[CLS] the red part is sgc8 aptamer for targeting cem cells B-material . [SEP]
[CLS] c ) tem imaging of mg - rnc with sizes of about 100 nm . [SEP]
[CLS] d ) biostability of mg - rnc comparing with the free lssdna . [SEP]
[CLS] the " time " in the inset refers to the incubation B-technique time in serum . [SEP]
[CLS] e ) the cytotoxicity B-property of mg - rnc1 @ dox nanoparticles B-nanoparticle on targeted cem cells B-material and nontargeted ramos cells B-material . [SEP]
[CLS] f ) the distribution of mg - rnc1 @ dox , mg - rnc2 @ dox ( without targeted aptamer sequences ) , and free dox in the main organs after intravenous injection . [SEP]
[CLS] from left to right are the hearts , lungs , livers , spleens , kidneys , and tumors B-material . [SEP]
[CLS] the pseudocolor indicates the fluorescence B-property intensity of dox . [SEP]
[CLS] all panels are reproduced with permission . [SEP]
[CLS] copyright 2018 . american chemical society [SEP]
[CLS] a ) illustration of the design principle : the rca product acts as a cocarrier for sirna delivery . [SEP]
[CLS] b ) agarose B-technique gel I-technique electrophoresis I-technique analysis of pei / sirna and pei / rca - sirna polyplexes . c ) the in vivo rnai test by the intratumoral injection of pei / sirna and pei / rca - sirna polyplexes . [SEP]
[CLS] all panels are reproduced with permission . [SEP]
[CLS] copyright 2019 . american chemical society . [SEP]
[CLS] a ) illustration of the preparation of rca - agncs . [SEP]
[CLS] b ) illustration of rca - agncs with higher photostability for detecting ros / rns . [SEP]
[CLS] c ) titration curves of the rca - agncs with an increase in • oh concentration ; the green emissions were excited at 440 nm and the red emissions were excited at 560 nm . [SEP]
[CLS] d ) using the rca - agncs as a ratiometric fluorescent B-property probe to monitor the dynamic levels of ros / rns in lipopolysaccharide - treated a549 cells B-material at various incubation B-technique times . [SEP]
[CLS] all panels are reproduced with permission . [SEP]
[CLS] copyright 2018 , american chemical society [SEP]
[CLS] 6 . the structural formula of commonly used hydrophobic B-property moieties of dna amphiphiles B-property . [SEP]
[CLS] a ) lipids B-material : cholesterol , monoacyl lipid B-material , and diacyl lipid B-material . [SEP]
[CLS] b ) molecules with [UNK] - conjugated systems : poly [ fluorene - phenylene ] derivative ( pfp ) , polythiophene derivative ( pt ) , and oligo - p - phenyleneethynylene derivative ( ppe ) . [SEP]
[CLS] c ) strongly hydrophobic B-property polymers B-material : polynorbornene ( pnb ) derivative grafted with aromatic rings and polystyrene ( ps ) . [SEP]
[CLS] d ) biodegradable B-property polymers B-material : polylactic acid ( pla ) , polycaprolactone ( pcl ) , and polylactic acid - glycolic acid ( plga ) . [SEP]
[CLS] e ) stimulus - responsive polymers B-material : temperature - responsive poly ( n - isopropylacrylamide ) ( pnimap ) and ph - responsive polyacrylic acid ( paa ) . [SEP]
[CLS] f ) other hydrophobic B-property molecules : polyphosphorylated hexaethylene ( he n ) , polypropylene oxide B-material ( ppo ) . [SEP]
[CLS] g ) typical aie molecule : tetraphenylethylene ( tpe ) . [SEP]
[CLS] 7 . several strategies for constructing fluorescence B-property biosensors based on dna amphiphiles B-property . [SEP]
[CLS] schematic illustration of the strategy of signal - on / off biosensors based on a ) dna - aie amphiphiles B-property and b ) dna - acq amphiphiles B-property . [SEP]
[CLS] c ) schematic representation of fluorescence B-property biosensors based on the self - assembly of dna amphiphiles B-property through hydrophobic B-property interactions I-property . [SEP]
[CLS] a ) schematic illustration of the strategy of telomerase activity detection based on the amphipathic bipolar cp - c - dna probe through changing the hydrophilicity B-property of the probe . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2015 , american chemical society . [SEP]
[CLS] b ) schematic representation of tpe - dna probe to detect mir - 21 with the assistance of exo iii . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2015 , american chemical society . [SEP]
[CLS] c ) upper panel : synthetic route of tpe - r - dna . [SEP]
[CLS] lower panel : schematic representation of tpe - r - dna probe to detect mnsod mrna . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , american chemical society . [SEP]
[CLS] d ) schematic illustration of tpe - dna probe for k + detection . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2017 , elsevier . [SEP]
[CLS] e ) the working scheme of ifs - tpe conjugates in response to ph changes , and the fluorescence B-property intensity of aie increased when the ph was switched from 7 . 0 to 5 . 0 . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , royal society of chemistry [SEP]
[CLS] a ) working principle of the switchable aptamer micelle flares formed by dna - diacyllipid conjugates . [SEP]
[CLS] b ) tem image of the switchable aptamer micelle flares after negative staining by 2 % aqueous uranyl acetate ( scale bar : 200 nm ) . [SEP]
[CLS] c ) the in vitro responses of switchable aptamer micelle flares to atp target . [SEP]
[CLS] the panels ( a ) - ( c ) are adapted with permission . [SEP]
[CLS] copyright 2013 , american chemical society . [SEP]
[CLS] d ) schematic illustration of the molecular structure of o1 - dtpe , e ) using the g - quadruplex structure as the molecular scaffold for aiegen self - assembly , and f ) the accurate control of aie effect by the g - quadruplex structure . [SEP]
[CLS] the panels ( d ) - ( f ) are adapted with permission . [SEP]
[CLS] copyright 2018 , royal society of chemistry . [SEP]
[CLS] 10 . [SEP]
[CLS] several strategies of constructing dna amphiphiles B-property to prove the in vivo performance of therapeutic nas . [SEP]
[CLS] a ) dna amphiphile B-property can selfassemble into 3d nanostructures such as micelle B-material and vesicle , which can enhance the biostability and intracellular delivery of nas . [SEP]
[CLS] b ) the micelles B-material formed from dna amphiphiles B-property contain a hydrophobic B-property environment that can accommodate the lipophilic B-property molecules through the physical encapsulation . [SEP]
[CLS] alternatively , the drug molecules are chemically conjugated to the hydrophobic B-property moiety of the dna amphiphile B-property . [SEP]
[CLS] c ) floxuridine , a typical anticancer B-property nucleoside drug , is embedded into the dna sequence by replacing the t - base due to their structural similarity . [SEP]
[CLS] benzyl bromide - modified drugs can react with phosphorothioate groups to achieve the direct chemical conjugation of drugs to dna without the need for additional drug carriers . [SEP]
[CLS] 11 . [SEP]
[CLS] a ) schematic representation of he 12 - luc - aso and ( he - heg ) 6 - luc - aso with different micelles B-material forming capability for the delivery of antiluciferase aso . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2015 , royal society of chemistry . [SEP]
[CLS] b ) schematic representation of the rna - polymer B-material amphiphile B-property for sirna delivery ; some chemical modifications , [UNK] - dabcyl , [UNK] - stilbene , [UNK] - dmab , [UNK] - phosphorylated , and [UNK] - hydroxyl , on the outer sirnas were investigated , and only dabcyl and stibenen could help to enhance the transfection B-property efficiency I-property of sirna . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , american chemical society [SEP]
[CLS] with the counterpart ps - b - peg , ps - b - dna sna showed ≈3 - fold enhanced cell B-material penetration ability , measured by fcm . [SEP]
[CLS] specific aptamers were introduced into the ps - b - dna sna with 10 % of the total surface dnas to obtain ps - b - dna / apt . [SEP]
[CLS] the cell B-material penetration ability of ps - b - dna / apt was determined to be 1 . 5 - fold stronger than that of ps - b - dna sna . [SEP]
[CLS] scavenger receptor B-material ( sr ) was proved to be a key receptor B-material for ps - b - dna sna penetrating cells B-material through competitive inhibitor experiments . [SEP]
[CLS] what ' s more , sr - mediated transcytosis is a promising strategy for crossing bbb . [SEP]
[CLS] therefore , the ability of ps - b - sna to cross bbb was evaluated , and the results indicated that ps - b - dna showed 4 . 5fold higher traversing efficiency than that of ps - b - peg ( figure 14c ) . [SEP]
[CLS] 14 . [SEP]
[CLS] a ) scheme of the preparation of nir - ii emitting organic snas and their application in brain tumor imaging . [SEP]
[CLS] b ) scheme of ps - b - dna synthesized by solid - phase " click " reaction . [SEP]
[CLS] c ) transcytosis efficiency for different samples over time . [SEP]
[CLS] d ) nir - ii fluorescence B-technique imaging I-technique of the mouse heads and isolated brains by using different nir - ii emitting materials under irradiation at 808 nm . [SEP]
[CLS] all panels are reproduced with permission . [SEP]
[CLS] copyright 2020 , wiley - vch . [SEP]
[CLS] 15 . [SEP]
[CLS] illustration of go - based dna sensor . [SEP]
[CLS] fluorophore - labeled ssdna is adsorbed by go and its fluorescence B-property is quenched ; after forming a dsdna with complementary target dna , it is desorbed from go and release the fluorescence B-property , achieving the fluorescence - based dna detection . [SEP]
[CLS] 16 . [SEP]
[CLS] label - free biosensors based on the ssdna - aunps B-nanoparticle complex . [SEP]
[CLS] a , b ) ssdna can prevent the aggregation of aunps B-nanoparticle while dsdna cannot . [SEP]
[CLS] c ) a colorimetric sensor for pb 2 + detection was designed by using the pb 2 + - related dnazyme . [SEP]
[CLS] d ) a sensing system could be designed by using conformational changes of dna in the presence of the specific analytes . [SEP]
[CLS] 17 . [SEP]
[CLS] a ) complexes were formed by pei and ssdna through the electrostatic interaction . [SEP]
[CLS] b ) the stability of the complex between pei and ssdna can be enhanced by the inducing of hydrophobic B-property interactions I-property through the modification of hydrophobic B-property moieties on the pei chain . [SEP]
[CLS] a ) ssdna shows stronger interaction with [UNK] - conjugated polyelectrolytes than dsdna because of the hydrophobic B-property interactions I-property . [SEP]
[CLS] b ) the aromatic ring of the [UNK] - conjugated polyelectrolyte backbone provides hydrophobic B-property forces , and the cationic B-material side chain provides electrostatic interactions . [SEP]
[CLS] c ) more efficient fret can be achieved from fluorescent B-property [UNK] - conjugated polyelectrolytes to the fluorophore - modified ssdna compared with dsdna . d ) the illustration of a sensor for cocaine detection based on [UNK] - conjugated polyelectrolytes / dna complex . [SEP]
[CLS] the panels ( c ) and ( d ) are reproduced with permission . [SEP]
[CLS] copyright 2010 , american chemical society [SEP]
[CLS] 19 . [SEP]
[CLS] a ) illustration of the artificial transmembrane channels prepared by dna origami technique . [SEP]
[CLS] the cylinder represents the dna double helix structure . [SEP]
[CLS] the red is the stem inserted into the cell B-material membrane . [SEP]
[CLS] the orange is cholesterol - modified dna used to anchor this artificial ion B-material channel into the lipid B-material bilayer I-material . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2012 , aaas . [SEP]
[CLS] b ) driven by hydrophobic B-property forces , a single layer of dna origami is folded into a double - layered structure . [SEP]
[CLS] the tem images show the dna origami sheets before and after folding . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2014 , wiley - vch . [SEP]
[CLS] c ) hydrophobic B-property molecules are hybridized to dna origami through dna tail to form a hydrophobic B-property framework , which can self - assemble into higher - order structures . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2016 , wiley - vch . [SEP]
[CLS] d ) dna origami was used as a template to mediate the formation of vesicles with the controlled size and shape . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2016 , nature publishing group . [SEP]
[CLS] e ) different quantized cage assemblies controlled by the length of the hydrophobic B-property polymer B-material ; dna cage - loop structures can be prepared by adjusting the hydrophobicity B-property of the dna amphiphiles B-property . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2016 , american chemical society . [SEP]
[CLS] f ) the hydrophobic B-property polymer B-material is packed into a dna cage by molecular hybridization . [SEP]
[CLS] the hydrophobic B-property environment in the cage can contain the guest molecule and release the guest molecule in the presence of specific dna sequence . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2013 , nature publishing group . [SEP]
[CLS] 20 . [SEP]
[CLS] a ) peg - b - pcl and dna - b - pcl can be coassembled into mixed micelles B-material . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2017 , american chemical society . [SEP]
[CLS] b ) thermal - responsive cell B-material - uptake of pnipam / dna hybrid micelles B-material . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , royal society of chemistry . [SEP]
[CLS] c ) the dna - tocopherol amphiphiles B-property can be inserted into the phospholipid bilayer of liposome B-nanoparticle through hydrophobic B-property interactions I-property to form liposomal B-nanoparticle snas , which show higher cellular uptake , more efficient gene knockdown , and higher biostability . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2014 , american chemical society . [SEP]
[CLS] d ) binary coassembly of pbd - b - peo and pma - b - dna into giant mixed polymersomes ; and the imaging of the formation of dna islands at the junction site of two polymersomes . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2016 , american chemical society [SEP]
[CLS] 21 . scheme of dna - b - pt and peg - b - pt coassembly into dnafunctionalized 2d nanoribbons . [SEP]
[CLS] on the right are the tem images of the dna - functionalized nanoribbons before and after the modification with aunps B-nanoparticle . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2014 , american chemical society [SEP]
[CLS] dedicated to the life and legacy of moritz f . kircher in the last two decades , dna has attracted significant attention toward the development of materials at the nanoscale for emerging applications due to the unparalleled versatility and programmability of dna building blocks . [SEP]
[CLS] dna - based artificial nanomaterials B-material can be broadly classified into two categories : dna nanostructures ( dna - nss ) and dna - functionalized nanoparticles B-nanoparticle ( dna - nps B-nanoparticle ) . [SEP]
[CLS] more importantly , their use in nanotheranostics , a field that combines diagnostics with therapy via drug or gene delivery in an all - in - one platform , has been applied extensively in recent years to provide personalized cancer treatments . [SEP]
[CLS] conveniently , the ease of attachment of both imaging and therapeutic moieties to dna - nss or dna - nps B-nanoparticle enables high biostability , biocompatibility B-property , and drug loading capabilities , and as a consequence , has markedly catalyzed the rapid growth of this field . [SEP]
[CLS] this review aims to provide an overview of the recent progress of dna - nss and dna - nps B-nanoparticle as theranostic agents , the use of dna - nss and dna - nps B-nanoparticle as gene and drug delivery platforms , and a perspective on their clinical translation in the realm of oncology . [SEP]
[CLS] cancer is the first or second leading cause of death in humans below the age of 70 in over 50 % of countries . [SEP]
[CLS] more alarmingly , the annual number of new cancer cases is projected to grow from 18 . 1 million in 2018 to 29 . 4 million in 2040 due to population growth , aging , and increasing pollution . [SEP]
[CLS] currently , cancer treatment options for solid tumors B-material include removal of cancerous tissue via surgery , chemotherapy , radiation therapy , immunotherapy , or a combination of some or all of these approaches . [SEP]
[CLS] once a solid tumor B-material without the presence of metastases B-event is diagnosed , the primary line of treatment in most cases is surgery , which improves the clinical outcome in the majority of cases . [SEP]
[CLS] however , complete surgical removal of cancer tissue is often not feasible due to i ) poor visual contrast between cancerous and healthy tissue and ii ) the fact that surgeons are often hindered in resecting the full tumor B-material extent because critical structures such as nerve and blood vessels run near or through the diseased tissue . [SEP]
[CLS] to alleviate this issue , molecular imaging agents are used to augment the contrast between healthy and diseased tissues , however , current molecular imaging B-technique techniques I-technique have significant drawbacks in detecting the true microscopic extent of cancer . [SEP]
[CLS] in addition , conventional chemotherapeutic agents are relatively nonselective , causing frequent severe side effects which can range from acute manifestations to chronic problems that deteriorate the quality of life of cancer patients . [SEP]
[CLS] other limitations of chemotherapy include the development of drug resistance . [SEP]
[CLS] it is therefore desirable to develop anticancer B-property drugs that can target cancer cells B-material in a more selective manner , thus reducing side effects while improving therapeutic efficacy . [SEP]
[CLS] taken together , the main goals for better cancer treatment and mitigation are : i ) early diagnosis , ii ) complete surgical removal of the macroscopic tumor B-material burden , and iii ) selective delivery of anticancer B-property drugs to destroy residual cancerous tissues . [SEP]
[CLS] with regards to cancer , the ideal therapy would deliver the correct treatment to the specific target in a localized and controlled manner , and in turn , minimize systemic side effects . [SEP]
[CLS] of course , this is an extremely challenging task , namely , due to tumor B-material heterogeneity between patients . [SEP]
[CLS] however , in recent years , the use of theranostics has emerged as a promising means to overcome such challenges . [SEP]
[CLS] theranostics combines diagnostics with therapeutics , enabling cancer to be diagnosed and www . advancedsciencenews . com www . advancedscience . com treated on an individual , personalized level , and allowing the therapeutic efficacy to be monitored noninvasively and in real time . [SEP]
[CLS] such platforms utilize contrast B-technique agents I-technique which can be tracked in vivo using at least one imaging modality , e . g . , magnetic B-property resonance imaging ( mri ) , positron B-technique emission I-technique tomography I-technique ( pet ) , computed B-technique tomography I-technique ( ct ) , surface - enhanced raman scattering ( sers ) , or fluorescence B-property [SEP]
[CLS] a therapeutic module such as chemotherapeutics , immunotherapies , or photosensitive B-property molecules [SEP]
[CLS] is also incorporated . [SEP]
[CLS] nanotheranostics makes use of nanomaterials B-material , e . g . , proteins B-material , polymers B-material , or dna as a host material B-material for imaging and therapeutic agents . [SEP]
[CLS] in recent years , substantial efforts have resulted in the production of a range of nanoplatforms with theranostic capabilities . [SEP]
[CLS] nanotheranostics have been explored extensively in oncology for applications , including the monitoring of intratumoral drug delivery , image - guided focal therapy , and monitoring of changes in the tumor B-material microenvironment ( tme ) as a response to therapy . [SEP]
[CLS] following treatment , the optimum nanotheranostic should , therefore , be thought of as a platform capable of providing longterm , patient - specific information on disease status . [SEP]
[CLS] as such , when designing nanotheranostics , researchers must go above and beyond to create a platform that offers several advantages over traditional approaches , a key requirement that will further support the translation of nanotheranostics into the clinic . [SEP]
[CLS] this can be achieved by taking advantage of and incorporating several unique functionalities inherent to nanomaterials B-material in order to deliver personalized medicine . [SEP]
[CLS] such features include : i ) rapid and highly selective accumulation within the tumor B-material ; ii ) the reporting of biochemical and morphological changes from the region of interest ; iii ) delivery of an effective therapeutic response ; ( iv ) safe and biodegradable B-property properties ; and ( v ) offering a benefit over traditional treatments by improving drug efficacy and consequently patient tolerance . [SEP]
[CLS] therefore , owing to the desire to incorporate all of these necessities , the design of nanotheranostic agents is far from simple . [SEP]
[CLS] it is well established that blood vessels in malignant lesions are more permeable to macromolecules ( size range of > 10 nm ) than those in healthy tissues , and these molecules are subsequently retained within the tumor B-material tissue due to impaired lymphatic clearance . [SEP]
[CLS] this phenomenon is referred to as " enhanced permeability and retention " ( epr ) effect or " passive targeting . [SEP]
[CLS] " macromolecules and nps B-nanoparticle that are surface - functionalized with moieties targeting the cancer microenvironment or cancer cells B-material can further enhance the intratumoral homing , which is termed as " active targeting . [SEP]
[CLS] " further , innovative strategies have also been developed to integrate anticancer B-property drug or drug combinations in the nanostructures via covalent and noncovalent chemistry . [SEP]
[CLS] in the last decade , a better understanding of tumor B-material biology and synthesis of versatile nanomaterials B-material such as polymers B-material and polymer B-material gels , lipids B-material , inorganic nps B-nanoparticle , and biomacromolecular scaffolds has aided the development of novel theranostic platforms for cancer . [SEP]
[CLS] the development of such materials is very diverse and extensively reviewed elsewhere . [SEP]
[CLS] one of the significant drawbacks of using engineered B-material nanomaterials I-material is the systemic and immune toxicity B-property , which raises significant concerns for real - life clinical applications . [SEP]
[CLS] as an alternative strategy , the last 20 years have seen enormous interest in the use artificial ( synthetic ) dna - based nanomaterials B-material . [SEP]
[CLS] dna , a genetic material B-material , possesses high biocompatibility B-property and low cytotoxicity B-property , which is optimal for biomedical applications . [SEP]
[CLS] other advantages of dna - based nanostructures are predictable intermolecular B-property interaction I-property and recognition properties ; intrinsic nanoscale size ( 2 nm in diameter and ≈0 . 3 nm / per base pair ) ; ease of chemical / enzymatic synthesis of specific sequences ; and optimum half - life in biological fluids . [SEP]
[CLS] further , selective chemical modifications B-event of I-event dna I-event structures with anticancer B-property drugs and imaging modalities provide flexibility in the design of theranostic nanoprobes B-nanoparticle . [SEP]
[CLS] in this review , we present recent applications of two different artificial dna nanostructures , namely , dna nanostructures ( dna - nss ) and dna - functionalized nanoparticles B-nanoparticle ( dna - nps B-nanoparticle ) , for the purpose of cancer theranostics . [SEP]
[CLS] in section 2 . 1 , we provide a brief introduction to dna - nss and dna - nps B-nanoparticle . [SEP]
[CLS] in section 2 . 2 , we cover the biological stability of dna - nss and dna - nps B-nanoparticle . [SEP]
[CLS] we summarize recent developments in the use of dna - nps B-nanoparticle and dna - nss in theranostic applications in sections 2 . 3 and 2 . 4 , respectively , and specifically review the current status of dna - nss and dna - nps B-nanoparticle as drug and gene delivery platforms in sections 2 . 5 and 2 . 6 , respectively . [SEP]
[CLS] in section 3 , we discuss the clinical translation of dna - ns and dna - nps B-nanoparticle and conclude in section 4 with our final thoughts on the future of the field . [SEP]
[CLS] we thematically classify artificial dna - nss into dna tile - based and dna origami - based nanostructures . [SEP]
[CLS] tile - based structures are self - assembled by hybridizing multiple unique dna single strands . [SEP]
[CLS] further , hierarchical assembly via " sticky end " cohesion emanates large dna structures . [SEP]
[CLS] in 1983 , kallenbach et al . proposed and reported the first artificial , immobile fourway junction , also known as holliday junction , which is transiently formed during genetic recombination . [SEP]
[CLS] using a similar strategy , the same group constructed three - , five - , six - , eight - , and 12 - way junctions . [SEP]
[CLS] however , due to substantial structural flexibility , the multi - arm junctions were not amenable to produce higher - order nanoscale structures . [SEP]
[CLS] to alleviate this issue , more robust dna double - crossover ( dx ) dna - nss were developed [SEP]
[CLS] in 1998 , the first example of a large - scale , 2d dna structure was fabricated using sticky - end cohesion two - arm dx tiles and visualized by an atomic force microscope ( afm ) . [SEP]
[CLS] this work opened up many more design opportunities and several tilebased dna - nss with diverse and intricate patterns , with or without defined boundaries , have been assembled . [SEP]
[CLS] concurrently , several cage - like polyhedral objects have been constructed from fixed numbers of dna tiles and using other design principles . [SEP]
[CLS] the dna tile - based designs lead to highly periodic and symmetric structures and therefore are not amenable to constructing nanostructures of arbitrary and well - defined sizes and shapes . [SEP]
[CLS] in a complementary approach , a single - stranded dna ( ss - dna ) tile ( sst ) - based dna - ns , also referred to as a " dna brick " was developed . [SEP]
[CLS] in this design principle , each ss - dna is defined diagrams demonstrate the bends at , and away , from crossovers ( second row ) . [SEP]
[CLS] colors illustrate the base - pair index along the folding path where red refers to the 1st base and purple refers to the 7000th base . [SEP]
[CLS] afm images of the dna origami structures are also shown ( third row ) . [SEP]
[CLS] all image are the same size of 165 nm × 165 nm . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2006 , springer nature . [SEP]
[CLS] introduction andby unique sequences located in specific locations of a 2d or 3d nanoobject [SEP]
[CLS] therefore , any particular dna strand can be excluded or vice versa , thus enabling one to build a library of dna objects with arbitrary sizes , shapes , and surface features in a high throughput manner . [SEP]
[CLS] in 2006 , rothemund developed the " dna origami " technology , which transformed the landscape of dna - nss ( figure 1 ) . [SEP]
[CLS] the dna origami uses hundreds of short ss - dna ( staple strands ) to fold a several thousand bases long ss - dna ( scaffold strand ) . [SEP]
[CLS] rothemund used the genomic ss - dna from the m13 bacteriophage ( 7249 nucleotides long ) as a scaffold strand and designed a set of ss - dna or " staple strands " which selectively hybridized to unique locations of the scaffold , folding it into a 2d nanoscale shape with a nearly quantitative yield of formation ( figure 1 ) . [SEP]
[CLS] consequently , numerous research groups successfully extrapolated dna origami technology to fabricate 3d objects with shapes , sizes , designer twists , and curvatures ( figure 2 ) . [SEP]
[CLS] higher - order or larger dna origami structures were also developed by several groups by using methods such as edge - toedge base - stacking interactions , sequence - specific sticky end cohesion , dna tiles , and utilization of longer scaffolds . [SEP]
[CLS] in recent developments , various research groups have devised both simple and complex wireframe architectures to create dna origami structures of complex 3d shapes ( figure 3 ) . [SEP]
[CLS] dna - functionalized nps B-nanoparticle ( dna - nps B-nanoparticle ) were first reported in 1996 by mirkin et al . in which the authors described a synthetic strategy that enabled the preparation of nucleic acid−np nanostructures consisting of densely functionalized and highly oriented dna covalently attached to the core B-material surface of gold B-material nps B-nanoparticle ( aunps B-nanoparticle ) ( figure 4 ) . [SEP]
[CLS] dna molecules with a thiol B-material end were grafted onto the aunp B-nanoparticle surface , giving rise to different properties such as a cooperative binding and subsequent sharp melting transitions , and resistance to nuclease degradation in comparison to the free dna molecules . [SEP]
[CLS] subsequently , the core B-material was replaced by various inorganic and polymeric materials such as ag , pd , fe 3 o 4 , quantum B-nanoparticle dots I-nanoparticle , nanoshells B-nanoparticle , proteins B-material , and polymers B-material with distinct optical , catalytic , and physicochemical properties . [SEP]
[CLS] strategies to remove the core B-material material B-material to form a hollow structure surrounded by an ss - dna shell B-material were also developed to improve biocompatibility B-property . [SEP]
[CLS] more recently , elegant strategies to graft dna onto metal B-material - organic framework nanoparticles B-nanoparticle have been devised . [SEP]
[CLS] li et al . reported an extremely simple approach for grafting dna onto lanthanide - doped up - conversion nanoparticles B-nanoparticle . [SEP]
[CLS] direct coordination of dna with metal B-material ions B-material such as fe 2 + resulted in spherical metal B-material - dna nanostructures for drug and gene delivery applications . [SEP]
[CLS] in parallel , dna - lipid / polymer B-material amphilites have been explored for self - assembled nanostructures of various shapes with dna nm close - ups , with the exception of the pentagonal rod ( 200 nm × 100 nm ) ; e , f ) ( ball and bunny ) are imaged using cryo - electron microscopy B-technique . [SEP]
[CLS] the au particle used for alignment is shown in ( f ) . [SEP]
[CLS] in ( f ) , the scale bar represents 50 nm . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2015 , springer nature . [SEP]
[CLS] a high - density dna - np B-nanoparticle is formed by using citrate - stabilized particles incubated B-technique in alkylthiol - functionalized dna in water B-material which consequently forms a low - density monolayer . [SEP]
[CLS] when the nps B-nanoparticle are incubated B-technique in aqueous solutions with successively higher concentrations of salt B-material ( typically 0 . 15−1 . 0 m ) and surfactants B-property over ≈12 h , successful formation of a high - density dna - np B-nanoparticle shell B-material is achieved . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2012 , american chemical society . molecules protruding out . [SEP]
[CLS] further , dna hybridizationbased strategies have been established to integrate dna - nss and dna - nps B-nanoparticle structures in a single nanoscale structure with superior / tuneable optical properties and novel functionalities . [SEP]
[CLS] for a nanomaterial B-material to be considered as a viable theranostics agent , it is essential to demonstrate biocompatibility B-property and it should , therefore , maintain long - term structural integrity in different physiological environments experienced on the path to cancerous sites so that no premature functional activation occurs . [SEP]
[CLS] thus , both dna - nss and dna - nps B-nanoparticle should exhibit a suitable half - life . [SEP]
[CLS] there has been increasing interest in understanding the structural stability of the nanostructures and fabrication of materials with enhanced stability . [SEP]
[CLS] in general , artificial dna nanostructures show enhanced stability compared to single - stranded and double - stranded dna in nucleasecontaining environments . [SEP]
[CLS] the last decade has seen many investigations that have aimed at assessing the biostability of dna - nss . [SEP]
[CLS] correct folding and formation of 3d dna nanostructures require high concentrations of divalent cations B-material ( such as ≈5 × 10 −3 - 20 × 10 −3 m of mg 2 + ) , which aids in reducing the repulsion of the negatively charged phosphate backbone of dna . [SEP]
[CLS] therefore , most complex dna - nss suffer from poor structural integrity in physiological conditions , which are associated with low amounts of bivalent cations B-material . [SEP]
[CLS] on the other hand , nuclease enzymes lead to a degradation of dna - nss upon incubation B-technique under physiological conditions . [SEP]
[CLS] mei et al . reported that 2d dna origami nanostructures were more stable in cell B-material lysates compared to natural single - and double - stranded dna and all wireframe dna origami - based structures have been shown to exhibit high stability in biological milieus . [SEP]
[CLS] walsh et al . demonstrated that dna - nss remain stable for at least 48 h after their cellular internalization . [SEP]
[CLS] shen et al . demonstrated that the complete degradation of dna origami nanotubes B-nanoparticle required at least 60 h using a double - stranded dna - selective fluorescent B-property probe . [SEP]
[CLS] further , simple end - modification B-event of I-event dna I-event strands in dna - nss significantly enhanced the serum stability . [SEP]
[CLS] recently , surana et al . developed a quantitative fluorescence - based assay to investigate structural stability and lifetime of dna - ns in the coelomocytes of the multicellular organism c . elegans . [SEP]
[CLS] using noninvasive pet , jiang et al . evaluated the stability and biodistribution of dna origami dna - nss in vivo . [SEP]
[CLS] interestingly , the authors discovered that dna origami structures accumulate in the kidney of healthy mice . [SEP]
[CLS] one of the advantages of dna - nss is the ease of structural modifications , which significantly enhances their structural stability . [SEP]
[CLS] several approaches have been investigated to alleviate stability concerns . [SEP]
[CLS] cassinelli et al . developed a click chemistry - based approach to lock dna helices of dna - nss , therefore enhancing the structural stability at low ionic strength . [SEP]
[CLS] methods to coat B-material dna - nss with cationic B-material capsid proteins B-material or with cationic B-material polymers B-material have been reported . [SEP]
[CLS] perrault et al . reported an elegant virus - inspired encapsulation strategy of dna - nss in liposomes B-nanoparticle , and therefore preventing the structure from nuclease degradation and increasing the time of circulation in vivo . [SEP]
[CLS] ponnuswami et al . demonstrated that dna - nss coated with oligolysine , a positively charged polymer B-material , exhibited enhanced stability in low salt B-material concentrations and increased resistance to dnase i digestion compared to the uncoated counterpart ( figure 5 ) . [SEP]
[CLS] more recently , glutaraldehyde - mediated crosslinking of the oligolysines has been shown to enhance the structural stability of the dna - nss up to 2 weeks in dnase i condition . [SEP]
[CLS] dna - nps B-nanoparticle have also been shown to possess enhanced nucleic B-material acid I-material stability in cellular environments by limiting the extent of degradation caused by endogenous nucleases . the dense negatively charged dna layer and unique shape are attributed to minimal enzymatic nucleic degradation and innate immune response . [SEP]
[CLS] patel et al . demonstrated that dicer and serum nucleases have a lesser favorability for blunt duplexes compared to those with 3 ′ overhangs of dna - nps B-nanoparticle . [SEP]
[CLS] genome - wide expression profiling showed that dna - nps B-nanoparticle induce a minimal biological response in hela cells B-material . [SEP]
[CLS] the high in vivo stability was attributed to the structural complexity of the dna - nps B-nanoparticle , which diminished the accessibility and activity of nucleases . [SEP]
[CLS] recently , chou et al . . [SEP]
[CLS] at low salt B-material concentrations , bare dna - nss rapidly become denatured and degraded in cell B-material medium containing 10 % fbs . [SEP]
[CLS] however , it is possible to override low - salt B-material - induced denaturation and nuclease degradation through coating B-material the dna - nss with various ratios of positively charged peptides B-material ( k10 or k10 - peg5k ) . [SEP]
[CLS] reproduced under the terms of the creative commons attribution license . [SEP]
[CLS] copyright 2017 , springer nature . [SEP]
[CLS] biological stability and cellular uptake of dna - nss and dna - npsdemonstrated an elegant strategy for the fabrication of superstructures of dna - nps B-nanoparticle for reduced macrophage sequestration and to improve tumor B-material accumulation and elimination . [SEP]
[CLS] nucleic B-material acids I-material are associated with a high negative charge that hinders the translocation across negatively charged lipid B-material bilayers I-material . [SEP]
[CLS] typically , cationic B-material polymers B-material such as polyethylenimine are often employed as transfection agents due to the capability of binding and condensing large nucleic B-material acids I-material into nanosized structures , thus aiding effective cellular uptake of nucleic B-material acids I-material . [SEP]
[CLS] in contrast , dna - nps B-nanoparticle are taken up very efficiently by almost all cell B-material types without any transfection agents . [SEP]
[CLS] however , since artificial dna - nss and dna - nps B-nanoparticle usually carry a negative charge , passive uptake into the cells B-material is not feasible due to the electrostatic repulsion and they are instead , internalized by an active energy - dependent transport pathway . [SEP]
[CLS] liang et al . demonstrated that tetrahedral dna - nss are uptaken by a caveolin - dependent pathway , and thereafter , are transported to the lysosomes using microtubules . [SEP]
[CLS] aptamer - modified dna - nss were shown to be internalized by the surface receptors and compartmentalized in endosomes and subsequently in acidic lysosomes . [SEP]
[CLS] there is a significant body of work that has investigated lysosomal escape strategies for better targeting of other organelles . [SEP]
[CLS] for example , chen et al . demonstrated endosomal escape of dna nanoribbons due to rigidity and high aspect ratio . [SEP]
[CLS] the intracellular fate of dna - nps B-nanoparticle is relatively better understood . [SEP]
[CLS] wu et al . demonstrated that dna - nps B-nanoparticle can traffic through the endocytic pathway into late endosomes , residing there for up to 24 h following incubation B-technique . [SEP]
[CLS] notably , the dna - nps B-nanoparticle were not observed to enter the lysosomes . [SEP]
[CLS] after assessing the fate of the np B-nanoparticle core B-material and dna shell B-material , the authors showed that the oligonucleotide shell B-material of the dna - np B-nanoparticle was recycled out of the cell B-material , while the np B-nanoparticle cores B-material remained inside . [SEP]
[CLS] theranostic approaches combine diagnostics with a therapeutic approach for the treatment of cancer . [SEP]
[CLS] the aim is to eliminate unnecessary invasive procedures such as biopsies and reducing delays in treatment , ultimately improving patient care and survival rates , while at the same time improving patient compliance by simplifying the clinical workflow . [SEP]
[CLS] for example , dna aptamers provide a means of selectively recognizing , targeting specific cells B-material and also can act as a drug delivery platform . [SEP]
[CLS] thus , owing to its high stability , controllable , and cost - efficient synthesis , dna can be used as a scaffold to engineer dna - nss for numerous biomedical applications , including those with a theranostic approach . [SEP]
[CLS] shu et al . constructed multifunctional rna nps B-nanoparticle using the three - way junction motif , thus manipulating the properties of rna for targeted imaging and therapy of triple negative breast cancer . [SEP]
[CLS] to precisely guide the anti - mirnas to the cancerous cells B-material , epidermal growth B-material factor I-material receptor I-material ( egfr ) aptamers were used as targeting B-material ligands I-material to internalize the rna nps B-nanoparticle into cancer cells B-material via receptor - mediated endocytosis B-event and then imaged using the fluorescent B-property agent , alexa674 . [SEP]
[CLS] the dna - nss demonstrated high rnase resistance and thermodynamic stability and in addition , remained intact after systemic injection . [SEP]
[CLS] strong binding and internalization into cancerous tissue with little or no accumulation in healthy organs 8 h after administration were also observed . [SEP]
[CLS] the authors noted the potential of incorporating drug molecules into such nanostructures for clinical applications involving cancer therapy . [SEP]
[CLS] doxorubicin B-material ( dox ) is a well - established anticancer B-property agent that disrupts the replication of cancer cells B-material through dna B-event intercalation I-event . [SEP]
[CLS] qd655 - labeled triangular dna origami structures loaded with dox were administered to tumor - bearing mice and , using noninvasive fluorescence B-technique imaging I-technique , tumor B-material growth in mice and antitumor efficacy of drug - loaded dna carriers was dynamically monitored . [SEP]
[CLS] the results demonstrated that the dna origami dual imaging and drug delivery system showed optimal anticancer B-property efficacy in vivo without observable systemic toxicity B-property . [SEP]
[CLS] moreover , it was shown that triangular dna - nss remained in the tumor B-material for a longer period compared to square and tubular structures . [SEP]
[CLS] the authors attributed the enhanced tumor B-material retention times to the shape of the ns . [SEP]
[CLS] further , the dna - nss offered additional layers of tightly packed double helices for dox binding , allowing a higher payload of dox to be delivered . [SEP]
[CLS] as a consequence of their compact structure , origami nanostructures can be loaded with high drug concentrations , allowing them to be digested slowly in vitro and in vivo , therefore reducing unintentional drug release during trafficking of the dna - nss to the tumor B-material site in vivo . [SEP]
[CLS] lei et al . [SEP]
[CLS] dna - ns - based theranosticsrecently used a dox - loaded dna nanotriangle - scaffold aptamer probe for cancer detection , imaging , and therapy in both in vitro and in vivo settings . [SEP]
[CLS] superior targeted binding affinity with an ultralow nonspecific background was observed , thus generating high contrast tumor B-technique imaging I-technique within an extended time window of 8 h . [SEP]
[CLS] moreover , as a consequence of a five - time improvement in dox loading capabilities in comparison to the control , the dna nanotriangle - scaffold aptamer demonstrated high antitumor efficacy in vivo with 81 . 95 % inhibition and no observable bodyweight loss , therefore indicating favorable biocompatibility B-property ( figure 6 ) . [SEP]
[CLS] dna dendrimers B-nanoparticle have also been developed as dual imaging and drug delivery platform . [SEP]
[CLS] the nanostructures were assembled from three - armed yshaped dna monomers B-material using an enzyme - free step - by - step basepairing assembly strategy . [SEP]
[CLS] the resulting structure served as a mechanism for cancer imaging and drug delivery through the incorporation of fluorophores , targeting dna aptamers and dox . [SEP]
[CLS] the structure displayed a high degree of biostability and biocompatibility B-property as well as high selectivity and high drug loading capacity , therefore making it a propitious candidate for applications involving dna - based theranostics . [SEP]
[CLS] dna nanorobots conjugated with dual functioning nucleolin targeting aptamers have been investigated for the delivery of thrombin . [SEP]
[CLS] such nanostructures are capable of selectively targeting the tumor B-material environment , triggering the dna nanorobot to open and release the therapeutic payload . [SEP]
[CLS] following the release of thrombin to the tumor B-material site , vascular occlusion of the tumor B-material vessels was observed , which in turn induced blockage of tumor B-material blood supply , and inhibition of tumor B-material growth to a high degree . [SEP]
[CLS] in vivo imaging demonstrated specific tumor B-material targeting with highintensity fluorescence B-property signal observed in the tumor B-material region 8 h post - injection . [SEP]
[CLS] furthermore , in comparison to the controls , tumor B-material volume was significantly smaller following treatment with the targeted thrombin nanotube B-nanoparticle , and the delivery of the dnathrombin nanorobot prevented metastasis B-event , thus showing excellent therapeutic potential . [SEP]
[CLS] inspired by the " envelope " of viral particles , perrault and shih encased a dna octahedron into a pe - gylated lipid B-material bilayer I-material to mimic virus - like particles . [SEP]
[CLS] the wireframe dna nanooctahedron with a diameter of ≈50 nm was composed of bundles of six long double helices engineered with 90°curvatures , allowing the lipid B-material bilayer I-material to be directly assembled around the dna octahedron . [SEP]
[CLS] the authors demonstrated the envelopment of dna nanostructures into pegylated lipids B-material , which were successfully protected against nuclease digestion . [SEP]
[CLS] activation of the immune system was decreased by two orders of magnitude below the controls , and pharmacokinetic bioavailability B-property was enhanced significantly by a factor of 17 . [SEP]
[CLS] therefore , the authors demonstrate the development of sophisticated , translationready dna nanodevices B-nanoparticle capable of being functionalized by targeting moieties to specifically target cancer tissue in vivo . [SEP]
[CLS] using a transdermal drug delivery approach , wiraja et al . utilized dna origami nanostructures to deliver dox for the treatment of melanoma in vivo . [SEP]
[CLS] specifically , the dox - loaded dna - ns generated more than two - fold improvement in drug accumulation and tumor B-material growth inhibition compared to topically applied free dox or dox loaded in liposomes B-nanoparticle and polymeric nps B-nanoparticle . [SEP]
[CLS] in vivo fluorescence B-technique imaging I-technique was used to demonstrate successful skin penetration of the dna - ns to the dermis . [SEP]
[CLS] several cancer types are associated with a mutated p53 gene , which is important in tumor B-material suppression . [SEP]
[CLS] research suggests that p53 gene expression can enhance the sensitivity of multidrug - resistant ( mdr ) tumors B-material to chemotherapeutics . [SEP]
[CLS] more recently , ding et al . designed a dna - based co - delivery system which contained a linear tumor B-material therapeutic gene ( p53 ) and dox for the combined therapy of mdr tumor B-material ( mcf - 7r ) using a muc1 aptamer - mediated targeted delivery . [SEP]
[CLS] the structure , which resembles a kite ( " nanokite " ) , exhibited superior anticancer B-property activity against mcf - 7r both in vitro and in vivo . [SEP]
[CLS] fluorescence B-technique imaging I-technique of the cy5 . 5 - labeled dna nanokite structure also indicated preferential uptake of the targeted muc1 aptamer in tumors B-material over the nontargeted group . [SEP]
[CLS] compared to the control , the nanocarrier was also shown to inhibit tumor B-material growth in vivo , demonstrating an almost 20 - fold higher level of p53 expression in tumors B-material treated with the dual - therapeutic p53 and dox - loaded dna - ns . [SEP]
[CLS] owing to its biocompatibility B-property , several studies have also explored the use of dna nanostructures as a carrier for photosensitizers B-property . [SEP]
[CLS] photodynamic therapy ( pdt ) is a promising technique that utilizes the sensitivity of a drug to a specific wavelength of light to create reactive oxygen B-material species ( ros ) , killing the cancerous cells B-material . [SEP]
[CLS] in addition to directly killing cancer cells B-material , pdt can shrink or destroy tumors B-material in two other ways ; photosensitizer induced damage of tumor B-material vasculature and activation of the immune system to attack tumor B-material cells B-material . [SEP]
[CLS] targeted pdt was demonstrated by tan et al . using aptamers to recognize cancerous cells B-material selectively . [SEP]
[CLS] toehold - mediated catalytic strand displacement caused by the overhanging catalyst B-property sequence on the aptamer resulted in the activation of the photosensitizer B-property molecule chlorin B-material e6 ( ce6 ) , which , in turn , enhanced the therapeutic effect . [SEP]
[CLS] importantly , through the use of a targeting sequence , catalytic amplification of ce6 took place close to the cancerous cells B-material , thus amplifying the local concentration of singlet oxygen B-material . [SEP]
[CLS] similarly , a dna - based nanoclaw has been designed for targeted cancer therapy using pdt . [SEP]
[CLS] consisting of an oligonucleotide backbone as the scaffold , ce6 was incorporated into the aptamer - based nanostructure which was capable not only of selectively targeting cancerous cells B-material but also administering a diagnostic signal such as fluorescence B-property as well as pdt . [SEP]
[CLS] the results demonstrate the advantage of using logic - based aptamer nanostructures for cancer detection and treatment applications . [SEP]
[CLS] although pdt is an fda ( the food and drug administration ) - approved method of treatment for certain cancers , its application remains limited due to d ) an inner strand , the dna nanostructure is formed . [SEP]
[CLS] in the free state , the dna nanotriangle , which is loaded with dox via the cg base pair regions is a flat , double helix - jointed structure with no fluorescence B-property since it is quenched . [SEP]
[CLS] once it encounters the target cell B-material , the aptamers disassemble and consequentially change shape . [SEP]
[CLS] this leads to fluorescence B-property emission and partial drug release with the remaining drugs being freed following internalization . [SEP]
[CLS] reproduced under the terms of the creative commons attribution license . [SEP]
[CLS] copyright 2018 , ivyspring international publisher . [SEP]
[CLS] the poor penetration depth of uv and visible light in tissue . [SEP]
[CLS] to account for this , liang et al . utilized a dna origami nanostructure for dual functioning imaging and pdt applications using the carbazole derivative 3 , 6 - bis [ 2 - ( 1 - methylpyridinium ) ethynyl ] - 9 - pentyl - carbazole diiodide ( bmepc ) . [SEP]
[CLS] the molecule exhibits low solubility B-property in aqueous samples . [SEP]
[CLS] however , the advantage of using such a molecule is its ability to be activated by near - infrared ( nir ) light which has better tissue penetration capabilities . [SEP]
[CLS] using dna as a carrier to overcome the issues associated with solubility B-property and quenching of fluorescence B-property due to aggregation , dna was used as a carrier to increase the density of bmepc molecules in the cellular environment . [SEP]
[CLS] through the use of bmepc as a photosensitizer B-property and imaging probe , increased fluorescence B-property emission and higher cell B-material apoptosis B-event were observed as a consequence of free radical production . [SEP]
[CLS] by utilizing coordination chemistry to induce self - assembly , chen et al . created novel dna nanoscale coordination polymers B-material ( ncps ) . [SEP]
[CLS] dna ncps were created by mixing ca 2 + , as1411 g quadruplex , and poly his - peg copolymer ( figure 7a ) . [SEP]
[CLS] in addition , photosensitive B-property ce6 and hemin were inserted into the dna - ns to enable targeted intranuclear delivery of ce6 which , following exposure to 660 nm light , induce the generation of mixing of the g - quadruplex structure and ca 2 + together with phis - peg yields ca - as1411 / ce6 / hemin @ phis - peg ( cach - peg ) ncps , which are ph responsive at ≈5 . 5 and were reduced to smaller g - quadruplex complexes in acidic environments of the lyso - and endosomes . [SEP]
[CLS] b ) the tumor B-material growth curves of 4t1 - tumor - bearing mice following each treatment as indicated . [SEP]
[CLS] tumors B-material were irradiated using 660 nm light - emitting diode light ( 5 mw cm −2 ) for 60 min . [SEP]
[CLS] c ) average tumor B-material weights collected from the each group following 14 days after each treatment . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , american chemical society . [SEP]
[CLS] ros inside the cell B-material nuclei [SEP]
[CLS] tumor B-material growth inhibition was observed to be most effective in groups treated in this manner compared to all other control groups ( figure 7b , c ) . [SEP]
[CLS] efficient tumor B-material accumulation was demonstrated in vivo using fluorescence B-property and single photon emission computed B-technique tomography I-technique ( spect ) imaging , supported by radiolabeling ce6 within the ncps with 99m tc 4 + ions B-material . [SEP]
[CLS] furthermore , the inhibition of antiapoptotic protein B-material bcell lymphoma 2 ( bcl - 2 ) expression by as1411 supported pdtinduced cell B-material apoptosis B-event . [SEP]
[CLS] this work offers an efficient means of achieving intranuclear delivery of photosensitizers B-property and downregulation of antiapoptotic proteins B-material for applications involving cancer theranostics . [SEP]
[CLS] dna - nps B-nanoparticle have also shown promise in overcoming the limitations associated with the lack of effective contrast B-technique agents I-technique in photoacoustic imaging . [SEP]
[CLS] by assembling gold B-material nanorods B-nanoparticle ( aunrs ) onto dna origami structures , a dna - based optoacoustic imaging system was reported by du et al . [SEP]
[CLS] the authors showed that the resulting aunr - based dna - ns hybrid demonstrated exceptional properties , serving as both a unique probe and an efficient contrast B-technique agent I-technique for in vivo optoacoustic imaging by generat - ing significant improvements in image quality and contrast . [SEP]
[CLS] simultaneous photothermal therapy ( ptt ) was achieved as a response to nir irradiation and thus inhibited the regrowth of tumors B-material and prolonged the survival rate of mice bearing 4t1tumors ( figure 8 ) . [SEP]
[CLS] in response to the tme , zhang et al . designed a self - assembling ce6 - fdna - dox hybrid capable of performing cancer cell - specific fluorescence B-technique imaging I-technique . [SEP]
[CLS] synergistic redox - responsive pdt and ph - triggered chemotherapy was also achieved through the release of dox in hepatocellular carcinoma . [SEP]
[CLS] here , the smart ce6 - fdna - dox therapeutic ns overcame unwanted side effects by switching the pdt " on " and " off " as necessary . [SEP]
[CLS] treatment outcomes were also improved due to the synergistic use of dox . [SEP]
[CLS] fluorescence B-property emission of dox from ce6 - fdna - dox was significantly greater , in comparison to the nontargeted control ( ce6 - fndna - dox ) ( figure 9a , b ) , indicating that smart dna - nss can be better incorporated into hepg2 cells B-material due to specific targeting . [SEP]
[CLS] in comparison to the control group ( phosphate - buffered saline , pbs ) , injection of ce6 - fdna , ce6 - fndnadox , or ce6 - fdnadox followed by 670 nm irradiation demonstrated noticeable delay of tumor B-material growth copyright 2016 , wiley - vch . [SEP]
[CLS] ( figure 9c ) [SEP]
[CLS] importantly , mice that were administered the dual therapy , i . e . , the ce6 - fdnadox treatment in combination with pdt ( 670 nm ) , exhibited significantly slower tumor B-material growth due to the synergistic therapeutic effects of pdt and chemotherapy . [SEP]
[CLS] also , no discernable weight loss was observed , indicating low toxicity B-property of the ce6 - fdnadox probe ( figure 9d ) . [SEP]
[CLS] other dna - nss responsive to the ph of the tumor B-material environment include one reported by di et al . for the controlled imaging of adenosine triphosphate ( atp ) in the extracellular tumor B-material matrix . [SEP]
[CLS] as a consequence of mild acidity ( lower ph ) , specific anchoring of aptamer units to the membrane of tumor B-material cells B-material induced off - on fluorescence B-property , thus enabling fluorescence B-technique imaging I-technique of extracellular atp with high signal to background ratios in solid tumors B-material . [SEP]
[CLS] relevant references with in vivo studies are summarized in table 1 . [SEP]
[CLS] in this emerging field , many studies have aimed at understanding simultaneous transfection and gene regulation using dna - nps B-nanoparticle . [SEP]
[CLS] in contrast with conventional approaches to gene delivery , dna - nps B-nanoparticle do not require cationic B-material transfection agents or additional structural modifications to aid cellular entry . [SEP]
[CLS] in 2006 , rosi et al . first demonstrated that dna - nps B-nanoparticle could de - liver " antisense " oligonucleotides to eukaryotic cells B-material combined with a tuneable enhanced green fluorescence B-property protein B-material ( egfp ) knockdown . [SEP]
[CLS] later , giljohann et al . reported the successful delivery of sirna molecules using a polyvalent rna - gold B-material np B-nanoparticle ( rna - au nps B-nanoparticle ) in a human cancer cell B-material line . [SEP]
[CLS] the rna - au nps B-nanoparticle exhibited a greater half - life than free dsrna , were capable of entering cells B-material without the use of transfection agents , and exhibited high gene knockdown capabilities in vitro . [SEP]
[CLS] there are many more examples of in vitro gene silencing reviewed elsewhere . [SEP]
[CLS] dna - nps B-nanoparticle have been utilized for the targeting of several genes such as bcl2l12 , mir - 182 , ganglioside gm3 synthase , egfr , malat - 1 in vivo . [SEP]
[CLS] jensen et al . evaluated an rna interference ( rnai ) - based nanotheranostic for the neutralization of oncogene expression in glioblastoma multiforme ( gbm ) . [SEP]
[CLS] dna - nps B-nanoparticle consisted of aunps B-nanoparticle grafted with densely packed and highly oriented si - rna duplexes . [SEP]
[CLS] excitingly , the nps B-nanoparticle crossed the blood - brain barrier B-property ( bbb ) to accumulate throughout the tumor B-material mass in gbm mouse models ( figure 10 ) . [SEP]
[CLS] the nps B-nanoparticle were designed to target the oncoprotein bcl2like12 ( bcl2l12 ) , an effector caspase , and p53 inhibitor overexpressed in gbm relative to a healthy brain . an efficient knockdown of endogenous bcl2l12 mrna and protein B-material levels was observed , and glioma cells B-material underwent therapy - induced apoptosis B-event by enhancing effector caspase and p53 activity . [SEP]
[CLS] later , using a similar approach , nps B-nanoparticle were copyright 2017 , wiley - vch . [SEP]
[CLS] developed to deliver sirna and mirna to intracranial gbm tumor B-material sites . [SEP]
[CLS] a reporter xenograft model that was able to stably co - express optical reporters for luciferase and an nir fluorescent B-property protein B-material ( irfp670 ) was developed to evaluate efficacy in vivo . using noninvasive optical B-technique imaging I-technique , knockdown of the dna repair protein B-material o6 - methylguanine - dna - methyltransferase ( mgmt , linked to drug resistance in gbm ) , by nps B-nanoparticle composed of mgmt - targeting sirna duplexes was quantitatively assessed . [SEP]
[CLS] a systemic administration of nps B-nanoparticle via single tail vein injection was shown to be capable of robust intratumoral mgmt protein B-material knockdown . [SEP]
[CLS] further , np B-nanoparticle biodistribution and pharmacokinetics revealed rapid intratumoral uptake and retention , therefore im - proving the antitumor B-event activity I-event of co - administered temozolomide ( tmz ) . [SEP]
[CLS] these nps B-nanoparticle exhibited no observable toxicity B-property , as corroborated by histopathology and blood chemistry assessment . [SEP]
[CLS] the topical application of dna - nps B-nanoparticle offers potential therapeutic advantages for the treatment of skin diseases . [SEP]
[CLS] zheng et al . demonstrated that dna - nps B-nanoparticle freely penetrate keratinocytes in vitro ( e . g . , mouse skin and human epidermal tissue ) within hours of post - application despite the fact that the intact epidermal barrier B-property typically precludes entry of gene - suppressing therapy . [SEP]
[CLS] significantly , the nps B-nanoparticle were delivered in a commercially available moisturizer or pbs and importantly , no barrier B-property - disrupting or transfection agents , such as liposomes B-nanoparticle , peptides B-material , or viruses rna nanostructure fluorescence B-property delivery of mirna - based therapeutics [ 79 ] dna origami triangle fluorescence B-technique imaging I-technique and therapy of breast cancer [ 80 ] dna nanotriangle fluorescence B-property bioimaging and drug delivery of dox [ 64 ] dna octahedron fluorescence B-property assessment of immune activation [ 83 ] different 2d and 3d origami structure fluorescence B-property transdermal delivery of dox [ 85 ] dna origami triangle fluorescence B-property combined therapy for mdr - resistant breast cancer [ 90 ] dna nanogel fluorescence B-property and pet intranuclear delivery of photosensitizers B-property , down - regulation of antiapoptotic proteins B-material , and modulation of an unfavorable tme [ 91 , 92 ] dna origami triangle - aunr hybrid optoacoustic imaging improved optoacoustic and excellent photothermal therapeutic properties [ 93 ] [SEP]
[CLS] double stranded dna hybrid fluorescence redox - responsive pdt and ph - triggered dox release [ 94 ] [SEP]
[CLS] streptavidin dna hybrid nanomachine fluorescence controlled imaging of atp were required . [SEP]
[CLS] nps B-nanoparticle were designed to target egfr and were > 100fold more potent than sirna delivered with commercial lipid B-material agents in vitro . [SEP]
[CLS] furthermore , they completely abolished egfr expression , suppressing downstream erk phosphorylation , and reduced epidermal thickness by almost 40 % . [SEP]
[CLS] as such , dna - nps B-nanoparticle were shown to be very promising agents for topical gene therapy of neoplastic , inflammatory , and genetic disorders of the skin . [SEP]
[CLS] recently , aptamers , specific sequences of dna or rna molecule selected for binding specific targets , have been shown to be promising platforms for targeting cancer biomarkers B-property with high selectivity and specificity . [SEP]
[CLS] aptamers have multiple benefits over monoclonal antibodies B-material such as i ) lower molecular weight , ii ) low immunogenicity B-property , iii ) inexpensive synthesis via solid - phase phosphoramidite chemistry , and iv ) available chemical modifications for attachment to the np B-nanoparticle surface . [SEP]
[CLS] specifically , gold B-material nanomaterials B-material have been extensively investigated as core B-material materials for aptamer - targeted theranostic applications . [SEP]
[CLS] aptamer dna - nps B-nanoparticle have been used in both in vitro and in vivo applications . [SEP]
[CLS] nicholls et al . reported a gold B-material np B-nanoparticle functionalized with altered deoxythymidine oligonucleotides bearing gd ( iii ) chelates and a fluorescent B-property cy3 moiety to enable the visualization of transplanted human neural stem cells B-material in vivo . [SEP]
[CLS] the dna - nps B-nanoparticle exhibited improved t1 relaxivity and excellent cellular uptake . [SEP]
[CLS] in vivo , mris were corroborated with histological studies . [SEP]
[CLS] the dna - nps B-nanoparticle thus offer new opportunities of visualizing transplanted cells B-material using mri as a tool to derive biologically relevant information . [SEP]
[CLS] melancon et al . developed aptamer - coated hollow gold B-material nps B-nanoparticle targeted to egfrs . [SEP]
[CLS] egfr - targeting rna aptamers were attached to a thiol - terminated single - stranded dna grafted to hollow gold B-material nps B-nanoparticle . [SEP]
[CLS] the pharmacokinetics , biodistribution , and micro - spect / ct imaging of in - labeled targeted dna - nps B-nanoparticle were evaluated in vivo in oral cancer mouse models . [SEP]
[CLS] micro - spect / ct imaging further confirmed tumor B-material homing of dna - nps B-nanoparticle . [SEP]
[CLS] later , li et al . demonstrated the fabrication of a fluorescent B-property and ct dual - modal dna - np B-nanoparticle , conjugated with diatrizoic acid and nucleolin - targeted aptamers . [SEP]
[CLS] the fluorescent B-property np B-nanoparticle conjugates were utilized as a molecular contrast B-technique agent I-technique to thus reveal the tumor B-material location in the human lung adenocarcinoma cell B-material line ( cl1 - 5 ) in tumor - bearing mice by ct imaging . [SEP]
[CLS] further - more , the orange - red fluorescence B-property emitted from the conjugates in the lung adenocarcinoma could be visualized by the naked eye ( figure 11 ) . [SEP]
[CLS] following tumor B-material resection , the tumor B-material ' s fluorescence B-property signal was significantly enhanced compared to that of the control as a result of tumor - specific accumulation of the dna - nps B-nanoparticle . [SEP]
[CLS] shi et al . designed a theranostic nanoprobe B-nanoparticle by conjugating aptamer probes onto au @ ag / au nps B-nanoparticle and utilizing them in image - guided cancer therapy of lung cancer mouse models . [SEP]
[CLS] in this study , the au @ ag / au nps B-nanoparticle with a large absorption crosssection from 400 to 1100 nm functioned as the fluorescence B-property quencher I-property and optical heater with a higher capacity for hyperthermia compared to aunrs . [SEP]
[CLS] by incorporating a target - specific signal alteration mechanism , the dna - nps B-nanoparticle substantially improved the imaging contrast with a shortened detection time and improved ptt potency . [SEP]
[CLS] ye et al . reported aptamer - functionalized cu - au alloy nanostructures for in vivo cancer theranostics . [SEP]
[CLS] due to the excellent thermal conductivity and lower cost of copper B-material ( cu ) , cu - au alloy nps B-nanoparticle were successfully synthesized in one - pot synthesis method . [SEP]
[CLS] the nps B-nanoparticle demonstrated superior thermo - optical properties , including a broad and intense nir absorption band , and demonstrated superior heating performance using incident light of different wavelengths and stability against melting . [SEP]
[CLS] by using a human leukemia ccrf - cem cancer cell B-material line , selective fluorescence B-technique imaging I-technique and nir photothermal therapy were demonstrated using cy5 - labeled aptamer - coated cu - au alloy nps B-nanoparticle . [SEP]
[CLS] pal et al . developed a strategy to functionalize sers nps B-nanoparticle with a dna aptamer to target mucin1 ( muc1 ) in human breast cancer . [SEP]
[CLS] muc1 - targeted sers nps B-nanoparticle were co - injected with nonspecific sers nps B-nanoparticle with different spectral signatures in a breast cancer xenograft mouse models . [SEP]
[CLS] a two - tumor mouse model with differential expression of muc1 was used to demonstrate that the targeted sers nps B-nanoparticle accumulate in the tme via active targeting of muc1 ( figure 12 ) . [SEP]
[CLS] recently , pal et al . also reported a dna - based strategy for multimodal nps B-nanoparticle with complementary fluorescence B-property and sers modalities for cancer imaging and therapy using mouse models of ovarian and glioblastoma . [SEP]
[CLS] this year , di et al . reported an up - conversion dna - np B-nanoparticle - based nanodevice B-nanoparticle which could be activated using two independent nir lights for programmable activation of aptamer - targeting modules and copyright 2013 , american association for the advancement of science . a photosensitizer B-property . [SEP]
[CLS] the authors showed excellent spatiotemporal target activation and photodynamic antitumor effect in vivo . [SEP]
[CLS] relevant references with in vivo studies are summarized in table 2 . [SEP]
[CLS] it is clear that important advancements have been made in developing effective dna - ns and dna - nps B-nanoparticle for theranostic applications . [SEP]
[CLS] however , it is also imperative to highlight recent progress that has investigated the use of these nanomaterials B-material exclusively in drug and gene delivery applications . [SEP]
[CLS] to be considered as an efficient drug and gene delivery system , both dna - nss and dna - nps B-nanoparticle must be designed to deliver the following features : i ) struc - tural stability in physiological conditions , ii ) predictable and welldefined structures , iii ) high loading capacity and the ability to be internalized by cells B-material , and iv ) excellent biocompatibility B-property . [SEP]
[CLS] dna - nss offer high loading capacity and can , therefore , be functionalized with a range of molecules , including pharmaceuticals , e . g . , dox , by means including covalent modification or intercalation . [SEP]
[CLS] in addition , alongside their excellent biocompatibility B-property and low toxicity B-property , dna - nss can be internalized into cancerous cells B-material . [SEP]
[CLS] as a consequence of these properties , dna - nss have emerged as promising candidates for drug and gene delivery platforms . [SEP]
[CLS] capable of suppressing gene expression , small interfering rna ( sirna ) has been investigated for disease therapy applications . [SEP]
[CLS] however , due to the fragility of sirna in plasma and reduced internalization without transfection agents , its therapeutic license [SEP]
[CLS] copyright 2015 , springer nature . [SEP]
[CLS] type of np B-nanoparticle core imaging modality applications [ 96a ] [SEP]
[CLS] 13 nm spherical aunp B-nanoparticle mri , fluorescence B-property , icp - ms ( ex vivo ) rnai therapy for gbm [ 97 ] 13 nm spherical aunp B-nanoparticle indirect imaging B-technique with I-technique fluorescence I-technique and bioluminescence B-property [SEP]
[CLS] real - time assessment of mgmt - targeting spherical nucleic B-material acids I-material [ 98 ] [SEP]
[CLS] 13 nm spherical aunp B-nanoparticle fluorescence B-property egfr sirna - based gene silencing [ 101 ] 13 nm spherical aunp B-nanoparticle fluorescence B-property and mri to detect transplanted human stem cell B-material [ 102 ] [SEP]
[CLS] hollow gold B-material nanospheres B-nanoparticle [UNK] - ct targeted imaging of egfr positive head and neck cancer [ 103 ] 2 . 4 nm aunp B-nanoparticle ct , fluorescence B-property nucleolin targeted imaging of lung cancer [ 104 ] [SEP]
[CLS] au @ au / ag np B-nanoparticle activatable fluorescence B-technique imaging I-technique and ptt of lung cancer [ 105 ] cu - au alloy nanostructure fluorescence B-technique targeted imaging I-technique and therapy of leukemia [ 106 ] 60 nm aunp B-nanoparticle with silica raman imaging targeted imaging of breast cancer [ 107 ] aunr with silver B-material and silica B-material shell I-material raman and fluorescence B-technique imaging I-technique targeted imaging I-technique and ptt of ovarian cancer and gbm [ 108 ] lanthanum - doped up - conversion nanoparticle B-nanoparticle with silica B-material shell I-material their accumulation within tissue was confirmed via identification of the " fingerprint " spectral signature of the sers nps B-nanoparticle using raman B-technique spectroscopy I-technique . reproduced with permission . [SEP]
[CLS] copyright 2017 , wiley - vch . [SEP]
[CLS] applications in vivo are limited [SEP]
[CLS] in spite of this , dna - nss have been shown to be effective vehicles B-material in the delivery of sirna for cancer therapy . [SEP]
[CLS] lee et al . developed folic B-material acid I-material - targeted selfassembled tetrahedral dna nanostructures ( tdns ) for the delivery of sirna into cells B-material to silence target genes in tumors B-material . [SEP]
[CLS] such tdns demonstrated robust gene silencing via both intratumor and systemic injection , as well as boasting four times longer blood circulation time than free sirna . [SEP]
[CLS] in addition , controlled gene silencing was achieved only when the ligands were assembled in a suitable spatial orientation . [SEP]
[CLS] due to their structural rigidity and high aspect ratio , which makes them capable of escaping endosomal entrapment without involving a proton pump , chen et al . explored the use of dna nanoribbons as effective sirna delivery vehicles B-material for mediating gene silencing without the need for cationic B-material transfection agents . [SEP]
[CLS] the authors demonstrated the down - regulation of surviving mrna expression and protein B-material production by 40 . 8 % and 45 . 2 % , respectively . [SEP]
[CLS] as such , the capability of the dna nanoribbons to escape from the endosomes opens up the possibility for the use as a delivery vehicle B-material for sirna and other biologics . [SEP]
[CLS] zhang et al . developed a novel sirna delivery system in which sirnas functioned as cross - linkers to drive the self - assembly of dna - grafted polycaprolactone brushes into spherical and nanosized hydrogels via nucleic B-material acid I-material hybridization . [SEP]
[CLS] this ensured that the sirnas were fully embedded and protected for systemic delivery to the site of interest . [SEP]
[CLS] the sirna - embedded nanogels demonstrated resistance to rnase degradation , enhanced cellular uptake , accumulation , and consequential sirna delivery to the tumor B-material site , thus resulting in gene silencing in vivo . [SEP]
[CLS] bujold et al . constructed dna " nanosuitcases " capable of encapsulating sirna constructs and releasing them upon recognition of an oligonucleotide trigger such as mrna or microrna . [SEP]
[CLS] importantly , the dna scaffold offered protection of the sirna cargo from specific cleavage and nuclease degradation . [SEP]
[CLS] aptamer - integrated dna dendritic nanostructures have also been investigated for efficient sirna delivery in which aptamers and sirna were hybridized to the outermost layer of dna dendritic nanostructures to enable the formation of aptamer - sirna complexes . [SEP]
[CLS] these complexes were investigated for the targeted delivery of sirna in vitro gene therapy applications . [SEP]
[CLS] dna " trojan horses , " self - assembled from floxuridine - containing dna strands , have been utilized to deliver chemotherapeutics into cancer cells B-material and tissues . [SEP]
[CLS] the trojan horse with buckyball B-nanoparticle architecture displays superior anticancer B-property capability over free drugs . [SEP]
[CLS] zhang et al . conjugated camptothecin ( cpt ) molecules by directly reacting with phosphorothioate ( ps ) modified dna to improve solubility B-property . [SEP]
[CLS] further , the self - assembled dna - nss exhibited a decreased tumor B-material growth rate in vivo . [SEP]
[CLS] recently , wu et al . constructed an egfr nanobody - conjugated dna - ns - based carrier of 56mess , a platinum B-material - based intercalator for targeted delivery in vivo ( figure 13 ) . [SEP]
[CLS] interestingly , by exploiting the base - pairing properties of dna , the double - bundle dna tetrahedron was able to arrange anti - egfr nanobodies in a highly organized manner to achieve successful targeting . [SEP]
[CLS] in addition , a dna - ns capable of delivering a vector of rna interference ( rnai ) and chemotherapeutic drug ( dox ) for treatment of mdr tumors B-material was reported by liu et al . owing to the high degree of arrangement associated with the dna , two linear small hairpin rna ( shrna ) transcription B-event templates were precisely organized in drug - loaded dna - ns , to synergistically inhibit tumor B-material growth in preclinical mouse models . [SEP]
[CLS] ma et al . reported an her2 reproduced with permission . [SEP]
[CLS] copyright 2019 , wiley - vch . [SEP]
[CLS] aptamer - targeted dna - ns for lysosomal degradation of tumorspecific protein B-material molecules and apoptosis B-event by selectively targeting her2 positive breast cancer [SEP]
[CLS] this was achieved by anchoring anti - her2 aptamers onto the exterior dna - ns resulting in prolonged circulation , higher stability , and improved performance in vivo compared to the free anti - her2 aptamer . [SEP]
[CLS] as well as being used as gene delivery vehicles B-material , dna - nss have also been investigated for the delivery of drug molecules , namely , intercalating drugs such as dox . [SEP]
[CLS] dna - nss are particularly appealing vehicles B-material for the delivery of such therapeutic molecules since they are capable of permeating the cell B-material membrane , biocompatible B-property , and can be adapted with targeting B-material ligands I-material such as aptamers . [SEP]
[CLS] as such , tdns have emerged as one of the most efficient dna - nss for drug delivery applications , since they can be assembled from four ss - dna molecules and internalized without the need for transfection agents . [SEP]
[CLS] tdns were used for the delivery of paclitaxel B-material ( ptx ) used in the treatment of cancers , including lung and ovarian . [SEP]
[CLS] however , despite the pharmaceutical potency of ptx , its therapeutic value is limited by mdr . [SEP]
[CLS] lin et al . utilized tdns for the successful delivery of ptx to nonsmall cell B-material lung cancer cells B-material and cells resistant to ptx . [SEP]
[CLS] importantly , the ability to override drug resistance and the downregulation of the mdr1 gene and p - glycoprotein in ptx - resistant cells B-material was observed . [SEP]
[CLS] tdns functionalized with the aptamer as1411 have also been utilized for targeting cells B-material overexpressing nucleolin . [SEP]
[CLS] a substantially higher number of aptamer - targeted tdns entered and accumulated in the nucleus of mcf - 7 cells B-material compared to nontargeted tdns , indicating the superiority of tumor - targeted drug delivery tdns over nontargeted tdns . [SEP]
[CLS] the authors envis - age that such a platform could be utilized for the selective delivery of chemotherapeutic agents to target cells B-material . [SEP]
[CLS] other dna - nss for the delivery of small - molecule chemotherapeutics include the use of di - block dna strands containing both normal phosphodiester segments ( podna ) , and phosphorothioate segments ( ps - dna ) directly grafted with multiple ptx drug molecules , which then assemble into amphiphilic B-property ptx - loaded spherical nucleic B-material acid I-material ( sna ) - like micellar nps B-nanoparticle ( ptx - snas ) ( figure 14a , b ) . [SEP]
[CLS] significantly , multifunctional ptxsnas demonstrated high drug loading ratios ( up to ≈53 % ) and achieved active - targeting to inhibit tumor B-material growth and reverse drug resistance in both in vitro and in vivo models . [SEP]
[CLS] ( figure 14c - e ) [SEP]
[CLS] zhao et al . tuned dna origami nanostructures for the optimal delivery of dox to human breast cancer cells B-material . [SEP]
[CLS] to rationally control and tailor drug release kinetics , the dna - nss were designed to exhibit varying degrees of global twist , which resulted in different amounts of relaxation in the double helix structure . [SEP]
[CLS] in addition to controlling the degree of drug encapsulation and release rate , increased cytotoxicity B-property and lowered intracellular elimination were reported in contrast to free dox . [SEP]
[CLS] similarly , halley et al . observed increased delivery and retention of daunorubicin , which , similar to dox , is capable of intercalation . [SEP]
[CLS] rod - like dna origami was used as a vehicle B-material for the delivery of daunorubicin to a leukemia cell B-material line exhibiting mdr . [SEP]
[CLS] in addition to dna origami structures , ultrathin 2d nanosheets of layered transition metal B-material dichalcogenides , e . g . , mos 2 , have attracted increasing attention recently due to their suitability in a range of applications , including biomedicine ( figure 15 ) . [SEP]
[CLS] by taking the advantage of sulfur B-material atom B-material defect vacancies on mos 2 , li et al . copyright 2019 , wiley - vch . [SEP]
[CLS] 15 . [SEP]
[CLS] schematic illustration of the formation of multilayer dox / d2 / mos2 - ns and subsequent intracellular release of dox in which atp - aptamers enable layer - by - layer assembly . [SEP]
[CLS] dox is loaded within the multilayer structure . [SEP]
[CLS] cellular uptake is achieved through endocytosis B-event and atp - induced dox release occurs in the cytosol . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2017 , american chemical society . [SEP]
[CLS] functionalized otherwise inert mos 2 nanosheets with sulfurterminated dna oligonucleotides . [SEP]
[CLS] dox was loaded into the dna / mos 2 nanosheet structure and was protected from dna intracellular enzymes . [SEP]
[CLS] however , in the presence of atp target molecules , the dna / mos 2 structure disassembled to induce the release of dox and cell B-material apoptosis B-event ( figure 15 ) . [SEP]
[CLS] as such , the authors present a drug release system , responsive to stimuli , for targeted chemotherapy , which may have other applications in the wider nanotechnology applications . [SEP]
[CLS] liu et al . developed a novel strategy for the fabrication of a dna - np B-nanoparticle for efficient delivery of sgrna / cas9 / antisense for targeting a tumor B-material - associated gene , plk1 . [SEP]
[CLS] the biocompatible B-property dna - np B-nanoparticle system demonstrated efficient inhibition of tumor B-material growth without inducing systemic toxicity B-property . [SEP]
[CLS] interestingly , due to the optical properties associated with metallic nps B-nanoparticle , the use of dna - nps B-nanoparticle to facilitate the light - triggered release of oligonucleotides has also been explored . [SEP]
[CLS] reich and colleagues functionalized hollow gold B-material nanospheres B-nanoparticle with two types of rna strands : one with biotin modification for cell B-material targeting and penetration ( scaffold rna ) and the other without biotin , i . e . , sirna . [SEP]
[CLS] flexible singlestranded rna was utilized to achieve dense surface - packing , followed by hybridization with the complementary rna strand , which maximized the assembly of the targeting peptide B-material for cellular uptake and sirna delivery . riley et al . demonstrated the release of sirna from silica B-material core I-material / gold B-material shell B-material nanospheres B-nanoparticle for the release of conjugated sirna upon excitation with either a pulsed or continuous - wave laser ( 808 nm ) . [SEP]
[CLS] the authors observed sirna release using either continuous - wave and pulsed irradiation ; however , the latter demonstrated a higher percentage of released duplexes . [SEP]
[CLS] on - demand gene silencing was achieved without the use of additional chemical modifications making these sirna - nanoshell B-nanoparticle conjugates desirable gene delivery candidates . [SEP]
[CLS] the use of dna - nps B-nanoparticle , such as aunps B-nanoparticle , has also been explored as drug delivery vectors for the treatment of cancer . [SEP]
[CLS] heuer - jungemann et al . designed a np B-nanoparticle probe that was able to accurately distinguish different cell B-material types based on their mrna signature . [SEP]
[CLS] the probe consisted of four parts : aunp B-nanoparticle core B-material , a fluorophore - tagged oligonucleotide ( sense strand ) attached to the aunp B-nanoparticle , a fluorophore - tagged oligonucleotide ( flare strand ) that was released from the nps B-nanoparticle in the presence of a specific target , and finally the dna intercalating anticancer B-property drug ( dox ) . [SEP]
[CLS] due to the proximity of the fluorophores to the aunp B-nanoparticle core B-material , the fluorescence B-property emission was quenched . [SEP]
[CLS] however , in the presence of the specific mrna target , the flare strand is displaced as a result of competitive hybridization , thus enabling the restoration of the fluorescence B-property signal since it is no longer in close proximity to the gold B-material np B-nanoparticle core B-material . [SEP]
[CLS] the drug is then simultaneously released from the nps B-nanoparticle where it can enter the cell B-material nucleus and interfere with genomic dna , resulting in apoptosis B-event . [SEP]
[CLS] furthermore , incorporation of a fluorescent B-property dye on the sense strands enables the dna - np B-nanoparticle to serve also as a self - reporting mechanism , ensuring that any fluorescent B-property emission observed from the flare strand is a result of the detection of target mrna rather than intracellular dna degradation . [SEP]
[CLS] more recently , the same group developed multiplexed dna - aunp B-nanoparticle dimers capable of entering cells B-material and coordinating the delivery of two different intercalating drugs , dox , and mitoxantrone , displacement is observed as an increase in fluorescence B-property at a wavelength specific to that of the fluorophore . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , american chemical society . [SEP]
[CLS] into the local cellular environment ( figure 16 ) [SEP]
[CLS] high selectivity and specificity were achieved using oligonucleotide sequences capable of capturing a specific mrna target . [SEP]
[CLS] therefore , the design of np B-nanoparticle assemblies that can perform multiplexed synergistic roles of sensing and drug delivery activated by specific biological fingerprints within cells B-material provides a means of improving the efficacy of therapeutic treatments by reducing nonspecific targeting and associated off - target toxicity B-property . [SEP]
[CLS] the development of a dna - np B-nanoparticle capable of co - delivering nucleic B-material acid I-material therapeutics ( short hair pin rna ) and chemotherapeutics ( dox ) for the treatment of mdr breast cancer has also been reported . [SEP]
[CLS] following administration of the dna - nps B-nanoparticle , significant inhibition of mdr1 gene expression in mdr cancer cells B-material was achieved , which in turn resulted in enhanced intracellular accumulation of dox and revoked drug resistance . [SEP]
[CLS] inhibition of tumor B-material growth in a xenograft mdr solid tumor B-material model was also observed . [SEP]
[CLS] stimuli - responsive dna - nps B-nanoparticle have also shown promise as drug delivery platforms . [SEP]
[CLS] song et al . developed a ph - responsive dna - aunp B-nanoparticle nanocarrier for the delivery of dox to cells B-material at a ph of less than 5 . 5 . [SEP]
[CLS] kim et al . evaluated the use of " dnafunctionalized np B-nanoparticle - loaded - nps B-nanoparticle " as a drug delivery platform . [SEP]
[CLS] in this instance , silica nps B-nanoparticle ( 150 nm ) were loaded with dnafunctionalized aunps B-nanoparticle ( 15 nm ) . [SEP]
[CLS] dna hybridization ensured efficient loading and drug release as a response to lower ph in the tme and intracellular environment . [SEP]
[CLS] such constructs were shown to offer prolonged circulation time as well as effective tumor B-material irradiation in mouse models of metastatic breast cancer ( figure 17 ) . [SEP]
[CLS] the use of nir light has also been exploited for controlled drug release applications , namely , due to the fact that nir light is capable of penetrating further into biological media in comparison to visible light . [SEP]
[CLS] using an nir continuous wave ( cw ) or pulsed lasers , halas et al . triggered the release of docetaxel ( dtx ) from dna - functionalized au nanoshells B-nanoparticle with a sio 2 core B-material . [SEP]
[CLS] the results demonstrated higher cytotoxicity B-property for cw versus pulsed - laser - induced dtx release from a dna host . [SEP]
[CLS] however , some nonspecific cell B-event death I-event was induced by the cw laser itself . [SEP]
[CLS] dna - functionalized superstructures based on a " coresatellite " architecture have also been designed for photothermal drug release applications . [SEP]
[CLS] aunrs were selected due to their high absorption cross - section and photothermal conversion ability in the nir region . [SEP]
[CLS] such design consisted of three main structural components : i ) central core B-material gold B-material aunr as structural scaffold , ii ) dna strands as molecular linkers , and iii ) peripheral 5 nm spherical gold B-material nps B-nanoparticle ( satellites ) coated in polyethylene glycol ( peg ) . [SEP]
[CLS] the presence of peg helped to improve biological stability in serum - containing media and reduced nonspecific interaction with other biomolecules . [SEP]
[CLS] dox loading and release kinetics were tuned through the use of linker strands with different designs . [SEP]
[CLS] in comparison to the control without laser irradiation , a 2 . 1 - fold increase in dox release was observed . [SEP]
[CLS] in addition to gold B-material aunrs , hairpin dna - functionalized nayf4 - sio 2 aunps B-nanoparticle have also been investigated for photothermal drug release applications ( figure 18a ) . [SEP]
[CLS] by combining single - band anti - stokes nir emission with the photothermal effect , the authors enabled the release of dox in vivo ( figure 18c ) . [SEP]
[CLS] this was achieved by utilizing the up - conversion effect , which refers to the process when two or more low energy incident photons are absorbed and then emitted as one photon with higher energy . [SEP]
[CLS] nayf4 @ sio 2 - au nanoconjugates were designed to feature both excitation ( 980 nm ) and emission ( 800 nm ) bands inside the nir window , thus enabling light penetration through depths of 8 mm in tissue phantoms . [SEP]
[CLS] in comparison to the controls , tumors B-material injected with the dox - loaded nanoconjugates , which were irradiated with a 980 nm laser , demonstrated a significant reduction in size , thus indicating successful photothermal drug release ( figure 18b , d ) . [SEP]
[CLS] therefore , by utilizing the depth penetration properties of nir figure 17 . illustration of the sequential events of the np B-nanoparticle - loaded np B-nanoparticle carriers comprising the prepared large - pored mesoporous silica np B-nanoparticle and drug - loaded dna - gold nps B-nanoparticle ( aunps B-nanoparticle ) . [SEP]
[CLS] loading into the pores and stimulus - responsive release of the small aunps B-nanoparticle in the intracellular environment were programmed by dna hybridizations . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , wiley - vch . light , such nanoconjugates offer a promising approach for deeptissue imaging and guided therapy . [SEP]
[CLS] recently , mou et al . developed an elegant strategy to co - deliver nucleic B-material acid I-material to regulate drugresistant genes and simultaneously deliver the drug to cancer tissue . [SEP]
[CLS] as a proof of concept , floxuridine was delivered in vivo to liver cancer mouse models . [SEP]
[CLS] relevant references with in vivo studies are summarized in table 3 . [SEP]
[CLS] current methods for treating cancer typically involve surgery in combination with chemotherapy and / or immunotherapy and radiation therapy . [SEP]
[CLS] however , traditional therapies , specifically small molecule chemotherapeutics such as cisplatin B-material , are associated with off - target effects and lack of tumor B-material specificity . [SEP]
[CLS] this translates into major side effects that not all patients can tolerate , limiting the recommended therapeutic dose that can be administered in order to elicit a therapeutic effect . [SEP]
[CLS] in addition , cancer is associated with a high degree of molecular heterogeneity between cancers of the same type , as well as between the primary tumor B-material and its metastatic foci within the same patient . [SEP]
[CLS] because of these known problems , major efforts have focused on developing more personalized therapies with the hope of increasing treatment efficacy while minimizing unwanted side effects . [SEP]
[CLS] nanoformulations such as liposomes B-nanoparticle have several clinical advantages over traditional therapies including , i ) reduced renal and / or hepatic circulation , ii ) a greater ability to accumulate at the side of interest , and iii ) less off - target accumulation which in turn reduces systemic side effects . [SEP]
[CLS] doxil , the first fdaapproved nanomedicine , enabled the delivery of dox to tumor B-material sites via passive targeting by liposomal B-nanoparticle encapsulation . [SEP]
[CLS] as it stands , the fda has approved numerous nanomedicines with several more currently being investigated in preclinical and clinical studies . [SEP]
[CLS] however , several drug - delivery nanoparticle B-nanoparticle systems are a mixture of differently sized molecules , a result of the difficulties in precisely controlling the size , shape , and placement of the molecules within the nanostructure itself . [SEP]
[CLS] moreover , scaled - up production of many nanoformulations is often challenging and is associated with high costs . [SEP]
[CLS] on the other hand , the strict base pairing principle associated with dna enables flexible structural manipulation in the nanometer regime at a potentially lower cost . [SEP]
[CLS] this has resulted in the fabrication of monodisperse , highly controllable drug delivery platforms to enable control of the shape and positioning of units on the surface of dna . [SEP]
[CLS] furthermore , owing to its already prominent presence in nature , dna offers high biocompatibility B-property and low cytotoxicity B-property and , combined with its high drug loading and theranostic capabilities , dna - nss and dna - nss offer many advantages over current clinically approved nanomedicines . [SEP]
[CLS] encouraged by the success of preclinical research , which validated the effectiveness of dna - nps B-nanoparticle for the treatment of cancer , significant work has focused on translating these nanostructures into the clinic . [SEP]
[CLS] exicure inc . , a clinical stage biotech company , utilizes spherical nucleic B-material acid I-material ( sna ) architecture to enable safe and effective delivery of therapeutics into cells B-material and tissues . [SEP]
[CLS] the first clinical trial ( nct03010046 ) utilizing this technology investigated the use of sna ast - 005 for the treatment of chronic psoriasis by targeting tumor B-material necrosis factor alpha ( [UNK] ) , an inflammatory cytokine . [SEP]
[CLS] the topically applied gel formulation was shown to meet safety requirements copyright 2017 , wiley - vch . [SEP]
[CLS] 3 . summary of dna - ns and dna - np B-nanoparticle as drug and gene delivery platforms and their application in vivo . [SEP]
[CLS] type of nanostructure applications [ 113 ] [SEP]
[CLS] dna tetrahedron with sirna targeted delivery and improved serum stability of sirna [ 124 ] [SEP]
[CLS] dna drug micellar nps B-nanoparticle gene regulation , imaging , and chemotherapy in ptx - resistant cell - xenografted mice [ 133 ] c dna - polylactide ( pla ) micelles B-material [SEP]
[CLS] targeted chemotherapy and gene knockdown in subcutaneous mcf7 / adr [ 135 ] [SEP]
[CLS] au np B-nanoparticle - loaded sio 2 nps B-nanoparticle delivery of chemotherapeutics throughout the tumor B-material [ 138 ] hairpin dna - functionalized nayf4 - sio 2 aunps B-nanoparticle [SEP]
[CLS] multimodal imaging and photothermal dox release and importantly , reduced [UNK] mrna expression in psoriasis plaques . [SEP]
[CLS] unfortunately , however , results from the study indicated that there was no statistically significant decrease in echolucent band thickness , a key indicator B-property of efficacy in psoriasis patients . [SEP]
[CLS] nonetheless , exicure is currently investigating several other sna based dna - ns at both the preclinical and clinical stage as therapeutic candidates in the fields of immuno - oncology , neurology , and dermatology . [SEP]
[CLS] na - gold B-material nps B-nanoparticle ( nu - 0129 ) capable of targeting the overexpression of bcl2l12 are undergoing phase 1 clinical trials to evaluate safety in patients with recurrent gbm or gliosarcoma ( nct03020017 ) . [SEP]
[CLS] results from a recent phase 0 trial indicated that the dna - np B-nanoparticle , nu - 0129 , was well tolerated in patients with gbm with no unexpected adverse side effects . [SEP]
[CLS] in addition , initial data also indicated that that nu - 0219 is capable of crossing the bbb . [SEP]
[CLS] ast - 008 is a toll - like receptor B-material 9 ( trl9 ) agonist designed to activate an immune response particularly when used in combination with immune checkpoint inhibitors and is currently undergoing phase 1b / 2 clinical trials ( nct03086278 and nct03684785 , respectively ) . [SEP]
[CLS] initial findings in patients with advanced solid tumors B-material indicated that administration of ast - 008 , either on its own or in combination with pembrolizumab , initiated cytokine and chemokine expression and immune cell B-material activation in patient blood to a desired level . [SEP]
[CLS] a phase 2 clinical trial is currently ongoing and aims to determine the recommended phase 2 dose of ast - 008 given in combination with pembrolizumab or cemiplimab in order to provide an estimation of efficacy . [SEP]
[CLS] although results from both preclinical and early clinical trials are encouraging , as with any new medicine , there are still several challenges associated with the clinical translation of dna - nss and dna - nps B-nanoparticle . [SEP]
[CLS] with regards to oncology , we believe the key factor in improving their clinical efficacy lies with improving their uptake into the tme by overcoming the associated biological barriers B-property . [SEP]
[CLS] although extremely challenging , research should continue to focus on i ) improving the intracellular delivery of dna - ns and dna - nps B-nanoparticle to the tumor B-material , ii ) improving stability to prevent premature degradation of dna - nss and dna - nps B-nanoparticle following intravenous injection , and iii ) regulate and improve biodistribution and pharmacokinetic profiles of these nanomedicines to enable maximum delivery of a payload to the tumor B-material site and improved efficacy . [SEP]
[CLS] of course , the need to overcome the barriers B-property associated with the aforementioned challenges is well understood and significant effort is underway to address them . [SEP]
[CLS] challenges include ability to ensure dna - nss and dna - nps B-nanoparticle maintain their stability at physiological temperature in an environment where magnesium B-material concentration , which plays a critical role in stabilizing the dna duplex , is decreased . [SEP]
[CLS] in addition , efforts are being made to ensure these nanoformulations evade dna and rna degrading enzymes and are not overwhelmed by protein B-material corona I-material in order to enhance their intracellular trafficking and delivery to the site of interest . [SEP]
[CLS] nanotheranostics has matured to a fascinating field , where three essential abilities crucial for improved cancer therapy can be realized : i ) noninvasive detection of cancer , ii ) detection of cancer progression , and iii ) incorporation of therapeutic modules . [SEP]
[CLS] with regards to preclinical settings , these all - important parameters can be assessed , as shown by the many studies described in this review . [SEP]
[CLS] dna - nps B-nanoparticle and dna - nss have opened up new avenues in this direction due to intelligent design , simple functionalization strategies , and low toxicity B-property concerns . [SEP]
[CLS] in this review , we discussed , in a balanced fashion , the recent advancements in the applications of such nanostructures . [SEP]
[CLS] dna - nss and dna - nps B-nanoparticle , both stemming from unique selfassembly properties of dna , have seen immense growth in the last two decades . [SEP]
[CLS] the tailored and customizable size , shape , and charge of dna - nss and dna - nps B-nanoparticle allows efficent intracellular delivery . [SEP]
[CLS] this is in stark contrast to dna or sirna - based approaches , which do not enter cells B-material without the help of transfection agents . [SEP]
[CLS] another advantage of dna - nss and dna - nps B-nanoparticle is their ease of attachment of imaging agents or therapeutic drugs . [SEP]
[CLS] moreover , densely packed dna helices can enhance the in vivo stability against dna - degrading enzymes , increasing the halflife of the nanostructures and thereby increasing the chance of reaching the sites of malignancy . [SEP]
[CLS] to further enhance the biostability , dna nanostructures can be cloaked with membranes to enhance such feature . [SEP]
[CLS] the above reports collectively attest that both dna - nps B-nanoparticle and dna - nss can potentially provide an excellent design platform for the construction of all - in - one imaging and novel drug delivery vehicles B-material with optimum biocompatibility B-property and therapeutic efficacy . [SEP]
[CLS] the programmability of dna - based designs enables judicious selection of nanostructures with various shapes and sizes tuned to the desired targeting paradigm , be it to optimize shape and size for passive targeting ( epr effect ) or active targeting via intelligent surface functionalization strategies . [SEP]
[CLS] it is important to note , however , that as a field , nanotheranostic agents are not without their limitations . [SEP]
[CLS] this is in part due to an underestimation of the effects of tumor B-material heterogeneity , which consequently has resulted in an over simplified pharmacokinetic model in which the tumor B-material is thought of simply as a leaky sponge that enables np B-nanoparticle accumulation following prolonged circulation in the blood . [SEP]
[CLS] significant efforts have therefore focused on inhibiting np B-nanoparticle uptake by the reticuloendothelial system and tuning renal - clearance , however , this approach neglects the importance of the effect of the vessel leakiness , blood flow , macrophage abundance and microvessel density , and variation between tumors B-material . [SEP]
[CLS] therefore , it is extremely important to consider all aforementioned factors when designing the ideal nanotheranostic for use in oncology , and going forward , we encourage researchers to not simply rely on the epr effect as an effective delivery method . [SEP]
[CLS] while the existing preclinical data involving dna - nps B-nanoparticle and dna - nss is undoubtedly encouraging results from current clinical trials highlight that challenges remain . [SEP]
[CLS] however , should these hurdles be overcome , there is a realistic hope that therapeutic efficacy can markedly improve . [SEP]
[CLS] emerging technologies such as immunomodulation B-property , i . e . , the use of dna or rna to elicit an immune response , also has the possibility to revolutionize the field of dna nanotheranostics . [SEP]
[CLS] by taking advantage of the favorable properties associated with nucleic B-material acid I-material scaffolds , we envisage that methods involving dna - immunomodulation B-property will generate novel , effective therapies , thus allowing researchers and clinicians to modulate cell B-material function with high precision . [SEP]
[CLS] moreover , by incorporating other small molecules such as imaging agents and chemotherapeutics into the dna - origami structure , dna - immunomodulation B-property has significant potential to drive the advancement of dna - nss and dna - nps B-nanoparticle as theranostic tools . [SEP]
[CLS] with further refinements of the nanostructure concepts described in this review , it is expected that cutting - edge materials will be translated into the clinics where they are poised to improve the management and outcome of cancer patients . [SEP]
[CLS] introduction to dna nanostructures ( dna - nss ) and dna - functionalized nanoparticles B-nanoparticle ( dna - nps B-nanoparticle ) [SEP]
[CLS] different nanoscale shapes developed by dna origami technology with dangling curves and loops representing unfolded sequences ( top row ) . diagrams demonstrate the bends at , and away , from crossovers ( second row ) . [SEP]
[CLS] colors illustrate the base - pair index along the folding path where red refers to the 1st base and purple refers to the 7000th base . [SEP]
[CLS] afm images of the dna origami structures are also shown ( third row ) . [SEP]
[CLS] all image are the same size of 165 nm × 165 nm . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2006 , springer nature . [SEP]
[CLS] 2 . dna origami nanostructures with complex 3d curvatures . [SEP]
[CLS] schematic representations of the hemisphere and corresponding transmission electron microscope ( tem ) images of a , d ) the hemisphere , b , e ) the sphere , and c , f ) the ellipsoid . [SEP]
[CLS] associated scale bars for the tem images in ( d ) - ( f ) are 50 nm . [SEP]
[CLS] figure 2 . g ) schematic representation of the nanoflask with expected dimensions . h ) afm images of the nanoflask in which the scale bar represents 75 nm . [SEP]
[CLS] i ) negatively stained tem images of the nanoflask , scale bar is 50 nm . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] [ 41c ] [SEP]
[CLS] copyright 2011 , american association for the advancement of science . [SEP]
[CLS] 3 . 3d meshes rendered in dna with different views of the 3d meshes serving as starting points for the initial design process . [SEP]
[CLS] figure 3 . a ) 3d meshes of different structures . b ) the front face of each design . [SEP]
[CLS] single dna strands are rendered as tubes . [SEP]
[CLS] c - e ) negative - stain dry - state tem images ( except for the ball and bunny in ( e ) and ( f ) , respectively ) of each of the structures . [SEP]
[CLS] scale bars are as follows : c ) 250 nm × 250 nm views ; d , e ) 100 nm × 100nm close - ups , with the exception of the pentagonal rod ( 200 nm × 100 nm ) ; e , f ) ( ball and bunny ) are imaged using cryo - electron microscopy B-technique . [SEP]
[CLS] the au particle used for alignment is shown [SEP]
[CLS] in ( f ) [SEP]
[CLS] in ( f ) , the scale bar represents 50 nm . [SEP]
[CLS] reproduced with permission . [ 43c ] copyright 2015 , springer nature . [SEP]
[CLS] synthesis of a dna - np B-nanoparticle . a [SEP]
[CLS] figure 4 . high - density dna - np B-nanoparticle is formed by using citrate - stabilized particles incubated B-technique in alkylthiol - functionalized dna in water B-material which consequently forms a low - density monolayer . [SEP]
[CLS] when the nps B-nanoparticle are incubated B-technique in aqueous solutions with successively higher concentrations of salt B-material ( typically 0 . 15−1 . 0 m ) and surfactants B-property over ≈12 h , successful formation of a high - density dna - np B-nanoparticle shell B-material is achieved . [SEP]
[CLS] reproduced with permission . [ 30c ] copyright 2012 , american chemical society . [SEP]
[CLS] schematic illustration of the fate of bare and coated dna - ns in buffers ( physiological ph ) at 37 °c containing either low salt B-material or 10 % fetal bovine serum ( fbs ) . [SEP]
[CLS] at low salt B-material concentrations , bare dna - nss rapidly become denatured and degraded in cell B-material medium containing 10 % fbs . [SEP]
[CLS] however , it is possible to override low - salt B-material - induced denaturation and nuclease degradation through coating B-material the dna - nss with various ratios of positively charged peptides B-material ( k10 or k10 - peg5k ) . [SEP]
[CLS] reproduced under the terms of the creative commons attribution license . [SEP]
[CLS] copyright 2017 , springer nature [SEP]
[CLS] 6 . schematic and theranostic activity of the dna nanotriangle structure [SEP]
[CLS] by conjugating the dna nanotriangle scaffold and multiple split activable aptamer probes ( saaps ) , via a - c ) three exterior strands and d ) an inner strand , the dna nanostructure is formed . [SEP]
[CLS] in the free state , the dna nanotriangle , which is loaded with dox via the cg base pair regions is a flat , double helix - jointed structure with no fluorescence B-property since it is quenched . [SEP]
[CLS] once it encounters the target cell B-material , the aptamers disassemble and consequentially change shape . [SEP]
[CLS] this leads to fluorescence B-property emission and partial drug release with the remaining drugs being freed following internalization . [SEP]
[CLS] reproduced under the terms of the creative commons attribution license . [SEP]
[CLS] copyright 2018 , ivyspring international publisher . [SEP]
[CLS] 7 . schematic representation of the synthesis of cach - peg and its effects in vivo . [SEP]
[CLS] figure 7 . a ) hemin and ce6 are inserted into the g - quadruplex structure . [SEP]
[CLS] mixing of the g - quadruplex structure and ca 2 + together with phis - peg yields ca - as1411 / ce6 / hemin @ phis - peg ( cach - peg ) ncps , which are ph responsive at ≈5 . 5 and were reduced to smaller g - quadruplex complexes in acidic environments of the lyso - and endosomes . [SEP]
[CLS] b ) the tumor B-material growth curves of 4t1 - tumor - bearing mice following each treatment as indicated . [SEP]
[CLS] tumors B-material were irradiated using 660 nm light - emitting diode light ( 5 mw cm −2 ) for 60 min . c ) average tumor B-material weights collected from the each group following 14 days after each treatment . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , american chemical society [SEP]
[CLS] 8 . demonstration of the efficiency of photothermal therapy using either aunr or dox - aunr . [SEP]
[CLS] figure 8 . a ) cell B-material viability B-property of I-property 4t1 - fluc tumor B-material cells B-material following administration of varying treatments followed by nir laser irradiation ( 808 nm , 1 . 5 w cm −2 , 3 min ) ( * * p < 0 . 01 , * * * p < 0 . 0001 ) . [SEP]
[CLS] b ) ir thermographic maps of each group of mice obtained 10 min after nir irradiation . [SEP]
[CLS] c ) bioluminescence B-property imaging ( bli ) of 4t1 - fluc - tumor - bearing mice intravenously injected with pbs ( control ) , aunr and d - aunr in combination with nir laser irradiation acquired prior to injection and at days 0 , 4 , 7 , 10 , and 17 . d ) survival rates of each group of mice following treatment were monitored 30 days post - injection . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2016 , wiley - vch [SEP]
[CLS] figure 9 . a ) fluorescence B-technique imaging I-technique of hepg2 tumor I-technique - bearing mice injected with either ce6 - fdnadox or ce6 - fndnadox . [SEP]
[CLS] time lapse fluorescence B-property images of ce6 in vivo confirm tumor B-material uptake . [SEP]
[CLS] b ) white light and fluorescence B-property images of different organs removed from hepg2 tumor - bearing mice 2 h post - injection of either ce6 - fdnadox or ce6 - fndnadox where ce6 is represented in red and dox in green . [SEP]
[CLS] c ) tumor B-material volume of mice ( n = 5 ) after single iv injection of the indicated treatments . [SEP]
[CLS] tumors B-material were imaged 2 h after injection by irradiation with 670 nm light ( 0 . 2 w cm −2 ) for 10 min . d ) average body weight of mice following each treatment ( n = 5 ) . [SEP]
[CLS] reproduced under the terms of the creative commons attribution license . [SEP]
[CLS] copyright 2017 , wiley - vch . [SEP]
[CLS] 10 . [SEP]
[CLS] dna - nps B-nanoparticle distributed throughout glioma tissue . [SEP]
[CLS] figure 10 . a ) quantification of dna - np B-nanoparticle uptake into orthotopic u87mg tumor B-material and adjacent normal tissue at 48 h post - injection using inductively coupled plasma mass spectrometry ( icp - ms ) . [SEP]
[CLS] b ) schematic of the synthesis of gd ( iii ) - functionalized dna - nps B-nanoparticle . [SEP]
[CLS] c ) accumulation of gd ( iii ) - dna - nps B-nanoparticle within the intracerebral lesion is confirmed using magnetic B-property resonance imaging ( mri ) , hematoxylin and eosin staining , and 3d reconstructions . [SEP]
[CLS] d ) localization of au , fe , and gd ( iii ) contents in coronal brain sections of mice injected intracranially with gd ( iii ) - dna - nps B-nanoparticle as confirmed by la - icp - ms . [SEP]
[CLS] e ) confocal fluorescence B-technique microscopy I-technique of coronal brain sections derived from tumor B-material - and nontumorbearing mice following saline or cy5 - dna - np B-nanoparticle injection . [SEP]
[CLS] reproduced with permission . [ 96a ] copyright 2013 , american association for the advancement of science . [SEP]
[CLS] 11 . [SEP]
[CLS] figure 11 . a - c ) x - ray image , axial ct image , and fluorescence B-property image of the cl1 - 5 tumor B-material - bearing mouse taken 30 min post - injection of aptamer - targeted aunps B-nanoparticle as indicated by the yellow circle , white arrow , and yellow circle , respectively . [SEP]
[CLS] d ) cl1 - 5 tumors B-material under white light ( top ) and uv light ( bottom ) . [SEP]
[CLS] e ) cl1 - 5 tumors B-material incubated B-technique without and with aptamers . [SEP]
[CLS] f ) calculated total photon fluxes . [SEP]
[CLS] reproduced under the terms of the creative commons attributionlicense . [SEP]
[CLS] copyright 2015 , springer nature [SEP]
[CLS] targeted photodynamic therapy of breast cancer adv . sci . 2020 , 7 , 2001669 © 2020 the authors . published by wiley - vch gmbh 2001669 ( 14 of 27 ) 21983844 , 2020 , 23 , downloaded from https : / / onlinelibrary . wiley . com / doi / 10 . 1002 / advs . 202001669 by enpc - ecole des ponts paristech , wiley online library on [ 19 / 12 / 2022 ] [SEP]
[CLS] see the terms and conditions ( https : / / onlinelibrary . wiley . com / terms - and - conditions ) on wiley online library for rules of use ; oa articles are governed by the applicable creative commons license [SEP]
[CLS] figure 12 . a ) schematic illustration of muc1 - functionalized sers nps B-nanoparticle . [SEP]
[CLS] b ) the muc1 aptamer - functionalized dna - nps B-nanoparticle were administered intravenously to human breast cancer tumor B-material - bearing mice and selectively targeted the tumors B-material over - expressing muc1 . [SEP]
[CLS] their accumulation within tissue was confirmed via identification of the " fingerprint " spectral signature of the sers nps B-nanoparticle using raman B-technique spectroscopy I-technique . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2017 , wiley - vch [SEP]
[CLS] 13 . [SEP]
[CLS] conceptual figure illustrating the use of a nanobody ( nb ) - conjugated dna - ns for the delivery of the intercalating chemotherapeutic , 56mess , where nb , nanobody ; tet , double - bundle dna tetrahedron ; nb - tet , nb - conjugated double - bundle dna tetrahedron ; nb - tet - 56mess , platinum B-material - drugloaded nbtet ; egfr , epidermal growth B-material factor I-material receptor I-material . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , wiley - vch . [SEP]
[CLS] 14 . [SEP]
[CLS] figure 14 . a , b ) schematic of the synthesis , self - assembly , and theranostic potential of the dna - nss for anticancer B-property treatment . [SEP]
[CLS] c , d ) in vivo tumor B-material targeting and biodistribution of each dna - ns formulation following administration . [SEP]
[CLS] e ) semiquantitative analysis of fluorescence B-property signals in tumors B-material at 1 , 4 , 8 , and 24 h time points . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , wiley - vch [SEP]
[CLS] 16 . dimer loaded with dox ( drug 1 ) and mitoxantrone ( drug 2 ) ( left ) . [SEP]
[CLS] both dox and mitoxantrone have fluorescent B-property properties , which are quenched due to the close proximity to the aunp B-nanoparticle core B-material . [SEP]
[CLS] drug release is achieved following binding of target mrna to the sense strand . [SEP]
[CLS] target mrna can bind to the corresponding sense sequence via competitive hybridization to induce displacement of the flare strand . [SEP]
[CLS] displacement is observed as an increase in fluorescence B-property at a wavelength specific to that of the fluorophore . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2018 , american chemical society [SEP]
[CLS] 18 . [SEP]
[CLS] figure 18 . a ) schematic illustration of dox drug release from hairpin - dna - modified nayf4 @ sio 2 - au nanoconjugates triggered by a photon 980 nm excitation . [SEP]
[CLS] b ) changes to tumor B-material volume following response to either of the four treatments where the three control groups indicate : only laser irradiation ( control 1 ) , without laser irradiation and treatment of nanoconjugates ( control 2 ) , and with treatment of dox - loaded dna - nps B-nanoparticle without irradiation ( control 3 ) . [SEP]
[CLS] c ) in vivo up - conversion imaging of a tumor I-technique - bearing mouse treated with the dna _ nps B-nanoparticle . [SEP]
[CLS] d ) pictures of the tumor bearing mice following treatment with therapy or controls . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2017 , wiley - vch [SEP]
[CLS] summary of dna - ns - based theranostics and in vivo applications . [SEP]
[CLS] in the past decade , ultrasmall luminescent B-property metal B-nanoparticle nanoparticles I-nanoparticle ( ulmnps , d < 3 nm ) have achieved rapid progress in addressing many challenges in the healthcare field because of their excellent physicochemical properties and biological behaviors . [SEP]
[CLS] with the sharp shrinking size of large plasmonic metal B-nanoparticle nanoparticles I-nanoparticle ( pmnps ) , the contributions from the surface characteristics increase significantly , which brings both opportunities and challenges in the application - driven surface engineering of ulmnps toward advanced biological applications . [SEP]
[CLS] here , the systematic advancements in the biological applications of ulmnps from bioimaging to theranostics are summarized with emphasis on the versatile surface engineering strategies in the regulation of biological targeting and imaging performance . [SEP]
[CLS] the efforts in the surface functionalization strategies of ulmnps for enhanced disease targeting abilities are first discussed . [SEP]
[CLS] thereafter , self - assembly strategies of ulmnps for fabricating multifunctional nanostructures for multimodal imaging and nanomedicine are discussed . [SEP]
[CLS] further , surface engineering strategies of ratiometric ulmnps to enhance the imaging stability to address the imaging challenges in complicated bioenvironments are summarized . [SEP]
[CLS] finally , the phototoxicity of ulmnps and future perspectives are also reviewed , which are expected to provide a fundamental understanding of the physicochemical properties and biological behaviors of ulmnps to accelerate their future clinical applications in healthcare . [SEP]
[CLS] ultrasmall luminescent B-property metal B-nanoparticle nanoparticles I-nanoparticle ( ulmnps , mainly including au , ag , and cu [SEP]
[CLS] introductionare typically classified as atomically precise metal B-material nanoclusters and metal B-nanoparticle nanoparticles I-nanoparticle with core B-material sizes below ≈3 nm . [SEP]
[CLS] the quantum confinement effect doi : 10 . 1002 / advs . 202103971 begins to appear , and the optical and electronic properties are typically dominated by electrons in discrete energy levels , such as semiconductor quantum B-nanoparticle dots I-nanoparticle ( qds ) , which are different from the delocalized electrons observed in plasmonic metal B-nanoparticle nanoparticles I-nanoparticle ( pmnps ) . [SEP]
[CLS] unlike semiconductor qds , the emission associated with ulmnps does not appear to be purely associated with the quantum confined metal B-material core B-material effect , but is highly dependent on the surface state chemistry , indicating that the emission origin of ulmnps is the result of complicated interactions between the metal B-material core B-material and the metal - ligand surface . [SEP]
[CLS] because surface chemistry plays a critical role in the luminescence B-property of ulm - nps B-nanoparticle due to the ligand - metal B-material charge transfer ( lmct ) mechanisms , tuning the surface chemistry is expected to serve as an efficient method to tune the luminescence B-property properties of ulmnps . [SEP]
[CLS] ulmnps with discrete energy states not only exhibits outstanding tunable optical properties , but also display unique physicochemical properties and biological behaviors , which act as distinctive bridge materials between the small molecules and traditional plasmonic metal B-nanoparticle nanoparticles I-nanoparticle ( pmnps ) to address many challenges in the healthcare field . [SEP]
[CLS] pmnps are often selectively transported to disease sites ( e . g . , tumors B-material ) with much higher concentrations and longer retention times than small molecules ( e . g . , fluorescent B-property dyes ) through the well - recognized enhanced permeability and retention ( epr ) effect . [SEP]
[CLS] however , unlike small molecules with fast clearance and low body retention , pmnps often severely accumulate in the reticuloendothelial system ( res ) organs ( e . g . , liver and spleen ) , which could lead to potential long - term toxicity B-property and severe systemic side effects . [SEP]
[CLS] the ulmnps with in vivo hydrodynamic diameters ( hds ) below the kidney filtration threshold ( kft , ≈5 . 5 nm ) , can pass through the glomerular filtration barrier B-property and be excreted from urine with high biocompatibility B-property , which shows the integrated advantages of both pmnps ( e . g . , high targeting efficiency , high signal output , and multiple modalities ) and small molecules ( e . g . , efficient excretion , low body retention , and high biocompatibility B-property ) , holding great potential in clinical translation . [SEP]
[CLS] with the sharp shrinking size from pmnps to ulmnps , the contributions from the surface increase significantly , which brings both opportunities and challenges for surface engineering . [SEP]
[CLS] specifically , ulmnps with the advantages of ultrasmall size scale provide distinctive building blocks for selfassembly into uniform nanostructures with controllable size and shape , rendering new properties and functions for advanced biological applications ranging from multimodal imaging to nanomedicine . [SEP]
[CLS] a slight change in the surface chemistry ( e . g . , surface coverage , charge , and hydrophobicity B-property ) of ulmnps can significantly affect their optical properties ( e . g . , absorption and emission ) and biological behaviors ( e . g . , cellular interaction , organ distribution and excretion pathways ) , which indicates great opportunities in the application - driven surface engineering of ulmnps for advanced biological applications . [SEP]
[CLS] however , some of the well - established surface engineering strategies from pmnps would hardly be adaptable to ulmnps with a unique metal B-material ( 0 ) core B-material and metal ( i ) - thiolate shell B-material structures , which reminds chemists to further disclose the unique surface engineering strategies for ulmnps . [SEP]
[CLS] in this review , we present an overview of our efforts in the surface engineering of ulmnps for biological targeting and imaging ( figure 1 ) , emphasizing the versatile strategies for surface engineering of ulmnps . [SEP]
[CLS] as the biological interactions between ulmnps and targets are highly related to surface functionalization , the surface functionalization strategies and principles for enhanced targeting abilities are first summarized . [SEP]
[CLS] we then systematically introduce the self - assembly strategies of ulmnps for fabricating multifunctional nanostructures for multimodal imaging and nanomedicine . [SEP]
[CLS] we will also discuss surface engineering strategies of ratiometric ulmnps to enhance the imaging stabilities and sensitivities to address the imaging challenges in complicated bioenvironments . [SEP]
[CLS] finally , we critically review the phototoxicity of ulmnps and present future perspectives to accelerate their clinical applications in disease diagnosis , monitoring , and treatment . [SEP]
[CLS] ulmnps are typically considered as a type of molecule - like optical nanoprobe B-nanoparticle with low nonspecific retention , high metabolic clearance , and fewer background tissue interactions . [SEP]
[CLS] the emergence of renal - clearable ulmnps could potentially address some of the safety concerns of the large pmnps with longterm nonspecific accumulation in res organs . [SEP]
[CLS] however , the rapid renal clearance of ulmnps raises other issues , including short blood circulation , weak epr effect , and low targeting efficiency . [SEP]
[CLS] surface functionalization of ulmnps is becoming crucial to further control their stability , circulation , retention , and interaction with biological environments . [SEP]
[CLS] the surface ligand exchange strategy has typically been applied to the functionalization of pmnps with various thiolate active - targeting B-material ligands I-material ( e . g . , glucose , dna , and antibodies B-material [SEP]
[CLS] ) for enhanced targeting and accumulation at the tumor B-material site [SEP]
[CLS] however , the functionalization of renal - clearable ulmnps should consider the following challenges and principles ( figure 2 ) : 1 ) the unique metal ( 0 ) core B-material and metal ( i ) - thiolate shell B-material structures make it difficult to develop a well - developed thiolate ligand exchange strategy ; 2 ) the effects of both the strong anchoring sites ( e . g . , - sh ) and weak anchoring sites ( e . g . , - cooh and - nh 2 ) of the surface ligands due to the ultrasmall size ; 3 ) maintaining in vivo hd below kft ( ≈5 . 5 nm ) for efficient renal clearance after circulation from the body ; and 4 ) enabling the surface resistance to serum protein B-material adsorption in biological environment to avoid fast res sequestration . [SEP]
[CLS] dna molecules offer powerful design techniques and have unique advantages for molecular recognition . [SEP]
[CLS] typically , dna molecules are modified on the surface of pmnps using alkanethiol - capped dna through a surface ligand exchange strategy for targeted drug delivery , disease diagnosis , and well - organized self - assembly . [SEP]
[CLS] however , alkanethiol - capped dna is difficult to modify to luminescent B-property gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) with a unique au ( 0 ) core B-material and au ( i ) - thiolate shell B-material structures using a similar ligand exchange strategy . [SEP]
[CLS] therefore , phosphorothioate ( ps ) - modified dna ( psdna ) with programmable sulfur - containing phosphodiester ( po ) backbones are ideal alternatives for increasing the affinity of luminescent B-property aunps B-nanoparticle . [SEP]
[CLS] the rest of the domain with po backbones that show weak affinity to the gold B-material surface act as a functional recognition domain by predictable watson - crick base pairing . [SEP]
[CLS] the dna molecules were designed with both ps domain as the template and reductant , and the po domain for molecular recognition , which were used for in situ conjugation of ultrasmall nir - emitting aunps B-nanoparticle ( psdna - aunps B-nanoparticle ) through a thermal reducing method ( figure 3 ) . [SEP]
[CLS] the psdna - aunps B-nanoparticle showed size - independent nir emission ( ≈810 nm ) with sizes that could be finely tuned from ≈1 . 3 to 2 . 6 nm through regulation of the ps length . [SEP]
[CLS] the psdna - aunps B-nanoparticle could be controlled to bear strict dna valence , such as one dna ( v1 ) and two dnas ( v2 , divalent ) , which were good building blocks for the fabrication of well - organized nanostructures . [SEP]
[CLS] the psdna - aunps B-nanoparticle were then demonstrated to facilely hybridize with a sgc8c aptamer ( apt - aunps B-nanoparticle ) for targeting ptk7 proteins B-material , overexpressed on the ccrf - cem cell B-material membranes , which showed enhanced specific targeting abilities . [SEP]
[CLS] since there are many well - established protocols in dna technologies , this in situ synthesis strategy would provide a facile pathway to enable the ulmnps with various new functionalities . [SEP]
[CLS] glycoconjugation is a promising strategy for the functionalization of nanoparticles B-nanoparticle with enhanced tumor - targeting efficiency . [SEP]
[CLS] cancer cells B-material overexpress glucose transporters and hexokinase that can recognize glucose - functionalized nanoparticles B-nanoparticle , which could increase the cellular uptake efficiency and alter the biodistributions . [SEP]
[CLS] meanwhile , 1 - thio - [UNK] - d - glucose ( tg ) is a derivative of glucose , in which the hydroxyl B-material group I-material at the c - 1 position is replaced with a thiol B-material group , which enhances the binding capability toward the metal B-material surface . [SEP]
[CLS] using tg as both the surface ligand and reducing B-property agent I-property , ultrasmall 810 nm emitting gold B-material glyconanoparticles ( augnps , ≈2 . 4 nm ) were successfully synthesized ( figure 4 ) , which showed resistance to serum protein B-material adsorption and maintained hd of 4 . 2 ± 0 . 8 nm below kft . [SEP]
[CLS] the ultrasmall nir - emitting augnps entered the cancer cells B-material through glucose transporters , and showed not only long blood circulation with enhanced tumor - targeting efficiency but also low accumulations in res organs with efficient clearance . [SEP]
[CLS] after comparison with those of the ultrasmall nonglycoparticles , glycoconjugation of ultrasmall augnps led to ≈10 times and ≈2 . 5 times greater increase in the efficiency of cancer cell B-material uptake and in vivo tumor B-material targeting , respectively . [SEP]
[CLS] the tumor targeting efficiencies of ultrasmall augnps ( ≈2 . 4 nm ) were higher than those of the renal - clearable nonglycoconjugated gs - aunps B-nanoparticle ( ≈2 . 5 nm ) or the nonrenal - clearable glycoconjugated large plasmonic augnps ( p - augnps B-nanoparticle , ≈13 nm ) . [SEP]
[CLS] the findings will provide guidance in the future design of clinically translatable nanomedicines with both high tumor - targeting efficiency and efficient clearance for the advanced diagnosis and treatment of cancer , including metastatic tumors B-material and brain tumors with blood - brain barrier B-property crossing capabilities . [SEP]
[CLS] the acidic tumor B-material microenvironment ( tme , ph 6 . 5 - 7 . 2 ) , resulting from the increased metabolism and activated anaerobic glycolysis of cancer cells B-material to produce acidic metabolites ( e . g . , lactate ) , has been well recognized as a promising target for tumor - specific theranostics . [SEP]
[CLS] since surface chemistry ( e . g . , hd and surface charge ) determines the nanoparticle B-nanoparticle biodistributions among the tumor B-material site and mps organs , the synergistic integration of the strategies of precise hd and surface charge regulation for the ulmnps is critical for targeting the acidic tme . [SEP]
[CLS] surface functionalization with controllable hd and surface chargeproteins binding with the fabricated apt - aunps B-nanoparticle . [SEP]
[CLS] e ) fluorescent B-property imaging of apt - aunps B-nanoparticle after incubation B-technique with living cells B-material . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , american chemical society . [SEP]
[CLS] the charge - reversal nir - emitting aunps B-nanoparticle were designed through tunable surface pegylation to precisely tune the hd and surface charge , as well as conjugation of ph - responsive imidazole groups to further control the charge - reversal of aunps B-nanoparticle at different ph values . [SEP]
[CLS] using the ultrasmall 810 nm emitting aunps B-nanoparticle ( core B-material size ≈1 . 7 nm ) with precise hds ( 2 . 4 - 4 . 2 nm ) , [UNK] - potential values ( −31 . 2 to −11 . 4 mv at ph 7 . 4 ) and comparable negativeto - positive ( [UNK] ph 6 . 5 - [UNK] ph 7 . 4 , ≈28 . 8 mv ) charge - reversal capabilities as model , it was observed that the ultrasmall charge - reversal aunps B-nanoparticle with high [UNK] - potential values and large hd show the high tumor - targeting specificity ( figure 5 ) . [SEP]
[CLS] the optimized ultrasmall charge - reversal aunps B-nanoparticle ( hd : 4 . 2 nm ; [UNK] - potential : −11 . 4 mv at ph 7 . 4 ) showed high tumor - targeting efficiencies ( ≈9 % id g −1 ) and extremely low accumulation in mps organs ( e . g . , liver ≈2 % id g −1 ) , resulting in high signal - to - noise ( s / n ) ratio values for the identification of cancer metastases B-event . [SEP]
[CLS] the small metastatic tumors B-material ( ≈1 mm ) in the liver were well distinguished noninvasively with high s / n ratios ( ≈4 . 6 ) , fast response ( 10 min ) , and long imaging window time ( > 6 h ) . [SEP]
[CLS] in addition to the liver cancer metastasis B-event , the tumors B-material in other mps organs , such as the spleen and lungs were also well imaged with s / n ratios of ≈5 . 2 and ≈4 . 5 , respectively . [SEP]
[CLS] therefore , the charge - reversal ulmnps through precise hd and charge regulation would provide a facile pathway to address the long - standing challenge in optical B-technique imaging I-technique of metastatic tumors B-material with metal B-nanoparticle nanoparticles I-nanoparticle , which holds great promise for metastatic cancer diagnosis in future clinical practice . [SEP]
[CLS] functionalization of nanoparticles B-nanoparticle with surface ligands is considered the most common strategy for controlling the stability , circulation , toxicity B-property , and biological interactions . [SEP]
[CLS] various surface ligands , such as peptides B-material , proteins B-material , and nucleic B-material acids I-material , can be conjugated to the au surface via anchoring groups , such as - sh , - nh 2 , and - cooh . [SEP]
[CLS] the binding between - sh and au has been long - term recognized because of the strong covalent B-property interaction I-property of au - s ( ≈40 kcal mol −1 ) , in contrast to the relatively weak interaction of au - o - cooh ( ≈2 kcal mol −1 ) or au - n - nh2 ( ≈8 kcal mol −1 ) . [SEP]
[CLS] therefore , the effects of the weak anchoring groups of - nh 2 and - cooh are ignored during surface functionalization . [SEP]
[CLS] using a typical thiolate peptide B-material , gsh with anchoring groups of - sh , - cooh , and - nh 2 , as an example , gsh - functionalized nir emitting aunps B-nanoparticle with controllable anchoring sites were synthesized in situ through ph regulation ( figure 6 ) . [SEP]
[CLS] in addition to the formation of strong s - au binding on the surface of aunps B-nanoparticle , the weak anchoring groups ( - nh 3 + and - cooh ) protonated at a low ph value ( e . g . , ph 2 . 0 ) would facilitate the formation of n - au anchoring sites with reproduced with permission . [SEP]
[CLS] copyright 2019 , royal society of chemistry . [SEP]
[CLS] the circle and arrow indicate the liver region and the tumor B-material location , respectively . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , american chemical society . more exposed surface - cooh , while deprotonation at a high ph value ( e . g . , ph 10 . 0 ) would enhance the formation of anchoring sites of coo - au and more exposed surface - nh 2 [SEP]
[CLS] the different weak anchoring sites of the aunps B-nanoparticle affected their behaviors significantly at both in vitro and in vivo levels : the more anchoring sites of coo - au and more exposed surface - nh 2 would increase cellular interactions and prolong blood circulation and retention , which would also affect the elimination pathways and increase the tumor - targeting efficiencies . [SEP]
[CLS] because the typical surface ligands for the functionalization of ulmnps contain multiple anchoring groups , the findings provide guidance for chemists to be aware of the significance of weak anchoring reproduced with permission . [SEP]
[CLS] copyright 2021 , wiley - vch . [SEP]
[CLS] sites in the functionalization of ulmnps toward future clinical translations . [SEP]
[CLS] self - assembly of ulmnps into ordered and well - defined nanoarchitectures with controllable sizes and shapes enable new properties and applications , which provides new opportunities to construct smart multifunctional nanostructures . [SEP]
[CLS] various nanoparticles B-nanoparticle have been used as different building blocks via self - assembly . [SEP]
[CLS] for pmnps , the localized surface plasmon resonances ( sprs ) of adjacent nanoparticles B-nanoparticle are coupled after assembly in close contact , resulting in the increase of the electric field in the gap and magnification of optical absorption , which generates various multifunctional biomedical applications ( e . g . , photothermal therapy , multimode imaging , and gene / drug delivery ) . [SEP]
[CLS] the emergence of ulmnps has overcome some of the challenges ( e . g . , instability , uncontrolled aggregation , and polydispersity ) faced in the self - assembly of pmnps . [SEP]
[CLS] with their enhanced stability and diversified surface functionalities , ulmnps offer an excellent platform for developing multifunctional nanostructures through both templateguided self - assembly ( e . g . , polymers B-material and peptides B-material ) and stimuliresponsive self - assembly ( e . g . , ph ) . [SEP]
[CLS] amphiphilic B-property block copolymers ( abcs ) can be used as powerful templates to self - assemble both small molecules ( e . g . , dox - orubicin and paclitaxel B-material ) and nanoparticles B-nanoparticle ( e . g . , aunps B-nanoparticle ) into core - shell architectures with distinct sizes and shapes . [SEP]
[CLS] the small molecules or nanoparticles B-nanoparticle were loaded inside the inner hydrophobic B-property core B-material with the hydrophilic B-property corona outer of the nanoassemblies , which was beneficial for prolonging circulation in blood for enhanced disease - targeting efficiency . [SEP]
[CLS] by taking advantage of the controllable size and shape of the abc template ( e . g . , pluronic f127 ) , a facile strategy was developed for the in situ fabrication of highly luminescent B-property cu nanoassemblies ( figure 7 ) . [SEP]
[CLS] owing to the crosslinking between the rigidity multidentate thiol B-material ligand and ultrasmall cunps ( ≈2 . 2 - 2 . 7 nm ) , the absolute quantum yield ( qy ) of the cunp assemblies was 7 . 3 % , which was higher than most of the reported ultrasmall cunps . [SEP]
[CLS] both the size of the cunp assemblies ( e . g . , ≈10 . 8 , 12 . 9 , and 18 . 8 nm ) and the dominant number ( e . g . , ≈2 , 3 , and 8 ) of encapsulated cunps in an assembly could be regulated by varying the block segments ( e . g . , f68 , f108 , and f127 ) . [SEP]
[CLS] the cunp assemblies were well covered by hydrophilic B-property peg chains on the surface , enabling the cunp assemblies with good water B-material solubility B-property , biocompatibility B-property , and high photophysical and structural stability in the harsh lysosomal acidic microenvironment ( e . g . , ph 4 . 5 - 6 ) , which holds great promise for biological applications . [SEP]
[CLS] the abc template - guided assembly for in situ fabrication of more highly controlled and stable cunp - based assemblies would stimulate more advanced downstream applications of unstable metal B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] in addition to the enhancements in stability and qy , tunable self - assembled shapes from spherical to elongated nanostructures could be achieved through growth regulation of aunps B-nanoparticle with amphiphilic B-property block copolymers ( figure 8a , b ) , pluronic f127 , copyright 2019 , american chemical society . [SEP]
[CLS] ) hd shows different interactions between proteins B-material and three kinds of nanoassemblies . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , springer nature . [SEP]
[CLS] c ) schematic illustration of in situ self - assembly process of aunps B-nanoparticle @ cp . [SEP]
[CLS] d ) colocalization analysis between aunps B-nanoparticle ( red channel ) ( aunps B-nanoparticle @ cp , left ; gs - aunps B-nanoparticle , right ) and gfp - labeled lysosomes ( green channel ) . [SEP]
[CLS] pearson ' s correlation coefficients were shown in yellow . [SEP]
[CLS] the reduced pearson ' s correlation coefficient of aunps B-nanoparticle @ cp between 1 and 6 h represents the effective lysosome escape process . [SEP]
[CLS] e ) cell B-material images after pcdna3 . 1 ( + ) - ires - gfp - p53 plasmid transfection with different vectors . [SEP]
[CLS] f ) transfection efficiencies . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , springer nature . [SEP]
[CLS] as a template [SEP]
[CLS] by tuning the reducing B-property agents I-property in the growth of luminescent B-property aunps B-nanoparticle ( ≈1 . 7 nm ) , the use of different reducing B-property agents I-property not only changed the emission spectra of the encapsulated aunps B-nanoparticle , but also adjusted the hydrophobic / hydrophilic environment of the amphiphilic B-property block copolymers to control the assembled nanostructures and the number of encapsulated aunps B-nanoparticle ( naunps ) . [SEP]
[CLS] the morphologies of au nanoassemblies ( aunas ) could be well - controlled from spherical to elongated nanostructures with different naunps ( ≈6 , 11 , and 26 ) , which showed emission wavelengths of 810 nm ( aunas - 810 ) , 610 nm ( aunas - 610 ) , and 520 nm ( aunas - 520 ) , respectively . [SEP]
[CLS] in the self - assembly of pmnps , the surface plasmon absorption of the formed nanostructures could red - shift even to the nir region depending on the interparticle distances and assembled morphology . [SEP]
[CLS] however , the emission wavelengths of the nanoassemblies are governed by the different amounts of au ( i ) species resulting from the different reducing abilities . [SEP]
[CLS] the au ( i ) species of the aunas were 40 . 81 , 36 . 53 , and 34 . 35 % for aunas - 520 , aunas - 610 , and aunas - 810 , respectively , further confirming that the valence states of the au atoms B-material affected the emission properties of the aunas , and the emission peak blue - shifted upon increasing the au ( i ) species . [SEP]
[CLS] the further evaluation of their biological behaviors showed that a spherical nanostructure with a more hydrophilic B-property surface and low values of naunps resulted in prolonged blood circulation and enhanced transport of aunps B-nanoparticle into the tumor B-material site as high as ≈25 . 3 % id g −1 , which was much higher than most of the previously reported nanoparticles B-nanoparticle , such as pegylated 2 . 5 nm aunps B-nanoparticle ( 8 % id g −1 ) and upconversion nps B-nanoparticle ( 13 . 6 % id g −1 ) . [SEP]
[CLS] in contrast , an elongated nanostructure with a more hydrophobic B-property surface and high values of naunps resulted in short blood circulation and low tumor - targeting efficiency , which were different from the abc micelles B-material for transport of small molecular drugs ( e . g . , paclitaxel B-material ) . [SEP]
[CLS] the observation that the morphologies of the aunas played a significant role in governing the in vivo transport behaviors are expected to offer essential insights into the further design of biocompatible B-property nanoassemblies as efficient nanocarriers / nanovectors for future clinical translation . [SEP]
[CLS] owing to their multifunctionality , biocompatibility B-property , and biodegradability B-property , synthetic peptides B-material are also perfect templates for the self - assembly of nanoparticles B-nanoparticle into biological systems for regenerative medicine , drug delivery , and cancer therapy . [SEP]
[CLS] the biodegradation B-property of peptide - guided assemblies is of great importance in achieving accurate and body - clearable smart multifunctional nanostructures , and further reducing several concerns regarding biosafety problems . [SEP]
[CLS] a well - designed cyclopeptide ( cp ) containing three arginines B-material and four alanines B-material in a cyclic ring and one cysteine B-material on the terminal of the branched chain , could spontaneously form controllable nanofibers B-nanoparticle , which were utilized as templates for the in situ assembly of nir - emitting aunps B-nanoparticle into well - controlled 1d nanostructures ( aunps B-nanoparticle @ cp ) ( figure 8c - f ) . [SEP]
[CLS] the self - assembled aunps B-nanoparticle @ cp exhibited a high emission peak at 810 nm with a qy of more than 5 . 9 % . [SEP]
[CLS] the aunps B-nanoparticle @ cp were also revealed to show unique capability of rapid cellular interaction , efficient lysosome escape , as well as fast cellular efflux as compared to the unassembled ones , which resulted in high gene transfection efficiencies in the construction of hela cell B-material line with ≈7 . 5 - fold overexpression of p53 protein B-material . [SEP]
[CLS] in addition , the unique 1d aunps B-nanoparticle @ cp nanostructures were demonstrated to be robust and efficient in vivo nanovectors , showing high diffusibility after intratumoral injection and could be eliminated through both renal and hepatic metabolic pathways with extremely low nonspecific accumulation in healthy organs . [SEP]
[CLS] this cp - templated in situ self - assembly strategy offers a facile and feasible pathway to generate highly luminescent B-property ulmnp - based 1d nanostructures with multifunctionalities for both optical tracking and delivery . [SEP]
[CLS] among the environmental stimuli ( e . g . , ph , redox , and hypoxia ) , the ph gradients have been widely applied to design smart responsive nanoassemblies . [SEP]
[CLS] to design phresponsive functional nanoassemblies that could change their structures and properties under the stimuli of ph gradients , the typical strategy is the construction of reversible protonation / deprotonation systems with ph - responsive moieties ( e . g . , imidazole , polyhistidine , and polyamines ) . [SEP]
[CLS] by co - coating B-material with ph - responsive zwitterionic imidazole groups , the renal - clearable 800 nm emitting pegylated aunps B-nanoparticle ( pmiz - aunps B-nanoparticle , ≈1 . 9 nm ) showed both charge - reversal ( [UNK] - potential values : −10 . 9 ± 1 . 0 mv at ph 7 . 4 ; 17 . 4 ± 1 . 6 mv at ph 5 . 5 ) and self - assembly ( hds : ≈3 . 5 nm at ph 7 . 4 ; ≈1048 . 7 nm at ph 5 . 5 ) capabilities toward acidic ph microenvironments , resulting in the enhanced ultrasound intensities at low ph values ( figure 9a - d ) . [SEP]
[CLS] the ultrasound signal of the ph - responsive renal - clearable luminescent B-property aunps B-nanoparticle was demonstrated for both noninvasive fluorescence B-property and ultrasound B-technique imaging I-technique of early kidney injury . [SEP]
[CLS] the enhancement from both ph - induced tubular reabsorption and in situ self - assembly of ultrasmall luminescent B-property aunps B-nanoparticle in tubular cells B-material of the injured kidney showed enhanced ultrasound signals for noninvasive imaging of kidney injury . [SEP]
[CLS] the discovery of tubular reabsorption of self - assembled ulmnps in acidic kidneys provided uniqueness in synergistic fluorescence B-property and ultrasound B-technique imaging I-technique for future early diagnosis and treatment of renal diseases . [SEP]
[CLS] the integration of a sensitive emission response with a highly ph - responsive charge conversion system would also enable selfassembled luminescent B-property aunps B-nanoparticle with more applications for both intracellular delivery and optical B-technique imaging I-technique . [SEP]
[CLS] using a conventional cationic B-material polymer B-material chitosan B-material ( cs ) with isoelectric point of 6 . 5 as a template , ph - responsive self - assembled luminescent B-property aunps B-nanoparticle with reversible ph - dependent swelling and compacting structures at physiological ph range ( ph 6 . 5 - 7 . 4 ) were constructed ( figure 9e - h ) . [SEP]
[CLS] the ultrasmall aunps B-nanoparticle formed compacted nanostructures ( ≈23 . 5 nm ) with qy increasing from 3 . 6 % to 9 . 4 % at low ph values ( e . g . , ph < 6 . 5 ) . [SEP]
[CLS] however , the compacted nanostructures could transform to swelled nanostructures with weak emissions reversibly at high ph values ( e . g . , ph 7 . 4 ) , with a fast and sharp emission transition from ph 7 . 5 to 6 . 5 . [SEP]
[CLS] the phresponsive self - assembly of the luminescent B-property aunps B-nanoparticle not only increased the emission properties ( > 4 - fold ) but also changed the surface charge and assembled size for enhanced cellular internalization ( ≈15 - fold ) . [SEP]
[CLS] the self - assembled aunps B-nanoparticle @ cs showed not only strong capability for endosomal escape but also a drastic fluorescence B-property response from an acidic lysosome to a neutral cytoplasm , which provided a feasible pathway for intracellular delivery and optical visualization of the intracellular tracking pathway . [SEP]
[CLS] the most reported ulmnps rely on the change in the emission intensity as a recognition signal , which would be easily interfered by target - independent factors ( e . g . , photobleaching , instrumental stability , environmental conditions , and probe concentration ) and might cause inaccurate sensing and imaging results . [SEP]
[CLS] in quantitative imaging , dual - emissive probes with ratiometric ability are more attractive than single - emissive ones because of the built - in calibration feature from the two different emissive wavelengths to avoid irrelevant influence factors ( figure 10 ) . [SEP]
[CLS] there are two common design strategies for achieving ratiometric fluorescent B-property probes based on fluorescence B-property resonance energy transfer ( fret ) , intermolecular charge transfer ( ict ) or excited - state intramolecular proton transfer ( esipt ) principles : one is to introduce a second fluorophore to a fluorescent B-property molecule / nanoparticle B-nanoparticle , and then utilize their separate responses as a signal response group / reference ; the other is to embed two different fluorescent B-property molecules / nanoparticles B-nanoparticle together [SEP]
[CLS] copyright 2021 , wiley - vch [SEP]
[CLS] e ) self - assembled aunps B-nanoparticle @ cs at different ph values [SEP]
[CLS] the inset photo shows obvious fluorescence B-property enhancement after assembly . [SEP]
[CLS] f ) schematic diagram of the endocytosis B-event and lysosome escape of aunps B-nanoparticle @ cs . [SEP]
[CLS] g ) subcellular distribution of aunps B-nanoparticle at different time points . [SEP]
[CLS] representative microscopic cellular images after incubation B-technique of aunps B-nanoparticle @ cs ( red ) and overlay with gfp - labeled lysosomes ( green ) with the colocalization analysis . [SEP]
[CLS] h ) dark - field ( df ) - stem images of cells B-material incubated B-technique with aunps B-nanoparticle @ cs ( 20 . 0 × 10 −9 m ) for 1 h ( left ) and 8 h ( right ) . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , american chemical society . [SEP]
[CLS] to form a hybrid nanostructure that enables ratiometry . [SEP]
[CLS] however , these construction strategies often involve many sophisticated coupling / chemical modification processes , and even tedious multistep synthesis / purifications . [SEP]
[CLS] an ideal ratiometric ulmnp is that a single nanoparticle B-nanoparticle can intrinsically emit dual emissions without the conjugation of an additional fluorophore . [SEP]
[CLS] therefore , the design of intrinsic dual - emissive metal B-material nanoprobes B-nanoparticle is of great importance for ratiometric imaging in complicated bioenvironments . [SEP]
[CLS] with the sharp shrinking in size , the contributions of the surface would significantly increase towards the optical and physicochemical properties of the nanoparticles B-nanoparticle . [SEP]
[CLS] for instance , with an increase in the density , rigidity , or electron - donating capability of the surface ligands , ultrasmall aunps B-nanoparticle showed a large enhancement in the emission qys . [SEP]
[CLS] through a systematic investigation of gsh - coated aunps B-nanoparticle ( gs - aunps B-nanoparticle ) emitting at 600 and 810 nm with identical sizes of ≈2 . 5 nm , it was discovered that the different emission wavelengths were independent of the size of the aunps B-nanoparticle but highly dependent on the surface coverage , with a high surface coverage resulting in strong au ( i ) - ligand charge transfer and 600 nm emission , whereas a low surface coverage led to weak charge transfer and 810 nm emission . [SEP]
[CLS] inspired by the surface coverage governed size - independent emissions , the dual - emissive gs - aunps B-nanoparticle with emissions at both 600 and 810 nm were then synthesized through strictly controlled surface coverage by fine - tuning the gsh / haucl 4 ratio in a synthetic process . [SEP]
[CLS] the integration of both 600 and 810 nm emission centers in a single aunp B-nanoparticle was then discovered to cause a synergistic effect : the dual - emissive gs - aunps B-nanoparticle were sensitive to ph changes in a ratiometric manner , which was attributed to the strong energy transfer between the two emission centers in the same ultrasmall particle ( 2 . 5 nm ) . [SEP]
[CLS] single 810 nm emissive gs - aunps B-nanoparticle showed negligible emission response towards ph changes , whereas single 600 nm emissive gs - aunps B-nanoparticle exhibited a slight increase during ph changes from 5 . 0 to 9 . 0 . [SEP]
[CLS] in addition , the mixture of single 810 nm emissive aunps B-nanoparticle and single 600 nm emissive aunps B-nanoparticle showed independent emission responses to ph changes , significantly different from the results observed from dual - emissive gs - aunps B-nanoparticle . [SEP]
[CLS] these differences demonstrate that the dual emissions of dual - emissive gs - aunps B-nanoparticle are from one single ultrasmall nanoparticle B-nanoparticle with two coupled emission centers that show strong energy transfer . [SEP]
[CLS] the above findings open the possibility for the design of ultrasmall ratiometric ph nanoindicators by controlling the surface coverage of the ulmnps . [SEP]
[CLS] the dual - emissive aunps B-nanoparticle showed unique advantages in subcellular imaging and tracking ( figure 11a - f ) . [SEP]
[CLS] to enhance the and ratiometric 3d images of living cells B-material after incubation B-technique with 09cr - aunps B-nanoparticle ( up ) and 09gs - aunps B-nanoparticle ( bottom ) . [SEP]
[CLS] e ) the percentages of statistical ratio values > 3 in ratiometric 3d images . f ) colocalization percentages of aunps B-nanoparticle and lysotracker green . [SEP]
[CLS] red channel : 810 nm . green channel : 615 nm . [SEP]
[CLS] blue channel : lysotracker green . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , american chemical society . [SEP]
[CLS] g ) a snap shot of an mps - aunp B-nanoparticle with a few ethylenediamine ( eda ) molecules in molecular dynamics ( md ) simulations . [SEP]
[CLS] sticks show mps , and yellow balls show au atoms B-material . [SEP]
[CLS] blue ( n ) , cyan ( c ) , and white ( h ) balls represent eda . [SEP]
[CLS] h ) luminescence B-property spectra of mps - aunps B-nanoparticle with different eda concentrations . i ) ratio values of mps - aunps B-nanoparticle after interaction with different amines B-material . [SEP]
[CLS] insert showed statistical ratio values of mps - aunps B-nanoparticle upon adding of different amines B-material ( 1 . 0 × 10 −3 m ) . j ) luminescent B-property and ratiometric images of glass catfish wrapped with mps - aunps B-nanoparticle under 37 °c at different storage time . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , wiley - vch . [SEP]
[CLS] cellular interaction , the dual - emissive aunps B-nanoparticle were designed by co - introduction of gsh and cr 8 ( cr - aunps B-nanoparticle ) , a cell - penetrating peptide B-material that has both a thiol B-material for binding aunps B-nanoparticle and a cellpenetrating region across the plasma membrane . [SEP]
[CLS] the cr - aunps B-nanoparticle with dual emissions at both 810 and 615 nm served as powerful fluorescent B-property probes for imaging their cellular interactions . [SEP]
[CLS] distinct from the single - emitting cr - aunps B-nanoparticle ( 810 or 615 nm ) that showed lower ph dependency , the emission intensity at 810 nm of the dual - emitting cr - aunps B-nanoparticle increase while the intensity at 615 nm decreased with extracellular ph values from 7 . 4 to 5 . 8 . [SEP]
[CLS] in addition , the ph - dependent differences in the intensity at 810 and 615 nm could be clearly displayed by the ratiometric image of the ratio value of i 615 nm / i810 nm ( r 615 / 810 nm ) , which demonstrates the great potential of ph - responsive dual - emitting cr - aunps B-nanoparticle as ratiometric nanoprobes B-nanoparticle for fluorescent B-property ph imaging of living cells B-material . [SEP]
[CLS] in addition , the ph - responsive dual emissions of the cr - aunps B-nanoparticle could be applied for ratiometric imaging to indicate the subcellular location and endocytosis B-event pathways of the nanoprobe B-nanoparticle . [SEP]
[CLS] due to the significant acidity of lysosomes ( e . g . , ph 4 . 5 - 5 . 5 ) , the high r 810 / 615 nm values ( > 3 ) in the ratiometric images could indicate the lysosome during endocytosis B-event . [SEP]
[CLS] compared with the results of gs - aunps B-nanoparticle , the lower percentage ( r 810 / 615 nm > 3 ) was attributed to the capabilities of cr - aunps B-nanoparticle in a direct cytomembrane cross without undergoing endocytosis B-event . [SEP]
[CLS] therefore , the surface - coverage - regulated dual - emissive aunps B-nanoparticle could serve as powerful self - calibration ultrasmall nanoindicators for the investigation of cellular ph values , subcellular distributions , and cellular interaction mechanisms . [SEP]
[CLS] in addition to the intrinsic emissions governed by the surface coverage that are regulated by tuning the molar ratios between surface ligands and metal B-material precursors during the synthesis , the emission of pre - formed ulmnps is also highly sensitive to surface coverage change induced through introduction of second ligand . [SEP]
[CLS] for example , after introduction of tetraoctylammonium ( toa ) cations B-material to the surface of au 22 ( sg ) 18 , the au 22 clusters showed a high qy emission ( > 60 % ) , resulting from the rigidification of the au ( i ) - thiolate shell B-material after toa binding . [SEP]
[CLS] surface coverage also establishes the interfaces between the aunp B-nanoparticle core B-material and the outer surroundings with various biomolecules , playing important roles in biomedical applications ( e . g . , targeting , sensing , and imaging ) . [SEP]
[CLS] the elucidation of stimuli - responsive surface coverage regulation from exotic targeting molecules at the ultrasmall gold B-material surface is of great importance . [SEP]
[CLS] using the typical electron - rich amines B-material as examples , it was discovered that the nucleophilic amines B-material could bind to electrophilic ultrasmall thiolate gold B-material surface , which resulted in the formation of a high - energy emission ( ≈600 nm ) from the intrinsically low - energy emitting ( ≈810 nm ) aunps B-nanoparticle ( figure 11g - j ) . [SEP]
[CLS] through systematic investigation of both the structures of the exotic amine B-material molecules and surface chemistries of the ultrasmall aunps B-nanoparticle , it was demonstrated that the electrophilic surface of the aunps B-nanoparticle provided a pathway to donate electron directly to the gold B-material surface from the nucleophilic amines B-material with low steric hindrance . [SEP]
[CLS] the formed highenergy emission as well as the decreased low - energy emission resulted from the energy transfer between the two emissions in one single ultrasmall aunp B-nanoparticle synergistically made the ultrasmall aunps B-nanoparticle robust ratiometric nanoprobes B-nanoparticle for quantitative assessment of the amine molecule interactions . [SEP]
[CLS] the induced highenergy emission showed an average lifetime of 0 . 66 μs under excitation at 405 nm , further indicating the 600 nm emission resulted from the amine B-material interaction was involved with lmct . [SEP]
[CLS] such stimuli - responsive emission transformed from single - emissive aunps B-nanoparticle to dual - emissive ones , providing a platform to develop stimuli - responsive ratiometric nanoprobes B-nanoparticle based on ulmnps for quantitative bioimaging and sensing in complicated biological systems . [SEP]
[CLS] in addition to the biomolecules , metal B-material ions B-material can also stimulate the creations of new emissive centers on the surface of ultrasmall luminescent B-property aunps B-nanoparticle with low surface coverage . [SEP]
[CLS] under the stimulus of trace amounts of ag ( i ) ions B-material , dual - emissive bimetallic ag @ gs - aunps B-nanoparticle with emissions at both 705 and 810 nm were formed from a single 810 nm emitting gs - aunp B-nanoparticle . [SEP]
[CLS] the ag ( i ) ions B-material could interact with the au ( 0 ) core B-material of the 810 nm emitting gs - aunps B-nanoparticle , resulting in both an antigalvanic reaction and formation of ag ( i ) - carboxylate B-material shell B-material on the surface of aunps B-nanoparticle to generate a new 705 nm emission peak in the preformed 810 nm emitting gs - aunps B-nanoparticle . [SEP]
[CLS] the dual - emissive ag @ gs - aunps B-nanoparticle with both 705 and 810 nm emissions showed unique linearly ratiometric ph - dependent emissions . [SEP]
[CLS] the carboxyl B-material groups B-nanoparticle of the surface ligands were important in the formation of dual - emissive ag @ gs - aunps B-nanoparticle , and it could be adapted to other thiolate surface ligands with carboxyl B-material groups I-material besides gsh , which demonstrates the generality of this strategy in the formation of dual - emissive ulmnps toward ratiometric measurements . [SEP]
[CLS] phototoxicity is a crucial consideration for live fluorescence B-technique imaging I-technique , which is mainly caused by excited fluorescent B-property probes that can generate reactive oxygen B-material species , heat , and dna damage . [SEP]
[CLS] the ulmnps typically show fluorescence B-property lifetimes within the microsecond time scale with triplet excited states involved in emission , resulting in the generation of singlet oxygen B-material ( 1 o 2 ) due to the enhanced probability of energy transfer between triplet electrons from the probe and adjacent 3 o 2 . [SEP]
[CLS] in disease diagnosis , the probe should generate low levels of 1 o 2 to reduce the side effects of phototoxicity , but disease therapy requires high efficiency in 1 o 2 generation . [SEP]
[CLS] these contradictory requirements on the level of 1 o 2 generation from ulmnps naturally raise a fundamental question of how to bidirectionally regulate the 1 o 2 generation ability based on the different application requirements . [SEP]
[CLS] because many factors influence the phototoxicity ( e . g . , excitation wavelength , intensity , exposure time , surface chemistry of the probe , probe concentration , cell B-material type , and age ) , we focus on the discussion on concentration and their subcellular distribution as well as the related strategies for bidirectional regulation of phototoxicity , based on the unique properties of ulmnps . [SEP]
[CLS] the capability to control the subcellular distribution of ulmnps not only provides unique advantages for the enhanced targeting , but also offers valid ways to fundamentally understand the cellular interaction mechanism and phototoxicity . [SEP]
[CLS] the concentration of nanomedicine significantly affects the theragnostic efficiency and systematic toxicity B-property . [SEP]
[CLS] various nanomedicines showed concentration - dependent biological behaviors , which resulted in different diagnostic effects and systematic toxicity B-property at both in vitro and in vivo levels . [SEP]
[CLS] to unveil the concentrationdependent subcellular distribution of luminescent B-property aunps B-nanoparticle , the nir - emitting aunps B-nanoparticle co - coated with gsh and a cell - penetrating peptide B-material cr 8 ( cr - aunps B-nanoparticle ) served as versatile nanomedicines to indicate their interactions with living cells B-material ( figure 12 ) . [SEP]
[CLS] cr - aunps B-nanoparticle showed strong cellular membrane - binding capacities in high concentration regions ( e . g . , 500 × 10 −9 m ) , whereas higher ) . [SEP]
[CLS] e ) light illumination and fbs medium ( li : + / fbs : + ) . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , wiley - vch . [SEP]
[CLS] subcellular distribution and phototoxicitymitochondria - targeting abilities and more relative endocytosis B-event efficiencies were observed in the low concentration regions ( e . g . , 1 × 10 −9 m ) . [SEP]
[CLS] at high concentrations of cr - aunps B-nanoparticle , more cr - aunps B-nanoparticle were confined to the membrane , rather than endocytosis B-event into the cell B-material . [SEP]
[CLS] the modification of a cell - penetrating peptide B-material with a local positive charge would help the cr - aunps B-nanoparticle accumulate around the mitochondria at low concentrations . [SEP]
[CLS] however , the exact mechanism for concentration dependent accumulation in mitochondria is still unclear . [SEP]
[CLS] it would be feasible to achieve concentrationdependent cell B-material membrane labeling and subcellular targeting with well - designed ulmnps . [SEP]
[CLS] furthermore , this concentrationdependent subcellular distribution resulted in photocytotoxicity for tumor B-material cells B-material in a peculiar low - concentration region due to the mitochondria - targeting generation of 1 o 2 . [SEP]
[CLS] these results not only demonstrate the unique concentration - dependent biological behaviors of the ulmnps , but also expand the understanding of ulmnps toward future theranostics and biotoxicity . [SEP]
[CLS] for organic photosensitizers B-property , the efficiency of 1 o 2 generation can be controlled by tuning the charge . [SEP]
[CLS] the increase of positively charged binding sites of organic photosensitizers B-property favors 3 o 2 adsorption , which leads to the rapid and efficient formation of 1 o 2 . [SEP]
[CLS] this rule could also be applied to ulmnps . [SEP]
[CLS] it was discovered that ultrasmall pegylated aunps B-nanoparticle with high surface charge showed higher 1 o 2 production capability than those of aunps B-nanoparticle with low surface charge ( figure 13 ) . [SEP]
[CLS] general strategies for the controllable 1 o 2 production of ultrasmall aunps B-nanoparticle through facile surface charge regulation by thiolated ligand exchange ( e . g . , positively charged cysteamine ; negatively charged thioglycolic acid ) or deprotonation / protonation ( e . g . , ph regulation ) have been realized . [SEP]
[CLS] interestingly , the surface charge of the aunps B-nanoparticle would affect the subcellular distributions : the introduction of negative charge would enhance the distribution of the nps B-nanoparticle in lysosomes , but the increase in positive charge would drive the nps B-nanoparticle to be significantly distributed in mitochondria . [SEP]
[CLS] the cytotoxicity B-property of the 1 o 2 generation was also demonstrated to be highly controllable through surface charge manipulation resulting from the synergistic effects of 1 o 2 generation capability and subcellular distribution . [SEP]
[CLS] therefore , the bidirectional regulation strategy of 1 o 2 generation from ultrasmall aunps B-nanoparticle has significant implications for the deep understanding of the photocytotoxicity of nanomedicines , as well as the future design of nanosized metal B-material nanomedicines for specific disease diagnosis and treatment . [SEP]
[CLS] in summary , we briefly summarize our recent progress in the surface engineering of ulmnps for biomedical applications , such as biological targeting and imaging . [SEP]
[CLS] our research attempts reproduced with permission . [SEP]
[CLS] copyright 2020 , wiley - vch . [SEP]
[CLS] to take advantage of the unique and fascinating surface features of ulmnps to improve their targeting capabilities , multifunctionalities , and quantitative imaging stabilities , as well as bidirectionally regulate the toxicities B-property based on the different application requirements . [SEP]
[CLS] with the help of surface functionalization strategies , the ulmnps could be designed to avoid long - term nonspecific accumulation in mps organs ( e . g . , liver and spleen ) with efficient renal clearance , which not only reduces possible background interference signals in advanced imaging applications ( e . g . , metastatic cancer in mps organs ) , but also minimize potential long - term toxicity B-property and systemic side effects , a prerequisite for future clinical translation . [SEP]
[CLS] with both template - guided and stimuli - responsive self - assembly strategies , the ulmnps have been shown to be excellent platforms for developing multifunctional nanostructures in multimodal imaging ( e . g . , fluorescence B-property and ultrasound ) and nanomedicine ( e . g . , nanovectors ) . [SEP]
[CLS] with the discovery of surface coverage - regulated dual emissions from ulmnps , ulmnps could be easily designed as ratiometric nanoprobes B-nanoparticle without additional fluorophores for quantitative bioimaging and sensing in more complicated biological systems . [SEP]
[CLS] the observation of unique concentration - dependent subcellular distribution biological behaviors of the ulmnps and the developed bidirectional regulation strategies of phototoxicity would further expand our understanding of ulmnps toward future theranostics and biotoxicity . [SEP]
[CLS] despite substantial progress made from the worldwide research groups , research on ulmnps in biomedical application is still in its early stages , and many key issues and challenges remain unsolved . [SEP]
[CLS] for example , the fundamental understanding of the correlation between the physicochemical properties of ulm - nps B-nanoparticle and related biological behaviors is still unclear . [SEP]
[CLS] because the biological behaviors of ulmnps ( e . g . , cellular interaction , organ distribution , and excretion pathways ) are much more sensitive to their surface chemistry ( e . g . , surface coverage , charge , and hydrophobicity B-property ) than those of the pmnps , high - quality ulm - nps B-nanoparticle with atomically precise and highly controlled surface functionalization are required to address this issue . [SEP]
[CLS] the luminescent B-property mechanism of ulmnps should then be investigated in detail and systematically . [SEP]
[CLS] because the surface chemistry plays a significant role in governing their optical properties , a thorough investigation of the optical mechanism would sustainably develop ulmnps with much more fascinating features ( e . g . , tunable optical properties and multifunctionities ) for advanced bioapplications . [SEP]
[CLS] in addition , methodologies for integration with other irradiations without the tissue penetration depth limit should be further developed . [SEP]
[CLS] future surface engineering strategies ( e . g . , self - assembly and metal B-material ion B-material doping ) are also needed to endow the ulmnps with more theranostic functions , such as mri , ct imaging , and radiotherapy . [SEP]
[CLS] furthermore , in addition to the cancer and renal diseases , enormous efforts should be made to explore the application of ulmnps in other serious diseases , such as immunological , cardiovascular , and neurological diseases . [SEP]
[CLS] finally , the exact underlying mechanisms of physiological interactions of ulmnps , from cells B-material to various organs in different animal models , should be further investigated . [SEP]
[CLS] with continued development and interdisciplinary cooperation , we believe that ulmnps will find clinical applications in addressing many key issues related to healthcare . [SEP]
[CLS] 1 . surface engineering strategies of ultrasmall luminescent B-property metal B-nanoparticle nanoparticles I-nanoparticle ( ulmnps ) for biological targeting and imaging . [SEP]
[CLS] 2 . surface functionalization challenges and principles of ulmnps . [SEP]
[CLS] 3 . surface functionalization of dna molecules . [SEP]
[CLS] a ) schematic diagram of phosphorothioate ( ps ) - modified dna ( psdna ) . [SEP]
[CLS] b ) effect of ps length on the core B-material size control of psdna - aunps B-nanoparticle ( from ≈1 . 3 to 2 . 6 nm with ps length increase ) . [SEP]
[CLS] c ) the uv - vis and fluorescence B-property spectra of psdna - aunps B-nanoparticle . [SEP]
[CLS] d ) schematic diagram of the protein B-material tyrosine B-material kinase 7 ( ptk - 7 ) proteins B-event binding I-event with the fabricated apt - aunps B-nanoparticle . [SEP]
[CLS] e ) fluorescent B-property imaging of apt - aunps B-nanoparticle after incubation B-technique with living cells B-material . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , american chemical society . [SEP]
[CLS] glycoconjugation . [SEP]
[CLS] a ) schematic diagram of the in situ synthetic process of 810 nm emitting gold B-material glyconanoparticles ( augnps ) . [SEP]
[CLS] b ) the core B-material - size distributions ( 2 . 4 ± 0 . 4 nm ) and hd ( 4 . 2 ± 0 . 8 nm ) of the augnps . [SEP]
[CLS] c ) high performance liquid B-technique chromatography I-technique ( hplc ) chromatograms demonstrated that augnps showed negligible serum protein B-material absorptions ( incubation B-technique conditions : 10 % ( v / v ) fetal bovine serum ( fbs ) incubation B-technique for 30 min ) . [SEP]
[CLS] d ) fluorescent B-property imaging after intravenous ( iv ) injection of augnps into mda - mb - 231 tumor bearing nude mice . [SEP]
[CLS] the arrows point to the tumor B-material area . [SEP]
[CLS] e ) comparison of tumor targeting efficiency between augnps and control group gs - aunps B-nanoparticle at different post - injection ( p . i . ) time points . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , royal society of chemistry . [SEP]
[CLS] 5 . surface functionalization of controllable surface charges and hds . [SEP]
[CLS] a ) schematic diagram of the 800 nm emitting aunps B-nanoparticle with the similar core B-material size but different hds and charges . [SEP]
[CLS] na , not available . [SEP]
[CLS] b ) precise regulation of [UNK] - potential values and hds of the aunps B-nanoparticle . c ) total clearance efficiency ( ce ) calculated from both urine and feces , and the ratio values between urine and feces ( r urine / feces ) of the aunps B-nanoparticle within 168 h p . i . d ) relationships among the biodistributions in liver and tumor B-material ( at 6 h p . i . ) , hds and [UNK] - potential values of aunps B-nanoparticle . [SEP]
[CLS] e ) in vivo fluorescent B-property images of the mouse iv injection with different aunps B-nanoparticle at different times points of p . i . ( left ) and ex vivo liver tissue fluorescent B-property images of the liver - metastasis B-event ( at 6 h p . i . ) . [SEP]
[CLS] the circle and arrow indicate the liver region and the tumor B-material location , respectively . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , american chemical society . [SEP]
[CLS] 6 . effects of the weak anchor sites . [SEP]
[CLS] a ) flow diagram of in vivo behaviors of the aunps B-nanoparticle with different anchoring sites . [SEP]
[CLS] b ) 3d - fluorescent B-property images of the cells B-material incubated B-technique with 11b - aunps B-nanoparticle a ) , 11a - aunps B-nanoparticle b ) , 07b - aunps B-nanoparticle c ) , and 07a - aunps B-nanoparticle d ) for 3 h . scale bar , 10 μm . [SEP]
[CLS] c ) in vivo fluorescent B-property images of tumor - bearing mice at different p . i . time points . [SEP]
[CLS] the arrow represents the location of tumor B-material , and the circle indicates the area of bladder . [SEP]
[CLS] the 11b - aunps B-nanoparticle and 07b - aunps B-nanoparticle with more anchoring sites of - cooh were synthesized at ph 10 . 0 with molar ratio of gsh to haucl 4 ( r gsh / haucl4 ) at 1 . 1 and 0 . 7 , respectively . [SEP]
[CLS] the 11a - aunps B-nanoparticle and 07a - aunps B-nanoparticle with more anchoring sites of - nh 2 were synthesized at the corresponding conditions except at ph 2 . 0 . reproduced with permission . [SEP]
[CLS] copyright 2021 , wiley - vch [SEP]
[CLS] template - guided cu nanoassemblies . [SEP]
[CLS] a ) schematic illustration of the controllable encapsulated cunp number in one cu nanoassembly using different block copolymers as templates . [SEP]
[CLS] b ) high - angle annular dark field ( haadf ) - scanning B-technique transmission I-technique electron I-technique microscopy I-technique ( stem ) images ( up ) , and the analysis of encapsulated quantity and size of the cunp assemblies . [SEP]
[CLS] c ) excellent stability of the crosslinked cunp assemblies at low temperature a ) and with dilutions b ) . [SEP]
[CLS] d ) overlay of bright and fluorescence B-property field , and 3d images of the cunp assemblies at different cellular incubation B-technique time points a ) . b ) colocalization analysis between green fluorescent B-property protein B-material ( gfp ) - labeled lysosomes and the cunp assemblies . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , american chemical society . [SEP]
[CLS] template - guided au nanoassemblies . [SEP]
[CLS] a ) controllable shape and loading of the aunas with different reducing B-property agents I-property . [SEP]
[CLS] b ) hd shows different interactions between proteins B-material and three kinds of nanoassemblies . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , springer nature . [SEP]
[CLS] c ) schematic illustration of in situ self - assembly process of aunps B-nanoparticle @ cp . [SEP]
[CLS] d ) colocalization analysis between aunps B-nanoparticle ( red channel ) ( aunps B-nanoparticle @ cp , left ; gs - aunps B-nanoparticle , right ) and gfp - labeled lysosomes ( green channel ) . [SEP]
[CLS] pearson ' s correlation coefficients were shown in yellow . [SEP]
[CLS] the reduced pearson ' s correlation coefficient of aunps B-nanoparticle @ cp between 1 and 6 h represents the effective lysosome escape process . [SEP]
[CLS] e ) cell B-material images after pcdna3 . 1 ( + ) - ires - gfp - p53 plasmid transfection with different vectors . [SEP]
[CLS] f ) transfection efficiencies . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , springer nature . [SEP]
[CLS] stimuli - responsive functional nanoassemblies . [SEP]
[CLS] a ) scheme of ph - induced aggregation and ultrasound contrast . [SEP]
[CLS] b ) ultrasound B-technique imaging I-technique of pmiz - aunps B-nanoparticle at different ph values . [SEP]
[CLS] ultrasound B-technique imaging I-technique c ) and fluorescence B-technique imaging I-technique d ) of kidney injury with pmiz - aunps B-nanoparticle . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2021 , wiley - vch . [SEP]
[CLS] e ) self - assembled aunps B-nanoparticle @ cs at different ph values . [SEP]
[CLS] the inset photo shows obvious fluorescence B-property enhancement after assembly . [SEP]
[CLS] f ) schematic diagram of the endocytosis B-event and lysosome escape of aunps B-nanoparticle @ cs . g ) subcellular distribution of aunps B-nanoparticle at different time points . [SEP]
[CLS] representative microscopic cellular images after incubation B-technique of aunps B-nanoparticle @ cs ( red ) and overlay with gfp - labeled lysosomes ( green ) with the colocalization analysis . [SEP]
[CLS] h ) dark - field ( df ) - stem images of cells B-material incubated B-technique with aunps B-nanoparticle @ cs ( 20 . 0 × 10 −9 m ) for 1 h ( left ) and 8 h ( right ) . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , american chemical society . [SEP]
[CLS] 10 . features of dual - emissive ulmnps . [SEP]
[CLS] a ) comparison of dual - emissive ulmnps and single - emission ulmnps . [SEP]
[CLS] dual - emissive ulmnps can convert fluorescence B-property intensity signals that are susceptible to environmental interferences ( e . g . , environmental conditions , instrumental stability , and probe concentration ) into more stable and reliable ratio signals . [SEP]
[CLS] b ) comparison of dual - emissive ulmnps and traditional dual - emissive materials . [SEP]
[CLS] the construction strategy of dual - emissive ulmnps is more convenient than that of the traditional dual - emissive materials . [SEP]
[CLS] 11 . [SEP]
[CLS] ratiometric ulmnps . [SEP]
[CLS] a ) cr - aunps B-nanoparticle with different surface coverage and emissions . [SEP]
[CLS] the cr - aunps B-nanoparticle shows dual - emission at the ratio of ligand to au of 0 . 9 . [SEP]
[CLS] b ) the ph - dependent fluorescent B-property spectra of dual - emitting 09cr - aunps B-nanoparticle at different ph values . [SEP]
[CLS] the fluorescence B-property between 615 and 810 nm shows different ph responses . [SEP]
[CLS] c ) fluorescent B-property images at different emission channels of living cells B-material after treatment of 09cr - aunps B-nanoparticle . [SEP]
[CLS] d ) fluorescentand B-property ratiometric 3d images of living cells B-material after incubation B-technique with 09cr - aunps B-nanoparticle ( up ) and 09gs - aunps B-nanoparticle ( bottom ) . [SEP]
[CLS] e ) the percentages of statistical ratio values > 3 in ratiometric 3d images . f ) colocalization percentages of aunps B-nanoparticle and lysotracker green . [SEP]
[CLS] red channel : 810 nm . [SEP]
[CLS] green channel : 615 nm . [SEP]
[CLS] blue channel : lysotracker green . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , american chemical society . [SEP]
[CLS] g ) a snap shot of an mps - aunp B-nanoparticle with a few ethylenediamine ( eda ) molecules in molecular dynamics ( md ) simulations . [SEP]
[CLS] sticks show mps , and yellow balls show au atoms B-material . [SEP]
[CLS] blue ( n ) , cyan ( c ) , and white ( h ) balls represent eda . [SEP]
[CLS] h ) luminescence B-property spectra of mps - aunps B-nanoparticle with different eda concentrations . i ) ratio values of mps - aunps B-nanoparticle after interaction with different amines B-material . [SEP]
[CLS] insert showed statistical ratio values of mps - aunps B-nanoparticle upon adding of different amines B-material ( 1 . 0 × 10 −3 m ) . j ) luminescent B-property and ratiometric images of glass catfish wrapped with mps - aunps B-nanoparticle under 37 °c at different storage time . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , wiley - vch [SEP]
[CLS] 12 . concentration - dependent subcellular distribution of ulmnps and the related phototoxicity . [SEP]
[CLS] a ) schematic diagram of concentrationdependent subcellular distribution and 1 o 2 production of ulmnps . [SEP]
[CLS] b ) cellular imaging of aunps B-nanoparticle at different concentrations ( 100 × 10 −9 and 1 × 10 −9 m ) and their colocalization analysis with mitored . [SEP]
[CLS] scale bar : 10 μm . [SEP]
[CLS] c ) transmission B-technique electron I-technique microscopy I-technique ( tem ) , stem images enlarged from yellow frame and edx through white arrow of cytomembrane and mitochondria of cells B-material incubated B-technique with 05cr - aunps B-nanoparticle of 100 × 10 −9 and 1 × 10 −9 m . d ) generated rate of 1 o 2 from cr - aunps B-nanoparticle at different concentrations . [SEP]
[CLS] cell B-material viability B-property of I-property hela I-property cells I-property after incubation B-technique with cr - aunps B-nanoparticle ( 0 . 001 × 10 −9 - 1000 × 10 −9 m ) : light illumination and fbs - free medium ( li : + / fbs : − ) . [SEP]
[CLS] e ) light illumination and fbs medium ( li : + / fbs : + ) . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , wiley - vch . [SEP]
[CLS] 13 . bidirectional regulation of phototoxicity . [SEP]
[CLS] a ) the relationship between [UNK] - potential values and the 1 o 2 generation rate ( abda degradation kinetics ) of aunps B-nanoparticle after bidirectional regulation by negatively charged thioglycolic acid ( tga ) or positively charged cysteamine ( ca ) at different ligand to gold B-material ratios . [SEP]
[CLS] b ) cell B-material viabilities of hela cells B-material treated with aunps B-nanoparticle after regulation with tga and ca using ligand exchange strategies at different ligand to au ratios under light irradiation . [SEP]
[CLS] fluorescent B-property images ( left ) and colocalization analysis ( right ) of aunps B-nanoparticle ( red channel ) and lyso - tracker green c ) or mito - tracker green d ) ( green channel ) in hela cells B-material . [SEP]
[CLS] scale bar : 10 μm . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , wiley - vch . [SEP]
[CLS] the binding B-event of I-event plasma I-event proteins I-event to nanomedicines is widely considered detrimental to their delivery to tumors B-material . [SEP]
[CLS] here , the design of oxpt / sn38 nanoparticle B-nanoparticle containing a hydrophilic B-property oxaliplatin B-material ( oxpt ) prodrug in a coordination polymer B-material core B-material and a hydrophobic B-property cholesterol - conjugated sn38 prodrug on the lipid B-material shell B-material for active tumor B-material targeting is reported . [SEP]
[CLS] oxpt / sn38 hitchhikes on low - density lipoprotein ( ldl ) particles , concentrates in tumors B-material via ldl receptor - I-event mediated endocytosis B-event , and selectively releases sn38 and oxpt in acidic , esterase - rich , and reducing tumor B-material microenvironments , leading to 6 . 0 - and 4 . 9 - times higher accumulations in tumors B-material over free drugs . [SEP]
[CLS] by simultaneously crosslinking B-event dna I-event and inhibiting topoisomerase i , oxpt / sn38 achieved 92 - 98 % tumor B-material growth inhibition in five colorectal cancer tumor B-material models and prolonged mouse survival by 58 - 80 days compared to free drug controls in three human colorectal cancer tumor B-material models without causing serious side effects . [SEP]
[CLS] the study has uncovered a novel nanomedicine strategy to co - deliver combination chemotherapies to tumors B-material via active targeting of the ldl receptor B-material . [SEP]
[CLS] nanomedicines have been actively pursued to enhance tumor B-material control and reduce treatment side effects by improving pharmacokinetics and tumor B-material deposition of drug payloads . [SEP]
[CLS] the conventional approach modifies the surfaces of 20 - 200 nm doi : 10 . 1002 / advs . 202201614 nanoparticles B-nanoparticle with hydrophilic B-property molecules such as polyethylene glycol ( peg ) to reduce plasma protein B-event binding I-event , thus minimizing mononuclear phagocytic system ( mps ) uptake after systemic injection . [SEP]
[CLS] such longcirculating nanoparticles B-nanoparticle are believed to accumulate in tumors B-material via the enhanced permeability and retention ( epr ) effect that arises from a leaky vasculature and reduced lymphatic drainage in tumors B-material . [SEP]
[CLS] however , the epr hypothesis has been recently challenged , in part due to a low success rate of nanomedicines in the clinical setting . [SEP]
[CLS] chan and coworkers first demonstrated plasma protein B-material coronas I-material on gold B-nanoparticle nanoparticles I-nanoparticle and liposomes B-nanoparticle , and established the transport of gold B-nanoparticle nanoparticles I-nanoparticle to the tumor B-material via an active uptake process through endothelial cells B-material in tumors B-material but not via the epr effect . [SEP]
[CLS] these findings have raised questions on whether and how nanoparticles B-nanoparticle alter pharmacokinetics to enhance tumor B-material deposition of drug payloads and call for new strategies to overcome the limitations of current nanomedicine platforms . [SEP]
[CLS] as key transporters of hydrophobic B-property molecules in plasma , lipoproteins are known to interact with certain non - pegylated nanoparticles B-nanoparticle . [SEP]
[CLS] lipoproteins are categorized into chylomicrons , very low - density lipoproteins ( vldl ) , low - density lipoproteins ( ldl ) , and high - density lipoproteins ( hdl ) based on their flotation densities and electrophoretic B-property mobilities I-property . [SEP]
[CLS] among these lipoproteins , ldl plays a dominant role in the transfer of cholesterol and cholesteryl esters from liver tissues to peripheral cells B-material by ldl receptor B-material ( ldlr ) - mediated endocytosis B-event . [SEP]
[CLS] in many tumor B-material cells B-material , ldlr is highly expressed and has enhanced activity . [SEP]
[CLS] thus , ldl - mimicking nanoparticles B-nanoparticle have been developed to deliver various chemotherapies to tumors B-material , but this approach has only been moderately effective . [SEP]
[CLS] here we report the design of tumor - responsive nanoscale coordination polymer B-material ( ncp ) core - shell nanoparticles B-nanoparticle for the enhanced delivery of oxaliplatin B-material ( oxpt ) and sn38 , an active metabolite of irinotecan , to tumors B-material by targeting ldlr . [SEP]
[CLS] chemotherapy regimens containing multiple drugs , including folfox ( folinic acid , fluorouracil , and oxaliplatin B-material ) , folfiri ( folinic acid , fluorouracil , and irinotecan ) , and irox ( irinotecan and oxaliplatin B-material ) , are the backbone in the treatment of metastatic colorectal cancer ( mcrc ) which has a dismal 5 - year survival rate of 12 % . [SEP]
[CLS] however , these treatments have narrow therapeutic windows with severe side effects . [SEP]
[CLS] for example , 30 % of patients treated with the irox regimen experienced severe neutropenia and 18 % of patients had severe neuropathy , likely due to their non - selective distribution to bone marrow and peripheral nerves , respectively . [SEP]
[CLS] in this work , we designed an oxpt / sn38 nanoparticle B-nanoparticle with the ncp core B-material built from a hydrophilic B-property prodrug of oxpt and a lipid B-material bilayer I-material shell B-material containing a hydrophobic B-property prodrug of sn38 . [SEP]
[CLS] the cholesterol - sn38 conjugate with a cleavable acetal linker ( chol - sn38 ) binds to ldl and allows oxpt / sn38 to hijack the cholesterol transport function of ldl to actively deliver both prodrugs to tumors B-material via ldlr - mediated endocytosis B-event . [SEP]
[CLS] in tumors B-material , chol - sn38 was selectively activated to release sn38 via both acid - and esterase - catalyzed hydrolysis whereas the core B-material of oxpt / sn38 preferentially released oxpt via disintegration in acidic endo / lysosomes followed by reduction by ascorbate and other intracellular reductants . [SEP]
[CLS] prolonged circulation and active transport of tumor - responsive oxpt / sn38 significantly increased tumor B-material deposition of both oxpt and sn38 , with 4 . 9 - and 6 . 0 - times higher tumor B-material areas under curves ( aucs ) compared to free drugs . [SEP]
[CLS] as a result , oxpt / sn38 showed strong antitumor efficacy in both murine and human subcutaneous tumor B-material models without causing serious side effects such as neutropenia , hepatotoxicity , and nephrotoxicity . [SEP]
[CLS] we conjugated the potent topoisomerase i inhibitor sn38 to cholesterol via an acid - sensitive and enzymatically cleavable acetal linker to form chol - sn38 ( figures s1 - s4 , supporting information ) . [SEP]
[CLS] we also introduced an acid - sensitive trimethylsilyl ( tms ) group to chol - sn38 at the o - 20 position of sn38 to disrupt the strong [UNK] - [UNK] stacking of planar sn38 moieties and facilitate the formation of a stable lipid B-material coating B-material . [SEP]
[CLS] oxpt / sn38 was prepared in two steps ( figure 1a ) . [SEP]
[CLS] first , pt ( dach ) ( oxalate ) ( bisphosphoramidic acid ) ( oxpt - bp ) was copolymerized with zn 2 + ions B-material in the presence of dopa in a reverse microemulsion containing triton x - 100 / hexanol / cyclohexane to form dopa - capped ncp particles ( oxpt - bare ) . [SEP]
[CLS] chol - sn38 was then incorporated into the lipid B-material layer together with cholesterol , dopc , and dspe - peg 2000 on the surface of oxpt - bare to form core - shell oxpt / sn38 particles . [SEP]
[CLS] oxpt / sn38 particles with an oxpt : sn38 molar ratio of 1 : 1 . 8 were used for all studies in this work . [SEP]
[CLS] oxpt - bare particles were characterized as spherical by transmission B-technique electron I-technique microscopy I-technique ( tem , figure 1b ) with a z - average diameter of 49 . 0 nm and a polydispersity index ( pdi ) of 0 . 17 by dynamic B-technique light I-technique scattering I-technique ( dls , figure 1c and table s1 , supporting information ) . [SEP]
[CLS] oxpt - bare showed high stability in tetrahydrofuran at room temperature ( figure 1d ) . [SEP]
[CLS] oxpt / sn38 particles showed spherical morphology ( figure 1e ) with a z - average diameter of 111 . 6 nm and pdi of 0 . 15 ( figure 1f and table s1 , supporting information ) . [SEP]
[CLS] oxpt / sn38 particles were stable in pbs containing 5 mg ml −1 bovine serum albumin ( bsa ) with no changes in size and pdi at 37 °c ( figure 1g ) . [SEP]
[CLS] monotherapy particles with oxpt - bp in the core B-material , oxpt ncp , and with chol - sn38 on the shell B-material , znp / sn38 , were prepared in the absence of chol - sn38 or with pyrophosphate replacing oxpt prodrug , respectively . [SEP]
[CLS] other control particles used in the present study were similarly prepared and characterized ( figures s5 and s6 and table s1 , supporting information ) . [SEP]
[CLS] ldl binds and transports cholesterol and cholesteryl esters to peripheral cells B-material . [SEP]
[CLS] chol - sn38 is strongly bound to ldl with an association constant ( k a ) of 4 . 97 × 10 5 m −1 by isothermal titration calorimetry ( itc , figure 2a ) . [SEP]
[CLS] titration of ldl with oxpt / sn38 led to exothermic binding with a k a of 3 . 34 × 10 4 m −1 . [SEP]
[CLS] both chol - sn38 and oxpt / sn38 showed a 1000 times lower affinity to albumin ( figure 2b , figure s7 , and table s2 , supporting information ) . [SEP]
[CLS] we further determined the binding of two control particles , oxpt ncp without cholesterol and chol - sn38 micelle B-material ( without the ncp core B-material ) , to ldl . [SEP]
[CLS] while chol - sn38 micelle B-material showed similar binding affinity to ldl as oxpt / sn38 , oxpt ncp without cholesterol showed 10 times lower binding affinity to ldl ( figure 2c , figure s7 , and table s2 , supporting information ) . [SEP]
[CLS] these results suggest that cholesterol and chol - sn38 mediate the binding of ncp to ldl . [SEP]
[CLS] molecular dynamics ( md ) simulations were next performed to elucidate atomic - level interactions between ldl and chol - sn38 ( figure s8 and table s3 , supporting information ) . [SEP]
[CLS] we computed the potential of the mean force for transferring chol - sn38 from bulk water B-material to the lipid B-material core B-material of ldl . [SEP]
[CLS] the potential of mean force for chol - sn38 significantly decreased at almost every location in ldl and decreased the most by ≈80 kj mol −1 at the interface of the hydrophobic B-property core B-material and hydrophilic B-property shell B-material of 1 - palmitoyl - 2 - oleoyl - snglycero - 3 - phosphocholine ( popc ) and 1palmitoyl - 2 - hydroxy - sn - glycero - 3 - phosphocholine ( lyso - pc ) ( figure 2d ) . [SEP]
[CLS] in contrast , the potential mean force for sn38 did not decrease until approaching the center of the hydrophobic B-property core B-material of ldl . [SEP]
[CLS] these results confirm attractive hydrophobic B-property / hydrophobic B-property interactions I-property between chol - sn38 and the ldl core B-material . [SEP]
[CLS] we then determined the binding kinetics of chol - sn38 , oxpt / sn38 , and sn38 to ldl in rat plasma by lc - ms . [SEP]
[CLS] while chol - sn38 quickly bound to ldl in the plasma and reached equilibrium with 74 . 5 % chol - sn38 binding to ldl within 1 h , oxpt / sn38 showed slower transfer of chol - sn38 to ldl but reached a similar level in 3 h ( figure 2e ) . [SEP]
[CLS] in contrast , sn38 showed only 17 . 1 % binding to ldl . [SEP]
[CLS] the distributions of sn38 , chol - sn38 , and oxpt / sn38 in various lipoproteins in rat plasmas were quantified by lc - ms . [SEP]
[CLS] the lipoproteins were separated based on their densities by nabr gradient ultracentrifugation . [SEP]
[CLS] sn38 was mainly distributed in albumin ( 68 . 8 % ) and only slightly distributed in ldl ( 16 . 8 % ) , while chol - sn38 was mostly distributed to ldl ( 85 . 8 % ) with < 1 % distribution in albumin ( figure 2g and table s4 , supporting information ) . [SEP]
[CLS] interestingly , chol - sn38 in oxpt / sn38 was efficiently transferred to lipoproteins , with 74 . 1 % in ldl , 19 . 4 % in vldl , and 6 . 1 % in hdl but < 1 % in albumin . [SEP]
[CLS] in vivo pharmacokinetics of oxpt / sn38 on sd / cd rats showed long blood circulation of chol - sn38 with a half - life ( t 1 / 2 ) of 9 . 7 h and an area under the curve ( auc 0→t ) of 1874 . 6 μg h ml −1 ( table s5 , supporting information ) . [SEP]
[CLS] we also assayed the distribution of chol - sn38 in different plasma proteins B-material at each time point and found an auc 0→t of 871 . 6 μg h ml −1 for ldlbound chol - sn38 , which represented 46 . 5 % of the total chol - sn38 auc 0→t ( figure 2h ) . [SEP]
[CLS] taken together , these studies show strong binding of chol - sn38 to ldl and the transfer of chol - sn38 from oxpt / sn38 to ldl in plasma . [SEP]
[CLS] apo b - 100 protein B-material in ldl is a strong ligand for ldlr and is responsible for the efficient transfer of cholesterol to peripheral cells B-material via ldlr - mediated endocytosis B-event . [SEP]
[CLS] we determined the adsorption of apo b - 100 to znp control ncp by bca assay . [SEP]
[CLS] as znp concentration increased , the amount of adsorbed apo b - 100 increased ( figure 2f ) , with 80 . 7 % apo b - 100 captured by 10 mg znp particles . [SEP]
[CLS] the apo b - 100 binding capacity decreased to 26 . 4 % for znp particles without cholesterol . [SEP]
[CLS] we mixed znp ncp with mouse plasma and then analyzed the adsorption of apo b - 100 on znp particles by western blot . [SEP]
[CLS] we found that apo b - 100 was adsorbed on znp particles , and the level of apo b - 100 adsorption correlated with znp concentration ( figure s9 , supporting information ) . [SEP]
[CLS] the uptake of ncp particles by tumor B-material cells B-material via ldlr - mediated endocytosis B-event was confirmed using fluorescently B-property labeled ldl ( dil - ldl ) and cholesterol - pyropheophytin a ( chol - pyro ) - loaded ncp as a surrogate for chol - sn38 in the shell B-material and chlorin B-material e6 ( ce6 ) loaded ncp as a surrogate for oxpt in the core B-material ( figure s10 , supporting information ) . [SEP]
[CLS] the uptake levels of both chol - pyro ncp and ce6 ncp by murine crc mc38 cells B-material decreased with ldlr blockade by an anti - ldlr antibody B-material ( [UNK] - ldlr ) in a doseproportional manner ( figure 3a , b ) . [SEP]
[CLS] with ldlr blockade , dil - ldl uptake by mc38 cells B-material decreased by a similar percentage as chol - pyro ncp and ce6 ncp . [SEP]
[CLS] compared to wildtype ( wt ) mc38 cells B-material , ldlr knockout ( ko ) mc38 cells B-material showed much lower uptake of chol - pyro ncp ( 5 % ) and ce6 ncp ( 15 % ) ( figure 3c and figure s11 , supporting information ) . [SEP]
[CLS] these results suggest that ldlr - mediated endocytosis B-event plays a major role in the cellular uptake of ncps . [SEP]
[CLS] we also visualized co - localization of chol - pyro ncp and lyso - tracker and determined cellular uptake levels of chol - pyro by confocal B-technique laser I-technique scanning I-technique microscopy I-technique ( clsm , figure 3d , e and figure s12 , supporting information ) . [SEP]
[CLS] after 24 h incubation B-technique , cholpyro ( red ) colocalized with endo / lysosomes with a pearson ' s r value of 0 . 91 ( figure 3f ) . [SEP]
[CLS] to investigate drug accumulation in tumors B-material by ldlrmediated endocytosis B-event , we determined chol - pyro fluorescence B-property signals in tumor B-material slices from mc38 - bearing c57bl / 6 mice 24 or 48 h after intravenous injection of 200 μg of chol - pyro ncp with and without intratumoral injection of 1 μg [UNK] - ldlr . [SEP]
[CLS] the administration of [UNK] - ldlr decreased chol - pyro signals by 52 . 9 % and 60 . 2 % at 24 and 48 h time points , respectively ( figure 3g and figure s13 , supporting information ) . [SEP]
[CLS] the tumors B-material were also digested into single cell suspensions for flow cytometric analysis of intracellular chol - pyro signals as a function of ldlr blockade . [SEP]
[CLS] flow cytometric results showed that chol - pyro levels decreased by 67 . 7 % and 69 . 0 % in tumor B-material cells B-material with ldlr blockade at 24 and 48 h time points , respectively ( figure 3h , i ) . [SEP]
[CLS] we next quantitatively determined drug accumulation in mc38 tumors B-material following intravenous injection 3 . 5 mg kg −1 oxpt / sn38 ( based on oxpt equivalents ) with and without concurrent intratumoral injection of 1 μg [UNK] - ldlr ( figure 3j , k and table s6 , supporting information ) . [SEP]
[CLS] without ldlr blockade , oxpt / sn38 exhibited a pt auc 0→t of 290 . 3 h μg ml −1 and an sn38 auc 0→t of 50 . 8 h μg ml −1 in the tumors B-material , which are 4 . 9 times that of free oxpt and 6 times that of free irinotecan at equivalent oxpt and sn38 doses , respectively . [SEP]
[CLS] with ldlr blockade , the oxpt auc 0→t decreased by 72 % and the sn38 auc 0→t decreased by 90 % , lowering the drug accumulation levels to those of free oxpt and irinotecan , respectively . [SEP]
[CLS] while oxpt / sn38 maintained intratumoral drug concentrations above ic 50 values for ct26 and mc38 murine crc cells B-material and for ht29 , hct116 , and sw480 human crc cells B-material for 72 h ( tables s7 and s8 , supporting information ) , oxpt / sn38 with ldlr blockade , like the free drugs , failed to maintain intratumoral drug concentrations above ic 50 values beyond 24 h . [SEP]
[CLS] these results demonstrate that oxpt / sn38 significantly increases intratumoral oxpt and sn38 concentrations by targeting the ldlr through binding and transferring chol - sn38 to ldl in vivo . [SEP]
[CLS] we next examined the ph - triggered release of sn38 from chol - sn38 in endo / lysosomes . [SEP]
[CLS] at ph = 4 . 7 , chol - sn38 from oxpt / sn38 was hydrolyzed at both the 20 - o - tms and carbonate B-material linkages B-property to release sn38 in 95 % yield in 72 h ( figure 4a , b , d , and figure s14 , supporting information ) . [SEP]
[CLS] in contrast , a negligible amount of sn38 was released from oxpt / sn38 at ph = 7 . 4 in 72 h . [SEP]
[CLS] as carboxylesterase in tumor B-material cells B-material contributes to the release of sn38 from irinotecan , we tested the release of sn38 from oxpt / sn38 in pbs with 10 unit ml −1 esterase . [SEP]
[CLS] chol - sn38 was gradually hydrolyzed to sn38 - tms ( figure 4c ) , which was further converted to sn38 . [SEP]
[CLS] these results indicate a dual activation mechanism for the release of sn38 from oxpt / sn38 in tumors B-material : the 20 - otms and carbonate B-material linkages B-property of chol - sn38 are successively hydrolyzed at low ph to yield sn38 and the carbonate B-material and sn38 - tms linkages B-property are hydrolyzed by the esterase and via proton / tms exchange , respectively , to generate sn38 . [SEP]
[CLS] the tumoractivated release of sn38 could potentially minimize normal tissue exposure to sn38 . [SEP]
[CLS] the ncp core B-material formed by zn 2 + ions B-material and oxpt - bp is known to disintegrate in acidic environments . [SEP]
[CLS] at ph = 7 . 4 , oxpt / sn38 released less than 6 % pt over a course of 48 h . [SEP]
[CLS] at ph = 4 . 7 , oxpt / sn38 particles quickly disintegrated to release pt ( dach ) ( oxalate ) ( biscarbamate ) ( oxpt - bc ) ( figure 4e , g ) . [SEP]
[CLS] in the presence of 5 mm ascorbate , the released oxpt - bc was efficiently reduced to form oxpt ( figure 4e , f ) . [SEP]
[CLS] these results demonstrate the triggered release of both sn38 and oxpt from oxpt / sn38 in cancer cells B-material . [SEP]
[CLS] in vitro cytotoxicity B-property of oxpt / sn38 and monotherapy control particles was tested in crc cells B-material ( tables s7 and s8 , supporting information ) . [SEP]
[CLS] oxpt / sn38 showed strong synergy of the two drugs in ct26 and mc38 cells B-material , lowering oxpt ic 50 values by three - to four - folds compared to oxpt ncp and halving chol - sn38 ic 50 values compared to znp / sn38 . [SEP]
[CLS] oxpt / sn38 showed similar synergistic cytotoxicity B-property in ht29 , hct116 , and sw480 cells B-material , and was more cytotoxic B-property than either oxpt or irinotecan . [SEP]
[CLS] interestingly , although sn38 - tms showed potent cytotoxicity B-property with ic 50 values of 2 . 52 and 3 . 04 μm for ct26 and mc38 cells B-material , respectively , this cytotoxicity B-property likely came from sn38 generated from hydrolysis of sn38 - tms , since analogous sn38 derivates 20 - o - tert - butyl - sn38 ( sn38 - t bu ) and 20 - o - boc - sn38 ( sn38 - boc ) showed no cytotoxicity B-property in ct26 and mc38 at concentrations up to 300 μm ( figures s15 and s16 , table s7 , supporting information ) . [SEP]
[CLS] alexa fluor 488 - annexin v and pi staining showed both oxpt and sn38 induced programmed B-event cell I-event death I-event by apoptosis B-event / necrosis . [SEP]
[CLS] the combination of oxpt and sn38 increased the percentages of both early apoptotic annexin v + / pi − cells B-material and late apoptotic / necrotic annexin v + / pi + cells B-material ( figure 5a and figure s17 , supporting information ) . [SEP]
[CLS] mc38 cells B-material treated with oxpt ncp and znp / sn38 showed 32 . 6 % and 53 . 9 % s - phase arrest , respectively ( figure 5b , d and figure s18 , supporting information ) . [SEP]
[CLS] in comparison , 25 . 3 % cells B-material were in s - phase for pbs control . [SEP]
[CLS] oxpt / sn38 showed a stronger s - phase arrest of 63 . 8 % . [SEP]
[CLS] oxpt / sn38 particles thus effectively caused dna damage and inhibited dna B-event replication I-event for antiproliferative effects . [SEP]
[CLS] we next evaluated mitochondrial disruption caused by oxpt / sn38 through flow B-technique cytometry I-technique analysis of jc - 1 staining and clsm imaging of cytochrome c release . [SEP]
[CLS] treatment of mc38 cells B-material with oxpt / sn38 for 24 h resulted in a fivefold increase in the depolarization of the mitochondrial membrane B-property potential I-property ( mmp ) compared to pbs control ( figure 5c , e and figure s19 , supporting information ) and the release of cytochrome c from mitochondria with a pearson ' s r value of 0 . 27 between mitotracker ( red fluorescence B-property ) and fitc - conjugated anti - cytochrome c antibody B-material [SEP]
[CLS] the in vivo anticancer B-property efficacy of oxpt / sn38 was evaluated in five subcutaneous tumor B-material models . [SEP]
[CLS] when the tumors B-material reached 80 - 120 mm 3 in volume , mice were intravenously injected with the indicated treatments every three days ( q3d ) . [SEP]
[CLS] oxpt / sn38 , oxpt plus irinotecan , oxpt ncp , and znp / sn38 were dosed at 3 . 5 mg kg −1 oxpt equivalent and 15 . 9 mg kg −1 chol - sn38 ( equivalent to 6 . 2 mg kg −1 sn38 ) , 3 . 5 mg kg −1 oxpt and 20 . 2 mg kg −1 irinotecan ( 11 . 7 mg kg −1 sn38 equivalent ) , 3 . 5 mg kg −1 oxpt equivalent , and 15 . 9 mg kg −1 chol - sn38 ( equivalent to 6 . 2 mg kg −1 sn38 ) , respectively ( figure 5g , h , jl and figures s22 , s23 , and s25 , supporting information ) . [SEP]
[CLS] in all tested models , oxpt / sn38 led to significantly greater tumor B-material growth curves of g ) mc38 tumors B-material in c57bl / 6 mice and h ) ct26 tumors B-material in balb / c mice . [SEP]
[CLS] pbs , oxpt plus irinotecan , oxpt ncp , znp / sn38 , or oxpt / sn38 was i . v . injected once every 3 days ( q3d ) at doses of 3 . 5 mg kg −1 oxpt or equivalent , 20 . 2 mg kg −1 irinotecan ( 11 . 7 mg kg −1 sn38 equivalent ) , and / or 15 . 9 mg chol - sn38 / kg ( 6 . 2 mg kg −1 sn38 equivalent ) to mc38 model for up to 8 doses and to ct26 model for up to 6 doses , n = 6 . i ) survival curves of ht29 model and j ) tumor B-material growth curves of ht29 , k ) hct116 , and l ) sw480 models in nude mice after q3d treatment with pbs , oxpt / sn38 , or oxpt plus for up to 16 doses . [SEP]
[CLS] the doses are the same as mc38 and ct26 models . [SEP]
[CLS] data are expressed as means ± sd . [SEP]
[CLS] the data were analyzed by one - way anova with tukey ' s multiple comparison test . [SEP]
[CLS] inhibition / regression of tumor B-material growth with minimal toxicity B-property as judged by comparable body weight gain , normal histology of major organs including heart , liver , spleen , lung and kidney , and normal liver and renal function tests ( figure s27 , supporting information ) . [SEP]
[CLS] mc38 tumor - bearing c57bl / 6 mice following 8 intravenous injections of oxpt / sn38 showed 92 . 2 % tumor B-material growth inhibition ( tgi ) . [SEP]
[CLS] despite a 1 . 9 - fold higher sn38 equivalent dose , oxpt plus irinotecan provided a modest tgi of 22 . 3 % . [SEP]
[CLS] oxpt ncp and znp / sn38 only slightly inhibited tumor B-material growth with tgi values of 66 . 9 % and 16 . 4 % , respectively . [SEP]
[CLS] the mice tolerated the treatments well with stable body weights for all groups ( figure s24a , supporting information ) . [SEP]
[CLS] neutropenia is the most serious side effect of the irox regimen with 30 % mcrc patients experiencing severe neutropenia after repeated doses of oxpt plus irinotecan . [SEP]
[CLS] blood samples were collected from mc38 tumor - bearing c57bl / 6 mice following 8 intravenous injections of pbs or oxpt / sn38 , or oxpt plus irinotecan . [SEP]
[CLS] the absolute neutrophil count ( anc ) significantly decreased in mice treated with oxpt plus irinotecan but slightly increased for mice treated with oxpt / sn38 compared to pbs control ( figure s28a , supporting information ) . [SEP]
[CLS] thus , oxpt / sn38 treatment prevented the dose - limiting toxicity B-property of severe neutropenia in the irox regimen . [SEP]
[CLS] we also determined liver and kidney functions by measuring the levels of alanine B-material aminotransferase ( alt ) , aspartate aminotransferase ( ast ) , and serum creatinine levels in the sera of c57bl / 6 mice after 8 and 15 doses of pbs or oxpt / sn38 ( figure s28b - d , supporting information ) . [SEP]
[CLS] the ast and alt levels for mice treated with oxpt / sn38 slightly increased over pbs control but remained within the normal ranges . [SEP]
[CLS] the creatinine level remained unchanged in mice treated with oxpt / sn38 ( 0 . 24 ± 0 . 07mg dl −1 ) compared to those treated with pbs ( 0 . 22 ± 0 . 01 mg dl −1 ) . [SEP]
[CLS] these results indicate that repeated doses of oxpt / sn38 did not cause hepatotoxicity and nephrotoxicity in mice . [SEP]
[CLS] in the ct26 model , oxpt / sn38 , oxpt plus irinotecan , oxpt ncp , and znp / sn38 showed tgi of 90 . 9 % , 27 . 6 % , 51 . 6 % , and 29 . 1 % , respectively . [SEP]
[CLS] thus oxpt / sn38 showed a strong synergy of the two drugs with enhanced anticancer B-property efficacy compared to the other treatment groups . [SEP]
[CLS] the treatments were well tolerated with no differences in body weights among the groups . [SEP]
[CLS] oxpt / sn38 showed excellent anticancer B-property efficacy in mouse tumor B-material xenograft models of human colorectal adenocarcinomas . [SEP]
[CLS] tumor - bearing mice were intravenously injected with pbs , oxpt / sn38 , or oxpt plus irinotecan for 16 doses . [SEP]
[CLS] for ht29 model , oxpt / sn38 regressed the tumors B-material showing a tgi of 99 . 4 % at the pbs endpoint . [SEP]
[CLS] oxpt plus irinotecan slightly inhibited the tumors B-material to provide a tgi of 32 . 2 % . [SEP]
[CLS] after the cessation of oxpt / sn38 treatment on day 46 , growth inhibition of ht29 tumors B-material persisted for another 12 days but tumors B-material eventually resumed growth after day 57 . [SEP]
[CLS] oxpt / sn38 and oxpt plus irinotecan treatments extended the median survival from 33 days for pbstreated control group to 94 and 36 days , respectively ( figure 5i and figure s26 , supporting information ) . [SEP]
[CLS] znp / sn38 at a dose of 36 mg kg −1 chol - sn38 also effectively inhibited ht29 tumor B-material growth with a tgi of 87 . 0 % ( figure s29 , supporting information ) . [SEP]
[CLS] the mice in all groups tolerated the treatments well . [SEP]
[CLS] oxpt / sn38 also showed potent antitumor efficacy against hct116 and sw480 tumor B-material xenografts . [SEP]
[CLS] for hct116 model , oxpt / sn38 regressed tumors B-material to afford a tgi of 97 . 6 % at the pbs endpoint on day 21 , while oxpt plus irinotecan showed a tgi of 75 . 8 % . [SEP]
[CLS] oxpt / sn38 and oxpt plus irinotecan extended mouse survival from 20 days for pbs group to 106 and 41 days , respectively . [SEP]
[CLS] for sw480 model , oxpt / sn38 regressed tumors B-material to yield a tgi of 96 . 9 % at the pbs endpoint on day 17 while oxpt plus irinotecan gave a moderate tgi of 57 . 9 % . [SEP]
[CLS] oxpt / sn38 and oxpt plus irinotecan extended mouse survival from 20 days for pbs group to 112 and 32 days , respectively . [SEP]
[CLS] with unique pharmacokinetics and pharmacodynamics , excellent antitumor efficacy in multi - ple crc tumor B-material models , and good safety profiles , oxpt / sn38 is primed for clinical testing on crc patients . [SEP]
[CLS] to evaluate the role of ldlr - mediated endocytosis B-event on anticancer B-property efficacy , we determined tgi of intravenously injected oxpt / sn38 with concurrent ldlr blockade by intratumorally injecting 1 μg [UNK] - ldlr on a q3d schedule ( figure 6a , and figure s30a , supporting information ) . [SEP]
[CLS] while [UNK] - ldlr slowed tumor B-material growth with a tgi of 40 . 2 % over the immunoglobin B-material g ( igg ) control , concurrent ldlr blockade significantly weakened the anti - tumor efficacy of oxpt / sn38 treatment with a tgi of 51 . 6 % compared to a tgi of 91 . 2 % for oxpt / sn38 treatment with control igg injection . [SEP]
[CLS] the results were corroborated by the tumor B-material weights at the endpoint : oxpt / sn38 treatment with concurrent igg injection showed a tgi of 92 % while oxpt / sn38 treatment with concurrent ldlr blockade showed a tgi of 57 % ( figure s30b , c , supporting information ) . [SEP]
[CLS] we confirmed the role of ldlr in oxpt / sn38 anti - tumor B-material efficacy using ldlr ko mc38 tumor B-material cells B-material . [SEP]
[CLS] the anticancer B-property efficacy of oxpt / sn38 was nearly abrogated in ldlr ko mc38 tumors B-material with no significant difference between oxpt / sn38 and pbs groups ( figure 6b and figures s31 and s32 , supporting information ) . [SEP]
[CLS] we further examined in vivo cytotoxicity B-property in tumor B-material cells B-material through histopathological analysis ( figure 6c ) . [SEP]
[CLS] h & e staining showed severe necrosis in mc38 tumors B-material treated with oxpt / sn38 but much less necrosis in mc38 tumors B-material treated with oxpt / sn38 and [UNK] - ldlr . [SEP]
[CLS] tunel and caspase 3 ihc staining showed strong apoptosis B-event induced by oxpt / sn38 , but greatly reduced apoptosis B-event when ldlr was blocked . [SEP]
[CLS] these results show that ldlrmediated endocytosis B-event is key to the tumor B-material uptake of oxpt / sn38 in vivo and plays a crucial role in antitumor efficacy . [SEP]
[CLS] for decades , cytotoxic B-property anticancer B-property drugs such as sn38 ( logp = 3 . 37 ) have been modified with hydrophilic B-property groups to render them more soluble B-property in an aqueous solution . [SEP]
[CLS] conversion of water - insoluble sn38 into water - soluble B-property irinotecan hydrochloride ( logp = −0 . 45 ) represents one of the most successful examples of anticancer B-property drug design . [SEP]
[CLS] binding of hydrophobic B-property chemotherapeutics to plasma proteins B-material presents an alternative approach and can actively target highly expressed receptors . [SEP]
[CLS] for example , nab - paclitaxel B-material ( albumin - paclitaxel nanoparticles B-nanoparticle ) showed better efficacy than paclitaxel B-material in some tumors B-material , presumably via targeting the gp60 transcytosis pathway in endothelial cells B-material and binding B-event to secreted I-event protein I-event , acidic and rich in cysteine B-material ( sparc ) in the tumor B-material extracellular matrix . [SEP]
[CLS] in this work , we uncovered a novel strategy to co - deliver combination chemotherapies via active targeting of ldlr in tumors B-material . [SEP]
[CLS] we conjugated sn38 to highly hydrophobic B-property cholesterol ( logp = 7 . 02 ) through a labile acetal linkage B-property to hijack ldl for tumor B-material targeting . [SEP]
[CLS] systemically injected nanotherapeutics have long been shown to prolong blood circulation over their parent drugs , which are believed to operate through the epr effect . [SEP]
[CLS] core - shell ncp particles allowed the loading of both hydrophilic B-property oxpt - bp and hydrophobic B-property chol - sn38 . [SEP]
[CLS] we discovered strong binding of chol - sn38 to ldl and transfer of chol - sn38 from oxpt / sn38 to ldl for active transport to tumors B-material via ldlr - mediated endocytosis B-event . [SEP]
[CLS] sn38 was selectively released inside tumor B-material cells B-material via acid - and esterase - catalyzed hydrolysis . [SEP]
[CLS] on the other hand , the ncp core B-material of oxpt / sn38 adsorbed ldl for tumor B-material targeting via the ldlr pathway and released oxpt in tumors B-material via disintegration in acidic endo / lysosomes and reduction by ascorbate and other intracellular reductants . [SEP]
[CLS] as a result , oxpt / sn38 significantly increased tumor B-material deposition of oxpt by a factor of 4 . 9 over oxpt and sn38 by a factor of 6 . 0 over irinotecan at equivalent doses . [SEP]
[CLS] irox is one of the standard chemotherapy regimens for mcrc due to the synergistic action of oxpt and sn38 on crc cells B-material . [SEP]
[CLS] by increasing intratumoral oxpt and sn38 concentrations , oxpt / sn38 maximized the synergy between oxpt and sn38 on murine and human crc cells B-material . [SEP]
[CLS] oxpt / sn38 simultaneously crosslinked dna with oxpt and inhibited topoisomerase i with sn38 , resulting in severe dna damage , inhibition B-event of I-event dna B-event replication I-event , and disruption of mitochondrial membranes . [SEP]
[CLS] oxpt / sn38 achieved > 92 % tgi of mc38 and ct26 tumor B-material models and > 97 % tgi of ht29 , hct116 , and sw480 tumor B-material models . [SEP]
[CLS] oxpt / sn38 also prolonged mouse survival by 61 - 92 days compared to pbs control and by 58 - 80 days compared to oxpt plus irinotecan in these tumor B-material models . [SEP]
[CLS] in summary , we developed a novel strategy to actively target tumors B-material via ldlr - mediated endocytosis B-event with core - shell ncps for enhanced drug accumulation in tumors B-material . [SEP]
[CLS] with tumor - responsive release of sn38 and oxpt , oxpt / sn38 exhibited 6 . 0 - and 4 . 9times higher tumor B-material aucs over free drugs and achieved 92 - 98 % tgi in five crc tumor B-material models and significantly prolonged mouse survival over oxpt plus irinotecan without causing neutropenia , hepatotoxicity or nephrotoxicity . [SEP]
[CLS] our study uncovers a novel nanomedicine strategy to co - deliver combination chemotherapies by actively targeting ldlr in tumors B-material and presents a promising clinical strategy for the treatment of mcrc . [SEP]
[CLS] materials , cell B-material lines , and animals : all starting materials were purchased from sigma - aldrich and fisher ( usa ) , unless otherwise noted , and used without further purification . [SEP]
[CLS] 1 , 2 - dioleoyl - snglycero - 3phosphate ( dopa ) , 1 , 2 - dioleyl - sn - glycero - 3 - phosphocholine ( dopc ) , cholesterol , and 1 , 2 - distearoyl - sn - glycero - 3 - phosphoethanolamine - n - [ amino ( polyethylene glycol ) 2000 ] ( dspe - peg 2k ) were purchased from avanti polar lipids B-material ( usa ) . [SEP]
[CLS] murine mammary carcinoma cells B-material mc38 and ct26 were purchased from american type culture collection ( rockville , md , usa ) and cultured in dulbecco ' s modified eagle ' s medium ( dmem ) and rpmi - 1640 medium ( gibco , grand island , ny , usa ) , respectively , supplemented with 10 % fetal bovine serum , 100 u ml −1 penicillin g sodium B-material , and 100 g ml −1 streptomycin sulfate in a humidified atmosphere containing 5 % co 2 at 37 °c . [SEP]
[CLS] human colon cancer cells B-material ht29 , hct116 , and sw480 were purchased from american type culture collection ( rockville , md , usa ) and cultured in mccoy ' s 5a ( for ht29 ) or dmem ( for hct116 and sw480 ) , supplemented with 10 % fetal bovine serum , 100 u ml −1 penicillin g sodium B-material , and 100 g ml −1 streptomycin sulfate in a humidified atmosphere containing 5 % co 2 at 37 °c . [SEP]
[CLS] balb / c female mice ( 6 weeks , 18 - 22 g ) , c57bl / 6 female mice ( 6 weeks , 18 - 22g ) , athymic female nude mice ( 6 weeks , 18−22 g ) , and sd / cd female rats ( 6 weeks , 160 - 200 g ) were provided by harlan laboratories , inc ( usa ) . [SEP]
[CLS] the study protocols ( # 72 334 and # 72 408 ) were reviewed and approved by the institutional animal care and use committee ( iacuc ) at the university of chicago ( phs assurance # d16 - 00322 ( a3523 - 01 ) ) . [SEP]
[CLS] preparation and characterization of oxpt / sn38 : oxpt - bare was synthesized according to the previously reported method with minor modifications . [SEP]
[CLS] briefly , an aqueous solution of oxpt - bp ( 30 mg , 150 mg ml −1 ) was added to a 5 ml of 0 . 3m triton x - 100 / 1 . 5m 1 - hexanol in cyclohexane and stirred vigorously for 15 min in the presence of dopa ( 30 mg , 200 mg ml −1 in chcl 3 ) . [SEP]
[CLS] an aqueous solution of zn ( no 3 ) 2 ( 60 mg , 600 mg ml −1 ) was added to a 5 ml of 0 . 3m triton x - 100 / 1 . 5m 1 - hexanol in cyclohexane and stirred vigorously for 5 min . [SEP]
[CLS] the zn ( no 3 ) 2 - containing microemulsion was added dropwise to the pt - containing microemulsion and stirred vigorously for 30 min at room temperature . [SEP]
[CLS] after the addition of 10 ml ethanol , oxpt - bare was obtained by centrifugation at 11 628 × g . [SEP]
[CLS] the resulting pellet was washed twice with thf / ethanol and finally redispersed in thf . [SEP]
[CLS] oxpt loadings in the particles were determined by icp - ms ( agilent 7700 × , agilent technologies , usa ) after digestion with nitric acid . [SEP]
[CLS] oxpt / sn38 was prepared by adding a thf solution ( 80 μl ) of dopc , cholesterol , dspe - peg 2k , chol - sn38 ( 3 : 3 : 1 . 5 : 1 ) , and oxpt - bare to 500 μl of 30 % ( v / v ) ethanol / water B-material at room temperature . [SEP]
[CLS] the mixture was stirred at 1700 rpm for 1 min . [SEP]
[CLS] thf and ethanol were completely evaporated and the solution was allowed to cool down to room temperature . [SEP]
[CLS] the particle size and zeta B-property potential I-property were determined by dls using a zetasizer ( nano zs , malvern , uk ) . [SEP]
[CLS] tem ( tecnai spirit , fei , usa ) was used to determine the morphology . [SEP]
[CLS] oxpt / sn38 was mixed with saturated nacl solution and 1 % triton x - 100 solution , followed by ethyl acetate extraction . [SEP]
[CLS] the mixture was vortexed and centrifuged at 10 000 × g for 5 min , the supernatant was analyzed by lc - ms ( agilent 6540 , agilent technologies , usa ) to determine chol - sn38 loading . [SEP]
[CLS] preparation and characterization of control ncp particles : znp bare or oxpt - bare particles were synthesized according to the previously reported method with minor modifications . [SEP]
[CLS] briefly , an aqueous solution of oxpt - bp or sodium B-material pyrophosphate ( 30 mg , 150 mg ml −1 ) was added to a 5 ml of 0 . 3m triton x - 100 / 1 . 5m 1 - hexanol in cyclohexane and stirred vigorously for 15 min in the presence of dopa ( 30 mg , 200 mg ml −1 in chcl 3 ) . [SEP]
[CLS] an aqueous solution of zn ( no 3 ) 2 ( 60 mg , 600 mg ml −1 ) was added to a 5 ml of 0 . 3m triton x - 100 / 1 . 5m 1 - hexanol in cyclohexane and stirred vigorously for 5 min . [SEP]
[CLS] the zn ( no 3 ) 2 - containing microemulsion was added dropwise to the pt - containing microemulsion and stirred vigorously for 30 min at room temperature . [SEP]
[CLS] after the addition of 10 ml ethanol , bare particles were obtained by centrifugation at 11 628 × g . [SEP]
[CLS] the resulting pellet was washed twice with thf / ethanol and finally re - dispersed in thf . [SEP]
[CLS] ncps were prepared by adding a thf solution ( 80 μl ) of dopc , cholesterol , dspe - peg 2k , chol - drugs ( chol - sn38 or chol - pyropheophytin a ) ( 3 : 3 : 1 . 5 : 1 ) , and bare particles to 500 μl of 30 % ( v / v ) ethanol / water B-material at room temperature . [SEP]
[CLS] the mixture was stirred at 1700 rpm for 1 min . [SEP]
[CLS] thf and ethanol were completely evaporated and the solution was allowed to cool down to room temperature . [SEP]
[CLS] the particle size and zeta B-property potential I-property were determined by dls using a zetasizer ( nano zs , malvern , uk ) . [SEP]
[CLS] tem ( tecnai spirit , fei , usa ) was used to observe the morphology . [SEP]
[CLS] stability of chol - sn38 on oxpt / sn38 particles : 2 ml of oxpt / sn38 particles with a chol - sn38 concentration of 20 ppm was incubated B-technique in pbs at 37 °c . [SEP]
[CLS] at 0 , 1 , 3 , 5 , 8 , 24 , 48 , and 72h , 20 μl aliquot from the solution was harvested , mixed with 100 μl of 0 . 5 % triton x - 100 saturated nacl solution and 100 μl of ethyl acetate , vortexed for 1 min , and then centrifuged for 5 min at 14 000 rpm . [SEP]
[CLS] the organic fraction was collected and analyzed by lc - ms . [SEP]
[CLS] isothermal titration calorimetry analysis : itc experiments were performed with an itc200 calorimeter ( microcal malvern ) at 37 °c . [SEP]
[CLS] the lipoprotein or albumin concentration ( in pbs ) in the microcalorimeter cell B-material and the concentrations of chol - sn38 ( pbs with 3 % dmso ) or each of the nanoparticles B-nanoparticle ( based on chol - sn38 or corresponding components in pbs ) in the syringe were adjusted to 15 and 200 μm , respectively . [SEP]
[CLS] a first injection of 0 . 4 μl was followed by 20 injections of 2 μl at intervals of 150 s . [SEP]
[CLS] the data were analyzed according to the one binding - site model using the microcal origin software provided by the manufacturer ( originlab corporation , northampton , ma 01060 ) . [SEP]
[CLS] in silico md simulations : md simulations were carried out according to previously published procedures . a slice of spherical ldl particle with 10 % of its full volume was used to reduce the system size and make it computationally tractable on the atomic level . [SEP]
[CLS] the lipid B-material composition in table s2 , supporting information , corresponds to typical human ldl particles and the first coarse - grained ldl model . [SEP]
[CLS] the system was simulated with periodic boundary conditions as in the simulation set up of lipid B-material bilayer I-material . [SEP]
[CLS] apob protein B-material was not included in the simulation because the hydrophobic B-property core B-material provides the main interactions between ldl and chol - sn38 . [SEP]
[CLS] the lipid B-material force field was used as it was considered one of the best all - atom force fields for lipid B-material systems . [SEP]
[CLS] for md simulations , the ldl - like slice was designed as follows . [SEP]
[CLS] topologies for lyso pc , cholesterol oleate , and glycerol oleate were acquired from automated topology builder ( atb ) and repository and modified manually by combining pieces of popc and cholesterol . [SEP]
[CLS] initial topology of sn38 and chol - sn38 were generated by acpype topology generator . [SEP]
[CLS] the structure of sn38 was optimized by gaussian09 at the b3lyp / 6 - 31 + + g ( d ) level of theory and esp partial charges were added to the initial topology . [SEP]
[CLS] the cholesterol tail of chol - sn38 was adjusted to match the lipid B-material force field . [SEP]
[CLS] first , the hydrophobic B-property core B-material of a random mixture of cholesterol , cholesterol oleate , and glycerol trioleate was generated by packmol and lyso pc and popc were added to each side of the core B-material . [SEP]
[CLS] the system was then solvated on both sides and equilibrated for 200 ns with a time step of 2 fs . [SEP]
[CLS] gromacs 5 . 1 . 4 package was used to implement all md simulations . [SEP]
[CLS] tip3p water B-material model was used . [SEP]
[CLS] all simulations were performed in npt conditions with a constant pressure of 1 bar and temperature of 320 k maintained by v - rescale thermostat and berendsen barostat , respectively . [SEP]
[CLS] potentials of mean force ( pmfs ) of transporting sn38 and chol - sn38 to the center of ldl slice were computed by umbrella sampling simulations . [SEP]
[CLS] the center masses of sn38 and chol - sn38 were restrained by harmonic potential characterized by a 2000 kj mol −1 nm −2 force constant at different distances from the center of the slice . [SEP]
[CLS] nine sampling windows were selected with a step interval of 1 nm along z axis . [SEP]
[CLS] each window was sampled for 200 ns and the last 100 ns were used for statistical analysis . [SEP]
[CLS] pmfs were obtained by the weighted histogram technique in the gromacs package . [SEP]
[CLS] lipoprotein binding studies : 20 ppm of sn38 , chol - sn38 , and oxpt / sn38 ( based on chol - sn38 ) were prepared in fresh rat plasma . [SEP]
[CLS] after incubation B-technique at 37 °c for 0 . 5 , 1 , 3 , or 5 h , 20 ul of plasma was pipetted and added to 20 ul of 2 × ldl precipitation buffer ( biovision . incorporated ) . [SEP]
[CLS] 10 min later , the mixture was centrifuged at 5000 rpm for another 10 min . [SEP]
[CLS] the supernatant was analyzed by lc - ms to determine the drug concentrations remaining in the solution ( unbound to ldl ) . [SEP]
[CLS] apob - 100 binding studies : 100 μl of 0 . 5 mg ml −1 apob - 100 was added to 50 μl of 0 , 25 , 50 , 100 , or 200 mg ml −1 of znp ncp or znp ncp without cholesterol and incubated B-technique at 37 °c for 3h . [SEP]
[CLS] unbound apo b - 100 was precipitated by 0 . 01 m acetic acid and immediately centrifuged at 4000 rpm for 5 min . [SEP]
[CLS] during this process , ncp particles remained in the solution . [SEP]
[CLS] the amount of apob - 100 in the supernatant was quantified by bca assay . [SEP]
[CLS] znp ncp was used here to avoid interference with the bca absorption signals at 562 nm . [SEP]
[CLS] 5 μl of ncp was mixed with 95 μl mouse plasma for 3 h . [SEP]
[CLS] 50 μl of the plasma solutions were added with 0 . 01 m acetic acid for precipitating the major unbound proteins B-material . [SEP]
[CLS] the samples were centrifuged at 4000 rpm for 5 min , and then the supernatant was analyzed by western blot . [SEP]
[CLS] drug distributions in rat plasma : the distribution of drugs in different plasma fractions was studied by ultracentrifugation of lipoproteins on the basis of their hydrated densities as previously described by cassidy et al . in brief , 300 μl rat plasma was slowly and gently added to the top of 150 μl of 1 . 006 g ml −1 nabr solution , and then centrifuged in an optima max - xp tabletop ultracentrifuge ( beckman instruments , inc . ) with a tla120 . 1 fixed angle rotor ( beckman instruments , inc . ) at 420 000 g at 4 °c for 1 . 5 h . [SEP]
[CLS] after the top layer of 150 μl vldl fraction was removed , 150 μl of 1 . 182 g ml −1 nabr solution was mixed with the remaining solution to separate the ldl fraction ( 1 . 006 g ml −1 < [UNK] < 1 . 063 g ml −1 ) at 420 000 g at 4 °c for 2 . 5 h . [SEP]
[CLS] after the top layer of 150 μl ldl fraction was removed , 150 μl of 1 . 478 g ml −1 nabr solution was mixed with the remaining solution to separate the hdl fraction ( 1 . 063 g ml −1 < [UNK] < 1 . 21 g ml −1 ) , and the remaining albumin fraction was centrifuged at 420 000 g at 4 °c for 4 h . [SEP]
[CLS] lipid - staining sudan black ( 0 . 01 % w / v ) was added to the 1 . 25 g ml −1 solution ( one tube ) to visualize all lipoprotein fractions . [SEP]
[CLS] in vivo drug distribution in plasma : sd / cd rats were i . v . injected with oxpt / sn38 at a dose of 9 . 13 mg kg −1 chol - sn38 and at 0 . 5 , 1 , 3 , 5 , 8 , or 24 h post - injection , rat plasma was harvested for lipoprotein separation by ultracentrifugation . [SEP]
[CLS] 20 μl of each fraction was analyzed by lc - ms . [SEP]
[CLS] in vitro cellular uptake : mc38 cells B-material seeded in six - well plates ( 5 × 10 4 cells B-material / well ) were starved by removing serum - containing medium and incubating B-technique in fbs - free dmem for 24h . [SEP]
[CLS] 100 ug of chol - pyro ncp or ce6 ncp ( core B-material labeled ) was i . v . injected into the tail veins of c57bl / 6 mice . [SEP]
[CLS] three hours later , the plasma was obtained and added to the serum - deprived cells B-material , which were incubated B-technique with pbs in the presence of 0 , 1 , or 10 μg ml −1 ldlr antibody B-material for 2 h . 24 h later , the cells B-material were harvested and analyzed by flow B-technique cytometry I-technique or confocal imaging . [SEP]
[CLS] cell B-material uptake of dil - ldl was similarly studied . [SEP]
[CLS] ldlr ko mc38 cells B-material were obtained following the protocol of ldlr crispr plasmids ( m ) ( santa cruz biotechnology ) . [SEP]
[CLS] wt or ldlr ko mc38 cells B-material seeded in six - well plates ( 5 × 10 4 cells B-material / well ) were serum starved by removing the culture medium and incubating B-technique in fbs - free dmem for 24h . [SEP]
[CLS] one hundred ug of chol - pyro ncp or ce6 ncp was i . v . injected into c57bl / 6 mice . [SEP]
[CLS] three hours later , the plasma was obtained and added into serum - deprived cells B-material . [SEP]
[CLS] 24 h later , cells B-material were harvested and analyzed by flow B-technique cytometry I-technique . [SEP]
[CLS] mc38 cells B-material seeded in six - well plates ( 5 × 10 4 cells B-material / well ) were starved by removing the culture medium and incubating B-technique in fbs - free dmem for 24h . [SEP]
[CLS] 100 ug of chol - pyro ncp was injected into c57bl / 6 mice . [SEP]
[CLS] three hours later , the plasma was obtained and added into serum - deprived cells B-material , which were incubated B-technique with 10 μg ml −1 igg or an anti - ldlr antibody B-material ( [UNK] - ldlr ) for 2 h . the cells B-material were then stained with lysotracker red dnd - 99 ( 50 nm ) for 1 h . [SEP]
[CLS] the cell B-material medium was replaced with fresh medium and cells B-material were incubated B-technique for another 30 min , and then fixed with 4 % paraformaldehyde for 10 min , stained with dapi for 5 min , and imaged with clsm ( leica sp8 ) . [SEP]
[CLS] ldlr expression on tumor B-material cells B-material : mc38 , mc38 ldlr ko , ct26 , ht29 , hct116 , or sw480 cells B-material were seeded in six - well plates ( 5 × 10 4 cells B-material / well ) . [SEP]
[CLS] 24 h later , the cells B-material were harvested and stained with primary anti - ldlr antibody B-material ( 1 : 100 ) for 1 h on ice . [SEP]
[CLS] the cells B-material were washed by pbs twice and stained with af488 secondary anti - rabbit antibody B-material ( 1 : 1000 ) on ice for 30 min . [SEP]
[CLS] the cells B-material were washed with pbs and analyzed by flow B-technique cytometry I-technique . [SEP]
[CLS] in vivo tumor B-material uptake : c57bl / 6 mice were subcutaneously injected with 2 × 10 6 mc38 cells B-material in the right flanks . [SEP]
[CLS] when the tumors B-material reached ≈100 mm 3 , mice were i . v . administrated pbs or 100 μg chol - pyro ncp . [SEP]
[CLS] 24 and 48 h later , tumors B-material were harvested . [SEP]
[CLS] frozen 5 μm tissue sections were prepared using a cryostat . [SEP]
[CLS] the sections were fixed in acetone for 10 min at −20 °c and stained with dapi for another 10 min . [SEP]
[CLS] the sections were then washed twice with pbs and imaged with clsm . [SEP]
[CLS] the freshly harvested tissues were treated with 1 mg ml −1 collagenase i ( gibco , usa ) for 1 h at 37 °c , and ground with the rubber end of a syringe . [SEP]
[CLS] cells B-material were filtered through nylon mesh filters and washed with pbs . [SEP]
[CLS] the single - cell suspensions were analyzed by flow B-technique cytometry I-technique . [SEP]
[CLS] in vivo biodistribution analysis : c57bl / 6 mice were subcutaneously injected in the right flank with 1 × 10 6 mc38 cells B-material . [SEP]
[CLS] when the tumors B-material reached ≈100 mm 3 , mice were intravenously ( i . v . ) administrated free oxpt or oxpt / sn38 at a dose of 3 . 5 mg kg −1 oxpt and 6 . 2 mg kg −1 sn38 equivalent ( oxpt : sn38 = 1 : 1 . 8 ) . [SEP]
[CLS] the livers , lungs , spleens , kidneys , and tumors B-material were collected at 1 , 3 , 8 , 24 , 48 , and 72 h post - injection . [SEP]
[CLS] the content of pt was quantified by icp - ms . [SEP]
[CLS] release of oxpt and sn38 from oxpt / sn38 : 100 ppm of oxpt / sn38 solution was prepared in ph = 7 . 4 or ph = 4 . 7 aqueous conditions . [SEP]
[CLS] at different time points , 200 μl aliquots were centrifuged with amicon ultra - 0 . 5 centrifugal filter unit ( nmwl : 30 kda ) at 14 000 × g for 10 min . [SEP]
[CLS] the filtrates were analyzed by icp - ms to determine the pt contents . [SEP]
[CLS] for the extraction of sn38 , 100 μl aliquots were added to 100 μl of saturated nacl solution , 5 μl of 20 % triton x - 100 aqueous solution , and 100 μl ethyl acetate . [SEP]
[CLS] the mixture was vigorously vortexed for 30 s and then centrifuged at 14 000 × g for 5 min . [SEP]
[CLS] the upper organic layer was analyzed by lc - ms to determine sn38 concentrations . [SEP]
[CLS] apoptosis B-event / necrosis analysis : mc38 cells B-material seeded in six - well plates ( 5 × 10 4 cells B-material / well ) were treated with oxpt , sn38 , oxpt plus sn38 , oxpt ncp , znp / sn38 , or oxpt / sn38 at 5 μm oxpt or / and 5 μm sn38 for 24 h . [SEP]
[CLS] the cells B-material were harvested , washed twice with ice - cold pbs , stained with alexa fluor 488 - annexin v and propidium iodide B-material ( pi ) for 15 min at room temperature in the dark , and then analyzed by flow B-technique cytometry I-technique ( lsr ii , bd , usa ) . [SEP]
[CLS] in vitro cytotoxicity B-property : colon cancer cells B-material were seeded in 96 - well plates at a density of 2 × 10 3 cells B-material per well and allowed to adhere for 24 h . [SEP]
[CLS] cells B-material were then treated with different concentrations of oxpt , sn38 , irinotecan , chol - sn38 , sn38 - tms , oxpt ncp , znp / sn38 , or oxpt / sn38 for 48 h . [SEP]
[CLS] cell B-material cycle assay : mc38 cells B-material seeded in six - well plates ( 5 × 10 4 cells B-material / well ) were treated with oxpt , sn38 , oxpt ncp , znp / sn38 , or oxpt / sn38 at 5 μm oxpt and / or 5 μm sn38 for 24 h . [SEP]
[CLS] the cells B-material were harvested , washed twice with ice - cold pbs , fixed with 70 % ethanol at 4 °c overnight , treated with rnase a for 45 min , and stained by pi for 30 min . [SEP]
[CLS] cell B-material cycle distributions ( go , g1 , s , and g2m ) were analyzed by flow B-technique cytometry I-technique . [SEP]
[CLS] depolarization of mitochondrial membrane B-property potential I-property : mc38 cells B-material seeded in six - well plates ( 5 × 10 4 cells B-material / well ) were treated with oxpt , sn38 , oxpt plus sn38 , oxpt ncp , znp / sn38 , or oxpt / sn38 at 5 μm oxpt and / or 5 μm sn38 for 24 h . [SEP]
[CLS] the cells B-material were stained with jc - 1 ( abcam , uk ) at a concentration of 10 μm at 37 °c for 30 min in the dark , harvested , washed twice with ice - cold pbs , and analyzed by flow B-technique cytometry I-technique . [SEP]
[CLS] cytochrome c release : after treatment with oxpt , sn38 , oxpt plus sn38 , oxpt ncp , znp / sn38 , or oxpt / sn38 at 5 μm oxpt and / or 5 μm sn38 for 24 h , mc38 cells B-material were sequentially stained with mitotracker red cmxros ( 100 μm ) for 15 min , fixed with 4 % paraformaldehyde for 10 min , permeabilized with 0 . 2 % triton x - 100 for 10 min , incubated B-technique with anti - cytochrome c ( ebioscience , diluted 1 : 100 ) for 2 h , stained with dapi for 10 min , and imaged with clsm ( leica sp8 , germany ) . [SEP]
[CLS] in vivo anticancer B-property efficacy : 2 × 10 6 cells B-material ct26 or mc38 cells B-material were subcutaneously injected into the right flank regions of 6 - week - old balb / c or c57bl / 6 mice , respectively . [SEP]
[CLS] seven days after cell B-material injection , pbs , oxpt plus irinotecan , oxpt ncp , znp / sn38 , and oxpt / sn38 were i . v . injected once every 3 days ( q3d ) at doses of 3 . 5 mg kg −1 oxpt or equivalent , 20 . 2 mg kg −1 irinotecan ( 11 . 7 mg kg −1 sn38 equivalent ) , and / or 15 . 9 mg chol - sn38 / kg ( 6 . 2 mg kg −1 sn38 equivalent ) to mc38 model for up to 8 doses and to ct26 model for up to 6 doses , n = 6 . [SEP]
[CLS] 5 × 10 6 cells B-material ht29 , hct116 , or sw480 were subcutaneously injected into the right flank regions of 6 - week - old athymic nude mice , respectively . [SEP]
[CLS] seven days after cell B-material injection , pbs , oxpt plus irinotecan , oxpt ncp , znp / sn38 , and oxpt / sn38 were i . v . injected once every 3 days ( q3d ) at doses of 3 . 5 mg kg −1 oxpt or equivalent , 20 . 2 mg kg −1 irinotecan ( 11 . 7 mg kg −1 sn38 equivalent ) , and / or 15 . 9 mg chol - sn38 / kg ( 6 . 2 mg kg −1 sn38 equivalent ) to each model for up to 16 doses , n = 6 . [SEP]
[CLS] tumor B-material growth was monitored by measurement with a digital caliper , with tumor B-material volumes calculated as ( width 2 × length ) / 2 . [SEP]
[CLS] tgi is defined as 1 - ( rtvt / rtvc ) where rtv = endpoint tumor B-material volume ) . [SEP]
[CLS] in vivo pharmacokinetics and biodistribution analysis : sd / cd rats were intravenously ( i . v . ) injected with oxpt / sn38 at an equivalent oxpt dose of 2 mg kg −1 . [SEP]
[CLS] the blood was collected at 5 min , 30 min , 1 h , 3 h , 5 h , 8 h , 24 h , or 48 h post - injection and immediately centrifuged at 604 × g for 10 min to harvest plasma samples . [SEP]
[CLS] 20 μl plasma was digested with concentrated nitric acid for 24 h and analyzed for pt concentration by icp - ms . [SEP]
[CLS] another 20 μl plasma was added to 5 μl 20 % triton x - 100 to disrupt the lipid B-material bilayer I-material of the nanoparticles B-nanoparticle . [SEP]
[CLS] chol - sn38 and its metabolites were extracted from plasma by adding 100 μl ethyl acetate , followed by centrifugation at 6708 × g for 10 min . [SEP]
[CLS] chol - sn38 and its metabolites were quantified by lc - ms . [SEP]
[CLS] c57bl / 6 mice were subcutaneously injected with 1 × 10 6 mc38 cells B-material into the right flanks . [SEP]
[CLS] when the tumors B-material reached ≈100 mm 3 , mice were i . v . administrated oxpt plus irinotecan or oxpt / sn38 at doses equivalent to 3 . 5 mg kg −1 oxpt and 6 . 2 mg kg −1 sn38 . [SEP]
[CLS] the livers , lungs , spleens , kidneys , and tumors B-material were collected at 1 , 3 , 8 , 24 , 48 , or 72 h post injection . [SEP]
[CLS] pt concentrations were quantified by icp - ms while chol - sn38 and sn38 concentrations were quantified by lc - ms . [SEP]
[CLS] liver and kidney toxicity B-property evaluation : mc38 tumor - bearing c57bl / 6c mice ( female , n = 6 ) were randomly assigned and i . v . injected with oxpt / sn38 once every 3 days for up to 15 doses . [SEP]
[CLS] on day 22 ( after 8 doses ) and day 43 ( after 15 doses ) , blood was harvested to measure ast , alt , and serum creatinine ( scr ) with ast activity assay kit , alt activity assay kit , and creatinine B-material assay kit ( sigma - aldrich , usa ) , respectively , using a microplate reader . [SEP]
[CLS] absolute neutrophil counts : blood samples were collected from mc38 tumor - bearing c57bl / 6 mice following 8 intravenous injections of pbs or oxpt / sn38 or 3 intravenous injections of oxpt plus irinotecan . [SEP]
[CLS] whole blood samples were added to bd trucount tubes and stained with cd45 , cd11b , and gr - 1 antibodies B-material for flow B-technique cytometry I-technique . [SEP]
[CLS] neutrophils were gated as sschi by fsc / ssc on the cd45 + population and were confirmed to be cd11b + and gr - 1 + . [SEP]
[CLS] hematoxylin and eosin ( h & e ) staining : healthy c57bl / 6 mice ( female , n = 6 ) were randomly assigned and treated with 8 total doses of oxpt / sn38 or oxpt and irinotecan given every three days . [SEP]
[CLS] mice were euthanized on day 22 after the first dose , and gross necropsies were performed . [SEP]
[CLS] the tissues of interest were collected , fixed with 4 % paraformaldehyde , embedded in paraffin , and cut into sections for hematoxylin and eosin ( h & e ) staining before undergoing histopathological examination with a panoramic midi ii digital slide scanner . [SEP]
[CLS] statistical analysis : the data of experiments including ldl binding studies , in vitro studies , and in vivo tumor B-material accumulation were expressed as mean ± standard deviation ( sd ) . [SEP]
[CLS] the above experimental data were repeated three times in parallel ( n = 3 ) and analyzed by one - way anova with tukey ' s multiple comparison test . [SEP]
[CLS] sample sizes ( n ≥ 5 ) were selected to ensure adequate statistical power with anova for efficacy studies . [SEP]
[CLS] student ' s t - tests were used to determine if the variance between groups was similar or between only two groups . [SEP]
[CLS] statistical analysis was performed using prism software ( version 7 . 0 , graphpad software ) . [SEP]
[CLS] * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . [SEP]
[CLS] animal experiments were represented as mean ± sd . [SEP]
[CLS] 1 . preparation and characterization of oxpt / sn38 . [SEP]
[CLS] figure 1 . a ) schematic illustration of two - step construction of core - shell oxpt / sn38 particles via copolymerization of oxpt - bp with zn 2 + ions B-material and coating B-material with chol - sn38 , cholesterol , dopc , and dspe - peg 2000 . [SEP]
[CLS] b ) tem image and c ) number - average diameter of oxpt - bare . d ) stability of oxpt - bare in thf at room temperature . e ) tem image and f ) number - average diameter of oxpt / sn38 . g ) stability of oxpt / sn38 in pbs with bsa ( 5 mg ml −1 ) at 37 °c . [SEP]
[CLS] oxpt / sn38 transfers chol - sn38 to ldl . [SEP]
[CLS] integrated binding heats upon injection of a ) chol - sn38 into an ldl or albumin solution , b ) oxpt / sn38 into an ldl or albumin solution , and c ) chol - sn38 micelle B-material or oxpt ncp without cholesterol into an ldl solution by itc . [SEP]
[CLS] figure 2 . d ) the pmf for transferring sn38 ( red line ) and chol - sn38 ( blue line ) from bulk water B-material to the lipid B-material core B-material of an ldl slice from md simulations . [SEP]
[CLS] the plots are superimposed onto a snapshot of an equilibrated ldl slice . [SEP]
[CLS] e ) time - dependent binding of sn38 or chol - sn38 to ldl and transfer of chol - sn38 from oxpt / sn38 to ldl in rat plasma . [SEP]
[CLS] f ) concentration - dependent apob - 100 binding to znp ncp with and without cholesterol , n = 3 . [SEP]
[CLS] g ) distributions of sn38 , chol - sn38 , and chol - sn38 from oxpt / sn38 in different lipoproteins of rat plasmas by ultracentrifugation separation . [SEP]
[CLS] h ) pharmacokinetic profile of chol - sn38 from oxpt / sn38 in rat plasma and its lipoprotein distribution after intravenous injection of oxpt / sn38 at a chol - sn38 dose of 14 . 4 mg kg −1 . [SEP]
[CLS] data are expressed as means ± sd by student ' s two - tailed t - test . [SEP]
[CLS] ldlr - mediated endocytosis B-event determines ncp particle uptake by tumor B-material cells B-material . [SEP]
[CLS] uptake of a ) chol - pyro ncp and b ) ce6 - ncp by mc38 cells B-material after ldlr blockade with 1 or 10 μg ml −1 [UNK] - ldlr . [SEP]
[CLS] the dil - ldl uptake served as control . [SEP]
[CLS] figure 3 . c ) cellular uptake of chol - pyro - ncp and ce6 - ncp on wt and ldlr ko mc38 cells B-material . [SEP]
[CLS] clsm d ) statistical analysis and e ) images of chol - sn38 uptaken by mc38 cells B-material 24 h after treatment with 10 μg ml −1 igg or [UNK] - ldlr . [SEP]
[CLS] scale bar : 20 μm . [SEP]
[CLS] f ) lysosome was stained with lysotracker and the colocalization of lysosome and chol - sn38 was evaluated based on pearson ' s r value . [SEP]
[CLS] g ) immunofluorescence analysis showing the in vivo tumor B-material uptake of chol - pyro ncp at 24 and 48 h with 1 μg of igg or [UNK] - ldlr . [SEP]
[CLS] scale bar : 20 μm . [SEP]
[CLS] h ) flow B-technique cytometry I-technique results and i ) mean fluorescent B-property intensity of chol - pyro ncp tumor B-material uptake at 24 and 48 h post i . v . injection with 1 μg of igg or [UNK] - ldlr . [SEP]
[CLS] time - dependent j ) pt and k ) sn38 accumulation after i . v . injection of oxpt ( 3 . 5 mg kg −1 ) plus irinotecan ( 6 . 2 mg sn38 / kg equivalent ) , oxpt / sn38 ( 3 . 5 mg oxpt / kg equivalent , 6 . 2 mg sn38 / kg equivalent ) to mc38 - bearing mice with and without 1 μg of intratumorally injected [UNK] - ldlr . [SEP]
[CLS] data are expressed as means ± sd . [SEP]
[CLS] the data were analyzed by one - way analysis of variance ( anova ; a , b ) with tukey ' s multiple comparison test , or student ' s two - tailed t - test ( c , d , i ) . [SEP]
[CLS] 4 . release of sn38 and oxpt from oxpt / sn38 . [SEP]
[CLS] figure 4 . a ) proposed sn38 release via acid - catalyzed hydrolysis and esterase - mediated cleavage . [SEP]
[CLS] percentages of chol - sn38 and released products from oxpt / sn38 at b ) ph = 4 . 7 in pbs and c ) ph = 7 . 4 pbs with 10 unit ml −1 esterase throughout 72 h at 37 °c . [SEP]
[CLS] d ) percentages of sn38 released from oxpt / sn38 particle when incubated B-technique in pbs at ph = 4 . 7 or ph = 7 . 4 throughout 72 h at 37 °c . e ) release of oxpt from oxpt / sn38 via hydrolysis and reduction by ascorbate . [SEP]
[CLS] f ) total pt and oxpt release profiles from oxpt / sn38 particles when incubated B-technique in pbs with ph = 4 . 7 and 5 mm ascorbate at 37 °c . g ) time - dependent pt release from oxpt / sn38 particle when incubated B-technique in pbs at ph = 4 . 7 or ph = 7 . 4 at 37 °c . [SEP]
[CLS] the inset showed the tem image of disintegrated oxpt / sn38 particles after incubation B-technique in pbs at ph = 4 . 7 and 37 °c for 5 h . [SEP]
[CLS] green fluorescence B-property ) ( figure5fand figuress20 and s21 , supporting information ) . [SEP]
[CLS] 5 . in vitro programmed B-event cell I-event death I-event and in vivo anticancer B-property efficacy . [SEP]
[CLS] figure 5 . a ) apoptosis B-event induced by oxpt plus sn38 or oxpt / sn38 . [SEP]
[CLS] after treatment , cells B-material were stained by alexa fluor 488 - labelled annexin v and pi and analyzed by flow B-technique cytometry I-technique . [SEP]
[CLS] b , d ) cell B-material cycle arrest caused by oxpt / sn38 . [SEP]
[CLS] treated cells B-material were fixed with 70 % ethanol overnight , treated with rnase a , stained with pi , and analyzed by flow B-technique cytometry I-technique , n = 3 . [SEP]
[CLS] c , e ) flow B-technique cytometry I-technique analysis with jc - 1 staining of mmps of mc38 cells B-material treated with pbs , oxpt plus sn38 or oxpt / sn38 , n = 3 . [SEP]
[CLS] f ) release of cytochrome c from mitochondria in mc38 cells B-material treated with oxpt plus irinotecan or oxpt / sn38 . [SEP]
[CLS] mitochondria ( red fluorescence B-property ) and cytochrome c ( green fluorescence B-property ) were stained by mitotracker red cmxros and anti - cytochrome c antibody B-material , respectively . [SEP]
[CLS] scale bar : 20 μm . [SEP]
[CLS] tumor B-material growth curves of g ) mc38 tumors B-material in c57bl / 6 mice and h ) ct26 tumors B-material in balb / c mice . [SEP]
[CLS] pbs , oxpt plus irinotecan , oxpt ncp , znp / sn38 , or oxpt / sn38 was i . v . injected once every 3 days ( q3d ) at doses of 3 . 5 mg kg −1 oxpt or equivalent , 20 . 2 mg kg −1 irinotecan ( 11 . 7 mg kg −1 sn38 equivalent ) , and / or 15 . 9 mg chol - sn38 / kg ( 6 . 2 mg kg −1 sn38 equivalent ) to mc38 model for up to 8 doses and to ct26 model for up to 6 doses , n = 6 . i ) survival curves of ht29 model and j ) tumor B-material growth curves of ht29 , k ) hct116 , and l ) sw480 models in nude mice after q3d treatment with pbs , oxpt / sn38 , or oxpt plus for up to 16 doses . [SEP]
[CLS] the doses are the same as mc38 and ct26 models . [SEP]
[CLS] data are expressed as means ± sd . [SEP]
[CLS] the data were analyzed by one - way anova with tukey ' s multiple comparison test . [SEP]
[CLS] ldlr - mediated endocytosis B-event determines the anticancer B-property efficacy of oxpt / sn38 . [SEP]
[CLS] figure 6 . a ) anticancer B-property efficacy of oxpt / sn38 with intratumorally injected 1 μg igg or [UNK] - ldlr on mc38 tumor - bearing c57bl / 6 mice at a dose of 3 . 5 mg oxpt / kg equivalent . n = 6 . [SEP]
[CLS] b ) anticancer B-property efficacy of oxpt / sn38 on wt and ldlr ko mc38 tumor B-material - bearing c57bl / 6 mice at a dose of 3 . 5 mg oxpt / kg equivalent . n = 6 . c ) h & e staining ( top ) , tunel ( middle ) , and caspase 3 ( bottom ) showing differences in apoptosis B-event and necrosis of mc38 tumors B-material after oxpt / sn38 treatments with intratumorally injected igg or [UNK] - ldlr . [SEP]
[CLS] scale bar : 100 μm . [SEP]
[CLS] d ) scheme showing oxpt / sn38 delivery by hitchhiking ldl and anticancer B-property mechanism . [SEP]
[CLS] once injected intravenously , oxpt / sn38 binds to ldl and transfers chol - sn38 to ldl . [SEP]
[CLS] ldl - bound oxpt / sn38 is taken up by cancer cells B-material through ldlr - mediated endocytosis B-event . [SEP]
[CLS] acidic endo / lysosomal environment triggers the release of oxpt and sn38 from oxpt / sn38 . [SEP]
[CLS] the released oxpt and sn38 synergistically inhibit dna B-event replication I-event by crosslinking B-event dna I-event bases and binding to topoisomerase i , respectively . [SEP]
[CLS] the dna damage causes mmp disruption , resulting in the early apoptosis B-event of cancer cells B-material . [SEP]
[CLS] data are expressed as means ± sd . [SEP]
[CLS] the data were analyzed by one - way anova with tukey ' s multiple comparison test . [SEP]
[CLS] ( d ) was created with biorender . com . [SEP]
[CLS] cell B-property viability I-property was determined by 3 - ( 4 , 5 - dimethylthiazol - 2 - yl ) - 5 - ( 3 - carboxymethoxyphenyl ) - 2 - ( 4 - sulfophenyl ) - 2h - tetrazolium assay ( promega , madison , wi ) according to the manufacturer ' s instructions . [SEP]
[CLS] youngeun kim and peng yin * abstract : the use of dna - based nanomaterials B-material in biomedical applications is continuing to grow , yet more emphasis is being put on the need for guaranteed structural stability of dna nanostructures in physiological conditions . [SEP]
[CLS] various methods have been developed to stabilize dna origami against low concentrations of divalent cations B-material and the presence of nucleases . [SEP]
[CLS] however , existing strategies typically require the complete encapsulation of nanostructures , which makes accessing the encased dna strands difficult , or chemical modification , such as covalent crosslinking B-event of I-event dna I-event strands . [SEP]
[CLS] we present a stabilization method involving the synthesis of dna brick nanostructures with dendritic oligonucleotides attached to the outer surface . [SEP]
[CLS] we find that nanostructures assembled from dna brick motifs remain stable against denaturation without any chemical modifications . [SEP]
[CLS] furthermore , densely coating B-material the outer surface of dna brick nanostructures with dendritic oligonucleotides prevents nuclease digestion . [SEP]
[CLS] dna nanotechnology enables the synthesis of rationally designed dna nanostructures of arbitrary geometric configuration , and such molecular - scale nanodevices B-nanoparticle can be used in biomedical applications , driving the field towards programmable and customizable nanomedicine . [SEP]
[CLS] examples of dna nanocages B-nanoparticle , capsules , and carriers have been studied as delivery vehicles B-material or diagnostic devices for drug delivery , cancer treatment , and immunotherapy . [SEP]
[CLS] nevertheless , more recent literature highlights the importance of the structural stability of dna - based materials under physiological conditions when using them in vitro and / or in vivo . [SEP]
[CLS] this is due to two main factors involved in degradation of dna nanostructures upon exposure to biological conditions : i ) denaturation caused by low divalent cation B-material concentration ( physiological salt B-material concentration approximately 0 . 04 - 0 . 8 mm mgcl 2 ) , and ii ) digestion caused by the presence of nucleases . [SEP]
[CLS] multiple strategies have been developed to chemically or physically prevent the dna nanostructures from falling apart in cellular media ( for example , 10 % fbs ) , and in general , encapsulation of dna nanostructures with different coating B-material moieties prolongs the survival time the longest published . for example , while most bare dna origami falls apart easily under physiological conditions , peg - oligolysines , lipid B-material molecules , or cationic B-material polymers B-material can be applied as a coating B-material material B-material to extend the half - life of dna origami by an order of up to approximately 100 . [SEP]
[CLS] such an encasing strategy , however , often covers the entire outer surface of dna origami and therefore in theory , makes it difficult to access the dna strands post - coating B-material . [SEP]
[CLS] ideally , one should be able to access the dna strands and nanostructures for binding without having to penetrate through a thick layer of overlaid material B-material . [SEP]
[CLS] in this work , we aim to structurally stabilize dna nanostructures with only oligonucleotide strands such that both biocompatible B-property stability and dna accessibility remain viable . [SEP]
[CLS] and in doing so , we present two findings : i ) nanostructures consisting of certain dna brick motifs remain structurally stable at low divalent salt B-material concentrations ( for example , 1 pbs ) , and ii ) functionalizing the outer surface of dna brick nanostructures with dendritic oligonucleotides prevents the digestion of nanostructures from nucleases due to putative steric hindrance ( figure 1 ) . [SEP]
[CLS] as a result , this strategy suggests that neither chemical protectants nor covalent base - pair interlocking is necessary to enhance the stability of dna brick nanostructures . [SEP]
[CLS] recently , the yin lab developed a strategy to assemble dna nanostructures of different sizes and shapes using short , synthetic oligonucleotide strands . [SEP]
[CLS] this assembly of dna bricks , which consist of four short binding domains arranged so that the bricks can interlock , does not require a scaffold . [SEP]
[CLS] and while the first generation of bricks were 32 nucleotides ( nt ) long , consisting of four 8 nt binding domains [SEP]
[CLS] , more recent developments investigated brick strands with longer binding domains ( 52 nt bricks with four 13 nt domains ; 74 nt bricks with two 18 nt and two 19 nt domains ) . [SEP]
[CLS] we discovered that dna brick nanostructures with binding domains of 13 nt or longer remain stable even when they are placed in 1 pbs with no divalent salt B-material . first , the stability of dna brick nanostructures was tested by removing cations B-material from solution . [SEP]
[CLS] three separate sets , each consisting of approximately two hundred strands of 32 , 52 , or 74 nt bricks , were assembled in 10 mm or 40 mm mgcl 2 ( note that assembly salt B-material conditions were chosen based on previous literature ; see the supporting information for a detailed protocol ) . [SEP]
[CLS] all sets of samples were divided into two subsets for comparison : i ) maintaining the overall mgcl 2 concentration at 10 mm or 40 mm as control , and ii ) replacing the buffer with 1 pbs via incubating B-technique the assembled structures in 1 pbs at 37 8c for 1 hour , then using filtration to remove any excess divalent salt B-material in solution . [SEP]
[CLS] to note , 3d dna origami with a binding length of 8 nt was included for comparison ( see the supporting information for design and assembly conditions ) . [SEP]
[CLS] gel B-technique electrophoresis I-technique results show the dissociation of structures in 1 pbs for 32 nt brick nanostructures and 3d dna origami ( lanes 5 and 14 , respectively , in figure 2 a ) , while 52 and 74 nt brick nanostructures remained stable , implying the significance of binding - domain length when designing dna nanostructures ( lanes 8 and 11 , respectively , in figure 2 a ) . [SEP]
[CLS] this experiment was repeated with a longer incubation B-technique time ( 24 hours ; supporting information , figure s2 ) and differently sized brick nanostructures ( supporting information , figure s3 ) , and results indicate that all 52 nt brick nanostructures remain stable regardless of their overall size . [SEP]
[CLS] transmission B-technique electron I-technique microscopy I-technique ( tem ) data also verified the structural stability of the dna brick nanostructures in 1 pbs ( figure 1 b ) and demonstrated that they retain their structure even after 4 days of storage at room temperature ( supporting information , figure s4 ) . [SEP]
[CLS] though it is expected that longer complementary binding lengths lead to stronger interlocking between base - pairs , all results emphasize the importance of binding - domain length as a contributing factor in stabilizing dna nanostructures . [SEP]
[CLS] this finding also helps explain why most 3d dna origami structures with average binding - domain lengths of 7 - 10 basepairs degrade when the global divalent salt B-material concentration is low ( 0 - 0 . 8 mm mgcl 2 ) . [SEP]
[CLS] dna brick nanostructures that remain stable even in 1 pbs were still susceptible to nucleases . [SEP]
[CLS] after confirming that dna brick nanostructures were stable against low salt B-material denaturation without the requirement of any additional stabilization techniques , the same 52 nt brick nanostructures were tested against nuclease digestion ( supporting information , figure s5 ) . [SEP]
[CLS] we followed a dnase i titration assay used in previous literature , and brick nanostructures were incubated B-technique with varying concentrations of dnase i at 37 8c for 1 hour , then characterized via gel B-technique electrophoresis I-technique . [SEP]
[CLS] a decrease in band intensity as well as a slight shift in band position were found ( figure 3 and supporting information , figure s5 ) , suggesting that 52 nt bricks fall apart at a dnase i concentration above approximately 5 u ml a1 . [SEP]
[CLS] the nuclease resistance of dna brick nanostructures is not high enough when compared to that of chemically stabilized or encapsulated dna origami , and therefore an additional method is required to stabilize dna brick nanostructures against nuclease digestion . [SEP]
[CLS] we envisioned that densely functionalizing the outer surface of dna brick nanostructures with additional oligo - nucleotide strands would stabilize them against enzymes . this was mainly inspired by spherical nucleic B-material acids I-material ( snas ) , which are formed by organizing nucleic B-material acids I-material radially around a nanoparticle B-nanoparticle core B-material . [SEP]
[CLS] snas can enter cells B-material without transfection reagents , and once inside the cell B-material , the nucleic B-material acid I-material components of snas resist nuclease degradation , leading to longer intracellular lifetimes . [SEP]
[CLS] because bare dna brick nanostructures alone were not stable against nucleases , we hypothesized that introducing surrounding oligonucleotides in high density would prevent enzyme accessibility and therefore stabilize them against digestion . [SEP]
[CLS] because only a limited number of overhang strands can be designed into a given dna brick nanostructure , the surface density could be increased by attaching dendritic oligonucleotides . [SEP]
[CLS] to do this , 52 nt dna brick nanostructures were carefully designed to have approximately 100 protruding overhang strands to which dendritic oligonucleotides can be attached ( figure 1 b ) . [SEP]
[CLS] dendritic oligonucleotides were synthesized by incorporating a trebler phosphoramidite , a branching reagent that can be incorporated in regular dna synthesis protocols . [SEP]
[CLS] integrating one trebler modifier enables each single - stranded dna to branch into three separate single - stranded arms ( 3 ) , while including two repeated trebler moieties enables the dendritic oligonucleotide to have single stranded arms ( 9 ) . [SEP]
[CLS] 3 or 9 dendrimers B-nanoparticle can hybridize with the overhang strands to systematically increase the total number of available singlestranded oligonucleotides on the outermost surface . [SEP]
[CLS] dna brick nanostructures were tested against nuclease digestion at four different surface oligonucleotide densities : i ) bare , with no protruding strands from the brick nanostructures , ii ) single , with approximately 100 protruding overhang dna strands , iii ) triple , with 3 dendrimers B-nanoparticle attached , and iv ) nonuple , with 9 dendrimers B-nanoparticle attached ( figure 3 a ) . [SEP]
[CLS] all samples were exposed to different concentrations of dnase i , ranging from 0 to 100 u ml a1 . [SEP]
[CLS] these concentrations were chosen based on previous literature and a dnase i titration assay was used to characterize nuclease resistance . [SEP]
[CLS] gel analysis shows that dna brick nanostructures have an increased resistance to dnase i at higher oligonucleotide density on the outer surface . [SEP]
[CLS] some samples show the formation of multimeric ( for example , dimer ) structures , however , this phenomenon is found in most synthesized brick structures ( including bare ones ) [SEP]
[CLS] , and [SEP]
[CLS] therefore we do not believe the multimeric structures themselves dramatically increase the overall structural stability . [SEP]
[CLS] to calculate the number of oligonucleotides available on the outer surface , fluorescence B-property measurements were performed ( supporting information , figures s7 and s8 ) . [SEP]
[CLS] the estimated numbers of available dna strands , relatively close to the expected numbers with some error , indirectly validate the accessibility of dna sequences at the outer surface of dna brick nanostructures . [SEP]
[CLS] lastly , in vitro studies were carried out on dna brick nanostructures incubated B-technique in 10 % fbs cellular media for different lengths of times . [SEP]
[CLS] to compare the effect of dendritic brushes , bare and nonuple dna brick nanostructures were tested in 10 % fbs ( supporting information , figure s9 ) . [SEP]
[CLS] results reveal a shorter survival time for bare dna brick nanostructures while nonuple brick nanostructures survived up to 30 hours without significant degradation ( figure 4 a and supporting information , figure s10 ) . [SEP]
[CLS] cellular uptake studies were also performed by incubating B-technique nonuple dna brick nanostructures in hek293t cells B-material . [SEP]
[CLS] nonuple dna brick nanostructures were fluorescently B-property labeled via hybridizing complementary cy5 - ssdna to the single - stranded region of the dendritic oligonucleotides . [SEP]
[CLS] in situ imaging results show a clear difference in fluorescence B-property between cy5 - ssdna only versus cy5 - dna brick nanostructures ( figure 4 b ) , as well as successful uptake of the dna brick nanostructures inside the cells B-material . [SEP]
[CLS] in conclusion , dna brick nanostructures with bindingdomain lengths of 13 nt or longer are stable against denaturation in low divalent salt B-material concentration and attaching dendritic oligonucleotides to the outer surface of dna brick nanostructures stabilizes them against nuclease digestion . [SEP]
[CLS] dendritic - oligonucleotide - coated dna brick nanostructures do not require chemical base - pair interlocking techniques or encapsulation methods yet still display structural stability in cellular media as well as accessibility of dna sequences at the surface . [SEP]
[CLS] as a result , dna brick nanostructures offer a promising alternative to dna origami . [SEP]
[CLS] furthermore , unlike spherical nucleic B-material acids I-material in which dna strands are coated in an isotropic manner , these dna brick nanostructures can become asymmetrically functionalized with precise location control , which will have important implications as research efforts shift to the use of multifunctionalized nanomaterials B-material . [SEP]
[CLS] dna nanostructures are constructed by a ) attaching dendritic oligonucleotides to the outer surface of dna brick nanostructures via b ) hybridization to the complementary , protruding overhang strands . [SEP]
[CLS] structural stability test against low salt B-material denaturation . [SEP]
[CLS] figure 2 . a ) 32 , 52 , and 74 nt brick nanostructures and 3d origami were assembled at either 10 or 40 mm mgcl 2 ( lanes 4 , 7 , 10 , and 13 ) . [SEP]
[CLS] assembled samples were incubated B-technique at 37 8c in 1 pbs for 1 hour , then spin - filtered to completely remove any remaining salt B-material in solution ( lanes 5 , 8 , 11 , and 14 ) . [SEP]
[CLS] all samples were characterized via agarose B-technique gel I-technique electrophoresis I-technique . [SEP]
[CLS] b ) tem images were taken to confirm the preservation of structures . [SEP]
[CLS] scale bars indicate 50 nm in length . [SEP]
[CLS] structural stability test against nuclease digestion . [SEP]
[CLS] figure 3 . a ) dna brick nanostructures with four different surface densities were studied : bare ( pink ) , single ( blue ) , triple ( orange ) , and nonuple ( green ) . [SEP]
[CLS] all assembled dna brick nanostructures were incubated B-technique at 37 8c for 1 hour with varying dnase i concentrations ( 0 to 100 u ml a1 ) . [SEP]
[CLS] b ) incubated B-technique samples were characterized via gel B-technique electrophoresis I-technique ( lane 1 : control ; lane 2 : 5 u / l of dnase 1 ; lane 3 : 0 u / l of dnase 1 ; lane 4 : 50 u / l of dnase 1 ; lane 5 : 100 u / l of dnase 1 ) . [SEP]
[CLS] c ) gel band intensities from each gel were quantified via a gel - imaging software , then normalized such that the intensity of the control is always 1 . [SEP]
[CLS] data from three separate gel results were averaged , and standard deviations were calculated . . [SEP]
[CLS] testing stability in cellular media ( 10 % fbs ) and conducting cellular uptake . [SEP]
[CLS] figure 4 . a ) bare ( blue circles ) and nonuple ( orange squares ) dna brick nanostructures were assembled , then incubated B-technique in 10 % fbs at 37 8c for varying lengths of time ( 0 , 1 , 2 , 8 , 12 , 30 hours ) . [SEP]
[CLS] normalized band intensities , from three separately conducted gel B-technique electrophoresis I-technique experiments , were averaged and plotted against time . [SEP]
[CLS] b ) nonuple dna brick nanostructures ( 200 nm ) were incubated B-technique with complementary cy5 - ssdna ( 2 mm ) to fluorescently B-property label the structures . [SEP]
[CLS] hek293t cells B-material were incubated B-technique with 200 nm fluorescently B-property labeled nonuple dna brick nanostructures , and as control , a separate set of hek293t cells B-material were incubated B-technique with 2 mm cy5 - ssdna . [SEP]
[CLS] both sets were characterized via an inverted fluorescence B-technique microscopy I-technique using the cy5 and bright - field channels . [SEP]
[CLS] circulating microrna ( mirnas ) have been proven as potential biomarkers B-property for disease diagnosis . [SEP]
[CLS] herein , this work reports the design of a chemiluminescence B-property resonance energy transfer ( cret ) based multicolor sensing nanoplatform with a portable smartphone as mini - detector for simultaneous and sensitive analysis of multiple circulating mirnas . [SEP]
[CLS] universal multicomponent nucleic B-material acid I-material enzyme functionalized gold B-nanoparticle nanoparticle I-nanoparticle ( mnazyme @ aunp B-nanoparticle ) nanoprobes B-nanoparticle activated by the addition of different target mirnas were constructed . [SEP]
[CLS] as a result , spherical nucleic B-material acid I-material enzyme ( snazyme ) with high catalytic activity for the luminol - h 2 o 2 chemiluminescence B-property or cret reaction was produced . [SEP]
[CLS] it enables the construction of a multicolor sensing nanoplatform for simultaneously visualizing multiple mirnas based on chemiluminescence B-property imaging arrays ( clia ) . [SEP]
[CLS] in this way , acute myocardial infarction ( ami ) related biomarkers B-property , mir - 133a and mir - 499 , were detected with high sensitivity and selectivity in human serum . [SEP]
[CLS] micrornas ( mirna ) , endogenously expressed non - coding rnas with 18 - 23 nucleotides in length , play a critical role in different physiological and pathologic B-event processes I-event . [SEP]
[CLS] aberrant expressions of various mirnas have been demonstrated to be linked to various cancers and human diseases . [SEP]
[CLS] among them , acute myocardial infarction ( ami ) , the first cause of death for cardiovascular diseases ( cvds ) , is undergoing an annual increase of morbidity and mortality worldwide in recent decades . [SEP]
[CLS] generally , it is hard to obtain definite diagnosis of disease based on the single - biomarker B-property level . [SEP]
[CLS] recently , mounting evidence has proved the remarkable elevation of cardiacenriched multiple mirnas in peripheral circulation after the onset of ami , such as mir - 133a , mir - 499 , mir - 1 and mir - 208 , which makes these circulating mirnas a class of potential hallmarks for noninvasive diagnose and management of ami . [SEP]
[CLS] given their low abundance and big interference in complex matrix , there is an urgent need to develop robust approaches for ultrasensitive and simultaneous detection of multiple amirelated circulating mirnas . [SEP]
[CLS] recently , chemiluminescence B-property ( cl ) has attracted wide interest in developing sensing strategies for biomarker B-property profiling , due to the merits of low detection limit ( lod ) , wide detection range , and no need for excitation light source , and so on . [SEP]
[CLS] however , the vast majority of existing methods demonstrate their application in single - biomarker B-property detection . [SEP]
[CLS] simultaneous detection of multiple disease - associated biomarkers B-property remains a challenge . [SEP]
[CLS] the emergence of spatial - resolved chemiluminescent B-property imaging arrays ( clia ) is recently revolutionizing the traditional cl - based methods for biomarkers B-property detection due to the merits of visualization , high throughput and multiple detection . [SEP]
[CLS] at present , although significant progress has been made in clia - based chemiluminescence B-property imaging enzyme immunoassay ( cieia ) for multiple disease - related antigens detection , most of them need sophisticated , high - cost and time - consuming enzyme - labeling process and the charge coupled device ( ccd ) as detectors . [SEP]
[CLS] hence , developing a portable and universal cl imaging platform is of great significance for simultaneous multi - mirna imaging analysis for facile disease diagnosis . [SEP]
[CLS] in this work , we present a cret - based multicolor sensing nanoplatform coupling with clia for simultaneously visualizing multiple mirnas in circulating blood ( scheme 1 ) . [SEP]
[CLS] a universal mnazyme @ aunp B-nanoparticle nanoprobe B-nanoparticle was constructed for enzyme - free signal amplification , which consists of hairpin dna - functionalized aunp B-nanoparticle nanoconjugate ( hpdna @ aunp B-nanoparticle ) and multicomponent nucleic B-material acid I-material enzyme ( mnazyme ) including two subunits ( s1dna and s2dna ) . [SEP]
[CLS] both subunits involve a recognition domain , a partial catalytic core B-material and a substrate - binding domain . [SEP]
[CLS] the target mirna can hybridize with the recognition domains and thereby guides the assembly of mnazyme subunits into a stable sandwich structure ( mirna - mnazyme - hpdna ) with a catalytic core B-material on the aunp B-nanoparticle surface . [SEP]
[CLS] this activates the mnazyme to catalyze the cleavage of rna site of hpdna , accompanied by the release of g - rich sequence blocked in the stem of hpdna . [SEP]
[CLS] ultimately , spherical nucleic B-material acid I-material enzyme ( snazyme ) is produced in the presence of hemin , acting as a nanocatalyst with high catalytic activity for the luminol - h 2 o 2 cl reaction with a smartphone as the portable detector . [SEP]
[CLS] based on chemiluminescence B-property resonance energy transfer ( cret ) , a multicolor imaging method integrating with spatial - resolved cl array for simultaneous mirna detection is developed . [SEP]
[CLS] theoret - ically , our proposed mnazyme @ aunp B-nanoparticle nanoprobe B-nanoparticle can be initiated by any interested nucleic B-material acid I-material inputs just with simple modification of the recognition domains in the nanoprobe B-nanoparticle , which makes it a powerful and universal tool for biomarkers B-property detection . [SEP]
[CLS] the transmission electron microscope ( tem ) image in figure 1a shows that uniform and spherical aunp B-nanoparticle with an average diameter of 14 . 7 1 nm was prepared . [SEP]
[CLS] the size distribution scheme 1 . schematic illustration of the cl imaging strategy for simultaneous detection of multiple mirnas based on a universal smartphone sensing platform [SEP]
[CLS] 1 . tem images of ( a ) bare aunp B-nanoparticle , ( b ) linker @ aunp B-nanoparticle , and ( c ) mnazyme @ aunp B-nanoparticle nanoprobe B-nanoparticle ; ( d ) zeta B-property potential I-property , ( e ) uv a visible spectra , and ( f ) dls of bare aunp B-nanoparticle , linker @ aunp B-nanoparticle , and mnazyme @ aunp B-nanoparticle nanoprobe B-nanoparticle . [SEP]
[CLS] histogram can be found in figure s1 . [SEP]
[CLS] functionalization of linker strands and mnazyme has no influence on the morphology of aunp B-nanoparticle , but increases the monodispersity ( figure 1b - c ) , which can be explained by the increased electrostatic repulsion demonstrated by the zeta B-property potential I-property analysis in figure 1d . [SEP]
[CLS] after the conjugation of linker and mnazyme , the average hydrodynamic diameter increases from ~ 18 to 25 nm , and ultimately reaches 38 nm ( figure 1f ) . [SEP]
[CLS] in addition , uv - vis absorption B-technique spectroscopy I-technique can offer direct evidence for the successful immobilization of dna on aunp B-nanoparticle ( figure 1e ) . [SEP]
[CLS] bare aunp B-nanoparticle had a typical characteristic peak at ~ 520 nm ( black line ) . [SEP]
[CLS] upon the modification of linker strands , a distinct characteristic peak of dna at 260 nm can be observed ( blue line ) . [SEP]
[CLS] further hybridization of mnazyme with linker strands on aunp B-nanoparticle surface results in an increase of the peak height ( red line ) . [SEP]
[CLS] all abovementioned data firmly prove the successful preparation of mnazyme @ aunp B-nanoparticle nanoprobe B-nanoparticle . [SEP]
[CLS] the average number of linker strands loading on single aunp B-nanoparticle was estimated to be ~ 94 by the fluorescence - based method ( figure s2 ) . [SEP]
[CLS] to affirm the feasibility of the proposed program , native polyacrylamide B-technique gel I-technique electrophoresis I-technique ( page ) analysis was carried out to demonstrate the target mirna - responsive autonomous cyclic cleavage reaction of free dna nanoprobe B-nanoparticle . [SEP]
[CLS] as a proof of concept , mir - 499 was chosen as the model target for the following experiment . [SEP]
[CLS] as shown in figure 2a , two separate bands corresponding to s1dna ( lane 1 ) and s2dna ( lane 2 ) respectively can be observed ( lane 3 ) , indicating the mnazyme subunits are unable to form stable duplex and activate the catalytic activity . [SEP]
[CLS] however , s1dna and s2dna can stably coassembly with hpdna , forming an inactive probe structure , which can be proved by the disappearance of both s1dna and s2dna bands and appearance of a new obviously slower band in the presence of hpdna ( lane 6 - 8 ) . [SEP]
[CLS] upon addition of target in the presence of mg 2 + , an obvious cleavage product band ( lane 9 ) in accordance with that of g4 reference ( lane 4 ) is observed , while no cleavage product appears in the control system without rna site ( lane 10 ) . [SEP]
[CLS] further experiment was carried out to visually validate the respond of mnazyme @ aunp B-nanoparticle nanoprobe B-nanoparticle to the target by agarose B-technique gel I-technique electrophoresis I-technique ( age ) . [SEP]
[CLS] as shown in figure 2b , progressively slower bands can be observed in lane 1 - 3 , corresponding to linker @ aunp B-nanoparticle , hpdna @ aunp B-nanoparticle , mnazyme @ aunp B-nanoparticle nanoprobe B-nanoparticle , respectively , which indicates the sequential immobilization of dna on the aunp B-nanoparticle surface . [SEP]
[CLS] in the presence of target , mnazyme is activated and finally results in the generation of g - quadruplex product on the aunp B-nanoparticle surface . [SEP]
[CLS] therefore , a smaller nanoconjugate is formed , identified by the presence of a band with higher mobility B-property ( lane 5 vs lane 3 ) . [SEP]
[CLS] when the hpdna without rna site is employed in the control system , however , an obvious band with lowest mobility B-property is observed ( lane 4 ) . [SEP]
[CLS] this is attributed to the formation of synergistically stabilized sandwich structure ( mirna - mnazyme - hpdna ) with bigger size on the aunp B-nanoparticle surface , which is the intermediate B-property structure during the amplification process , but no cleavage reaction happens . [SEP]
[CLS] the chemiluminescence B-property spectroscopy B-technique was used to obtain quantitative data ( figure 2c ) . [SEP]
[CLS] when target is present , a dramatically higher cl signal is generated than when the target is absent ( figure 2c , red line vs blue line ) . [SEP]
[CLS] this can be attributed to the initiation of amplification reaction by target and the generation of snazyme with high catalytic activity for luminol - h 2 o 2 cl reaction . [SEP]
[CLS] in addition , we can observe that maximum emission wavelength is at ~ 440 nm , indicating that luminophore is excited intermediate B-property 3 - aminophthalate ( 3 - ap * ) . [SEP]
[CLS] to achieve the determination of mirna in instrument - free and point - of - care fashion , we utilized a smartphone as mini - detector to record the change of cl intensity . [SEP]
[CLS] we can observe visually bright blue colored light emission in the presence of target and a weak light emission background signal in the absence of target , which was consistent with the results of luminescence B-property spectroscopy B-technique analysis . [SEP]
[CLS] it was worth mentioning that the glow - type kinetic behavior of cl system for imaging analysis is critically important . [SEP]
[CLS] undoubtedly , flash - type cl reaction can cause a great difference of final signal even if a slight delay of exposure time , deterrent to the detection reproducibility and accuracy , and this can be avoided by the glow - type one . [SEP]
[CLS] as shown in figure 2d , the cl reaction is triggered instantaneously upon the addition of co - reactants , and cl intensity rapidly rises to the peak value and keeps stable within 2 min , which is suitable for imaging detection with smartphone . [SEP]
[CLS] consequently , 60 s is set as the exposure time to capture the cl images in detection zone . [SEP]
[CLS] all results mentioned above favorably demonstrate that our constructed nanoplatform is feasible and promising for imaging analysis of mirna with smartphone as mini - detector . [SEP]
[CLS] in order to acquire the best detection performance , several major experimental parameters were optimized . [SEP]
[CLS] the cl reaction conditions were firstly optimized . [SEP]
[CLS] the concentrations of luminol ranged from 0 . 01 to 5 mm were screened . [SEP]
[CLS] as shown in figure 3a , cl intensity increased with luminol concentration increasing until 0 . 5 mm . [SEP]
[CLS] further increasing the concentration of luminol will result in the decrease in imaging brightness and cl intensity . [SEP]
[CLS] thus , the optimal concentration of luminol is 0 . 5 mm . [SEP]
[CLS] we examined the effect of the h 2 o 2 concentrations on the cl signals . [SEP]
[CLS] as shown in figure 3b , with the increase of h 2 o 2 concentration from 0 . 1 to 2 mm , the cl intensity and imaging brightness increases accordingly . [SEP]
[CLS] however , the concentration over 2 mm inhibits the catalytic activity of snazyme . [SEP]
[CLS] therefore , h 2 o 2 concentration is selected as 2 mm in the following experiments . [SEP]
[CLS] furthermore , snazyme achieves the best catalytic performance at ph 12 . 0 ( figure 3c ) . [SEP]
[CLS] further increase of ph results in the decrease of cl signal , which is possibly ascribed to the collapse of g4 structure in snazyme under strong alkaline condition and the caused destruction of catalytic activity . [SEP]
[CLS] the following experiments are carried out at this optimal ph . [SEP]
[CLS] different from the mir - 499 detection , to better discriminate the signals from mir - 133a , we introduced fluorescein as energy acceptor of chemiluminescent B-property luminol for mir - 133a detection on the arrays . [SEP]
[CLS] on the one hand , there is a well overlap between the absorption spectrum of fluorescein and the cl emission spectrum of luminol , and on the other hand , the concentration of luminol reaches the mm level , which greatly shortens the donor - acceptor molecular distance . [SEP]
[CLS] therefore , efficient cret from luminol to fluorescein is directly initiated by physically mixing . [SEP]
[CLS] cyan - to green - color cl emission can be achieved by adjusting the molar ratio of luminol and fluorescein ( figure 3d ) . [SEP]
[CLS] moreover , the luminescent B-property intensity decreased gradually while increasing the fluorescein concentration . [SEP]
[CLS] this can be due to the fact that too high concentration of fluorescein can result in strong quenching of fluorescence B-property although higher cret efficiency can be obtained ( figure 3e ) . [SEP]
[CLS] as a compromise condition , the molar ratio of luminol and fluorescein is set to be 4 : 1 , with green colored light emission emerging , which is clearly distinguished by the blue light from mir - 499 analysis . [SEP]
[CLS] although aunp B-nanoparticle is well known to show strong quenching effect on fluorescent B-property molecule close to it , both mnazyme @ aunp B-nanoparticle nanoprobe B-nanoparticle and the free fluorescein sodium B-material formed under alkaline condition are negatively charged , making it difficult to approach each other , thus , the quenching effect of aunp B-nanoparticle on fluorescence B-property of fluorescein is greatly reduced and strong green luminescent B-property intensity is still generated . [SEP]
[CLS] furthermore , the incubation B-technique time of mirna with the sensing nanoprobe B-nanoparticle has a critical impact on the amplification reaction . [SEP]
[CLS] as seen in figure 3f , the signal ratio in the presence to absence of target maximized after 3 h , indicating that the cleavage reaction finished completely at this time , and most of g - rich dna caged in hpdna is released to form snazyme . [SEP]
[CLS] thus , this optimal reaction time is utilized in the following study . [SEP]
[CLS] under the above - optimized conditions , the cl responses to mir - 499 and mir - 133a at different concentrations were tested . [SEP]
[CLS] 4a and figure 4d show the increase of visualized imaging brightness and the quantitative intensity corresponding to the increased mirna concentration . [SEP]
[CLS] the calibration curves show the cl intensity is well linearly related to the mirna concentration varying in the range of 1 . 0 × 10 a 7 - 1 . 0 × 10 a 14 m ( figure 4b and figure 4d ) . [SEP]
[CLS] the regression equation is g = 8 . 312 c + 132 . 472 ( r 2 = 0 . 9986 ) for mir - 499 detection , g = 6 . 981 c + 110 . 518 ( r 2 = 0 . 9947 ) for mir - 133a detection . [SEP]
[CLS] where c is the logarithm of mirna concentrations and g is the imaging gray value . [SEP]
[CLS] the lod was 0 . 53 fm and 0 . 22 fm for mir - 499 and mir - 133a detection ( s / n = 3 ) , respectively . [SEP]
[CLS] the detection results are compared with previously reported methodologies , as listed in table s2 . [SEP]
[CLS] the sensitivity of our proposed sensing strategy is comparable or even superior to that of most methods , and the linear detection range is wider , which imply the prospect in the sensitive detection of low concentration of mirna biomarkers B-property . [SEP]
[CLS] the high sensitivity may be attributed to several major factors : ( i ) mnazyme can function as a powerful tool for signal amplification . [SEP]
[CLS] trace amounts of mirna can initiate the liberation of copious cleaved products , finally yielding amplified cl signal . [SEP]
[CLS] ( ii ) functionalization of mnazyme on gold B-material core B-material increases the local concentration of mnazyme , target - boosted cleavage reaction is confined on the aunp B-nanoparticle surface , which might be conducive to the improved amplification efficiency . [SEP]
[CLS] ( iii ) separation function of gold B-material core B-material is greatly helpful to reduce the background signal from unbound hemin in supernatant , then a higher ratio of signal to background can be achieved . [SEP]
[CLS] several ami - related mirnas were employed as potential interferences to evaluate the selectivity and specificity of the nanoprobe B-nanoparticle for target detection . [SEP]
[CLS] as shown in figure 4c and 4f , only the presence of target can bring about a remarkable increase of cl intensity , whereas no obvious responses are observed even in the presence of a high concentration of interveners . [SEP]
[CLS] furthermore , the cl response for mixture containing target mirna and other three interveners has hardly any difference compared with that for only target , which suggests the negligible cross - reactions . [SEP]
[CLS] these data reveal that the developed biosensor has good selectivity and anti - interference ability . [SEP]
[CLS] the storage stability of the nanoprobe B-nanoparticle was investigated . [SEP]
[CLS] as shown in figure s3 , the cl response of the nanoprobe B-nanoparticle to target mirna has no distinct change after seven days of storage , which is comparable to that of freshly prepared nanoprobe B-nanoparticle . [SEP]
[CLS] we also synthesized seven batches of mnazyme @ aunp B-nanoparticle nanoprobe B-nanoparticle to test the reproducibility ( figure s4 ) . [SEP]
[CLS] the consistence of different batches is determined by the subtle distinction of cl intensity . [SEP]
[CLS] the relative standard deviations ( rsd ) of intra - and inter - assays are 1 . 05 % and 2 . 13 % , respectively , indicating the good precision of proposed biosensor . [SEP]
[CLS] in addition , the stability of aunp B-nanoparticle probe is well known to be adversely affected by high ionic concentration in serum . [SEP]
[CLS] we incubate B-technique the mnazyme @ aunp B-nanoparticle with nacl at diverse concentrations , and negligible change in the uv - vis absorption peak is observed even after the incubation B-technique with 1 m nacl ( figure s5a ) , showing good tolerance of the nanoprobe B-nanoparticle to salt B-material . [SEP]
[CLS] this result is in accordance with the that of age analysis ( figure s5b ) , in which no clear distinction in bands shifts can be seen . [SEP]
[CLS] we also compare the cl response of the nanoprobe B-nanoparticle to target spiked in buffer and 10 % human serum , respectively ( figure s6 ) . [SEP]
[CLS] 97 % of cl intensity in buffer is retained while carrying out the same experiment in 10 % serum , which lays a good foundation for the accurate detection in real sample . [SEP]
[CLS] these excellent properties can be ascribed to that the densely modified oligonucleotides layer in mnazyme @ aunp B-nanoparticle nanoprobe B-nanoparticle increases the resistance of aunp B-nanoparticle against aggregation and oligonucleotides against serum degradation , in line with the previously reported studies . [SEP]
[CLS] in addition , the resultant snazyme is transferred to perform cl reaction in buffer rather than initial serum , which contributes to the reduced interference in heterogeneous matrix and the improved signal reproducibility . [SEP]
[CLS] all the above investigations suggest the good stability of mnazyme @ aunp B-nanoparticle nanoprobe B-nanoparticle and the potential application in detection of clinical samples . [SEP]
[CLS] recovery experiments were carried out to evaluate the reliability of the sensing nanoplatform . [SEP]
[CLS] based on the great selectivity of the nanoprobes B-nanoparticle for their respective targets , as demonstrated in figure 4c and figure 4f , mixture solutions containing different concentration of multiple target mirnas are respectively spiked into two aliquots of healthy human serum for simultaneous visual detection on the imaging array ( row 1st for mir - 499 , row 2nd for mir - 133a ) . [SEP]
[CLS] to further outstand the multiplexing capabilities of clia analysis , the signals from all five serum samples are captured on one picture , rather than batch - by - batch , which facilitates the high - throughput quantification of multiple samples . [SEP]
[CLS] as shown in figure 5a , the detection zones for mir - 499 and mir - 133a show blue and green colored cl emission , respectively . [SEP]
[CLS] the quantitative results in figure 5b show that higher cl intensity yielded corresponding to the increase of mirna concentration , which is consistent with the changes in brightness in figure 5a . [SEP]
[CLS] as listed in table 1 , satisfactory recovery rates ( 95 % - 111 % ) are obtained , and the rsd are below 7 . 96 % . [SEP]
[CLS] all as - mentioned results demonstrate the good accuracy of our proposed sensing nanoplatform for multiple mirnas detection in serum . [SEP]
[CLS] we tried to directly detect the endogenous circulating mirna in human serum . [SEP]
[CLS] firstly , in order to verify the practicability of our sensing platform for detection of endogenous mirna , we prepared mnazyme @ aunp B-nanoparticle nanoprobe B-nanoparticle for mir - 16 detection by simply tuning the recognition sequence . [SEP]
[CLS] mir - 16 is a kind of nondiagnostic mirna with moderate abundance in plasma or serum and broadly used as an endogenous control . [SEP]
[CLS] s7a shows that the luminescent B-property brightness increased with the increase of mir - 16 concentration , and the brightness was well linearly related to the mir - 16 concentration in the range of 1 . 0 × 10 a 7 - 1 . 0 × 10 a 14 m . the regression equation is g = 8 . 602 c + 140 . 219 ( r 2 = 0 . 9876 ) , and the lod is calculated as 0 . 88 f / m ( s / n = 3 ) . [SEP]
[CLS] as seen in figure 6a , input of mir - 16 results in an obviously enhanced cl signal , and favorable specificity for mir - 16 against other interveners can be achieved . [SEP]
[CLS] these results suggest the feasibility of our proposed nanoplatform to selectively detect mir - 16 and the high university for different mirnas analysis . [SEP]
[CLS] recent studies suggested that circulating mirnas are stably present in circulation by extracellular vesicles enclosure , such as exosomes , or forming complexes with associated proteins B-material , which protect them from the rnase activity . [SEP]
[CLS] this feature is beneficial to their high stability but deterrent to the hybridization with dna probe . [SEP]
[CLS] to make it possible to directly detect endogenous mirnas in serum , according to previously reported protocols , tween 20 and proteinase k ( pk ) were employed to release mirnas from exosome and protein B-material in serum , respectively , and the abundance of mir - 16 in the serum lysate was determined . [SEP]
[CLS] as shown in figure 6b , the cl signal obtained from treated serum is higher than that obtained from untreated one , attributed to the successful release of mir - 16 from the protection of exosome or protein B-material for the initiation of amplification reaction . [SEP]
[CLS] therefore , our method is potential for detection of endogenous mir - 16 . [SEP]
[CLS] furthermore , the cl signal obtained from serum lysate is higher than that from 100 fm mir - 16 spiked serum , suggesting the concentration of mir - 16 in serum is more than 100 fm , which is in agreement with previously reported concentration range in plasma ( 200 750 fm ) . [SEP]
[CLS] to further figure out the mechanism underlying the remarkable stability of mir - 16 in serum , the serum samples from healthy human were treated by tween 20 and pk , respectively , and the responses to the serum lysate were determined simultaneously . [SEP]
[CLS] as shown in figure 6c , addition of the processing reagents leads to negligible change of cl signal , suggesting the reagents have little impact on the sensing nanoplatform . [SEP]
[CLS] the nanoplatform has no obvious response to the treatment of serum with only tween 20 . [SEP]
[CLS] in contrast , after treatment of pk , a higher cl signal comparable to that obtained from the serum treated by both tween 20 and pk is obtained . [SEP]
[CLS] these results suggest that the majority of mir - 16 is associated with protein B-material complex rather than exosome , and the formation of protein B-material complex might be the major protection mechanism for the high stability of mir - 16 . [SEP]
[CLS] this is in good consistence with the previous studies , which reported that mir - 16 is exclusively associated with argonaute2 ( ago2 ) protein B-material complex . [SEP]
[CLS] mir - 16 and mir - 133a were then chosen as the dual targets to perform simultaneous detection experiments , based on the fact that mir - 133a can usually reach a low but relatively higher abundance than mir - 499 after onset of ami . [SEP]
[CLS] as shown in figure 6d , comparable levels of mir - 16 are readily determined in all the serum samples collected from healthy humans and ami patients . [SEP]
[CLS] moreover , though our sensing nanoplatform may be unable to meet the need to directly quantify the extreme low abundance of endogenous mirnas related to ami , especially in diluted serum , a slightly higher cl signal was still obtained from serum samples from all ami patients compared to all healthy humans , which is in accordance with the detection results of mir - 499 in serum ( figure s7 ) . [SEP]
[CLS] this can be ascribed to the fact that there are more than 20 , 000 proteins B-material and nucleic B-material acids I-material in serum , the nanoprobe B-nanoparticle for mir - 133a and mir - 499 is not selective enough for their family members , which are all released after treatment . [SEP]
[CLS] all the same , our proposed nanoplatform is still of great prospect for simultaneous detection of endogenous mirnas in serum by further lowering the lod and improving selectivity , which can be achieved by combining with other amplification strategy with higher specificity to convert the target mirna into lots of universal dna inputs for mnazyme @ aunp B-nanoparticle probe . [SEP]
[CLS] in summary , we designed a cret based multicolor sensing nanoplatform with a portable smartphone as the mini - detector for simultaneous and sensitive detection of multiple circulating mirnas . [SEP]
[CLS] we construct universal mnazyme @ aunp B-nanoparticle nanoprobes B-nanoparticle for different target mirnas detection related to ami . [SEP]
[CLS] the nanoprobe B-nanoparticle can be activated for the amplified cleavage reaction once the addition of target and snazyme with high catalytic activity for luminol - h 2 o 2 cl reactions and luminol - fluorescein - h 2 o 2 cret reaction is generated . [SEP]
[CLS] blue and green colored imaging signals corresponding to two different targets are generated . [SEP]
[CLS] it enables the construction of a multicolor sensing platform for simultaneously visualizing multiple mirnas by combining with clia . [SEP]
[CLS] with mir - 499 and mir - 133a as the model targets , the sensing nanoplatform not only exhibits high sensitivity and specificity , but also high universality for different target mirna detection by simply tuning the recognition sequence of the nanoprobe B-nanoparticle . [SEP]
[CLS] furthermore , the nanoplatform shows great practicability in simultaneously detecting endogenous circulating mirnas and gives an insight into the mechanism underlying the remarkable stability of mir - 16 in serum . [SEP]
[CLS] our proposed sensing nanoplatform opens a door for simultaneous and sensitive cl imaging analysis of multiple biomarkers B-property related to specific diseases . [SEP]
[CLS] ( a ) native page analysis of mnazyme amplification reaction . [SEP]
[CLS] lane 1 : s1dna ; lane 2 : s2dna ; lane 3 : s1dna + s2dna ; lane 4 : g4 reference ; lane 5 : hpdna ; lane 6 : s1dna + hpdna ; lane 7 : s2dna + hpdna ; lane 8 : s1dna + s2dna + hpdna ; lane 9 : mirna + s1dna + s2dna + hpdna with rna - cleaving site ; lane 10 : mirna + s1dna + s2dna + hpdna without rnacleaving site ; the blue rectangle marks the band position of cleavage product ( b ) age analysis of mnazyme @ aunp B-nanoparticle amplification reaction . [SEP]
[CLS] lane 1 : linker @ aunp B-nanoparticle ; lane 2 : hpdna @ aunp B-nanoparticle ; lane 3 : mnazyme @ aunp B-nanoparticle ; lane 4 : mnazyme @ aunp B-nanoparticle without rna - cleaving site in the presence of target ; [SEP]
[CLS] figure 2 . lane 5 : mnazyme @ aunp B-nanoparticle with rna - cleaving site in the presence of target . [SEP]
[CLS] ( c ) measurement of cl spectra with ( red line ) and without ( blue line ) target mirna ; ( d ) cl kinetic curve in the presence of target . [SEP]
[CLS] the mirna concentration is 100 nm . [SEP]
[CLS] effects of the concentration of ( a ) luminol and ( b ) h 2 o 2 on luminescent B-property brightness and quantitative intensity ; ( c ) effects of ph of br buffer on luminescent B-property brightness and quantitative intensity ; effects of the molar ratio of luminol and fluorescein on ( d ) luminescent B-property brightness and quantitative intensity and ( e ) cret spectra ; ( f ) effects of the target mirna initiated amplification reaction time on cl intensity . [SEP]
[CLS] 4 . cl images captured on the imaging array and quantitative intensity for different concentration of ( a ) mir - 499 and ( d ) mir - 133a detection ; the calibration curves for ( b ) mir - 499 and ( e ) mir - 133a detection ; the specificity analysis of the mnazyme @ aunp B-nanoparticle nanoprobe B-nanoparticle for ( c ) mir - 499 and ( f ) mir - 133a detection [SEP]
[CLS] 5 . ( a ) image and ( b ) quantitative cl intensity for the simultaneous detection of multiple mirnas spiked healthy human serum on cl imaging array . [SEP]
[CLS] ( a ) the specificity analysis of the mnazyme @ aunp B-nanoparticle nanoprobe B-nanoparticle for mir - 16 detection ; ( b ) mir - 16 detection of the mnazyme @ aunp B-nanoparticle nanoprobe B-nanoparticle in ( 1 ) untreated serum ; ( 2 ) serum treated by pk and tween 20 ; ( 3 ) 100 fm mir - 16 spiked serum ; ( c ) simultaneous detection of endogenous mir - 16 in two serum samples . [SEP]
[CLS] ( 1 ) te blank ; ( 2 ) te treated by pk and tween 20 ; ( 3 ) serum ; ( 4 ) serum treated by tween 20 ; ( 5 ) serum treated by pk ; ( 6 ) serum treated by pk and tween 20 ; ( d ) simultaneous detection of endogenous mir - 16 and mir - 133a in serum samples from healthy humans and ami patients . [SEP]
[CLS] detection results of the recovery study . [SEP]
[CLS] abstract : rational and generalisable methods for engineering surface functionality will be crucial to realising the technological potential of nanomaterials B-material . [SEP]
[CLS] nanoparticlebound dynamic covalent exchange combines the errorcorrecting and environment - responsivef eatures of equilibrium processes with the stability , structuralp recision , and vast diversity of covalentc hemistry , d efining an ew and powerful approachf or manipulatings tructure , function and properties at nanomaterial B-material surfaces . [SEP]
[CLS] dynamic covalent nanoparticle B-nanoparticle ( dcnp ) building blockst hus present aw hole host of possibilities for constructing adaptive systems , devices andm aterials that incorporateb oth nanoscale and molecular functional components . [SEP]
[CLS] a tt he same time , dcnps have the potential to reveal fundamental insights regarding dynamic and complex chemical systems confined to nanoscale interfaces . [SEP]
[CLS] tremendous synthetic and analytical advances over more than two decades have openedu panew region of chemical space on the nanoscale , leadingt oe xcitement on account of the extraordinary properties observed for myriadt ypes of nanoparticles B-nanoparticle ( nps B-nanoparticle ) and other materials in this size regime . [SEP]
[CLS] virtually any potentiala pplication of nanomaterials B-material will require careful control over aw ide range of features , as well as integration with any number of other components . [SEP]
[CLS] however , s trategies for functionalising and assembling these new chemical entitiesh ave not kept pace with advances in their synthesis and there is now ap ressing need to bridget his capability gap if the technological potentialo fn anomaterials is to be realised . [SEP]
[CLS] the chemical nature of nanoscale surfaces is inherently critical to the behaviour of all nanomaterials B-material . [SEP]
[CLS] the canonical example is am onolayer - stabilised nanoparticle B-nanoparticle ( np B-nanoparticle ) , in which an inorganic B-material core I-material is stabilisedb yasurface - bound layer of molecular ligands . [SEP]
[CLS] the properties of these multicomponent systems are defined both by the material B-material , size and shape of the inorganic B-material core I-material and by the characteristics of the surface - bound species ( figure 1a ) . [SEP]
[CLS] furthermore , the ligand shell B-material provides ah andle for attaching other components - be they molecules , surfaces or other nanomaterials B-material . [SEP]
[CLS] despite al ong history , the controlled synthesis of nps B-nanoparticle of anys ort remains challenging and the range of compatible surface functionalities restricted . [SEP]
[CLS] ' ligand ex - change ' , w hereby temporary surface - bound molecules are replaced in their entirety ( figure 1b , i ) h as been widely exploited for some systems , yet still presentss everal practical challenges , hasl imited scope for linking to complex functionalities or non - molecular components , and is not applicable to all core B-material materials . consequently , r obusta nd predictable methods for modifying np B-nanoparticle - bound surfaces peciesi napostsynthetic fashion will be criticalf or manipulatingn ps , tuning their properties , and assembling devicesa nd materials from these nanoscale buildingb locks . [SEP]
[CLS] ideally these methodologies should be independentoft he underlying nanomaterial B-material and therefore generalisable across aw ide array of nanostructures that can have moleculesa ttached to the surface . [SEP]
[CLS] the currents trategies for in situ modification of np B-nanoparticle - bound molecules ( figure 1b , i i - iv ) have considerable drawbacks that prevent them offering universal solutions . [SEP]
[CLS] e xploitingn oncovalent interactions between biomolecules - and , in particular , h ybridisation to single - stranded oligonucleotides - has proven particularly successful , leading to myriadn p - based devicesa nd materials . [SEP]
[CLS] however , t he structurala nd chemical stabilityo f biomolecules is only maintained within tightly defined conditions ; c omplex high molecular weighta rchitecturesl imit the scope for structural and chemical variation ; a nd molecularlevel characterisation can be extremely challenging . [SEP]
[CLS] nonbiomolecular approaches have the potential to draw upon the full diversity of synthetic chemistry for optimisings tructure , function andp roperties . [SEP]
[CLS] innovative designst hat exploit noncovalent interactions for np B-nanoparticle functionalisation , aggregation , and surfacei mmobilisation , have recently been explored , but still cannotm atch the stability , specificity , a nd selectivity of dna . [SEP]
[CLS] stimuli - responsive molecular switchesi ncorporatedw ithin np B-nanoparticle ligand shellsh ave recently produced an umber of impressive dynamic np B-nanoparticle systems , in particularf or control of np B-nanoparticle aggregation . [SEP]
[CLS] the challenges for this relativelyu nexplored strategy include avoiding degradation processes that lead to switch fatigue , and extending the switching phenomena to properties beyondt he aggregation state . [SEP]
[CLS] covalent modification of np B-nanoparticle surface functionality is an obvious attractive alternative and has indeed been intensively ex - plored . [SEP]
[CLS] owever , t ypical examples rely on kinetically controlled reactions , often inspired by mild , robust and high - yielding protocols developed for bioconjugation applications . [SEP]
[CLS] these only offer one - shot transformations and fail to match the programmability of oligonucleotide approaches . [SEP]
[CLS] dynamic covalent bonds - which , under appropriate conditions , can form and break many times over , w hile under alternative conditions can be kinetically inert - offer au nique solution to these issues ( figure 1b , v ) , and the concept of dynamic covalent nanoparticle B-nanoparticle ( dcnp ) buildingb locks opens up ah ost of possibilities for repeatedly switching - and subtly tuning - np B-nanoparticle functionalisation and properties , and for controlling np B-nanoparticle self - assembly . [SEP]
[CLS] processes in whichc ovalentb onds are formed reversibly under conditions of thermodynamic controlh ave for decades been recognised as important in certainb ranches of chemistry , i ncluding carbohydrates B-material tereochemistry and polymer B-material synthesis . [SEP]
[CLS] inspired by the prospecto fe xtending the newly established principles of supramolecular chemistry into the molecular world - and enabled by advances in analyticalt echnology that made feasible the characterisation of mixtures - int he late 1990s , pioneering groups developed dynamic covalent chemistry as am eans of combining the strength , d irectionality and potential for kinetic stability of covalentb onds , with the benefits of error - correcting self - assembly , p roduct stabilityc ontrol , and stimuli - responsiveness . [SEP]
[CLS] creating equilibrating mixtures of molecular species that could adapt in response to molecular recognition interactions or environmental changes afforded an ew strategy for template - induced selection of optimised supramolecular hosts , guestsa nd catalysts B-property , or forh ighyield assembly of complex molecular architectures . [SEP]
[CLS] the intervening years have seen rapid progress , leading to severalremarkable achievements , including selection of unforeseen macrocyclic receptors from dynamic combinatorial libraries , construction of three - dimensional molecular cages and capsules , preparation of hitherto inaccessible interlockedm olecular architectures , crystallisation of infinite covalento rganic frameworks , self - replication from ad ynamic mixture of competing molecules , ando peration of sophisticated artificial molecular nanomachines . [SEP]
[CLS] logicale xtensions of these concepts have led to the self - assembly of responsive and adaptive materials , including dynamic covalentp olymers and selfselectings mall molecule ' dynablocks ' . [SEP]
[CLS] characterisation of inherently dynamic and multicomponent chemicals ystems is af ormidable challenge in any setting , even more so when movinga way from the familiar solutionstate environmenti nto condensed phases or on to surfaces . [SEP]
[CLS] dynamicc ovalent processes occurring at interfaces have been correspondingly demanding to establish . [SEP]
[CLS] p ioneering studies exploitede merging characterisation techniques including atomic B-technique force I-technique microscopy I-technique and confocal fluorescence B-technique microscopy I-technique to visualise erasable molecular patterns and directed surface diffusiono fc ovalently attachedm acromolecules , operating by reversible condensation and hydrolysiso fi mines on self - as - sembled monolayers ( sams ) . [SEP]
[CLS] more recently , p h - controlled patterningofi mine sams was achieved through dynamic covalent selection from am ixture of small - molecule amines B-material or aminecontaining proteins B-material . [SEP]
[CLS] multiple orthogonal dynamicc ovalent processes have since been combined to construct an impressive series of chemically responsive multicomponent surface architectures in which several coaxially aligned photoconductive channels can be grown normal to the surface with control over patterning parallel to the substrate . [SEP]
[CLS] enabled by developments in scanning B-technique probe I-technique microscopy I-technique , potentially reversible covalentr eactions have also been investigated for the constructiono fe xtended noncovalent and covalent atomically thin 2d networks at solid surfaces under vacuum , at the solid / liquid interface and at the liquid / air interface [SEP]
[CLS] recently , d ynamic covalente xchange was demonstratedi no ne such system , where the free energy of surface physisorption can act as ad riving force for constituent selection and amplification from adynamic library of imines B-material . [SEP]
[CLS] the emergence of surface - confined dynamic covalent systems now suggests several compellingp ossibilities , including spatiotemporalc ontrol over the exchange process , leading to the prospect of creatings urface patterns or achieving compartmentalised chemical behaviour spanning severall ength scales . [SEP]
[CLS] these studies have helped to reveal the often considerable influenceo ft he unique surfacem icroenvironment when chemicalprocesses are confined to interfaces . [SEP]
[CLS] these contemporaneous developments for both nanomaterials B-material and dynamic molecular systemsn ow set the stage for extending dynamic covalent reactions onto nanosurfaces . [SEP]
[CLS] intermediate B-property in size between macromolecules and extended surfaces , applyingr eversiblec ovalentr eactions to nanosurfaces allows the principles of equilibrium control to be appliedt oa chieve responsive and adaptive behaviour on the nanoscale , while exploiting the precisiona nd diversity of synthetic molecular structures . [SEP]
[CLS] working with nanosized chemical entitiesi ntroduces several new challenges , but also opens up excitingp ossibilitiesf or elucidating fundamental features of chemical processest aking place at interfaces that are just not availablef or extended substrates . [SEP]
[CLS] in ah andful of cases , c argoes have been attached to and then released from nanomaterials B-material through formation and cleavage of the same covalent bond . [SEP]
[CLS] for example , inspired by stationaryp hases developed for affinity chromatography B-technique , b oronic acids have been associated with magnetic B-property nps B-nanoparticle and used to isolate and enrich polyhydroxylated biomolecules from biological samples followed by release for analysis by mass spectrometry . [SEP]
[CLS] boronic B-material acid functionalised gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) have also been used to reversibly trap molecular cargoes in the pores of silica solids that display polyhydroxyl surface functionality ; c argo release being achieved by lowering ph , consistent with am echanism involving ph - switchedb oronate ester condensation / hydrolysis . [SEP]
[CLS] as imilara pproachh as been used fort he reversible gating of mesoporous silica nano - particles ( msnps ) , where the silica surface was functionalised with boronic B-material acids and the capping unit was the polyhydroxylated glycoprotein insulin . [SEP]
[CLS] moreover , i mine linkages B-property have been used to attach and detach hydrophobic B-property dendrons on the surfaceo fs ilica - coated superparamagnetic B-property microspheres and both hydrazones B-material and oximes have been used for loading and releaseo fs imple carbonyl - containing units on msnps . [SEP]
[CLS] ta king place on sparsely - functionalised irregular surfaces , direct characterisation of the molecular processes in such systems is af ormidable challenge . [SEP]
[CLS] these examples all exploit efficient interconversion of functional groups through high - yielding condensation and hydrolysis reactions . [SEP]
[CLS] undere ach set of conditions , formation or cleavage of ac ovalent bond is essentially irreversible and is therefore conceptually similar to other covalents urfacem odifications . [SEP]
[CLS] however , d ynamic covalent exchange processes would allow subtle and responsive tuning of the np B-nanoparticle surfacef unctionality , c ontrolled by thermodynamic differences between an umber of exchanging species , and exhibiting constitutional adaptation in response to myriad parameters and interactions . [SEP]
[CLS] dynamic covalentn anoparticle ( dcnp ) buildingb locks therefore introduce ap owerful new approach to nanomaterial B-material surface engineering , and the construction of responsive np B-nanoparticle - based devices , assemblies and materials . [SEP]
[CLS] myriad dynamic biomolecular reaction and recognition processes take place on phospholipid bilayers ; f or example , disulfide exchange with exofacial thiols B-material on membrane - associated proteins B-material can mediate signalling pathways , viral entry and nonendosomal cellular B-event uptake I-event processes I-event . [SEP]
[CLS] in addition to the considerable analytical challenges , achieving dynamic covalent reactions on artificial nanoscale membranes must also overcome the often limited stabilityo fs elf - assembled amphiphile B-property structures . [SEP]
[CLS] nevertheless , otto and co - workers successfully demonstrated dynamic covalent thioester exchange on the surfaceo f large ( d % 200 nm ) unilamellar B-material vesiclesa I-material ssembled from egg phosphatidyl choline and 10 mol % o famembrane - anchored thioester derivative 1 ( figure 2 ) . [SEP]
[CLS] surfacec onfinementw as found to retard the dynamic covalentt hioester exchange reactions only to am odest extent and equilibrium control was maintained . [SEP]
[CLS] significantly , t hioester libraries equilibratedo n these liposome B-nanoparticle surfaces exhibited markedly differentc ompositions compared to bulk solution . [SEP]
[CLS] f or example , when amphiphilic B-property bis - thioester 1 was equilibrated with dithiol 2 , ahigh proportion of linear compounds ( formed by cross - linking several membrane - anchored units ) w as observed , in contrastt o the small monomeric and dimeric products formed in solution ( figure 2b ) . [SEP]
[CLS] this can be attributed to the higherl ocal concentration of 1 at the interface , compared to when at the same overall concentration in bulk solution . [SEP]
[CLS] our group first explored the dcnp concept by using aunps B-nanoparticle ( d % 3 . 0 nm ) bearing ah ydrazone - terminated surface monolayer ( aunp B-nanoparticle - 3 ; f igure 3 ) . [SEP]
[CLS] on introducing an aldehyde B-material exchange unit , such as 4 , t ogether with an acid catalyst B-property , the monolayer composition couldb er eliably and reversibly modified through dynamic covalent hydrazone B-material exchange . the single - component monolayer achievese xtremelyh igh surfaced ensities of exchangeable units , which couldb eq uantitativelym odified ; o r alternatively , m ixed monolayers could be accessed with compositions defined by the thermodynamic stabilityo fe ach hydrazone B-material and the reaction conditions . [SEP]
[CLS] simply removing the acid catalysta fforded kinetically stable products that could be isolated , purified , characterised , and stored without further changes to the monolayer . [SEP]
[CLS] in contrast to larger and sparsely functionalised nanosurfaces - which behave in many respects as curved analogueso f 2d extendeds ams - these small colloidal nps B-nanoparticle , stabilisedb y ad ense , single - component monolayer , a re perhaps better considered as au nique category of 3d sams , with many features akin to large macromolecules [SEP]
[CLS] this pseudomolecular nature allowsa nalytical tools such as nmr spectroscopy B-technique to be exploited for in situ characterisation of surface - bound structures and processes ; o pportunities that are not applicable to analogous 2d systemso rs parselyf unctionalised larger nps B-nanoparticle . [SEP]
[CLS] monitoring dcnp hydrazone exchange by fnmr spectroscopy B-technique allowed both np B-nanoparticle - bound and unbound speciest ob eq uantified in real time ( figure 3b , c ) . [SEP]
[CLS] as might be intuitively expected , the surface - confined process proceeded more slowly than analogous solution - phase reactions . [SEP]
[CLS] however , t he retardation was relatively modesta nd dynamic covalente xchange was found to be significantly faster than analogous ligand exchange processes at the auasb ond , which of course also first require each adaptedf rom ref . [ 45 ] under the termsofc c - by 2 . 0 . new ligand to be synthetically prepared . [SEP]
[CLS] the ability to characterise molecular - level details promises aq uantitative understanding of np B-nanoparticle - bound dynamic covalent reactions that will underpin rational syntheticm ethods for manipulating dcnps with predictabilitya nd precision . [SEP]
[CLS] one of the advantages of the dcnp concept is the ability to exploit the vast toolboxo fs ynthetic organicc hemistry to tune virtually any aspect of the surface - bound molecular structure , properties or reactivity . [SEP]
[CLS] w hereas hydrazone - exchange reactions tend to reach equilibrium on at imescale of minutes to hours under acid - or nucleophile - catalysed conditions , the reaction between ab oronic acid and various dihydroxy compounds to yield boronatee sters occurs extremelyr apidlyi nt he presence of lewis bases . [SEP]
[CLS] hydrazones B-material and boronate B-material esters therefore represent two chemically orthogonal dynamic covalent functional groups with an attractive contrasti nk inetic characteristics . [SEP]
[CLS] we recently developed aunp B-nanoparticle - 7 ( d % 3 . 4 nm ; f igure 4 ) , stabilised by ah omogenous monolayer of structurally simple boronic B-material acids , which reacts with 1 , 2 - diols in the presence of al ewis base to provide np B-nanoparticle - bound boronate B-material esters . [SEP]
[CLS] in situ analysisb y 19 fnmr spectroscopy B-technique revealed that boronate B-material ester formation and dynamic covalent exchange ( when more than one diolcontaining exchange unit is introduced ) occurs rapidly and reversibly , a chieving high surface - saturation concentrations . [SEP]
[CLS] mixed boronate B-material ester monolayers display pathway - independent compositions that adapt to changes in the mixtureo fe xo - genouslyi ntroduced diols , confirming that dynamic covalent exchange on the np B-nanoparticle surfacer emains an equilibrium process under thermodynamic control ( figure 4 ) . [SEP]
[CLS] independently , otto and co - workers have exploited kinetically facile imine B-material exchange to achievet emplate - driven dynamic covalentn pf unctionalisation ( figure 5 ) . [SEP]
[CLS] on treating aunp B-nanoparticle - 12 / 13 ( d % 11 . 7 nm ) , which is stabilised by am ixed monolayer incorporating approximately 10 mol % a ldehyde B-material ligand 12 , w ith mixtures of simple primary B-material amines I-material , negligible np B-nanoparticle - bound imine B-material formation was observed . [SEP]
[CLS] however , o ni ntroducing short ( 16mer ) oligonucleotides , selective uptake of amines B-material from solution was observed . [SEP]
[CLS] this could be explained by thermodynamic stabilisation of np B-nanoparticle - bound imines B-material as ar esult of multivalent interactions between the imine - functionalised np B-nanoparticle surface and the oligonucleotide template . [SEP]
[CLS] significantly , t he degree of functionalisation , a nd the composition of np B-nanoparticle - bound imine B-material libraries , depended on the oligonucleotide sequence employed ( figure 5b ) : the dynamic covalent ligand shell B-material can adaptt oo ptimise binding to each specific dna template . [SEP]
[CLS] very recently , the same approach was extendedt ot he formation of npbound hydrazones B-material , w hereby mixtures of two different npbound hydrazones B-material were obtainedd epending on the dna template sequence . [SEP]
[CLS] this second - generation system also has the advantage of employing an eutral surface monolayer , w hich minimises nonspecific binding to the anionic oligonucleotide templates . [SEP]
[CLS] to gether , t heses tudies establish that dynamic molecular exchange chemistry can be used to selectively control np B-nanoparticle functionalisationt hrougha daptive surfacem onolayers , as well as pointingt ot he potential insights - and considerable chal - ( red ) . [SEP]
[CLS] in situ analysis by fnmr spectroscopy B-technique revealsidentical mixed monolayer equilibria are reached via two different routes ( r = n - methylmorpholinium ) [SEP]
[CLS] adapted from ref . [ 48 ] with permission from the royal society of chemistry . [SEP]
[CLS] 3 . a ) dynamically reconfigurable aunp B-nanoparticle - bound hydrazone B-material monolayers ; b ) in situ characterisation by [SEP]
[CLS] fnmr spectroscopyallows molecular - level characterisationo fmonolayer composition ; c ) real - time tracking of dynamic covalent exchanger eactions [SEP]
[CLS] adaptedw ith permission from ref . [ 46 ] , copyrightwiley - vch verlag gmbh & c o . kga , weinheim . lenges - associated with characterising dynamic covalentp rocesses in this unconventional environment . the diversity of abiotic synthetic chemistry may now be exploited to develop aw hole series of dcnp building blockst hat would constitute au niversal set of environment - responsive ' nanoparticle B-nanoparticle synthons ' with optimised reactivity and properties for aw ide range of applications . [SEP]
[CLS] mild methodsf or postsynthetic np B-nanoparticle property control are highly desirable and would have significant benefits for nanomaterial B-material handling and processing . [SEP]
[CLS] we reportedh ow hydrazone B-material exchange can be used to achieve reversible and stimuli - responsive control over dcnp physicochemical properties by appropriate choiceo fm olecular exchange unit ( figure 6 ) . [SEP]
[CLS] aunp B-nanoparticle - 3 was colloidally stable only in polar B-material aprotic I-material solvents I-material , yet hydrazone B-material exchange with hydrophobic B-property aldehyde B-material 14 produced aunp B-nanoparticle - 15 , w hich exhibited colloidal stability in less polar B-material organic I-material solvents I-material . [SEP]
[CLS] likewise , e xchange with charged aldehyde B-material 16 produced aunp B-nanoparticle - 17 , w hich was colloidally stable in water B-material . [SEP]
[CLS] i nc ontrast to alternative approaches such as ligand exchange or noncovalent encapsulation , dcnpsc ombine excellent stability in each state with complete reversibility ; a ny one state can be accessed from any of the others , a nd each state corresponds to as ingle , covalently bound entity , w ith no requirement for weakly associated stabilisers or surfactants B-property . [SEP]
[CLS] in ar ecent study , t akahara , otsuka and co - workers prepared large silica nps B-nanoparticle ( d % 100 nm ) functionalised with polymer B-material brushesb earing approximately 10 mol % a lkoxyamine side chains ( np B-nanoparticle - 18 , f igure 7 ) . [SEP]
[CLS] the dynamic covalentr adicalc rossover reaction of alkoxyamines was then exploited to graft on appropriately functionalised polymere xchange units , such as poly ( 4 - vinylpyridine ) derivative p19 . a lthought he extent of exchange was very limited ( ca . 2 . 2 % a sq uantified by xps analysis ) , subsequentq uaternisation of the pyridine units rendered silica np B-nanoparticle - 18ap19me + + dispersible in water B-material . [SEP]
[CLS] r eaction with the small - molecule alkoxyamine 20 recovered both the xps signature and solvent compatibility properties of the initial sample , consistentw ith af ully reversible dynamic covalent exchange process . [SEP]
[CLS] molecule - directed assembly of dcnp building blocks structuralc ontrol over nanoparticle B-nanoparticle assemblies is important for defining an umber of emergenta nd collective properties when severaln ps are broughtt ogether , a nd is therefore of crucial importance to many innovativen pa pplications , from sensing to catalysis ; o ptoelectronics to thermoelectrics . [SEP]
[CLS] furthermore , integrating nanostructures with components from existing technologies , such as microelectronics or optics , requires the controlled assembly and patterning of nps B-nanoparticle across several size scales . [SEP]
[CLS] although np B-nanoparticle ' superlattices ' may be assembled , driven by nonspecific dispersion forces on controlled solvent evaporation , these tend to form close - packed structures with arrangements that are specified by the size and shape of the np B-nanoparticle building blocks . [SEP]
[CLS] independent control over np B-nanoparticle building block characteristics and assembly structure through chemistry - ledd esign of interparticle linkersh as been exemplified by panel ( b ) adapted with permission from ref . [ 49 ] , copyright w iley - vch verlag gmbh & c o . kga , weinheim . [SEP]
[CLS] adapted with permission from ref . [ 46 ] , copyrightwiley - vchv erlag gmbh & c o . kga , weinheim . [SEP]
[CLS] the emergence of ordered , non - close - packed np B-nanoparticle arrays connected by oligonucleotide hybridisation . [SEP]
[CLS] however , a ll of the attendant issues with regards to dna stabilitya nd customisability remain , and assembly at surfaces is still very much in its infancy . [SEP]
[CLS] despite severaln otable advances exploiting nonbiomolecular linkers , all - covalent strategies tend to encounter kinetic traps , whereas noncovalenta pproaches can require very specific conditions or complex molecular designs to produce well - defined kinetically stable structures . [SEP]
[CLS] dcnp buildingb locks have the exciting potentialt os elf - assemble under error - correcting , thermodynamically controlled conditions , yet be connected by structurally unambiguous , stable and diverse covalentl inkers . [SEP]
[CLS] exploiting dynamic covalent exchange of bilayer - confined thioesters , ravoo and coworkers reported the reversiblea ssembly of liposome B-nanoparticle aggregates . [SEP]
[CLS] liposomes B-nanoparticle ( d % 100 nm ) constructedf rom soy bean lecithin and2 5mol % t hioester 21 were treated with dithiol 22 , resultingi nc ovalent cross - links between the vesicles ( figure 8 ) . [SEP]
[CLS] although the assembly morphology has not been determined , dynamic B-technique light I-technique scattering I-technique indicated the formation of polydisperse aggregates over ap eriod of several hours , which could subsequently be disruptedb ya ddition of an excess of monofunctional thiol B-material 23 to drive the dynamic covalent exchange process back towards the starting thioester . [SEP]
[CLS] an opticals ensor for assessing enantiomeric excess of chiral diols has been demonstrated , based on enantioselective disruption of colloidally stable aggregates produced from saccharide - functionalised aunps B-nanoparticle in the presence of inorganicb orate anions B-material . [SEP]
[CLS] aggregation , which produces an easily detectible optical signature on account of changes to the aunp B-nanoparticle - localised surfacep lasmon resonance , wasa ttributed to the formation of dynamic covalent spiroborate cross - links between np B-nanoparticle - bound vicinal diols . [SEP]
[CLS] these linkages B-property are subsequently disrupted on introduction of the small - molecule diol analytes . [SEP]
[CLS] combining organic molecule linkers with monolayer - stabilised dcnps , w er ecently demonstratedt he dynamic covalent assembly of extended andr esponsive np B-nanoparticle aggregates by using boronic acid - functionalised aunp B-nanoparticle - 7 ( figure 4a nd 9 ) in combination with ditopic linkerst hat can themselves be varied to tune the aggregate morphologyi namodularf ashion [SEP]
[CLS] in the presence of ab ifunctionald iol linker , d ynamic covalent boronate B-material ester cross - links are formed between the dcnps . [SEP]
[CLS] t he re - figure 8 . reversible liposomecross B-nanoparticle - linkingb ydynamicc ovalent thioester exchange [SEP]
[CLS] [ 55 ] [SEP]
[CLS] adaptedf rom ref . [ 55 ] with permission from the royal society of chemistry . [SEP]
[CLS] dynamic covalent polymer brush - functionalised silica nps B-nanoparticle . [SEP]
[CLS] alkoxyamine exchange can be used to reversiblyattach macromolecular poly ( 4 - vinylpyridine ) side chains p19 . subsequentquaternisation of pyridinenitrogen atoms B-material results in ahydrophilic B-property np B-nanoparticle coating B-material that can subsequently be removed by dynamic covalent exchange with small molecule 20 . [SEP]
[CLS] sulting extended aggregates eventually precipitate from solution , producing low - density open networks that are consistent with adiffusion - limited aggregation process . [SEP]
[CLS] assembly is quantitative and switchable : t he dncp building block and molecular linker do not interactuntil addition of alewis base to stabilise the np B-nanoparticle - bound boronate B-material esters , thus initiating assembly . [SEP]
[CLS] emarkably , d espite being linked by covalent bonds , the resulting solid - state aggregates can then be completely disassembled by introducing ac ompetitive monofunctional diol ( figure 9 ) . [SEP]
[CLS] interestingly , d cnp aggregate morphologyw as found to vary depending on the nature of the bifunctional linker , s uggesting that morphology can be tuned through structural modification of as mall - molecule component . with the ability to characterise np B-nanoparticle - bound dynamic covalent processes in situ ( vide supra ) , this offersastraightforward and rational approach to varying morphological parameters over aw ider range than is possible using biomolecular systems . [SEP]
[CLS] this modular strategy might also be extended to incorporate additional chemical , physicalo rs tructural features within the molecular linker design . [SEP]
[CLS] t he diversity of complementary and orthogonal dynamic covalente xchange processes points the way to yetmore sophisticated assemblies and devices that incorporate severald ifferentd cnp building blocks , designed to be selectively reactive with each other and / or with specific species . [SEP]
[CLS] the dcnp building block concept thus suggests severali ntriguing avenues towards responsive , reconfigurable and truly multifunctionalh ybrid nanomaterials B-material in which both molecular and nanoscale components are precisely arranged , and combine to define emergent system properties . [SEP]
[CLS] one only has to look to biology to understand the potential for producing remarkablef unctional materials or complex chemicaln etworks by nanoscale confinement of dynamic molecular systems on interfaces or within compartments . [SEP]
[CLS] advan - ces in both synthetic and analytical technology are now allowing chemists to consider emulating some of these extraordinary systems , even if only at ac omparatively rudimentary level . [SEP]
[CLS] dynamic covalent reactions offer unique advantages in their combination of specific and selectivet hermodynamically controlled reactivity , w ithin structurally robusta nd unambiguous small - molecule covalent structures that are synthetically and analytically tractable . [SEP]
[CLS] c onferringd ynamic covalent reactivity on the vast array of nanomaterials B-material that can now be generated with increasingly precise control over chemical composition , size , shape and dispersity , w ill therefore establish av ersatile new category of nanochemical synthon : t he dcnp building block . [SEP]
[CLS] whether on soft or hard nanosurfaces , combining the errorcorrecting and environment - responsive features of equilibrium processes with the stability and structurald iversity of covalent chemistry amounts to a ' best of both worlds ' solution to the problemo fe ngineering surface functionality . [SEP]
[CLS] t he basic concept is largely independent of the nanomaterial B-material itself , and so can ultimately be generalised across ar ange of materials , shapes and sizes . [SEP]
[CLS] this is in starkc ontrastt ol igand - exchange strategies , which by definition must be specific for each different nanomaterial B-material - molecule interaction ; d ynamic covalent exchange of simple units on the periphery of as tabilising monolayer can occur significantly faster under milder conditions and avoids the necessity for multistep synthesis of each ligand from scratch . [SEP]
[CLS] the advantages of an onbiomoleculara pproach include the ability to readily vary structural and chemical parameters , and operate under aw ide range of conditions . [SEP]
[CLS] the ever - increasing number of well - characterised dynamic covalent reactions affords ad iversity of complementary ando rthogonal reactivities , spanning ah uge range of kinetic behaviours and operating conditions . [SEP]
[CLS] furthermore , with the full gamuto fs ynthetic small - molecule chemistry at our disposal , the possibilities for augmenting both structurea nd properties through the supporting molecular scaffold are almost limitless . [SEP]
[CLS] for all thesereasons , dcnps can provide at oolbox of universal figure 9 . boronate B-material ester - driven dcnpassembly and disassembly on sequential addition of abifunctional linker ( blue ) , followed by am onofunctional capping unit ( pink ) . [SEP]
[CLS] adapted from ref . [ 48 ] with permission from the royal society of chemistry . [SEP]
[CLS] nanoscale buildingb locks that can be predictably modified , combined , and assembled to suit numerous of applications . [SEP]
[CLS] the pseudomolecular nature of colloidallys table nps B-nanoparticle - at least at the lower end of the size - scale - means that although the analyticalc hallenges are significant , they are not insurmountable . [SEP]
[CLS] severals tudies discussed herein have demonstrated that these systems can reveal direct insighti nto chemical processeso ccurring at interfaces , something that hasp roven extremelyc hallenging in other settings . [SEP]
[CLS] c onsequently , d cnps constitute au nique platform on which to study surface - bound molecular behaviour . [SEP]
[CLS] u nderstanding the subtle and complex network of interactions governing reactivity within np B-nanoparticle - bound monolayers will be crucial to arriving at rational andp redictable synthetic methodologies for working with dcnps , for example by quantifying and understanding the influence of surface confinement on reactionk inetics . [SEP]
[CLS] at the same time , such fundamentalq uestions are equallyr elevant to better understanding av ariety of other surface - confined molecular processes , from heterogeneousc atalysis to cell - surface recognition . [SEP]
[CLS] the profound influence of the surface - bound microenvironmento nt he thermodynamics of equilibrating dynamic covalent systemsh as already been demonstrated , and points the way towards templated nanosurfaces that array numerous copies of severalf unctionalities across an anoscale surface area , thus providing synthetic analogues form odelling or perturbing large - area multivalenti nteractions that are at the heart of numerousnatural processes . [SEP]
[CLS] the essential role that surface - bound species play in defining ar ange of nanomaterial B-material properties meanst hat dynamic covalentm odification heralds ap owerful , environment - responsive route to controlling system - level behaviours , including physicochemical properties or microscopic and mesoscopic assembly structures . [SEP]
[CLS] we have shown that this approach can be used to reversibly switch and tune np B-nanoparticle solvent compatibility using very simple molecular exchange units , or else to assemble covalently linked np B-nanoparticle aggregates that are responsive to specific chemical stimuli . [SEP]
[CLS] there is rich potential for creating adaptivea nd reconfigurable devicesa nd materialsi nw hich structural features and physicochemical properties can be tuned in am odular fashion by rational selection of molecular and np B-nanoparticle components that are assembled in thermodynamically controlled error - correcting processes and mayb ep erturbed by externalstimuli . [SEP]
[CLS] mastering complex systems that arise throught he interaction of dynamic chemical processes , reactionn etworks and structures is one of the next grand challenges facing synthetic , analytical and theoretical chemists in the coming decades ; dynamic covalent chemistry and nanomaterial B-material buildingb locks will undoubtedly play significant roles in realising this . althought he concept of ar ational ' heterosupramolecular chemistry ' whereby supramolecular chemistry principles could be applied to achieve molecular - level control over nanoparticles B-nanoparticle was proposed at least two decades ago , it is only now that synthetic and analytical capabilities have reached al evel of maturity that allows such aspirations to be met . [SEP]
[CLS] dynamic covalentr eactions are being exploited in all sorts of original ways , including in systemst hat are coupled to external energy sources , such as out - of - equilibrium chemical reactions , transmembrane concentration gradients or repeatedly appliede xternals timuli . [SEP]
[CLS] we can expect that cross - fertilisation between each of these themes will serve as inspiration for ever more innovative designsa nd complexb ehaviours on the nanoscale . with the ability to revealf undamental insights , a nd transformative potential as an enabling technology , d cnps will contributet ot his future chemical synthetic science , whereby molecular , n anoscale and macroscopic building blocks may be combined with equalp recision , t oc reate complex , responsive and adaptive systems operating across several size scales . [SEP]
[CLS] 1 . a ) calculated structure of am onolayer - stabilised np B-nanoparticle ( 5 nm pbs core B-material capped with oleicacid ligands ) and the crucial features that are determined by the ligand shell B-material ; b ) cartoonrepresentation of strategies for postsynthetic np B-nanoparticle surface engineering : i . ligand exchange ; ii . host - guestcomplexation ; i ii . stimuli - responsive molecularswitches ; iv . kinetically controlled covalent reactions ; v . dynamic covalent reactions ( highlighted ) . [SEP]
[CLS] panel ( a ) adapted from ref . [ 8 ] with permission from aaas . [SEP]
[CLS] figure 2 . a ) schematic representation of abilayer - templated dynamic combinatorial library formedb yt hioester exchange between surface - activeb isthioester 1 and dithiol 2 in the presenceo fl ipid bilayer vesicles ; b ) hplc tracesc omparing library compositions produced in bulk aqueouss olution ( top trace ) a nd at the lipid B-material bilayer I-material interface ( bottom trace , 10 mm egg phosphatidylc holine , 1 . 0 mm 1 and 1 . 0 mm 2 ) . [SEP]
[CLS] adaptedf rom ref . [ 45 ] under the termsofc c - by 2 . 0 . [SEP]
[CLS] 4 . np B-nanoparticle - bound boronate B-material ester formationa nd dynamic covalent exchange on sequential reaction of aunp B-nanoparticle - 7 with catechols 8 ( blue ) and 9 ( red ) . [SEP]
[CLS] in situ analysis by19 fnmr spectroscopy B-technique revealsidentical mixed monolayer equilibria are reached via two different routes ( r = n - methylmorpholinium ) . [SEP]
[CLS] adapted from ref . [ 48 ] with permission from the royal society of chemistry . [SEP]
[CLS] figure 4 . a ) dynamically reconfigurable aunp B-nanoparticle - bound hydrazone B-material monolayers ; b ) in situ characterisation by19 fnmr spectroscopyallows molecular - level characterisationo fmonolayer composition ; c ) real - time tracking of dynamic covalent exchanger eactions . [SEP]
[CLS] adaptedw ith permission from ref . [ 46 ] , copyrightwiley - vch verlag gmbh & c o . kga , weinheim . [SEP]
[CLS] 5 . a ) dna - templateddynamiccovalent aunp B-nanoparticle - bound imine B-material monolayers ; negligibleimine formation occursint he absence of dna ; b ) monolayer composition depends on the specific base pair sequence of the doublestranded dna template . [SEP]
[CLS] panel ( b ) adapted with permission from ref . [ 49 ] , copyright w iley - vch verlag gmbh & c o . kga , weinheim . [SEP]
[CLS] 6 . reversible control of dcnp solvent compatibility by dynamicc ovalent hydrazonee xchange ; s olvents : a = hexane , b = chloroform , c = tetrahydrofuran , d = methanol , e = n , n - dimethylformamide , f = water B-material . [SEP]
[CLS] adapted with permission from ref . [ 46 ] , copyrightwiley - vchv erlag gmbh & c o . kga , weinheim . [SEP]
[CLS] for aw ide range of nanomaterials B-material , surface - bound molecules play ac entral role in defining properties , and are key to integration with other components - be they molecules , surfaces , or other nanoparticles B-nanoparticle . [SEP]
[CLS] predictable and general methodsf or manipulatingt he surface monolayer are therefore crucial to exploiting this new region of chemical space . [SEP]
[CLS] this review highlights limitations of the few established methods for controlling nanoparticle - boundmolecular functionality , t hen focuses on emerging new strategies . [SEP]
[CLS] in particular , approaches that can achieve stimuli - responsive and reversiblem odification of surface - bound molecules in colloidal solution are examined , with an emphasis on using these methods to control nanoparticle B-nanoparticle properties such as solventc ompatibility , c atalytic activity and cytotoxicity B-property . [SEP]
[CLS] f inally , t he outstanding challenges and future potential forp recisely controlled nanoparticle - bound monolayersa re discussed . [SEP]
[CLS] monolayer - stabilized inorganic B-nanoparticle nanoparticles I-nanoparticle ( nps B-nanoparticle ) have been among the most intensively investigated nanomaterials B-material to emerge over the past quarterofacentury . [SEP]
[CLS] these new chemical entitiesc an often be manipulated as colloidal suspensions using existing molecular and macromolecular techniquesa nd infrastructure , and have engendered considerable interest on account of unique properties that are tunablea ccording to np B-nanoparticle material B-material , size and shape . [SEP]
[CLS] consequently , c onsiderable efforts have been madet oo ptimize synthetic control over these parameters . [SEP]
[CLS] yet , the core B-material nanomaterial B-material only partly determines np B-nanoparticle properties ; t he surface - bound molecular species are equally important in determining overall behavior , and control over this aspect of np B-nanoparticle structure remains comparatively underdeveloped . [SEP]
[CLS] irrespective of core B-material material B-material , the np B-nanoparticle - bound surfacel igands play acommon role : chemical passivation of coordinatively unsaturated surface sites , and steric or electrostatic stabilization of the materiali nn anoparticle form . [SEP]
[CLS] beyond this , the surface molecular monolayer is also crucial for addingf unctionand defining properties ( figure 1 ) . [SEP]
[CLS] for example , surface speciation of colloidal quantum B-nanoparticle dots I-nanoparticle ( qds ) strongly influences the intrinsic semiconductor electronic and optical properties that are key to applicationsf rom photovoltaics to lightingt echnology and fluorescenceb ioimaging . [SEP]
[CLS] for noblem etal nps B-nanoparticle , the dielectric environment defined by the ligand shell B-material affects the surface plasmon resonance features , and therefore their behaviora s sensors . [SEP]
[CLS] physicochemical properties that derive from interactions between an pa nd the surrounding matrix are also defined , in large part , by the surface - bound species . [SEP]
[CLS] nowhere is this more important than in the largen umber of potential ap - plications for monolayer - stabilized nps B-nanoparticle in biomedical fields , where cytotoxicity B-property , i mmunogenicity , b iodistribution , and pharmacokinetics are all determined by the np B-nanoparticle surfaced etails . [SEP]
[CLS] finally , n p - bound ligands present the opportunity to introduce entirely new functionality : f rom targeting B-material ligandst I-material ot herapeutics ; c atalysts to imaging probes . [SEP]
[CLS] for virtually all applications , it is now imperative that synthetic nanochemists develop robust and generalizable strategies for manipulatingt he npbound monolayer . [SEP]
[CLS] the de novo synthesis of new np B-nanoparticle entitiesw ith differing surface monolayers is tedious , and intrinsically limiting . [SEP]
[CLS] only as mall subset of monolayer functionalities will be compatible with any given synthesis conditions , while even subtle variations in ligand molecular structure can have as ignificant impact on the outcome of an ps ynthetic protocol . [SEP]
[CLS] far more attractive are postsynthetic methods , through which ar ange of systems can be accessedf rom as mall number of optimized buildingb locks , ideally with the ability to switch and tune properties in response to specific triggers . [SEP]
[CLS] directly replacing one np B-nanoparticle - bound species with another in a ' ligand exchange ' process has long been established as ap owerful methodf or postsynthetic np B-nanoparticle modification . [SEP]
[CLS] first developed by murray and co - workerso na unps , ligand exchange allows functionality to be incorporated independent of the np B-nanoparticle synthesis conditions , including the combinationo fs ev - eral different ligandst op roduce mixed monolayers , making it an attractive approach for modifyingahost of np B-nanoparticle properties . [SEP]
[CLS] despitew idespreada doption , however , t his strategy presents severals ignificant challenges . [SEP]
[CLS] exchange can often be slow , a nd complete replacement of one ligand for another is difficult to achieve . [SEP]
[CLS] for this reason , essentially irreversible replacement of aw eakly bound species with stronger ligands is often favored . [SEP]
[CLS] disrupting the np B-nanoparticle - ligand bond can be ar elativelyh arsh process , l eadingt ou nwanted changes in np B-nanoparticle properties , s ize distribution , or even np B-nanoparticle decomposition . [SEP]
[CLS] perhapsm ost significantly , t he mechanistic detailso fl igand exchange are specific to each particular nanomaterial B-material - ligand combination . [SEP]
[CLS] evidence points to ac omplexn etwork of reversible and irreversible processes , that often depend on sample history or other batch - to - batch differences . [SEP]
[CLS] despite continued development of innovative ligand exchangem ethodology on av ariety of np B-nanoparticle surfaces , this lack of understanding , and intrinsic system - dependentv ariability , h indersp rogress towardsp redictable and generalizable methods that may be adopted irrespectiveo for even blind to - theu nderlying nanomaterial B-material details . [SEP]
[CLS] in this focus review , w ed iscusse merging methods for tuning np B-nanoparticle properties in colloidal suspension , through postsynthetic manipulation of the surface monolayer , without requiring disruption of the np B-nanoparticle - monolayer bond . [SEP]
[CLS] t hrough selective examples , we highlight the strengthsa nd weaknesses of each strategy , w ith af ocus on approaches that may achiever eversible and stimuli - responsive propertyc ontrol . [SEP]
[CLS] c rucial to developing predictable methods , we examine evidencec orrelating phenomenologically observed property changes with underlying molecular changes , and we point to some of the outstanding challenges that must now be addressed . [SEP]
[CLS] although the scope here is restricted to colloidal suspensionso fs mall inorganic B-nanoparticle nanoparticles I-nanoparticle stabilized by as urface monolayer ( i . e . , m etal , metal B-material oxide I-material , and semiconductor nps B-nanoparticle ) , it should be noted that many of the same strategies may be extended to ( or in some cases were pioneered on ) other types of nanomaterial B-material ( e . g . , silica particles , carbon B-nanoparticle dots I-nanoparticle , fullerenes B-nanoparticle , polymer B-material nanoparticles B-nanoparticle or liposomes B-nanoparticle ) , as well as for creatingand controlling np B-nanoparticle assemblies , and properties in condensed phases . [SEP]
[CLS] electrostatics are particularly important in determining interactions between an pa nd the surroundingm atrix , s trongly influencing colloidal stabilitya nd np B-nanoparticle adhesion to exogenous molecules or surfaces . [SEP]
[CLS] reversible charges witching is readily achieved through ionization B-property of acidic or basic groups in the monolayer , s of or many applicationsc areful consideration must be given to maintaining an appropriate chargeu nder the expected range of operating conditions . [SEP]
[CLS] overall neutralm onolayers of zwitterionic ligands , for example , have been shown to exhibit excellent aqueous colloidal stability , r esistance to nonspecific binding in biological media and low cytotoxicity B-property . [SEP]
[CLS] re - cently , r ational design of acylsulfonamide ligand 1 provided aunpsd isplaying as harp transition between neutralz witterionic , andc ationic states aroundp h6 . 5 ( figure 2a ) . [SEP]
[CLS] congruent with the mildly acidic microenvironment surrounding many solid tumors B-material , the transition to positive surface charge enhanced np B-nanoparticle endocytosis B-event and cytotoxicity B-property , s uggesting promise for tumor - selectivetherapeutic or imaging applications . [SEP]
[CLS] manipulating monolayer charge state for nanoparticle B-nanoparticle property tuningterionic ) and cationic B-material surfacecharges at mildly acidic ph values . b ) mixed monolayers of permanently cationic B-material ( 2 ) and switchable carboxylic B-material acid I-material ( 3 ) l igands allow switchingb etween positive , neutral and negativesurface charges , accompanied by reversible modulation of colloidal stability . [SEP]
[CLS] neutral surfacec hargem ay alternativelyb ea ttained by combining two oppositely charged ligands in am ixed monolayer , a llowing fors witching between positive , neutrala nd negative charge states . [SEP]
[CLS] mixing permanently positivelyc harged tetraalkyl ammonium ligand 2 , w ith an excess of carboxylic B-material acid I-material 3 , p roduced ph - responsive aunp B-nanoparticle - 2 / 3 that could switch between highly positive and negative surfacec harges ( figure 2b ) . [SEP]
[CLS] the nps B-nanoparticle were colloidally stable in aqueous media at both low and high ph , but underwent reversible precipitation at an intermediate B-property ph , correspondingt os urfacec harge neutralization . interestingly , t he neutralization point could be tuned across aw ide ph range ( % 4t o % 7 ) according to both the ratio 2 : 3 and np B-nanoparticle radius of curvature . [SEP]
[CLS] this illustrates an important point : m olecular properties of monolayer constituents such as pk a can be strongly influenced by nanoscale features , including monolayer composition and np B-nanoparticle radius of curvature . [SEP]
[CLS] the mixed - charge monolayer allowed aunp B-nanoparticle - 2 / 3 , d isplaying an overall negative surfacec harge , to be internalized by cells B-material - counter to the commonly held understanding that cellular uptake requires overall positive surface charge . [SEP]
[CLS] it must be noted here that accurate characterization of absolute np B-nanoparticle surface chargeisc hallenging . [SEP]
[CLS] most commonly , l ight scattering is used to measure np B-nanoparticle electrophoretic B-property mobility I-property , w hich is then converted into z - potential . [SEP]
[CLS] howevert his conversion is fraughtw ith complications , while z - potential itself is only ap roxy for the attached surfacec harge , andi sh ighly sensitive to the measurement conditions . [SEP]
[CLS] electrochemical charging of redox - active monolayer constituents provides as imple - but relativelyu nder - exploited - means to reversibly modulate np B-nanoparticle surfacec harge . [SEP]
[CLS] in as imilar manner to pk a , r edox potentials are sensitive to both the nanoscopica nd molecular environment , presenting opportunities for sensing applications , or control of host - guesti nteractions ( see section3 . 1 ) . [SEP]
[CLS] one of the most highly developed approaches for postsynthetic np B-nanoparticle monolayer modification exploits reversiblea nd specific hybridization to np B-nanoparticle - bound single - stranded oligonucleotides . [SEP]
[CLS] introduced simultaneously by alivisatos , and mirkin , this has become one of the touchstonem ethods for colloidal np B-nanoparticle functionalization , leadingt od iversea pplicationsi ncluding sensing , g ene delivery , a nd np B-nanoparticle assembly . [SEP]
[CLS] detailed physicalorganic studies have revealed much aboutt he parameters determining the remarkable behavior of theses ystems , allowing np B-nanoparticle - bound oligonucleotide hybridizationt ob ee xploited in an increasingly predictable manner . [SEP]
[CLS] t ogether with the rapid , automated synthesis of oligonucleotides with any given sequence , dna - coated nps B-nanoparticle can genuinely now be considered as ' programmable atom B-material equivalents ' . [SEP]
[CLS] yet , the chemical and structural stabilityo fd ouble - stranded dna is only maintained within ar elativelyn arrow window of conditions . [SEP]
[CLS] furthermore , incorporation of non - nucleotide molecular functionality and in situ molecular - level characterization of these complex , oligomeric chemical structures is extremely challenging . [SEP]
[CLS] here , we will concentrate on newly emerging nonbiomolecular approaches . [SEP]
[CLS] encapsulating semiconductor qds in micelles B-material , or amphiphilic B-property polymers B-material , [SEP]
[CLS] intermolecular noncovalent monolayer modificationwas one of the earliest solutions for converting assynthesized hydrophobic B-property nanocrystals into biocompatible B-property fluorophores , while preserving their attractive opticalf eatures . drivenb yh ydrophobic interactions between the surfactant B-property and the lipophilic B-property np B-nanoparticle - bound ligands , bilayer - protected nps B-nanoparticle can similarly be prepared from any core B-material nanomaterial B-material bearing ahydrophobic surface monolayer . [SEP]
[CLS] polymeric amphiphiles B-property have provenp articularly convenient for incorporating aw ide range of functional groups B-nanoparticle on I-nanoparticle the I-nanoparticle bilayer surfacet I-nanoparticle hrought his mild , one - step approach . [SEP]
[CLS] however , encapsulationi se ssentially irreversible , the challenges of balancing long - term stability while avoidingn pc ross - linkingc an be significant , and the attendant increasei nn ps olvodynamic size is ad rawback form any applications . [SEP]
[CLS] small - molecule host - guest systemsp resent diversep ossibilities for addressable noncovalent np B-nanoparticle monolayer modification . [SEP]
[CLS] for example , colloidaln pp hase transfer hasb een achieved by maskingh ydrophobic np B-nanoparticle - bound ligands with water B-property - soluble B-property cyclodextrin ( cd ) macrocycles . [SEP]
[CLS] conversely , i ncorporating cds as one of the surface - bound ligands on qds has been used to create af luorescencet urn - ons ensorf or varioush ydrophobic small molecules , operating throught he displacement of aq uencher from the cd cavity . [SEP]
[CLS] yet , these early systemsi llustrate the major challenges faced by nonbiomolecular hostguest systems : o ptimizing complex stabilitya nd reversibility , while avoiding kinetic traps ; a nd achieving sufficient selectivity for application in complicated real - world environments . [SEP]
[CLS] recently , r otello and co - workersh ave successfully exploited the strong and selective recognition properties of cucurbit - figure 3 . competitive host - guest complexation between ada and cb [ 7 ] for selective np B-nanoparticle property switching . [SEP]
[CLS] a ) intracellular decomplexation of aunp B-nanoparticle - 4 • cb [ 7 ] revealscytotoxic aunp B-nanoparticle - 4 . [SEP]
[CLS] b ) intracellularremovalofc b [ 7 ] provides access to amonolayer - embedded catalyst B-property for prodruga ctivation . [SEP]
[CLS] [ n ] uril macrocyclest oa chiever emarkable control over aunp B-nanoparticle properties in live cells B-material ( figure 3 ) [SEP]
[CLS] diaminohexane - functionalized aunps B-nanoparticle were shown to form stronghost - guest interactions with cucurbit [ 7 ] uril ( cb [ 7 ] ) ; c lear evidence for an p - bound threadedh ost - guest complex was providedb y 1 hnmr spectroscopy B-technique . the resultinga unp - 4 • cb [ 7 ] complex ( figure 3a ) i s water soluble and readily taken up into the endosomes of human breast cancer cells B-material , with no apparent cytotoxicity B-property at concentrations 50 mm . [SEP]
[CLS] s ubsequenti ncubation B-technique with 1 - adamantylamine ( ada ) - a bioorthogonal competitive guest for cb [ 7 ] - resulted in endosomal escape of the nps B-nanoparticle and acytotoxic response very similar to that elicited by aunp B-nanoparticle - 4 alone ; p resumably the result of intracellular disruptiono ft he np B-nanoparticle - bound host - guest complex by ada . [SEP]
[CLS] this approach has now been extended to achieve intracellular control of ab ioorthogonal catalytic reaction ( figure 3b ) . [SEP]
[CLS] complex formationi nasimilar manner to before was used to prevent substrate access towards an organometallic catalysti ntercalated within the hydrophobic B-property regiono ft he monolayer ; s ubsequently , catalytic activity could be restored by competitive binding of cb [ 7 ] to ada . [SEP]
[CLS] kinetic analysisr evealed selective and reversiblec atalyst inhibition , and excellent agreement to cb [ 7 ] binding affinities measured independently by isothermalt itration calorimetry . [SEP]
[CLS] w ith this approach , intracellular activation of the anticancer B-property drug5fluorouracil ( 5fu ) was achievedo nco - localization of a5fu prodrug with anp - bound , cb - protected catalyst B-property and ada . [SEP]
[CLS] this elegant strategy suggests possibilities for tuning ab iological response according to local concentrations of np B-nanoparticle , c ompetitiveb inder and ( where relevant ) catalytic substrate . [SEP]
[CLS] there is excitingl ong - term potentialf or smart delivery of drugs or imaging agentsw ith spatiotemporal dose control , while more immediate applications include stimuli - responsive tools for chemicalb iology . [SEP]
[CLS] h owever , i nt hese proof - of - concept studies relativelyh ighc oncentrationso fc ompetitive binder B-property are incubated B-technique over several hours , and translation to in vivo settings still presents asignificant challenge [SEP]
[CLS] scrimin , p rins and co - workers have developed as eries of aunps B-nanoparticle functionalized with azacrown macrocycles , such as aunp B-nanoparticle - 5 ( figure 4 ) . [SEP]
[CLS] coordinationo fz n 2 + to the np B-nanoparticle - bound macrocycles affords active transphosphorylation catalystst hat mimic phosphodiester cleavage by ribonucleases . [SEP]
[CLS] as igmoidal increasei no bserved reaction rates with increasing metal B-material ion B-material loading indicates ac ooperative multinuclear catalytic site , which is facilitated by the multivalent nature of the np B-nanoparticle - bound monolayer , a nd leads to catalytic activities over 600 - fold greater than mononuclear molecular analogues . [SEP]
[CLS] the monolayer composition wasc haracterized by ac ombination of nmr analyses ands pectrophotometry . [SEP]
[CLS] y et , the structure of the active catalystc an still only be inferred on the basis of kinetic measurements . [SEP]
[CLS] although the evidences trongly suggestsacatalytic site involving two zn 2 + centers within the same monolayer , the prospect of several different catalytically active structures operating simultaneously , o ranon - random distribution of ligands in mixed np B-nanoparticle monolayers , cannot be ruled out . [SEP]
[CLS] detailed structural characterization of active sites - such as can be provided by crystallography for smallm olecule catalysts B-property or en - zymes - remains an important long - term challengef or npbound catalysts B-property . [SEP]
[CLS] the observation that aunp B-nanoparticle - 5 • zn 2 + also acts as ah igh - affinity host for small oligoanionsh as provided ap owerful , and syntheticallya daptable , system with which fundamentalf eatures of host - guest recognition at responsive multivalent nanoscale surfaces can be investigated . [SEP]
[CLS] reversibletuning of np B-nanoparticle valency can be achieved by adding and removing ( using ac ompetitive ligand ) z n 2 + from aunp B-nanoparticle - 6 . [SEP]
[CLS] the change in bindingc apacity ( i . e . , v alency ) a nd complexation kinetics has been monitored via emission quenching of fluorescent B-property anionic guests when in close proximity to the metal B-material surface , a ffording data consistent with at least two distinct anion B-material binding modes . [SEP]
[CLS] it was furthermore observed that aunp B-nanoparticle - 6 • zn 2 + exhibits am arked selectivity for binding diphosphates over dicarboxylates , while anion B-material binding to nonmetalated aunp B-nanoparticle - 6 is ph - dependent . [SEP]
[CLS] this multiparameter responsiveness hasa llowed increasingly complex behaviors to be achieved , including self - sorting of diphosphate ( 8 ) a nd tricarboxylate ( 9 ) g uests onto two different np B-nanoparticle monolayers : p hosphate - selective aunp B-nanoparticle - 6 • zn 2 + and nonselective cationic B-material aunp B-nanoparticle - 7 ( figure5 ) . [SEP]
[CLS] although ac omprehensive series of experiments strongly supportss elf - sorting , it is still not possible to directly distinguish binding of each fluorescent B-property guest to the different np B-nanoparticle surfaces . [SEP]
[CLS] switching betweent he sorted and unsorted states was achievedb yr emoval and addition of zn 2 + , w hile specific stimuli could trigger sequential release of the probes from their respective np B-nanoparticle - hosts . [SEP]
[CLS] i nr elated work , complexation to aunp B-nanoparticle - 6 • zn 2 + has been used for the dynamic combinatorial identification of metal - linked bis - nucleotide dimers from ap ool of lower - affinity monomers B-material , which could be developed either as am etal sensing platform , or for identifying optimal metal - bindingn ucleotide ligands . [SEP]
[CLS] selective modulation of catalytic activity based on zn 2 + complexation to aunp B-nanoparticle - bound azacrownl igands . [SEP]
[CLS] catalytic activities up to 600 - fold greater than analogous mononuclear molecular zn 2 + - azacrown catalysts B-property were achieved [SEP]
[CLS] nonbiomolecular systemso ffer several advantages over oligonucleotides in terms of structural simplicity , a daptability of molecular design and synthetic routes , anda pplicability under aw ider range of environmental conditions . [SEP]
[CLS] these pioneering designsa re slowly closing the gap on the exquisite specificity , selectivity and kinetic tunability exhibited by dna - functionalized nps B-nanoparticle , but in only ah andful of cases is binding strong enough that property changes can be achieved and maintained independento fa nu nbounde xcess of reagent . [SEP]
[CLS] significant challenges also remainindirectly linking structuralcharacterization of dynamic noncovalent complexes within np B-nanoparticle - bound monolayers to observedp roperty changes . [SEP]
[CLS] monolayerst hat respondt oas pecific stimulusb yu ndergoing ac onformational change presentaself - contained strategy to tune np B-nanoparticle properties withoutr equiring addition of exogenous species . [SEP]
[CLS] stimuli - responsive polymers B-material - such as those displaying lower critical solution temperature ( lcst ) behavior - are attractive ready - made systemsf or such strategies . [SEP]
[CLS] phase transfer across oil - water interfaces has been achievedf or aunps B-nanoparticle functionalized with randomcopolymers of 2 - ( 2 - methoxyethoxy ) ethyl methacrylate and oligo ( ethylene glycol ) m ethyl methacrylate . [SEP]
[CLS] however , t he polymers B-material witching behavior is significantly affectedb ya ttachment to the np B-nanoparticle surface , and bidirectional switching required careful adjustment of both temperature and aqueous phase ionic strength . [SEP]
[CLS] theoretical rationalization of these subtle effects based on thermodynamic considerations underlines the importance of understanding the influence of the nanosurface - bound environmento nm olecular behavior . [SEP]
[CLS] cross - linked thermosensitive polymers B-material display volume phase transitions between expanded and collapsed states , and have been used to modulate np B-nanoparticle catalytic activity and selectivity by controlling substrate diffusion towards the np B-nanoparticle surface . [SEP]
[CLS] for aunpsc oated with cross - linked poly ( n - isopropyl acrylamide ) , ad ecrease in np B-nanoparticle hydrodynamic diameter on increasing temperaturei ndicated polymer B-material collapse , and was correlated with as harp reduction in the rate of np B-nanoparticle - catalyzed ferricyanide reductionb yb orohydride . [SEP]
[CLS] in as imilars ystem , substrate selectivity could be alteredo np B-nanoparticle olymer phase transition ( figure 6a ) : reduction of hydrophilic B-property nitro - aromatic substrates being preferred at t < lcst ( swollen polymer B-material shell B-material ) , and hydrophobic B-property substrates reacting faster at t > lcst ( collapsed polymer B-material shell B-material ) . [SEP]
[CLS] the same strategy has been used to reversibly control the approacho fr aman - active and fluorescent B-property analytes towards aa unp surface ( figure 6b ) . [SEP]
[CLS] for ap robe molecule that does not interacts trongly with the np B-nanoparticle surface , intercalation into the swollenp olymer monolayer results in strong surface - enhanced fluorescence B-property ( sef ) , but weak surface - enhanced raman scattering ( sers ) ; c onversely , p olymer collapse resultsi nastrong sers signal butq uenching of the fluorescence B-property , presumably ar esult of confining the analyte closer to the np B-nanoparticle surface [SEP]
[CLS] the ability to capture probest hat have little or no affinity with the np B-nanoparticle surface , and to switch between two spectroscopic modalities , w ith an easily addressable , colloidally stable platform , suggestss everal opportunities for quantitative sensing in complex environments . [SEP]
[CLS] ecent exciting extension exploits photonic heating of hollow gold B-material nanoshells B-nanoparticle to simultaneously trigger the reversible polymer B-material phase - transition and monitor the concomitant sers responseu sing anear - ir light source . [SEP]
[CLS] the rich set of behaviors attained through nonspecific conformational changes suggest that an even greater level of sophistication may be reached by harnessing the more precise - figure 5 . [SEP]
[CLS] switchable noncovalent self - sorting on np B-nanoparticle - bound monolayers . [SEP]
[CLS] anionic guests 8 and 9 bind nonselectively to aunp B-nanoparticle - 6 and aunp B-nanoparticle - 7 , producing am ixed guest - monolayer state . [SEP]
[CLS] additionofz n 2 + produces aunp B-nanoparticle - 6 • zn 2 + , aselective host for phosphate 8 , l eading to as elf - sortedg uest - monolayer state . [SEP]
[CLS] chemnanomat 2016 , 2 , 87 - 98 www . chemnanomat . org ly defined stimuli - induced conformational , co - conformational or configurational changes achieved by modern - day artificial molecular machines . [SEP]
[CLS] in one of the first examples of amolecular - machine - controlled np B-nanoparticle device , aph - responsive oligonucleotide conformational switch was combined with fluorescent B-property qds to create ab iocompatible ph sensor ( figure 7a ) . [SEP]
[CLS] ar atiometric ( self - referencing ) o ptical response to changingp h was generated by modulating fcrster resonance energy transfer ( fret ) between the qd and aph - insensitive molecular fluorophore through large - amplitude proton - triggered folding / unfolding of the molecular machine . [SEP]
[CLS] excellent agreement between energy transfer efficiency and the physical dimensions of the sensor construct as ph is varied confirmedt he molecular - mechanical transduction mechanism . [SEP]
[CLS] this defines auniversal fluorescent B-property sensor design strategy whereby the analyte - responsive , a nd the opticalr eporting components may be optimized independently , a llowing the unique - but otherwise environment - insensitive - optical properties of qd fluorophores to be harnessed for tracking ph within individuale ndosomes of living cells B-material ( figure 7a ) . [SEP]
[CLS] however , h igh - resolutioni maging of very small qd populations ( in theory down to individual particles ) requires highly homogeneous probe behavior , a nd so methods for accessing such devicesw ith tightly controlled np B-nanoparticle : machine stoichiometry represent the next challenge . [SEP]
[CLS] stimuli - responsive changes in configurationo fi somerizable photoswitches also provideapowerful strategy for controlling np B-nanoparticle behavior . [SEP]
[CLS] t his area has recently been reviewed extensively elsewhere , and al arge proportion of these studies have been directed towards controlling np B-nanoparticle aggregation and np B-nanoparticle properties in condensed phases . [SEP]
[CLS] i nc olloidal suspension , t he opticalproperties of photochromic switches have been applied in an umber of instances to control emission from fluorescent B-property nps B-nanoparticle . [SEP]
[CLS] for example , fluorescencef rom cdse - zns core - shell qds functionalized with spiropyrans could be reversibly switched on and off by photoirradiation ( figure 7b ) . [SEP]
[CLS] this effect can be attributed to photoconversion between spiropyran ( 10 sp ) a nd merocyanine ( 10 mc ) i somersi nt he np B-nanoparticle - bound monolayer ; t he latter form absorbs strongly in the visible region ( l max = 588 nm ) , and can therefore quench qd emission ( l fl = 546 nm ) by fret . c onsistent with this mechanism , w eak red emission , characteristic of the merocyanine dye , is observed in the ' off ' state , and quenching efficiency depends on both the number of photoswitches in the monolayer and the spectral overlap between qd emission and meorcyanine absorption bands . [SEP]
[CLS] similar results have been achieved with fluorescent B-property gold B-material nanocrystals , and rare - earth upconverting nanophosphors . [SEP]
[CLS] these relatively simple examples only hint at the functionality that might be achieved by interfacing more sophisticated artificial molecular machinesw ith nps B-nanoparticle . [SEP]
[CLS] large - amplitude coconformational motions of mechanically interlocked molecular shuttlesh ave already been achieved within np B-nanoparticle monolayers , creatings ome of the most structurally advanced switchable np B-nanoparticle systemst od ate . [SEP]
[CLS] it now remains to properly understand the influence of the surface - confined environmento nm achine operation , and define the most appropriate properties and practical uses to be addressed with theseremarkable systems . [SEP]
[CLS] with the potential for accessing ah uge range of kinetically stable , well - defineds tructures , t hrough aw ide array of reactions , modifying the covalent connectivity of np B-nanoparticle - bound ligands in situ is ah ighly attractive strategy . [SEP]
[CLS] e arly work established that simple reactions such as ester or amide B-material coupling can directly modify reactive np B-nanoparticle - boundl igands , and subsequently aw ide range of protocols - many inspired by bioconjugation methods - have been adapted for functionalization of npbound monolayers . [SEP]
[CLS] these established techniques , however , each exploit essentially irreversible reactions , providing a " oneshot " opportunity to modify the monolayer , w ith no degree of error checking , and resulting in as tatistical product distribution that can be difficult to control . [SEP]
[CLS] incorporating orthogonally reactive ligandsi nt he ligand shell B-material can allow stepwise modification , but this in turn requires precise control of the initial figure 6 . nanoparticle B-nanoparticle property switching using thermosensitive polymer B-material ligand shells B-material . [SEP]
[CLS] kinetically controlled covalent monolayer modificationa ) switching substrate selectivity for ac atalytically active ' yolkshell ' aunp B-nanoparticle coated with poly ( n - isopropyl acrylamide ) . [SEP]
[CLS] b ) temperature controlled swelling of ap oly ( n - isopropyla crylamide ) l igand shellsimultaneously alters surface - enhanced raman scattering ( sers ) and surfaceenhanced fluorescence B-property ( sef ) effects for an intercalated dye . [SEP]
[CLS] monolayer composition . [SEP]
[CLS] stimuli responsiveness in these systems is limited to controlling reagent stoichiometry , orr eaction conditions to kinetically promote or inhibitr eaction progress . [SEP]
[CLS] af ew systemsh ave used bond - breaking reactions to achieve property changes in response to ap articulars timulus . [SEP]
[CLS] emrick and co - workersr eported the inversion of np B-nanoparticle - stabilized pickeringe mulsions , induced by the acid - promoted cleavage of ether B-material protecting B-technique groups I-technique ( figure 8a ) . [SEP]
[CLS] aunp B-nanoparticle - 11 0 . 9 12 0 . 1 , b earing am ixture of hydrophobic B-property ( 11 ) a nd hydrophilic B-property ( 12 ) l igands , was found to stabilizeawater - in - oil emulsion . acid initiated deprotection of 11 increases np B-nanoparticle hydrophilicity B-property , d isrupting the emulsion and leading to phase separation . adding more water B-material , with agitation , produced an oil - in - water emulsion , stabilized by the more hydrophilica B-property unp - 12 . a ppealingly , t his approach could also be photonicallyt riggered by including ap hotoacid generatorint he organic phase . [SEP]
[CLS] incorporating directly photocleavable units within the ligand design has been used to produce an umber of irreversibly switchable np B-nanoparticle systems . [SEP]
[CLS] for example , ortho - nitrobenzyl ligand 13 was used to create ' caged ' cdte / cds core / shell qd - 13 0 . 25 14 0 . 75 exhibiting highly quenched photoluminescence B-property on excitation at 405 nm . [SEP]
[CLS] irradiation at 365 nm causedt he photoluminescence B-property quantum yield to increase by over 400 - fold , ac onsequence of removing the aromatic substituent . [SEP]
[CLS] similar approaches have exploited enzymatic cleavage of peptidic linkers , or redox modification of energy - or electrontransfer quenchers on qd surfaces to irreversibly modify fluorescence B-property , thereby providing detection ands ensing functions . [SEP]
[CLS] approaches that combinet he reversibility and stimuli responsiveness displayed by noncovalent systemso rc onfigurational switches , with the stabilitya nd structurald iversity of covalent chemistry , w ould afford access to ad iverse range of self - contained np B-nanoparticle systemsw ith precisely controlled and reconfigurable monolayer features , a nd therefore properties . [SEP]
[CLS] dynamic covalent reactions have the potential to meet all these requirements . [SEP]
[CLS] applied to np B-nanoparticle - boundl igands , this would allow covalent bonds to be formed and broken reversibly , w ith product distributions that are predictably determined by thermodynamic stabilities , a nd responsive to reaction conditions . [SEP]
[CLS] however , achange in conditions ( e . g . , removalo facatalyst ) , can kinetically lock the products , allowing isolation , p urification , and application without fear of further changes . [SEP]
[CLS] largely exploited for their ability to operate under relatively mild conditions , there have been an umber of examples of conjugation to nps B-nanoparticle in ostensibly irreversible processes that in fact exploit potentially reversiblec ovalentb onds . [SEP]
[CLS] in particular , h ydrazone formationu nder pseudo - irreversible conditions has been developed as ac onvenientb ioconjugation method for incorporating as mall number of modifications on both aunp B-nanoparticle and qd monolayers . [SEP]
[CLS] conversely , a cid - catalyzed hydrazone B-material hydrolysis has been used to selectively releasen p - bound cargoes . [SEP]
[CLS] conceptually , h owever , t hese approaches are essen - figure 7 . [SEP]
[CLS] nanoparticle B-nanoparticle properties controlled by molecular switches . [SEP]
[CLS] a ) proton - triggered conformational switching modulates fret efficiency between aqd donor ( green ) a nd molecular acceptor ( red ) , producingaratiometric response that can be exploited for intracellular ph sensing . [SEP]
[CLS] b ) light - triggered spiropyran - merocyanine interconversion reversibly modulates fretf romaqd donor over several cycles . [SEP]
[CLS] adaptedw ith permission from references [ 41 ] , copyright 2 013 wiley - vch verlag gmbh & co . kgaa , w einheim and [ 43 ] , c opyright 2 005 , americanc hemical society . recently , o tto and co - workers reported the first example of template - driven dynamic covalentn pf unctionalization , creating optimal dna B-event - I-event binding I-event monolayers via selection from ad ynamic combinatorial library of aunp B-nanoparticle - bound imines B-material ( figure 9 ) . [SEP]
[CLS] am ixed monolayer of aldehyde B-material ligand 15 and cationic B-material ligand 16 wast reated in aqueous solution with an excess of three aromatic amines B-material ( 17 - 19 ) , chosen for their differing potential to form noncovalent interactions with dna . [SEP]
[CLS] using hplc to quantify the population of unbound amines B-material , i t could be inferred that negligible concentrations of imine B-material form in the absence of at emplate . [SEP]
[CLS] however , on addings hort oligonucleotides , selective uptake of different amines B-material was observed , with imine B-material product distributions dependento nt he base - pair sequence of the dna template . [SEP]
[CLS] the multivalent presentation of functionality on an ps urface , combined with rapid dynamic covalente xchange of monolayer components opens the way to combinatorial selection of binding surfaces for probingavariety of large - area noncovalent biomolecular interactions , which have proven particularly challenging to address using traditional host - guest systems . [SEP]
[CLS] in the same journal issue , we reported the first example of dynamic covalente xchange used to reversibly switch npbound dynamic covalentf unctionality between multiple kinetically stable states ( figure 10 ) . [SEP]
[CLS] aunps B-nanoparticle coatedw ith hydrazone terminated ligands could be modified at will by reaction with confocalm icroscopy images show emulsions with fitc - dextran dye labelling the aqueousphase . [SEP]
[CLS] adapted with permission from reference [ 50 ] , c opyright 2 013 wiley - vch verlag gmbh & co . kgaa , w einheim . b ) photocleavage of ortho - nitrobenzyl ligand 13 releases af luorescence quencher I-property , r estoring qd emission [SEP]
[CLS] 9 . dna - templated formationo fd ynamic covalent imine B-material aunp B-nanoparticle monolayers [SEP]
[CLS] monolayer composition dependsont he specific base pair sequence of the dna template introduced ; n egligible imine B-material formation is observed in the absence of dna [SEP]
[CLS] 10 . reversible dynamic covalent modificationo fanp ligand shellb y hydrazone B-material exchange , and concomitant solvent compatibility switching . [SEP]
[CLS] solvents : a = hexane , b = chloroform , c = tetrahydrofuran , d = methanol , e = n , n - dimethylformamide , f = water B-material . [SEP]
[CLS] adapted with permission from reference [ 58 ] [SEP]
[CLS] the structural changec ould be harnessed to produce ac oncomitantc hange in np B-nanoparticle solvent compatibility : aunp B-nanoparticle - 20 , c olloidally stable only in polar B-material aprotic I-material solvents I-material , was converted into aunp B-nanoparticle - 21 , w hich exhibits colloidal stabilityi nl ess polar B-material organic I-material solvents I-material , by reaction with hydrophobic B-property aldehyde B-material 22 . l ikewise , exchange with sulfonated aldehyde B-material 23 produced water - compatible aunp B-nanoparticle - 24 . t [SEP]
[CLS] focus reviewhe reversible exchange process allowed each state to be accessed from either of the other two , simply by selecting the appropriate aldehyde B-material exchange moiety ( 22 , 23 , o r25 ) , while the acid catalyst B-property and all other unbound molecular speciesc ould be easily removeda ta ny stage . [SEP]
[CLS] by incorporating appropriate fluorine B-material labels , and ensuring complete purification from unbound species , the exchange process could be monitored in real time by fnmr , providing direct quantitative assessment of reactivityi nt he np B-nanoparticle - bound monolayer . [SEP]
[CLS] althoughd ynamic covalent np B-nanoparticle monolayer functionalization is clearly still in its infancy , t hese first two examples illustrate ak ey advantage of this approach . [SEP]
[CLS] one system relies on kinetically andt hermodynamically unstabled ynamic covalent bonds , the other exploits reversible covalent exchange under specific conditions , to produce kinetically stable products that may be isolated , purified , characterized and stored . [SEP]
[CLS] the ability to access such aw ide range of reactivity , c ombined with the inherents tructurald iversity afforded by moderno rganic chemistry , s uggestst hat dynamic covalent approaches can provide an extremelyp owerful addition to the range of strategies for the reversible and responsive control of np B-nanoparticle - bound functionality and properties . [SEP]
[CLS] definingt he interface between ac olloidal np B-nanoparticle and the outside world , the surface - bound molecules are crucial to controlling ah ost of np B-nanoparticle properties ; p recise control overm onolayer structural detailsa tt he molecular , s upramolecular and nanoscale levels is therefore essential for virtually any potential application . [SEP]
[CLS] while well - established direct synthetic routes , ligand exchange , oligonucleotide hybridization , and amphiphile B-property encapsulation approaches will continuet op rovide essential solutions , methods forr eversible and stimuli - responsive postsynthetic manipulationo fn pp roperties using nonbiomolecular systems can lead to an as - yet unimagined array of smart , functional nps B-nanoparticle . [SEP]
[CLS] ap roper understanding of the multifarious parameters affecting molecular behavior within the np B-nanoparticle - bound monolayer will be essential to arrivinga tp redictable and generalizable strategies . [SEP]
[CLS] notwithstanding the significant challenges facing molecular - level characterization within dilute , heterogeneous and polydisperse systems , many of the examples discussed here illustrate how advances in synthetic strategy , p urification protocols , analyticalm ethods andt heoretical understanding are now combining to achieve unprecedented levels of information , and therefore link phenomenological behavior to structural details . [SEP]
[CLS] at the same time , complexity is one of the most exciting aspects of these multicomponent systems , p resenting possibilities that justd on ot exist in the solution - phase molecular world . [SEP]
[CLS] protonation state of surface - boundspeciesisanostensibly simple parameter that can have ap rofounde ffect on np B-nanoparticle properties ( section 2 ) , yet the nanoscale environmentp rovides unique opportunities for controlling np B-nanoparticle surface charge . [SEP]
[CLS] for example , exploiting the effect of local surface curvature on pk a , distinct charge ' patches ' can be created on nonspherical nps B-nanoparticle bearing an otherwise compositionally uniform monolayer . [SEP]
[CLS] in the complexe nvironment of an p - bound monolayer , s everal inter - relatedf actors can modifym olecular behavior , and therefore have the potentialt oi nfluence systemlevel properties . [SEP]
[CLS] analytical methods that can better probe molecular states with nanoscale resolution will be essential in fully understanding these effectsa nd exploiting the potentialt hat they offer for np B-nanoparticle property control . [SEP]
[CLS] mixed monolayers introduce furtheri mportantc hallenges , including improved control and analysis of ligand compositiona nd distribution ( which can vary from intimately mixed to fully phase - separated ) . [SEP]
[CLS] from this will come [SEP]
[CLS] truly multifunctional np B-nanoparticle systemsw here two or more surface - bound ligands each play an active role in definings timuliresponsive system properties . [SEP]
[CLS] advances in supramolecular design have allowed np B-nanoparticle - bound nonbiomolecular host - guest systemst ob egin to rival oligonucleotide hybridization , t ot he extentt hat they can control remarkable stimuli - responsive np B-nanoparticle systemsi nl iving cells B-material ( section 3 . 1 ) . [SEP]
[CLS] developing analytical methods capable of directly probing supramolecular structure and dynamics within the monolayer , o fd istinguishing different noncovalentb inding modes , and even different nano - locations will be key to driving future advances here . [SEP]
[CLS] having independently enjoyed similarly rapid development trajectories , recent examples suggestt hat the interface between artificial molecular machines and functional npsi sn ow ripe for exploiting ( section 3 . 2 ) . [SEP]
[CLS] understanding the interdependent relationships between np B-nanoparticle features , machine performance and system properties presentsf ormidable challenges , but there are undoubtedly great rewards to be had by combining the remarkable achievements of these two fields of modernsynthetic chemical technology . [SEP]
[CLS] while kinetically controlled covalent chemistry hasb een am ainstay of postsynthetic monolayer functionalization , the recent introduction of stimuli - responsive and dynamic covalent systemsthat combine the stabilitya nd diversity of covalent structures , with the tunable and adaptable behavior of equilibrium systems , promises as tep - change in capabilities ( section 4 ) . [SEP]
[CLS] these methodsw ill afford unprecedented control over monolayer composition , in response to either physicalo r chemicali nputs , using only well - definedc ovalents tructures and without disrupting the np B-nanoparticle - ligand interaction . [SEP]
[CLS] nondestructive methods for monitoring reaction processes in situ will be crucial to developing predictable methodology here ; j ust as molecular synthesis is underpinned by detailed mechanistic understanding , so too musts ynthetic methods on the nanoscale . [SEP]
[CLS] although the majority of examples discussed here involve aunps B-nanoparticle - in many respects the archetypal monolayer - stabilized np B-nanoparticle - strategies that manipulatet he monolayer in place are more readily generalizablet oawide range of nanomaterials B-material than those that attempt to adapt np B-nanoparticle synthetic routes , or to replace the surface - bound species in their entirety . [SEP]
[CLS] the ultimate goal to which synthetic chemists must aspire is as et of universal strategies , and ap redictable structure - property understanding , applicable irrespective of np B-nanoparticle material B-material , size , or shape . [SEP]
[CLS] when considered in totality , t he remarkable and unique properties possible for the core B-material nanomaterial B-material , combined with the vast diversity of modern - day molecular and supramolecular chemistry , a nd the multitude of parameters that can be called upon to manipulate surface - bound molecular behavior ( vide supra ) , suggest we are only just beginning to scratch the surface in terms of what can be achieved by postsynthetic manipulation of np B-nanoparticle - bound monolayers . [SEP]
[CLS] the excitingp ossibilities for controlling np B-nanoparticle properties and designing smart np B-nanoparticle devicesw ill play an important role in fully realizing the technological potential of monolayer - stabilized nanomaterials B-material . [SEP]
[CLS] 1 . cartoon representation of am onolayer - stabilized np B-nanoparticle and postsyntheticm ethods for ligand modification . [SEP]
[CLS] from top to bottom : l igand exchange , charge switching , h ost - guest complexation , molecular switches , irreversible covalent reactions , reversible covalent reactions . [SEP]
[CLS] monolayer molecular structure plays ac rucial role in determining avariety of important systemf eatures . [SEP]
[CLS] edwards received his mchem degree from the university of york in 2009 , s taying there to complete ap hd studying multicomponent supramolecular gels under the supervision of prof . david k . smith in 2013 . [SEP]
[CLS] following this he joined the group of dr euanr . k ay as ap ostdoctoral researchf ellow . h is current researchf ocuses on the controlled synthesis and functionalization of ligands tabilized nanoparticles B-nanoparticle . [SEP]
[CLS] euan kay received his mchem from the university of edinburgh in 2002 and completed his phd there in 2006 under the supervision of david a . leigh . [SEP]
[CLS] he was the recipient of a2 007 iupac prize for young chemists . [SEP]
[CLS] followingp ostdoctoral work in edinburgh , he joined prof . moungi bawendia tm it ( 2008 - 2010 ) . [SEP]
[CLS] since 2011 , he hasbeen aroyal society of edinburgh / scottish government personal research fellow at the university of st andrews . [SEP]
[CLS] his research interests focus on translating dynamic and stimuli - responsive ( supra ) molecular systems into the nanoworld . [SEP]
[CLS] manipulating np B-nanoparticle surface charge by proton transfer . a ) homogeneous monolayers of acylsulfonamide 1 allow switching between neutral ( zwit - terionic ) and cationic B-material surfacecharges at mildly acidic ph values . [SEP]
[CLS] figure 2 . b ) mixed monolayers of permanently cationic B-material ( 2 ) and switchable carboxylic B-material acid I-material ( 3 ) l igands allow switchingb etween positive , neutral and negativesurface charges , accompanied by reversible modulation of colloidal stability . [SEP]
[CLS] 016 the authors . published by wiley - vch verlag gmbh & co . kgaa , weinheim 92 focus review 2199692x , 2016 , 2 , downloaded from https : / / onlinelibrary . wiley . com / doi / 10 . 1002 / cnma . 201500146 by enpc - ecole des ponts paristech , wiley online library on [ 19 / 12 / 2022 ] . [SEP]
[CLS] see the terms and conditions ( https : / / onlinelibrary . wiley . com / terms - and - conditions ) on wiley online library for rules of use ; oa articles are governed by the applicable creative commons license [SEP]
[CLS] 2199692x , 2016 , 2 , downloaded from https : / / onlinelibrary . wiley . com / doi / 10 . 1002 / cnma . 201500146 by enpc - ecole des ponts paristech , wiley online library on [ 19 / 12 / 2022 ] [SEP]
[CLS] see the terms and conditions ( https : / / onlinelibrary . wiley . com / terms - and - conditions ) on wiley online library for rules of use ; oa articles are governed by the applicable creative commons license tially indistinguishable from the covalentb ond forming and breaking reactionsdiscussed in section4 . 1 . [SEP]
[CLS] covalent cleavage reactionsf or nanoparticle B-nanoparticle property control . [SEP]
[CLS] figure 8 . a ) acid - triggered cleavageo fahydrophobic tetrahydropyrangroup disrupts water - in - oil pickeringe mulsions stabilized by aunp B-nanoparticle - 11 0 . 9 12 0 . 1 ; subsequent addition of more watera nd agitation leads to an invertedano il - in - water emulsions tabilized by aunp B-nanoparticle - 12 . confocalm icroscopy images show emulsions with fitc - dextran dye labelling the aqueousphase . [SEP]
[CLS] adapted with permission from reference [ 50 ] , c opyright 2 013 wiley - vch verlag gmbh & co . kgaa , w einheim . b ) photocleavage of ortho - nitrobenzyl ligand 13 releases af luorescence quencher I-property , r estoring [SEP]
[CLS] qd emission . [ 51a ] [SEP]
[CLS] 10 . reversible dynamic covalent modificationo fanp ligand shellb y hydrazone B-material exchange , and concomitant solvent compatibility switching . [SEP]
[CLS] solvents : a = hexane , b = chloroform , c = tetrahydrofuran , d = methanol , e = n , n - dimethylformamide , f = water B-material . [SEP]
[CLS] adapted with permission from reference [ 58 ] , c opyright 2 015 wiley - vch verlag gmbh & co . kgaa , weinheim . [SEP]
[CLS] escaping from primary tumors B-material and entering into blood flow , circulating tumor B-material cells B-material ( ctcs ) contain significant information for both the original tumors B-material and metastasis B-event mechanisms . [SEP]
[CLS] ctcs detection has become an effective liquid biopsy of tumors B-material and shows great promise in early cancer detection , disease monitoring , prognosis and personalized medicine . [SEP]
[CLS] despite the urgent need from clinics , ctcs isolation from blood is still a huge challenge due to their extreme rareness in blood . [SEP]
[CLS] well - defined micro / nanostructures and nature - inspired hierarchical architectures offer unique avenues to address this challenge by matching well with the special physical properties of ctcs to sort cells B-material or forming local topographic interactions to strengthen cell B-event adhesion I-event , thereby improving the ctcisolation performance . [SEP]
[CLS] in this review , we first summarize researches on ctcs isolation with diverse micro / nanostructured substrates , which mainly include nanomaterials B-material , microfluidics , dna nanostructures , micromotors and in vivo detection devices . [SEP]
[CLS] in sequence , various ctc - isolation biomimetic architectures , which are inspired by different natural creatures ( e . g . , viruses , cells B-material , extracellular matrix [ ecm ] , plants , and animals ) , have been highlighted . [SEP]
[CLS] at last , remaining challenges and future perspectives in designing ctc - isolation platforms for clinical applications are also discussed . [SEP]
[CLS] indicative valuation information for early diagnosis , therapeutic efficacy , as well as cancer prognosis . [SEP]
[CLS] compared with routine clinical analysis , which is typically realized by surgical removal or tumor B-material biopsy to collect disseminated tumor B-material cells B-material , enumeration of ctcs from peripheral blood has emerged as a " liquid biopsy " and is readily operated in clinical application . [SEP]
[CLS] though urgently needed from clinics , the cellsearch r system is the only ctc - detection technique approved by the us food and drug administration ( fda ) until now . [SEP]
[CLS] however , its clinical application is severely restricted by the low capture efficiency and poor purity . [SEP]
[CLS] this is mainly attributed to the difficulty in reliable ctcs isolation as ctcs are ultrarare in the blood ( usually 1 - 100 ctcs cells B-material amongst 10 million leukocytes and 5 billion erythrocytes per ml ) . [SEP]
[CLS] therefore , highperformance ctc - isolation materials are highly desirable for promoting biological studies and clinical applications . [SEP]
[CLS] various approaches have emerged for efficiently separating rare ctcs from blood . [SEP]
[CLS] and these isolation strategies could be divided into biochemical methods and biophysical methods . [SEP]
[CLS] biochemical methods selectively isolate ctcs by recognizing unique biochemical markers ( epcam , her2 , and egfr ) expressed on ctcs surface . [SEP]
[CLS] biophysical methods separate ctcs utilizing the physical property differences of ctcs and blood cells B-material , such as cell B-material deformability , size , density , and adhesiveness . [SEP]
[CLS] although these strategies have made some progress in separating ctcs , they still have drawbacks and limitations . [SEP]
[CLS] specifically , the expression level of biochemical markers on the tumor B-material cells B-material can be changed because of epithelial to mesenchymal transition ( emt ) process and the heterogeneous nature of ctcs . [SEP]
[CLS] moreover , the size of some ctcs is found to be the same or even smaller than that of wbcs . [SEP]
[CLS] to maximize the isolation efficiency , some researchers developed a variety of dual - model separation strategies by combining biochemical and biophysical methods . [SEP]
[CLS] zhao et al . reported a multifunctional microbead - based density gradient centrifugation method for efficient isolation of ctcs . [SEP]
[CLS] they synthesized gelatin B-material nanoparticle - coated silicon B-material microbeads ( sio 2 @ gel mbs ) and modified them with anti - epcam and anti - cd146 antibodies B-material . [SEP]
[CLS] both epcampositive and epcam - negative ctcs could be obtained using the developed optimized gradient centrifugation medium after the cells B-material were incubated B-technique with sio 2 @ gel mbs , which could effectively increase the size and density of targeted cells B-material . [SEP]
[CLS] jiang et al . developed a fluorescent B-property microspheres - based separation platform by combining affinity - based microspheres attachment and size - based exclusion assay . [SEP]
[CLS] ctcs were first size - amplified and simultaneously labeled by antibody - functionalized fluorescent B-property polystyrene microspheres , then isolated by a pyramidal microcavity array . [SEP]
[CLS] these combinations of biochemical and biophysical methods show superior cell B-material isolation performances . [SEP]
[CLS] however , it is still challenging to efficiently isolate and subsequently profile ctcs because of the extreme rarity of ctcs in the bloodstream . [SEP]
[CLS] well - defined micro / nanostructures and nature - inspired hierarchical architectures may offer unique avenues to address these challenges . [SEP]
[CLS] for biochemical method , nanostructured substrates can promote the formation of local topographic interactions between nanoscaled cell B-material surface components ( e . g . , filopodia and microvilli ) and the underlying nanostructure topography , which is beneficial to improve the capture efficiency . [SEP]
[CLS] for biophysical method , the basic parameters of these structures can be precisely controlled to match well with the special physical properties of ctcs for sorting cells B-material accurately . [SEP]
[CLS] with the rapid advancement of micro / nanotechnology and biomimicry , various programmable materials based on well - defined micro / nanostructures and bio - inspired hierarchical architectures have been ingeniously designed for highly efficient separation of scarce ctcs from blood . [SEP]
[CLS] herein , this review will focus on the development of programmable materials for ctcs isolation ( figure 1 ) . [SEP]
[CLS] first , diverse micro / nanostructured substrates , which mainly include nanomaterials B-material , microfluidics , dna nanostructures , micromotors and in vivo detection devices , will be highlighted . [SEP]
[CLS] subsequently , we will summarize recent progress in ctcs isolation achieved by various multiscale biomimetic architectures , which are inspired by different natural creatures , such as viruses , cells B-material , ecm , plants , and animals . [SEP]
[CLS] lastly , remaining challenges and future perspectives in designing ctc - isolation platforms in clinical applications are also discussed . [SEP]
[CLS] with the rapid development of micro / nanotechnology , various micro / nanostructured B-material materials I-material have been constructed and show huge advantages in ctcs isolation . [SEP]
[CLS] on the one hand , microstructures and nanostructures could match well with the microscale morphology and nanoscale components ( e . g . , microvilli ) of the target cells B-material , respectively . [SEP]
[CLS] on the other hand , their high specific surface area can effectively increase the amount of recognition ligands on the substrate and the collision chances with target cells B-material . [SEP]
[CLS] in this section , we make an overview of the developed micro / nanostructured substrates , which mainly includes static capture materials ( nanomaterials B-material ) and dynamic capture materials ( microfluidics , dna nanostructures , micromotors and in vivo detection devices ) for efficient ctcs isolation . [SEP]
[CLS] and these representative micro / nanostructured substrates and their morphological , working mechanisms , cell B-material - isolation performances , and advantages / limitations are listed in table 1 . [SEP]
[CLS] nanomaterials B-material with well - defined nanoscale topography , which has wide applications in tissue engineering [SEP]
[CLS] various programmable materials based on well - defined micro / nanostructures ( left ) and nature - inspired hierarchical architectures ( right ) for ctcs isolation . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2012 , wiley - vch . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2012 , elsevier . reproduced with permission . [SEP]
[CLS] copyright 2012 , american chemical society . reproduced with permission . [SEP]
[CLS] copyright 2011 , wiley - vch . reproduced with permission . [SEP]
[CLS] copyright 2020 , american chemical society . reproduced with permission . [SEP]
[CLS] copyright 2014 , wiley - vch . reproduced with permission . [SEP]
[CLS] copyright 2020 , elsevier . reproduced with permission . [SEP]
[CLS] copyright 2013 , wiley - vch . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2017 , american chemical society . reproduced with permission . [SEP]
[CLS] copyright 2019 , wiley - vch due to their excellent ecm - mimicking property , can offer a unique route to address these challenges in ctcs isolation . [SEP]
[CLS] compared with the flat substrates , nanostructured substrates have the following advantages : ( 1 ) nanostructured substrates have a higher surface area to contact with ctcs ; ( 2 ) more capture agents can be loaded on the nanotextured substrates ; [SEP]
[CLS] ( 3 ) local topographic interactions of the underlying substrates and nanoscaled cell B-material surface components can be enhanced by nanostructured topography . [SEP]
[CLS] thus , various nanomaterials B-material have emerged for efficiently isolating ctcs . [SEP]
[CLS] silicon B-material - nanopillar ( sinp ) arrays were pioneeringly applied to rare - cell capture by wang et al . ( 2009 ) . [SEP]
[CLS] densely arrayed sinps ( 100 - 200 nm diameter and 1 - 20 μm length ) were produced onto silicon B-material wafers by a wet chemical etching method , followed by grafting biotinylated anti - epcam onto the substrates modified with streptavidin ( figure 2a ) . [SEP]
[CLS] the results revealed that the cells B-material capture yield on the sinp substrates ( 45 % - 65 % ) was 10 - fold higher than that on the flat si substrates ( 4 % - 14 % ) . [SEP]
[CLS] compared with the rounded cells B-material with few nanoscaled cellular protrusions adhered on the flat substrate , the cells B-material on the sinp substrates displayed many 100 - 200 nm f i g u r e 2 nanomaterial - based interfaces for efficient ctcs capture . [SEP]
[CLS] ( a ) nanopillars . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2009 , wiley - vch . ( b ) nanofibers B-nanoparticle . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2012 , wiley - vch . ( c ) hierarchical nanowires B-nanoparticle . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2016 , american chemical society . ( d ) dendrimers B-nanoparticle . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2011 , wiley - vch interdigitated cellular protrusions . this validated that enhanced local topographic interactions between sinps and nanoscaled cell B-material surface components played a main role in vastly increasing the capture performance of cells B-material . [SEP]
[CLS] different from the above reported vertically oriented sinps employed for ctcs enrichment , zhang et al . applied ultralong horizontally packed inorganic nanofibers B-nanoparticle into ctcs detection ( figure 2b ) . [SEP]
[CLS] densely packed calcinated tio 2 nanofibers B-nanoparticle ( tinfs ) with the size regime of 100 - 300 nm in diameter could be prepared by electrospinning the mixture solution of polyvinyl pyrrolidone ( pvp ) / titanium B-material n - butoxide ( tbt ) . [SEP]
[CLS] then the streptavidin - coated substrates were freshly grafted with biotinylated anti - epcam for testing capture performance of cancer cells B-material . [SEP]
[CLS] the capture efficiency of cells B-material on tinf substrate was more than 18 times than that of the flat si substrate after incubating B-technique with epcam - positive cells B-material ( bgc823 and hct116 ) . [SEP]
[CLS] and over 45 % of spiked cells B-material ( i . e . , hct116 ) maintained viable after isolating from the artificial blood samples . [SEP]
[CLS] it was observed that target cells B-material adhered on the tinfs surface exhibited completely outspread pseudopodia . [SEP]
[CLS] moreover , the sizes of the tinfs and the pseudopodia matched well to achieve sufficient contact and strong adhesion force , showing that the reformative substrate / cell B-material affinity was caused by the enhanced local topographic interactions between the cellar components and the horizontally packed tinfs . [SEP]
[CLS] combining horizontal with vertical ito nanowire B-nanoparticle branches , wang et al . further constructed a novel three - dimensional ( 3d ) fractal nanobiointerface for efficiently capturing cancer cells B-material . [SEP]
[CLS] higher efficiency ( 89 % vs . 67 % ) and shorter time ( 35 minutes vs . 45 minutes ) could be achieved using this fractal nanobiointerface when compared to ito nanowire B-nanoparticle array without branches . [SEP]
[CLS] what is more , environment scanning electron B-technique microscopy I-technique ( esem ) images showed that the cells B-material on the fractal nanobiointerface exhibited most filopodia ( figure 2c ) , which could be attributed to the combined action of the vertical and horizontal nanowire B-nanoparticle branches . [SEP]
[CLS] nanoscale poly ( amidoamine ) ( pamam ) dendrimer B-nanoparticle , which is well known for its easy deformability and the capability to preorganize / orient ligands , is thought to be an ideal mediator for forming multivalent binding effect , which was important to significantly improve the performance of ctcs isolation from peripheral blood . [SEP]
[CLS] as shown in figure 2d , the capture yield of cancer cells B-material on the dendrimer - immobilized surfaces ( 70 % ) was greatly enhanced than that on the pegylated surfaces ( 20 % ) for the simulated clinical samples , indicating that the multivalent binding effect of dendrimer B-nanoparticle played a key role in enhancing the cell B-material detection sensitivity . [SEP]
[CLS] this was mainly attributed to that the 3d structure of the dendrimer B-nanoparticle could effectively reduce the deformation energy ( entropy ) of dendrimer B-nanoparticle ligands to recognize their receptors , facilitating the formation of local multivalent binding . [SEP]
[CLS] moreover , capture efficiency could be increased by seven times by the addition of e - selectin , indicating that e - selectinmediated cell B-material rolling could synergistically cooperate with multivalent binding for substantially enhancing the tumor B-material cell B-material detection . [SEP]
[CLS] besides , many other types of nanomaterials B-material , such as nanodots , nanosheets , nanofilms , and nanoparticles B-nanoparticle , have also been introduced to enhance capture efficiency . [SEP]
[CLS] the microfluidic technique , which could precisely handle fluids in the microscale with high precision and sensitivity , has emerged as a promising tool for in vitro ctcs capture and isolation . [SEP]
[CLS] compared with conventional methods , microfluidic technique shows many advantages such as satisfactory sensitivity , automatic operation , and multiplexing capabilities . [SEP]
[CLS] first of all , the microfluidic technique with large surface - area - to - volume ratios could trap rare ctcs using a very small sample volume in a highly sensitive way . [SEP]
[CLS] moreover , it could simultaneously achieve sample collection , isolation , and analysis using a one - step process , significantly reducing the processing time and improving chances of capturing viable ctcs . [SEP]
[CLS] last but not the least , many advanced nanotechnologies and different functional units can be facilely integrated into microfluidic platforms for multiple analysis of ctcs . [SEP]
[CLS] recently , various ctc - isolation microfluidic devices have been developed . [SEP]
[CLS] microfluidic techniques mainly include " label - free strategies " and " affinity - based strategies . [SEP]
[CLS] " the label - free strategies achieve ctcs separation utilizing the physical property differences between normal blood cells B-material and ctcs . [SEP]
[CLS] size and deformability , which are considered as two commonly separation criteria , are usually employed in separating cells B-material . [SEP]
[CLS] compared with the size of normal blood cells B-material ( 2 - 20 μm ) , most epithelial - derived ctcs showed much larger cell B-material size ( 14 - 26 μm ) . [SEP]
[CLS] based on the size difference , hosokawa et al . constructed a microfluidic device to achieve size - based separation of ctcs . [SEP]
[CLS] a nickel B-material microfilter made up of 100 × 100 holes with 8 - 11 μm diameters was coupled to the microfluidic device ( figure 3a ) . [SEP]
[CLS] the capture yield of ctcs was approximately 97 % when the microfluidic device was applied to an artificial sample ( 10 - 100 lung carcinoma nci - h358 cells B-material per ml blood ) . [SEP]
[CLS] additionally , the viable cells B-material account for approximately 98 % after recovery , showing this platform could separate ctcs in a friendly way . [SEP]
[CLS] cell B-material deformability is another approach to improve the selectivity of distinguishing ctcs from hematological cells B-material . [SEP]
[CLS] due to having an enlarged nucleus , the nucleus - to - cytoplasm ratio and deformability of tumor B-material cells B-material are greater and less than that of leukocytes . [SEP]
[CLS] inspired by this , park et al . designed a deformability - based microfluidic device composed of a matrix of tapered constrictions to separate ctcs ( figure 3b ) . [SEP]
[CLS] the tapered constriction matrix was composed of 2048 columns and 32 rows . [SEP]
[CLS] the pore size of the constrictions remained unchanged along each row and increased ( from 2 to 18 μm ) from the top to the bottom of the matrix . [SEP]
[CLS] the infused cells B-material diagonally proceeded over the funnel matrix . [SEP]
[CLS] rbcs could only pass through the funnel top due to their high deformability . [SEP]
[CLS] while ctcs and white blood cells B-material ( wbcs ) proceeded diagonally before reaching an obstructive funnel row , then they proceeded horizontally to separate outlets . [SEP]
[CLS] the capture yield of ctcs on this microfluidic device could reach up to > 90 % for artificial samples , which were prepared by mixing cancer cells B-material into unprocessed normal blood samples . [SEP]
[CLS] and the capture yield of ctcs from clinical samples with this chip could be 25 times greater than that with the conventional cellsearch system . [SEP]
[CLS] the affinity - based strategies mainly utilize the recognition between expressed specific biomarkers B-property on the cancer cell B-material surface ( e . g . , cd45 and epcam ) and their corresponding aptamers or antibodies B-material coated on the surface of microfluidic platform . [SEP]
[CLS] and these strategies could be divided into positive selection which isolates ctcs by targeting biomarkers B-property expressed on cancer cells B-material ( epcam , her2 , and egfr ) and negative selection , which removes hematopoietic cells B-material by targeting antigens expressed on blood cells B-material ( cd45 ) . [SEP]
[CLS] one representative example of positive selection was the so - called ctc - chip , which was developed by toner ' s group . [SEP]
[CLS] the ctc - chip contained 78 , 000 microposts within 19 mm × 51 mm active capture area . [SEP]
[CLS] the dimensions of microposts and their gap were 100 μm × 100 μm and 50 μm , respectively . [SEP]
[CLS] and every three rows of the equilateral triangular arrays were vertically shifted by 50 μm throughout the chip , maximizing the chip - cells B-material interactions . [SEP]
[CLS] and the ctc - chip could successfully recognize ctcs from the blood of all the patients ( 115 / 116 ) with various metastatic cancers ( breast , f i g u r e 3 microfluidics - based " label - free strategies " ( a , b ) and " affinity - based strategies " ( c , d ) for ctcs isolation . [SEP]
[CLS] ( a ) microfluidics device equipped with microcavity array for size - selective ctcs recovery and sem image showing trapped mcf - 7 cells B-material on the microcavity array . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2010 , american chemical society . [SEP]
[CLS] ( b ) microfluidic ratchets with oscillatory flow for ctcs isolation based on deformability . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2016 , wiley - vch . ( c ) ctcs isolation on a microvortex - generating herringbone - chip . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2010 , national academy of sciences . [SEP]
[CLS] ( d ) negative enrichment of ctcs using 3d - printed microfluidic device integrated with a commercial membrane filter . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , royal society of chemistry pancreatic , prostate , lung , and colon cancer ) . [SEP]
[CLS] however , the interaction between ctc - chip substrate and target cells B-material were limited by laminar flow . [SEP]
[CLS] to overcome this problem and disrupt streamlines , they developed a high - throughput microfluidic by integrating herringbones or surface ridges into the device wall , which could maximize collisions between the antibody - modified microfluidic walls and target cells B-material . [SEP]
[CLS] the herringbone - chip consisted of a 1 ″ × 3 ″ glass slide fixed to a polydimethylsiloxane ( pdms ) structure , which contained eight microchannels with patterned chevrons or herringbones on their upper surface . [SEP]
[CLS] the height of the channels and grooves were 50 μm and 40 μm , respectively . [SEP]
[CLS] the angle between the axis of the channel and the herringbones was 45 • , and the principal wave vector was 2π / 100 μm . [SEP]
[CLS] and the herringbonechip could identity ctcs from the blood of patients ( 14 / 15 ) with metastatic prostate cancer . [SEP]
[CLS] this was mainly due to that the herringbone - induced microvortices could cause " shift " of cells B-material travel by disrupting the laminar flow streamlines , when compared with , thus increasing the collision frequency between cells B-material and chips than that of a traditional flat - walled microfluidic device ( figure 3c ) . [SEP]
[CLS] while the positive selection is highly effective , it usually suffers from a biased population due to tumor B-material heterogeneity and varying cell B-material expression induced by epithelial to mes - enchymal transition . [SEP]
[CLS] in order to solve this problem , the negative selection method to isolate ctcs is proposed . [SEP]
[CLS] it could selectively deplete wbcs by directly targeting their well - known overexpressed membrane biomarkers B-property ( e . g . , cd45 ) , thus achieving enrichment of ctcs from blood . [SEP]
[CLS] chu et al . developed a monolithic device using 3d printing technology by integrating a commercial membrane filter into immunoaffinity - based cell - capture microfluidic and for negative directly enriching ctcs from whole blood ( figure 3d ) . [SEP]
[CLS] the cell B-material - capture section consisted of 4 - 32 stacked microfluidic layers with 475 - μm pitch and 175μm height . [SEP]
[CLS] inside the microfluidic layers , 200 - μm microposts were separated by 200 μm from each other and shifted by 10 μm in every row to enable sufficient collision with wbcs , which may follow different flowlines in laminar flow . [SEP]
[CLS] followingly , a membrane filter with 3 μm - pore size was used to filter the leukodepleted blood for eliminating anucleated blood cells B-material . [SEP]
[CLS] this device could eliminate the need of rbcs - lysing sample preparation step . [SEP]
[CLS] and enriched tumor B-material cells B-material showed [UNK] % recovery rate after isolation from artificial samples , which were mixed with breast , prostate , or ovarian cancer cells B-material . [SEP]
[CLS] moreover , prostate cancer ctcs could also be directly isolated from a 10 ml clinical whole blood sample by this device . [SEP]
[CLS] f i g u r e 4 dna nanostructure - based platforms for ctcs isolation . [SEP]
[CLS] ( a ) 3d dna network for ctcs detection . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2012 , national academy of sciences . ( b ) nanovelcro chip coated with aptamer for capture and enzymatic release of ctcs . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2013 , wiley - vch . ( c ) a deterministic lateral displacement ( dld ) - patterned microfluidic chip ( aptdn - chip ) integrated with homogeneously orientated tetrahedral dna nanostructures ( tdns ) for enhancing the isolation performance of ctcs from whole blood . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , wiley - vch [SEP]
[CLS] as a naturally occurring biomacromolecule , dna has attracted considerable attention in ctcs detection due to some of their unique features , such as good affinity , high controllability , ease of synthesis , and inherent biocompatibility B-property . [SEP]
[CLS] additionally , exquisite dna nanostructures with various sizes , dimensions , and shapes can be formed due to the precisely programmable nucleotide base pairing of dna . [SEP]
[CLS] when used for ctcs isolation , dna molecules ( e . g . , aptamer ) have many advantages over antibodies B-material : ( 1 ) aptamers are more stable under various harsh conditions and can be easily modified with various chemical groups ; ( 2 ) aptamers are easier to recognize cell B-material membrane markers due to their small size , which could enhance the recognition performance in identifying distinct subpopulations ; and ( 3 ) cell B-material release can be achieved under mild conditions ( e . g . , nucleases or the complementary strand of an aptamer ) , which is beneficial to maintain cell B-material vitality . [SEP]
[CLS] as shown in figure 4a , a 3d dna network was prepared by rolling circle amplification ( rca ) to capture and isolate ctcs . [SEP]
[CLS] the diameter of dna network ranged from < 500 nm to 5 μm , which consisted of repeating adhesive aptamer domains , could extend as long as 20 μm into the solution and " wriggled " in 3d form . [SEP]
[CLS] significantly greater capture yield and higher cell B-material purity were shown on the multivalent dna networks than that on antibodies B-material and monovalent aptamers . [SEP]
[CLS] additionally , cell B-material release could be obtained after cleaving the dna networks using restriction enzymes , which guaranteed the cell B-material vitality for molecular analysis . [SEP]
[CLS] shen et al . designed a novel nanovelcro chip by grafting two dna - aptamers onto silicon B-material nanowires B-nanoparticle ( sinws ) in a stationary device setting ( figure 4b ) . [SEP]
[CLS] then microfluidic chaotic mixer was overlaid onto the sinws to increase the contact frequency of the substrate and flowing nsclc ctcs , thereby improving the cell B-material - capture efficiency . [SEP]
[CLS] the capture yield and release efficiency of cells B-material could exceed 80 % and 85 % , respectively . [SEP]
[CLS] and the recovered a549 cells B-material exhibited 78 % - 83 % viability , showing this method hardly disrupted the viability and functions of ctcs . [SEP]
[CLS] the orientation of aptamers is another important factor influencing their ctc - enrichment performance except for the loading amount . [SEP]
[CLS] and it has been reported that nanotopographic aptamers with upright - orientation are favorable for target interaction . [SEP]
[CLS] to address this issue , zhang et al . developed a novel aptdn - chip based on sub - 10 nm tdns as frameworks , which had a pendant aptamer at the top vertex . [SEP]
[CLS] as shown in figure 4c , the nanoscale tdns ensured that the aptamer orientated in a highly ordered upright form , which could effectively avoid the undesired orientation or local overcrowding effect . [SEP]
[CLS] combined with the dld principle - based triangular micropillar array , the aptdn - chip showed superior ctc - isolation performance . [SEP]
[CLS] in contrast to chip modified with a monovalent aptamer , approximate 60 % enhancement of the capture efficiency could be achieved on the aptdn - chip as a result of the high - precision dimension and rigid framework of tdns . [SEP]
[CLS] moreover , the chip showed 91 % cell B-property viability I-property and 83 % release efficiency as dnase i was readily accessible to the aptamer on the scaffolds , indicating the platform was available for downstream molecular analysis . [SEP]
[CLS] the methods mentioned above all adopted nuclease degradation to release cells B-material , which may cause damage to the cells B-material . [SEP]
[CLS] in contrast , complementary sequence displacement seems to be more efficient to keep the cell B-property viability I-property . [SEP]
[CLS] sun et al . prepared a functional biointerface based on electrospun chitosan B-material nanofibers B-nanoparticle . [SEP]
[CLS] poly ( carboxybetaine methacrylate ) ( pcbma ) brushes were grafted onto nanofiber B-nanoparticle surface to resist nonspecific adhesion B-event of I-event cells I-event and immobilize multivalent aptamers , ensuring the nanofibers B-nanoparticle capture highly pure ctcs with high efficiency . [SEP]
[CLS] furthermore , nondestructive release of the captured target cells B-material with high cell B-property viability I-property ( 90 . 5 % ) could be achieved by introducing a complementary sequence . [SEP]
[CLS] lin et al . further developed a multivalent dualaptamer - tethered rolling circle amplification ( ma - rca ) network for facile and effective capturing ctcs . [SEP]
[CLS] first they used rca to generate periodic sequence units onto a long dna scaffold , which was flowingly modified by interval hybridizing two different receptor - recognizing aptamers . [SEP]
[CLS] integration of multiple dual - aptamers made the long dna strand elastic and this allowed it effectively contact targeted cells B-material , thereby diminishing the cells B-material movement and tightly grasping them for imaging and analysis . [SEP]
[CLS] it is noteworthy that invertible capture and release of ctcs could be realized just by toehold - mediated strand displacement triggered by dna . [SEP]
[CLS] and this paves a new way of achieving dynamic capture and release of cells B-material in a noninvasive manner . [SEP]
[CLS] micromotors , which could move autonomously by effectively transforming various energy sources into driving powers , have paved a new way for various biomedical applications , such as precision surgery , targeted delivery , and motion - based biosensing disease marker . [SEP]
[CLS] for biosensing application , self - propelled micromotors could be used for capturing and transporting target substances by providing efficient fluid mixing . [SEP]
[CLS] inspired by this feature , balasubramanian et al . pioneeringly applied micromotor to ctcs isolation . [SEP]
[CLS] the micromotor was a reeling metal B-material sheet composed of platinum B-material , iron B-material , and gold B-material from the inside out ( figure 5a ) . [SEP]
[CLS] the inner platinum B-material layer could convert peroxide into water B-material and oxygen B-material , providing the driving force for the micromotor motion . [SEP]
[CLS] the sandwiched iron B-material layer , which was sensitive to an external magnetic B-property field , could steer the motion direction of the micromotor . [SEP]
[CLS] the outer gold B-material layer could be readily functionalized with anticarcinoembryonic antigen ( anti - cea ) that specifically target cea , which were overexpressed in about 95 % of pancreatic , gastric , and colorectal cancers . [SEP]
[CLS] as a result , target cancer cells B-material in both phosphate - buffered saline ( pbs ) and serum could be selectively captured and effectively transported by this micromotor . [SEP]
[CLS] the micromotor moved at the rate of 85 - 80 μm / s before and after capturing cancer cells B-material in serum environment , indicating the micromotor had a high towing force . [SEP]
[CLS] the specific recognition of the cea + cancer cells B-material to micromotors modified with anti - cea mab was proved by control experiments . [SEP]
[CLS] compared with the control groups ( group 1 : anti - cea mab - modified micromotors and the pancreatic cancer cells B-material without antigen [ cea− ] ; group 2 : sam - modified micromotors without the mab and the cea + pancreatic cancer cells B-material ) , only the micromotors modified with anti - cea mab could trap the target cea + cancer cells B-material . [SEP]
[CLS] furthermore , the micromotor could specifically identity target cancer cells B-material from cell B-material mixtures , offering a novel approach based on immunomicromachine for in vitro ctcs detection without sample preprocessing . [SEP]
[CLS] carbon B-nanoparticle nanotube I-nanoparticle ( cnt ) , which has a larger surface area and rich carboxyl B-material groups I-material due to its special hollow tube structures , is thought to be an ideal substrate for developing ultrafast supersensitive and biosensing systems . [SEP]
[CLS] inspired by this , banerjee et al . developed a chemically powered cnt - based micromotor for specific ctcs isolation . [SEP]
[CLS] the cnt - based micromotor [SEP]
[CLS] in vivo ctc detection technology , which can monitor the dynamic change of ctc number , may offer new tools for more in - depth cancer diagnosis and metastasis B-event research . [SEP]
[CLS] nowadays there are mainly two types of in vivo ctc detection technologies : gilupi cellcollector and in vivo flow B-technique cytometry I-technique ( ivfc ) . [SEP]
[CLS] gilupi cellcollector mainly consists of a bioclean stainless steel medical wire , which is modified with antibody B-material and placed in the vein for 30 minutes to enrich ctcs in vivo . [SEP]
[CLS] and the detection rates of ctcs are 70 % and 72 % for early and late cancer stages , respectively . [SEP]
[CLS] ivfc technology could monitor circulating cells B-material within living animals in real - time based on various contrast principles , such as fluorescence B-property excitation and emission , photothermal effect , photoacoustic effect . [SEP]
[CLS] while these two technologies have made some progress in ctcs detection , their further applications are limited by that they are unable to capture typical ctcs from blood . [SEP]
[CLS] vermesh et al . developed a new technology named mag - wire for intravascularly retrieving and enriching ctcs . [SEP]
[CLS] magwire , a flexible self - contained magnetic B-property wire , could be encapsulated in a standard intravenous catheter and readily inserted into a superficial blood vessel due to its small diameter , flexibility , and biocompatible B-property plastic sheath . [SEP]
[CLS] first the ctcs in the blood were labeled by injected magnetic B-property particles coated with antibody B-material . and the magnetic B-property fiber could capture the labeled cells B-material when the entire blood volume flowed past . [SEP]
[CLS] then the bound targets could be released into the buffer for downstream analysis when displacing the magnets from the magwire sheath . [SEP]
[CLS] and viable model ctcs could be captured by the in vivo labeling and pass within 10 seconds . [SEP]
[CLS] moreover , the capture efficiency was improved by 10 - 80 times and 500 - 5000 times than a 5 ml blood draw and the commercially available gilupi cellcollector , respectively . [SEP]
[CLS] cheng et al . developed a flexible 3d ctc - net probe for in vivo intravascularly capturing ctcs . [SEP]
[CLS] the ctc - net , which consisted of a 3d elastic poly ( dimethylsiloxane ) ( pdms ) scaffold composed of connected macropores with 40 - 250 μm size , could accommodate a large number of immobilized antibodies B-material and improve cell - substrate collision frequency . [SEP]
[CLS] the ctc - net was first functionalized with anti - epcam antibody B-material and then sticked into a blood vessel using an indwelling needle after compression . [SEP]
[CLS] finally , a 3d " fishing - net " structure for capturing ctcs could be formed in the vessel copyright 2020 , wiley - vch . [SEP]
[CLS] ( c ) immunomagnetic beads coated with platelet - leukocyte hybrid membrane for efficiently and specifically isolating ctcs . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , american chemical society . ( d ) a microfluidic chip decorated with aptamer - functionalized leukocyte membrane nanovesicles for efficient ctcs separation . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , american chemical society after the compressed ctc - net fully unfolded ( figure 5b ) . [SEP]
[CLS] extremely low concentration of rare ctcs ( less than 1 cell B-material / ml ) could be collected by the ctc - net , permitting diagnosis of early - stage tumors B-material . [SEP]
[CLS] moreover , captured ctcs could also be downstream analyzed due to the recompression and regain of the ctc - net . [SEP]
[CLS] the ctc - net could successfully isolate dozens of ctcs from tumor - bearing rats before cancer metastasis B-event , showing great potential in ctcbased early diagnosis , metastasis B-event research , and targeted therapy of cancer . [SEP]
[CLS] natural selection has resulted in the evolution of numerous materials and structures , which have been optimized for a broad variety of functions . [SEP]
[CLS] and this also provides a vast database of optimized solutions to technical problems with the survival of biological organisms , such as virus , cells B-material , plants , and animals . [SEP]
[CLS] in this section , various ctc - isolation biomimetic architectures inspired by nature creatures ( nanoscale viruses , microscale cells B-material and ecm , macroscale plants and animals ) are systematically summarized . [SEP]
[CLS] and these different morphological and unique features , working mechanisms , cell B-material isolation performances , and advantages / limitations of representative biomimetic architectures were listed in table 2 . [SEP]
[CLS] as the basic structural element and functional unit , cells B-material play key roles in various fundamental biological processes , such as the immune invading capability of red blood cells B-material ( rbcs ) , adhesive property of platelets to surgical sites and ctcs , and virus phagocytosis B-event by host cells B-material . [SEP]
[CLS] inspired by these , a variety of biomimetic platforms mimicking cell B-material morphologies or utilizing the functions of cell B-material membranes have been exploited for ctcs isolation . [SEP]
[CLS] inspired by leukocytes morphologies , meng et al . developed a unique micro / nano hierarchical biointerface by combining chemical B-technique vapor I-technique deposition I-technique ( cvd ) and thermal oxidation . [SEP]
[CLS] as shown in figure 6a particles ( lips ) coated with anti - epcam , played a key role in improving capture efficiency due to the following possible reasons : ( i ) microscale bump of rough lip substrates could touch the microspheric parts of the objective cells B-material ; ( ii ) perpendicular nanofibers B-nanoparticle perpendicular to the spherical parts could match well with the nanoscale components ( e . g . , microvilli ) on the cell B-material surface . [SEP]
[CLS] the capture efficiency of cells B-material on lip substrates ( 62 . 3 % ± 3 . 7 % ) was much higher than that on flat , mono - micro or nanobiointerfaces . [SEP]
[CLS] moreover , the cells B-material on lip substrates showed more lamellipodia and filopodia than the other three substrates . [SEP]
[CLS] furthermore , lip substrates showed 10 % higher capture efficiency than that of nanoscale sinws . [SEP]
[CLS] this revealed that more appropriate contact modes could be provided by the micro / nanoscale hierarchical substrates than flat , mono - micro , or nanobiointerfaces for capturing cancer cells B-material . [SEP]
[CLS] cell imprinting , an emerging technology combining shape matching and chemical recognition , could construct a microstructured interface for precisely matching the topological structures and chemical properties of the cells B-material surface . [SEP]
[CLS] recently , liu et al . developed a universal strategy for constructing an antibody - free hydrogel by combining boronate B-material affinity and cell imprinting ( figure 6b ) . [SEP]
[CLS] the cell - imprinted structures were fabricated by choosing human hepatocarcinoma smmc - 7721 cells B-material as the target cell B-material models . [SEP]
[CLS] with this artificial antibody B-material , high capture efficiency ( 90 % ) and release efficiency ( 99 % ) of cells B-material could be achieved on the cell - imprinted structures , and the released cells B-material showed high proliferation ability . [SEP]
[CLS] moreover , the artificial antibody B-material showed superior selectivity of the targeted ctcs , which could not only capture the smmc - 7721 cells B-material with an enrichment factor as high as 13 . 5 ± 3 . 2 ( n = 3 ) in the present of 1000 times of leukemia jurkat cells B-material as nontarget cells , but also trap the smmc - 7721 cells B-material from whole blood . [SEP]
[CLS] except for cellular morphology , cell B-material membrane with multiple functions has also attracted wide attention . [SEP]
[CLS] for example , platelets ( plts ) have recognition capability for ctcs and homologous wbcs show outstanding dispersibility in circulation . [SEP]
[CLS] inspired by these phenomena , rao et al . coated immunomagnetic beads with plt - wbc hybrid membrane ( hm - imbs ) and modified them with ctc - targeting antibodies B-material for isolating ctcs efficiently and specifically ( figure 6c ) . [SEP]
[CLS] the cell B-material capture efficiency of commercial imbs and hm - imbs was 66 . 68 % and 91 . 77 % , responsively . and their corresponding cell B-material purity was 66 . 53 % and 96 . 98 % . [SEP]
[CLS] this was mainly due to the enhanced binding ability of plts to cancer cells B-material and reduced homologous interactions between wbcs . [SEP]
[CLS] furthermore , the hm - imbs could identify pure ctcs in 19 of 20 ( 95 % ) patients with breast cancer . [SEP]
[CLS] dynamic lateral fluidity , another important property of biomembrane interface , can rearrange and localize membrane - bound com - ponents for multivalent ligand - receptor binding interactions with high affinity . [SEP]
[CLS] inspired by this , wu et al . developed a fluidic aptamer functionalized soft and highaffinity nanointerface microfluidic chip ( flash - chip ) for efficiently isolating ctcs . [SEP]
[CLS] the flash - chip was prepared by grafting leukocyte membrane nanovesicles functionalized with aptamer onto a microfluidic chip ( figure 6d ) . [SEP]
[CLS] the natural leukocyte membrane used for preparing biomimetic nanointerface had various biological functions . [SEP]
[CLS] first of all , recognition ligands can be laterally rearranged for high - affinity binding due to the membrane ' s fluidic nature . [SEP]
[CLS] and recruitment and accumulation of other targeted biomarkers B-property and recognition ligands will occur after a binding event takes place , achieving synergetic multivalent target binding . [SEP]
[CLS] second , nonspecific adhesion B-event of I-event blood I-event cells I-event can be greatly reduced because of the cell - resistant property of the leukocyte membrane . [SEP]
[CLS] third , cell B-material damage caused by interfacial collisions can be greatly reduced by the soft yet flexible nanovesicle layer , which serves as a cushion between the capture substrate and target cell B-material . [SEP]
[CLS] in addition , a capture platform was prepared by intergrating a dld - patterned microarray onto the microfluidic chip ( dld - chip ) with size - dictated interaction . [SEP]
[CLS] this microdevice functionalized with capture ligands could isolate highly pure target ctcs with high capture efficiency through increasing substrate - ctcs collision and decreasing substrate - blood cells B-material interaction . [SEP]
[CLS] compared with a chip functionalized with monovalent aptamer , this fluidic biomimetic nanointerface showed significant affinity increase by four orders of magnitude and sevenfold higher capture yield in blood . [SEP]
[CLS] moreover , low nonspecific adhesion B-event of I-event blood I-event cells I-event and high ctcs viability ( 97 . 6 % ) could be achieved by this soft nanointerface . [SEP]
[CLS] and this fluidity - reinforced multivalent binding strategy showed high potential in clinical applications as the chip could successfully detect ctcs in all cancer patient samples ( 17 / 17 ) . [SEP]
[CLS] as a natural phenomenon in biological systems , viral infection of mammalian cells B-material is a complex process driven by the synergistic effects of multiple biomolecules for efficient recognition . [SEP]
[CLS] inspired by the identifying mechanism of virus infection , wang et al . developed virus - mimetic magnetic B-property dna nanoclaws ( mdncs ) for specifically targeting and efficiently isolating ctcs ( figure 7a ) . [SEP]
[CLS] the mdncs were prepared by rca , which was followingly hybridized with a specific part of the tandem dna strand . [SEP]
[CLS] combining the flexibility of single - strand dna ( ssdna ) with the rigidity of double - strand dna ( dsdna ) , mdncs showed a high capture efficiency ( 82 . 3 % ± 7 . 1 % ) to mda - mb - 231 cells B-material , f i g u r e 7 ( a ) heterovalency magnetic B-property dna nanoclaws mimicking virus for capturing cells B-material . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , american chemical society . [SEP]
[CLS] ( b ) an engineered human fc - mannose - binding - lectin mimicking the innate immune mechanism for specific capture of ctcs . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2017 , wiley - vch . [SEP]
[CLS] ( c ) a magnetic B-property dynamic microbiointerface for selective capture and sugar - responsive release of cancer cells B-material from blood samples . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2013 , wiley - vch which was attributed to the loaded cell - targeting multiple antibodies B-material ( abs ) . [SEP]
[CLS] in addition , high capture efficiency and specificity from clinical samples could also be achieved by the rigid - flexible mdncs . [SEP]
[CLS] m13 bacteriophage is a wellknown virus for its large surface area ( 18 , 700 nm 2 ) , which has a width of 6 nm and length of [UNK] nm . [SEP]
[CLS] and the virion is mainly composed of the 2700 copies of the pviii coat B-material protein B-material , which account for 87 % of virus mass and 99 % of virus surface . [SEP]
[CLS] inspired by this , m13 bacteiophagebased " nanotentacle " - structured magnetic B-property particles were prepared for isolating ctcs from whole blood . [SEP]
[CLS] as shown in figure 7b , the m13 - bacteriophage immobilized to magnetic B-property particles was modified with peg and further conjugated with tethered monoclonal antibodies B-material specific for the epidermal receptor B-material 2 ( her2 ) . [SEP]
[CLS] the nanotentacle - structured magnetic B-property particles showed outstanding sensitivity to target [SEP]
[CLS] ecm is composed of dynamic meshwork of cross - linked proteins B-material and plays an important role in various cellular B-event processes I-event such as adhesion , migration , survival , and differentiation . [SEP]
[CLS] as the composition and organization of the ecm change with time which are caused by various external or internal biological stimuli , the dynamically changeable cell - ecm interactions can provide various architectural , mechanical , and biochemical signals for controlling relevant cell B-material behaviors . [SEP]
[CLS] as mimics of the natural ecms , dynamic biointerface is an emerging frontier in the field of biomaterial science and has been wildly applied in cell - based fundamental studies and tissue engineering . [SEP]
[CLS] recently it is found to be an ideal tool for the capture and release of ctcs . [SEP]
[CLS] pan et al . developed a novel dynamic biointerface by combining mussel - inspired peptide B-material mimics and invertible catecholboronate chemistry . [SEP]
[CLS] biomimetic peptides B-material containing a catechol - containing sequence and a cell - binding sequence at each end was designed . [SEP]
[CLS] the dynamic biointerface was obtained by binding mussel - inspired peptides B-material to a substrate grafted with polymers B-material containing phenylboronic acid ( pba ) via catechol - boronate B-material interactions . [SEP]
[CLS] the resultant biointerface could capture mcf - 7 cells B-material with high selectivity ( 98 . 1 % ± 0 . 7 % ) from the mixture ( 1 : 1 ) of mcf - 7 cells B-material and hl60 cells . [SEP]
[CLS] moreover , 99 % of the mcf - 7 cells B-material could be released in a sugar - responsive manner and the dynamic biointerface exhibited excellent reusability for capture and release of tumor B-material cell B-material . [SEP]
[CLS] subsequently , they constructed a magnetic B-property dynamic microbiointerface by grafting the biomimetic peptide B-material onto magnetic B-property microbead functionalized with pba - containing polymer B-material brushes . [SEP]
[CLS] as shown in figure 7c , magnetic B-property microbeads were first modified with 3 - methacryloxypropyltrimethoxysilane ( mpts ) , which could initiate the polymerization of hydrophilic B-property monomer B-material 2 - hydroxyethyl acrylamide ( heaa ) and saccharide - sensitive monomer B-material acrylamidophenylboronic acid ( aapba ) . [SEP]
[CLS] the hydrophobic B-property heaa polymer B-material brushes could guarantee the high purity of targeted cells B-material as it had good resistance to nonspecific cell B-event adhesion I-event . [SEP]
[CLS] the dynamic magnetic B-property platform not only showed selective cancer cell B-material capture ( [UNK] % ) and sugar - responsive release of them ( > 93 % ) in cell B-material culture medium , but also could isolate a decent number of target cells B-material ( [UNK] ) from artificial ctc blood samples ( 1 ml spiked with 100 cancer cells B-material ) . [SEP]
[CLS] in view of the high capture efficiency and remarkable selectivity , the ecm - inspired dynamic biointerfaces with reversible property and programmable features showed high potential for developing rare - cell detection platforms . [SEP]
[CLS] as mentioned above , nanostructures have been proved to play an important role in improving ctcs capture performance . [SEP]
[CLS] in view of the fact that the ctcs sizes are on the micrometer length scale , microstructures with nanotextures are thought to have the potential for further enhancing cell B-material capture . [SEP]
[CLS] on the one hand , more antibodies B-material can be immobilized on the microstructured topographies with a larger surface area , thereby improving the capture performance of ctcs by increasing the collision chances between antibodies B-material and membrane receptors . [SEP]
[CLS] on the other hand , microscale topographic structures could offer better size match and physical contact with the targeted cells B-material . [SEP]
[CLS] therefore , intricately hierarchical structures with both microstructures accommodating cell B-material size and nanostructures matching the cellular pseudopods have great potential in further enhancing ctcs capture . [SEP]
[CLS] various plants in nature ( such as straws , cactaceae , and rose petals , etc . ) have hierarchical structures with multiple levels . [SEP]
[CLS] inspired by nature , dou et al . fabricated a novel 3d hierarchically structured surface by mimicking the micro - and nanostructures of natural rose petals . [SEP]
[CLS] as shown in figure 8a , an imprint pattern - transfer technique was used to construct the rose petal - mimicking hierarchical surface , which was composed of microconcave / convex structures with 20 - 30 μm depths / heights and nanofolds with 500 - 600 nm widths . [SEP]
[CLS] then the hierarchical micro / nanostructures were grafted with anti - epcam by a disulfide bond for capturing ctcs and releasing cells B-material in a stimulus - responsive manner . [SEP]
[CLS] the cell B-material capture ability of these hierarchical substrates was six times higher than that of polydimethylsiloxane ( pdms ) surfaces functionalized with anti - epcam under static conditions at the concentration of 100 cells B-material / ml . compared with flat polydimethylsiloxane ( pdms ) surfaces functionalized with anti - epcam . [SEP]
[CLS] moreover , high release efficiency ( 85 % ) and ideal cell B-property viability I-property ( 98 % ) could be obtained after the substrates were treated with glutathione ( gsh ) . [SEP]
[CLS] inspired by the structure of straws , yang et al . constructed a microfluidic device integrated with hierarchical microtubes for simultaneously capturing and chemical manipulating cancer cells B-material in situ ( figure 8b ) . [SEP]
[CLS] the hierarchical spiky microstraw arrays ( hs - msa ) were prepared by repeated steps of material B-material depositions and etching , followed by nanospikes growth under hydrothermal conditions . [SEP]
[CLS] and high capture efficiency ( ≈84 % ) and strong specificity could be obtained using the anti - epcam - functionalized 3d f i g u r e 8 various plants / animals - inspired ctc - isolation platforms . [SEP]
[CLS] ( a ) bioinspired hierarchically structured surfaces mimicking the nano - and microstructures of natural rose petals for efficient ctcs isolation . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2017 , american chemical society . ( b ) microfluidic device integrated with hierarchical spiky microstraws for efficiently capturing and in situ manipulating cancer cells B-material . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , wiley - vch . ( c ) magnetic B-property microparticles functionalized with multivalent aptamers acting as " regenerative nanooctopus " for efficiently capturing target cells B-material from whole blood . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , american chemical society . ( d ) octopus - bioinspired multivalent aptamer - functionalized dld - patterned microfluidic chip ( ap - octopus - chip ) for improving ctcs isolation . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2019 , wiley - vch hs - msa micro / nanostructure for capturing cancer cells B-material . [SEP]
[CLS] after the hs - msa was integrated into a microfluidic device , extracellular drug could be delivered to the captured cells B-material in situ with ideal dose , spatial , and temporal controls through the inner fluidic conduits of hollow microstraws , achieving precise chemical regulation of the extracellular microenvironment . [SEP]
[CLS] the microfluidic device could simultaneously achieve both ctcs detection and cell B-material manipulation in situ , offering a new tool for highthroughput screening drugs in personalized therapy . [SEP]
[CLS] many animals in nature have evolved special structures for preying . [SEP]
[CLS] for example , multivalent trailing tentacles have been evolved by the octopus for hunting . [SEP]
[CLS] these long tentacles containing repeated B-material adhesive I-material units I-material ( e . g . , mucus ) could extend into the flow and maximized the touch with flowing targets , thereby enhancing the capture efficiency . [SEP]
[CLS] inspired by this , chen et al . developed a so - called " nanoocto - pus " device for capturing cells B-material from blood ( figure 8c ) . [SEP]
[CLS] the nanooctopuses consisted of a magnetic B-property microparticle ( mp ) acting as octopus head and grafted long singlestranded dna sequences mimicking tentacles . [SEP]
[CLS] and more than 500 repeating " suckers " of dna aptamer sequences contained in each dna sequence could specifically recognize target biomarker B-property proteins B-material on cell B-material membranes . [SEP]
[CLS] on the one hand , the binding affinity could be effectively improved by multivalent binding between the target cells B-material and the multimeric aptamer . [SEP]
[CLS] on the other hand , the aptamer accessibility to cell B-material receptors and steric hindrance from the particle surfaces could be respectively increased and decreased by the long tentacle dna strands ( [UNK] . 7 μm ) . [SEP]
[CLS] the capture yield and purity of target cells B-material in whole blood were 88 % ± 6 % and 96 . 7 % under the condition that the concentration of wbcs was 5000 times higher than that of ccrf - cem cell B-material . [SEP]
[CLS] the results showed that the nanooctopuses could be successfully applied to clinical samples , which was verified by that the nanooctopuses could isolate ptk7 - expressing cancer cells B-material ( 100 % detection ) from 33 aml patients . [SEP]
[CLS] song et al . developed an aptamer - tailed octopus chip ( ap - octopus - chip ) by combining octopus - mimicking nanostructures with a size - dictated immunocapture chip for improving capture efficiency . [SEP]
[CLS] they first synthesized the aunps B-nanoparticle and used the freeze - thaw method to immobilize thiolated aptamer on them . [SEP]
[CLS] on the one hand , local topographic interactions and the aptamer stability could be improved by aunp B-nanoparticle surface modified with the aptamer , which offered a rough interface and protected dna probes from nuclease degradation . [SEP]
[CLS] on the other hand , excess thiol B-material molecules with good biocompatibility B-property could disrupt the au - s bonds readily , achieving the effective release of target cells B-material with high cell viability I-property for further analysis . [SEP]
[CLS] then a dld - patterned microfluidic chip was integrated with the multivalent aptamer - functionalized aunps B-nanoparticle . [SEP]
[CLS] as shown in figure 8d , ctcs had full access to aunp B-nanoparticle - syl3c modified - micropillar as they could cross streamlines based on the dld principle . [SEP]
[CLS] while blood cells B-material had low contact chances as they stayed within the initial flow streamline due to their smaller size than ctcs . [SEP]
[CLS] the results showed that the multivalent aptamer - antigen binding affinity and the capture yield were improved 100 - fold and more than 300 % than that of a chip modified with monovalent aptamer . [SEP]
[CLS] moreover , up to 80 % release efficiency and 96 % cell B-property viability I-property could be obtained using a thiol B-material exchange reaction , showing this method was fully compatible with downstream analysis . [SEP]
[CLS] in summary , we have reviewed various advanced material B-material interfaces based on well - defined micro / nanostructures and nature - inspired hierarchical architectures for ctcs isolation . [SEP]
[CLS] despite the promising results achieved by these interfaces , most of them still stayed in the laboratory . [SEP]
[CLS] and there are many challenges to be addressed before these systems can be applied clinically . [SEP]
[CLS] first , the heterogeneity among ctcs ca not be ignored as most systems capture ctcs based on the affinity strategies . [SEP]
[CLS] the expressed biomarkers B-property on the ctcs membrane vary between different cancers and patients . [SEP]
[CLS] currently , epcam is a widely used targeted receptor B-material while it is not expressed in some malignant cells B-material , leading to false negatives or positives . [SEP]
[CLS] some researchers present the cocktail of the multiplex antibodies B-material method to overcome this obstacle , while their clinical application is severely limited by the expensiveness of antibodies B-material and complexity of surface modification . [SEP]
[CLS] future work may be focused on the screening of new affinity reagents , which are stable , cheap and can recognize a broad spectrum of ctc phenotypes . [SEP]
[CLS] additionally , the label - free methods seem to have greater potential for clinical application since they do not need any affinity reagents . [SEP]
[CLS] the purity is the main concern of these methods as the physical properties of some blood cells B-material are similar to that of ctcs . [SEP]
[CLS] fox example , marinnucci et al . reported that ctcs had the same or smaller size than leukocytes . [SEP]
[CLS] this will also inevitably introduce false positives results . [SEP]
[CLS] further efforts need to be devoted to searching for the unique properties of ctcs , which are completely different from that of other cells B-material . [SEP]
[CLS] finally , nature - inspired multiscale structures , which can achieve structural and functional integrity , may open new avenues to address the challenges of ctcs isolation . [SEP]
[CLS] although promising results and some progress have been achieved by these nature - inspired hierarchical materials in the light of detection sensitivity and capture efficiency , there is still a long way to go before the biomimetic technology comes into practical application . [SEP]
[CLS] current researches are mainly concentrated in improving the capture efficiency by just simulating the morphologies of nature creatures , while how to guarantee the purity and viability B-property of I-property cells I-property is often ignored . [SEP]
[CLS] thus , future endeavors should focus on the design of integrated multifunctional intelligent materials for ctcs isolation . [SEP]
[CLS] the authors acknowledge the financial support of the national natural science foundation of china ( nos . 51725303 , 51903214 , 21903065 , 52033007 ) , the science and technology project of sichuan province ( 2020yfsy0017 and 2021yfh0125 ) , key project of sichuan department of science and technology ( no . 2020yfsy0017 ) , and the fundamental research funds for the central universities ( nos . 2682020cx03 , 2682020cx54 ) . [SEP]
[CLS] the authors declare no conflict of interest . [SEP]
[CLS] shaobing zhou https : / / orcid . org / 0000 - 0002 - 6155 - 4010 [SEP]
[CLS] downloaded from https : / / onlinelibrary . wiley . com / doi / 10 . 1002 / viw . 20200023 by enpc - ecole des ponts paristech , wiley online library on [ 19 / 12 / 2022 ] . [SEP]
[CLS] see the terms and conditions ( https : / / onlinelibrary . wiley . com / terms - and - conditions ) on wiley online library for rules of use ; oa articles are governed by the applicable creative commons license ta b l e 1 summary of micro / nanostructured substrates for ctcs isolation yield ; r : release yield ; n / a : not available . [SEP]
[CLS] i g u r e 5 ( a ) selective binding and carrying target cancer cells B-material by the anti - cea mab - modified micromotors . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2011 , wiley - vch . [SEP]
[CLS] ( b ) flexible 3d net for intravascular fishing of ctcs . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] copyright 2020 , american chemical society system was constructed by loading fe 3 o 4 nanoparticles B-nanoparticle and transferrin ( tf ) onto the inner and outer surface . [SEP]
[CLS] and the cnt - based micromotor could efficiently propel itself by the thrust of o 2 bubbles formed by the decomposition of h 2 o 2 catalyzed by fe 3 o 4 nanoparticle B-nanoparticle . [SEP]
[CLS] cell B-material capture studies showed that tf - cnt - fe 3 o 4 particles could efficiently trap tfr + cancer cells B-material ( [UNK] % ) from an artificial ctc - like suspension in just 5 minutes . [SEP]
[CLS] and such self - powered micromotor provides a new approach for efficiently extracting ctcs from biological fluids . [SEP]
[CLS] downloaded from https : / / onlinelibrary . wiley . com / doi / 10 . 1002 / viw . 20200023 by enpc - ecole des ponts paristech , wiley online library on [ 19 / 12 / 2022 ] . [SEP]
[CLS] see the terms and conditions ( https : / / onlinelibrary . wiley . com / terms - and - conditions ) on wiley online library for rules of use ; oa articles are governed by the applicable creative commons license f i g u r e 6 cell - inspired biomimetic platforms for efficient isolation of ctcs . [SEP]
[CLS] ( a ) leukocyte - inspired particles based hierarchical biointerfaces for selectively isolating ctcs . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] 82 copyright 2014 , wiley - vch . [SEP]
[CLS] ( b ) hydrogel combining boronate B-material affinity with cell imprinting for ctcs isolation . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] , the hierarchical biointerface , which was assembled by leukocyte - inspired ta b l e 2 summary of nature - inspired hierarchical architectures for ctcs isolation yield ; r : release yield ; n / a : not available . [SEP]
[CLS] downloaded from https : / / onlinelibrary . wiley . com / doi / 10 . 1002 / viw . 20200023 by enpc - ecole des ponts paristech , wiley online library on [ 19 / 12 / 2022 ] . [SEP]
[CLS] see the terms and conditions ( https : / / onlinelibrary . wiley . com / terms - and - conditions ) on wiley online library for rules of use ; oa articles are governed by the applicable creative commons license [SEP]
[CLS] downloaded from https : / / onlinelibrary . wiley . com / doi / 10 . 1002 / viw . 20200023 by enpc - ecole des ponts paristech , wiley online library on [ 19 / 12 / 2022 ] . [SEP]
[CLS] see the terms and conditions ( https : / / onlinelibrary . wiley . com / terms - and - conditions ) on wiley online library for rules of use ; oa articles are governed by the applicable creative commons license cells B-material , which could isolate rare ctcs ( ≈25 cells B-material ) from whole blood . [SEP]
[CLS] and high capture yield ( > 90 % ) , cell B-material purity ( > 45 % ) , and viability ( > 84 % ) were obtained , showing its potential in clinical applications . [SEP]
[CLS] downloaded from https : / / onlinelibrary . wiley . com / doi / 10 . 1002 / viw . 20200023 by enpc - ecole des ponts paristech , wiley online library on [ 19 / 12 / 2022 ] . [SEP]
[CLS] see the terms and conditions ( https : / / onlinelibrary . wiley . com / terms - and - conditions ) on wiley online library for rules of use ; oa articles are governed by the applicable creative commons license [SEP]
[CLS] nanomedycyna jest obecnie jedna z najszybciej rozwijajacych sie dziedzin nanotechnologii , znajdujaca coraz wiecej zastosowan w walce z chorobami nowotworowymi . nanoczastki złota , ze wzgledu na swoje unikalne własciwosci optyczne i chemiczne , ciesza sie rosnacym zainteresowaniem naukowcow . [SEP]
[CLS] do zalet nanoczastek złota naleza miedzy innymi wysoki stosunek powierzchni do objetosci czastki , duze mozliwosci modyfikacji ich powierzchni , czy tez wystepowanie efektu wzmocnionej przepuszczalnosci i retencji , pozwalajacego na bardziej efektywna i selektywna akumulacje leku w miejscach zmienionych nowotworowo . w pracy krotko omowiono charakterystyke fizykochemiczna nanoczastek złota oraz przedstawiono ich wybrane zastosowania zarowno w diagnostyce , jak i terapii chorob nowotworowych . [SEP]
[CLS] czastki w skali nano ( tj . rzedu 10 - 9 m ) wyrozniaja sie wieloma zaletami w stosunku do makroczastek . [SEP]
[CLS] ich wyjatkowe własciwosci fizyczne i optyczne sprawiaja , ze znajduja zastosowanie nie tylko w medycynie , ale tez m . in . jako biosensory czy materiały do budowy urzadzen optoelektronicznych . [SEP]
[CLS] z uwagi na potrzebe ciagłego ulepszania znanych obecnie metod wczesnego wykrywania i leczenia chorob nowotworowych , duza czesc badan nad nanoczastkami dotyczy jednak ich potencjalnych zastosowan w medycynie . wczesna diagnoza nowotworow złosliwych jest trudna , a ich wykrycie czesto nastepuje dopiero w poznej fazie przerzutow , co znacznie zmniejsza skutecznosc leczenia . prezny rozwoj nanotechnologii w ostatnich latach znacznie sprzyja opracowywaniu nowoczesnych , bardziej wydajnych metod diagnostyki i terapii nowotworow . najwazniejszymi zaletami ( w przypadku terapii ) czastek w skali nano sa m . in . lepsza skutecznosc dostarczania lekow do chorych tkanek przy stosowaniu chemioterapii , zmniejszenie efektow ubocznych leczenia , a takze tworzenie nowych rodzajow terapii ( na przykład terapii fototermicznej ) czy celowanych terapii kombinowanych , ktore osiagaja znacznie lepsze efekty w walce z nowotworami . znanych jest juz wiele systemow opartych na nanoczastkach , ktore mozna wykorzystac jako nosniki lekow . istotne sa w tym przypadku biozgodnosc i biodystrybucja , na ktore duzy wpływ ma rozmiar nanoczastki - przykładowo [SEP]
[CLS] sposrod wszystkich nanoczastek metalicznych czastki złota wykazuja jedne z najbardziej unikalnych własciwosci , ktore pozwalaja na zastosowanie ich w wielu dziedzinach . najwazniejsze z tych własciwosci dotycza aktywnosci chemicznej , optyki , ich stabilnej natury oraz zdolnosci pochłaniania promieniowania x . w ostatnich latach pojawia sie coraz wiecej nowych metod syntezy nanoczastek złota o roznych rozmiarach i kształtach , zwiekszajac tym samym liczbe mozliwosci utworzenia roznorodnych nanokompozytow . [SEP]
[CLS] nanoczastki złota otrzymuje sie głownie w reakcjach w srodowisku cieczy poprzez redukcje haucl4 , choc istnieja bardziej zaawansowane metody pozwalajace na wieksza precyzje i modyfikacje , jesli chodzi o kształty i wielkosci syntezowanych nanoczastek . kwas chlorozłotowy po rozpuszczeniu zostaje szybko zmieszany , w tym samym czasie dodawany jest czynnik redukujacy i nastepuje redukcja jonow au 3 + do atomow złota na zerowym stopniu utlenienia . prowadzi to do nasycenia roztworu i rozpoczecia formacji aunps B-nanoparticle ( nanoczastek złota ) . [SEP]
[CLS] do roztworu dodaje sie takze czynnik stabilizujacy , ktorego rola polega na zapobieganiu agregacji czastek ( zwykle jest to substancja organiczna , ktora oddziałuje z powierzchnia metalu ) . [SEP]
[CLS] czynnik ten mozna pominac , np B-nanoparticle . przy syntezie aunps B-nanoparticle przez ablacje laserowa w cieczy . [SEP]
[CLS] najprostszym znanym sposobem otrzymania nanoczastek złota jest metoda turkevicha . w syntezie tej powstaja monodyspersyjne , sferyczne aunps B-nanoparticle zawieszone w wodzie , o srednicy około 10 - 20 nm . [SEP]
[CLS] kolejnym sposobem jest metoda brusta , zaproponowana we wczesnych latach ' 90 ubiegłego wieku , w ktorej otrzymywane sa złote nanoczastki w cieczach organicznych ( normalnie niemieszalnych z woda ) . [SEP]
[CLS] w syntezie tej powstaja nanoczastki o srednicy 2 - 6 nm . [SEP]
[CLS] metoda perraulta , opisana w 2009 roku przez perraulta i chana , polega na redukcji haucl4 hydrochinonem , w roztworze zawierajacym 15 - nanometrowe " ziarna " złotych nanoczastek . [SEP]
[CLS] otrzymywane aunps B-nanoparticle maja srednice w zakresie 30 - 300 nm . [SEP]
[CLS] metoda martina , wprowadzona w 2010 roku przez martina i eaha , pozwala otrzymac niemodyfikowane , niemalze monodyspersyjne nanoczastki złota zawieszone w wodzie . reakcja polega na redukcji haucl4 borowodorkiem sodu ( w srodowisku wodnym ) - mimo nieobecnosci czynnika stabilizujacego , aunps B-nanoparticle sa trwale zdyspergowane . srednica nanoczastek moze byc powtarzalnie i dokładnie kontrolowana - od 3 , 2 do 5 , 2 nm . [SEP]
[CLS] kolejna metoda otrzymywania nanoczastek złota jest sonoliza , po raz pierwszy opisana przez baigenta i mullera . w ich pracy ultradzwieki zapewniały energie dla zachodzacych procesow i umozliwiły wytworzenie złotych czastek o srednicy ponizej 10 nm . w innej pracy wykorzystujacej ultradzwieki przeprowadzono reakcje wodnego roztworu haucl4 z glukoza ( odpowiadajaca za kształt czastek ) , w ktorej reduktor stanowiły rodniki hydroksylowe i rodniki pirolizy cukru . uzyskane struktury miały kształt wstazek o szerokosci 30 - 50 nm i długosci kilku mikrometrow . [SEP]
[CLS] metoda navarro i in . powstała w oparciu o zmodyfikowana procedure turkevicha - frensa . modyfikacja polegała na dodaniu acetyloacetonianu sodu umozliwiajacego kompleksowanie i redukcje jonow au 3 + do au + . kolejnym krokiem było szybkie dodanie cytrynianu sodu , redukujacego au + do obojetnych atomow złota . tak przeprowadzona reakcja pozwoliła na maksymalna kontrole struktury nanoczastek , a zarazem zapewnienie prostoty syntezy . [SEP]
[CLS] synteza sakaia i in . wykorzystuje kopolimery blokowe w dwoch rolach . polega ona na redukcji jonow złota przez obecne w roztworze kopolimery blokowe , nastepnie tworzeniu złotych klastrow , adsorpcji kopolimerow na klastrach i dalszej redukcji jonow złota na powierzchni klastrow ( wzrost nanoczastek ) . [SEP]
[CLS] istnieja takze fotochemiczne metody otrzymywania nanoczastek oraz nalezace do dziedziny " zielonej " chemii , gdzie wykorzystuje sie naturalne fitochemikalia . przeprowadzone zostały juz syntezy z uzyciem takich materiałow jak czarna herbata , kardamon , cynamon , czy ekstrakt z aloesu . [SEP]
[CLS] złoto jest zwykle postrzegane jako metal B-material obojetny , ale wykazano , ze jego nanoczastki ( jako klastry ) o srednicy mniejszej niz 3 - 5 nm sa aktywne katalitycznie dla pewnych rodzajow reakcji . ta chemiczna aktywnosc moze sie zmieniac wraz ze zmiana rozmiaru czastek . [SEP]
[CLS] zauwazono , ze najbardziej aktywne sa atomy na rogach i krawedziach nanoczastek - efekt ten probowano wyjasnic przy uzyciu obliczen metoda funkcjonału gestosci . [SEP]
[CLS] atomy takie sa nisko skoordynowane i maja wieksza mozliwosc stworzenia wiazania chemicznego . w najwiekszej ilosci obecne sa w nanoczastkach o najmniejszych srednicach . [SEP]
[CLS] oddziaływanie aunps B-nanoparticle ze swiatłem silnie zalezy od srodowiska , modyfikacji powierzchni , rozmiaru i kształtu nanoczastek . zaleznosc od rozmiaru przejawia sie w roznym zabarwieniu roztworow z monodyspersyjnymi nanoczastkami ( od fioletu , poprzez roz do czerwieni i pomaranczy ) . [SEP]
[CLS] za te niezwykłe własciwosci optyczne odpowiedzialne jest zjawisko powierzchniowego rezonansu plazmonowego ( spr ) , polegajace na oscylacjach gestosci elektronowej w obszarze nanoczastek metali ( schemat zjawiska przedstawiono na ryc . 1 ) . chmura elektronowa jest rezonansowo wzbudzana przez fale swietlne o okreslonej długosci , co prowadzi do silnego rozproszenia promieniowania , w widmie absorpcyjnym pojawiaja sie wiec wyrazne pasma dla spr . podczas takich oscylacji plazmonowych dla sferycznych nanoczastek nastepuje harmoniczne przemieszczanie sie chmury elektronowej w stosunku do jadra nanoczastki . [SEP]
[CLS] ryc . 1 . schemat spr dla nanoczastek sferycznych . jak juz wspomniano , zjawisko spr silnie zalezy od wielkosci nanoczastek . [SEP]
[CLS] dla czastek o rozmiarze około 30 nm powierzchniowy rezonans plazmonowy powoduje absorpcje swiatła w spektrum własciwym dla barwy niebiesko - zielonej ( długosc fali około 450 nm ) , jednoczesnie wywołujac odbicie fal swiatła czerwonego ( długosc fali około 700 nm ) , co skutkuje czerwona barwa roztworu . [SEP]
[CLS] wraz ze wzrostem wielkosci nanoczastek , długosc fali absorpcyjnej zwiazanej z spr przesuwa sie w strone fal dłuzszych ( bardziej czerwonych ) , co skutkuje odbiciem fal niebieskich i niebieska lub fioletowa barwa roztworu . [SEP]
[CLS] zjawisko spr mozna dostosowywac rowniez poprzez zmiane kształtu nanoczastek - przykładowo , złote nanotrojkaty maja tendencje do absorpcji swiatła bliskiej podczerwieni przy fioletowej barwie roztworu . jedne z najciekawszych zsyntezowanych aunps B-nanoparticle , w kształcie owocu karamboli , wykazywały absorbcje swiatła czerwonego ( około 630 nm ) , przy niebiesko - zielonym zabarwieniu roztworu . na pozycje pasma plazmonowego maja wpływ takze stopien agregacji czastek oraz fluktuacje w stałej dielektrycznej srodowiska , ktore z kolei zaleza od stezenia uzytego do syntezy czynnika stabilizujacego . [SEP]
[CLS] złoto jako metal B-material o duzej liczbie atomowej z ( 79 ) silnie pochłania promieniowanie x . uwaza sie , ze jego oddziaływanie z kwantami rentgenowskimi skutkuje głownie emisja fotoelektronow i elektronow augera ( inne mozliwosci to m . in . zaistnienie efektu fotoelektrycznego , comptona , rozpraszanie rayleigha , czy emisja fotonow fluorescencji ) . [SEP]
[CLS] zasieg tych elektronow jest bardzo mały w porownaniu z zasiegiem fotonow , wyzwalana energia jest wiec deponowana w komorkach zawierajacych aunps B-nanoparticle lub bezposrednio przy atomach złota . rezultaty badan wrazliwosci aunps B-nanoparticle na promieniowanie x bywały bardzo rozne , dlatego przeprowadzono tez testy uwzgledniajace takie czynniki jak kształt , rozmiar , stezenie nanoczastek , typ linii komorkowej czy energie i rodzaj promieniowania . najbardziej wydajne okazały sie duze aunps B-nanoparticle o wysokim stezeniu molowym , absorbujace fotony o energii 50 kev - oszacowano , ze nanoczastki w takich warunkach mogłyby zwiekszyc dawke pochłanianych kwantow rentgenowskich az 6 razy . wrazliwosc złota na promieniowanie rentgenowskie sprawia , ze aunps B-nanoparticle mozna potencjalnie wykorzystac w roli kontrastu do tomografii komputerowej ( ct ) oraz w teleradioterapii . [SEP]
[CLS] ze wzgledu na swoje rozmiary , nanoczastki złota ulegaja rowniez efektowi wzmocnionej przepuszczalnosci i retencji ( epr ) . [SEP]
[CLS] efekt ten , obserwowany w przypadku czesci nowotworow , polega na zwiekszonej i dłuzej trwajacej akumulacji leku w tkankach zmienionych chorobowo , a jego istnienie powodowane jest nieszczelnoscia układu naczyniowego danego guza . w przypadku nanoczastek do terapii celowanych , koniugowanych z wybranymi biomolekułami wiazacymi sie z celami molekularnymi , efekt epr w sposob pasywny ( poprzez dyfuzje ) zwieksza efektywnosc gromadzenia aunps B-nanoparticle . [SEP]
[CLS] rosnace zainteresowanie zastosowaniem nanomateriałow ze złota w medycynie zrodziło obawy przed potencjalnie toksycznym efektem oddziaływania aunps B-nanoparticle z systemami biologicznymi . [SEP]
[CLS] kazdy projekt wykorzystujacy nanostruktury musi byc w tym kierunku szczegołowo zbadany przed dopuszczeniem go do uzytku . [SEP]
[CLS] niektore koniugaty nanoczastek złota z substancjami , takimi jak srodki kontrastowe , przeciwciała , peptydy , ligandy , leki i geny , sa juz z powodzeniem stosowane w leczeniu nowotworow . ograniczenia w stosowaniu aunps B-nanoparticle w terapii i diagnostyce wynikaja głownie z toksycznosci stosowanego w syntezie czynnika stabilizujacego , wiaza sie tez ze stabilnoscia nanoczastek w srodowisku biologicznym oraz farmakokinetyka . [SEP]
[CLS] jednym z najczesciej stosowanych czynnikow stabilizujacych jest ctab ( bromek cetylotrimetyloamoniowy ) , wykazujacy działanie toksyczne . obecnosc wolnych molekuł ctab w roztworze moze byc spowodowana niedostatecznym oczyszczeniem nanoczastek lub desorpcja surfaktantu z powierzchni aunps B-nanoparticle . stosowane sa wiec procedury majace na celu zmniejszenie toksycznosci i ustabilizowanie nanoczastek w srodowisku fizjologicznym poprzez miedzy innymi pokrywanie powierzchni aunps B-nanoparticle cienkimi filmami ( na przykład soli sodowej kwasu poli ( 4 - styrenosulfonowego ) ) czy dołaczenie do nich biokompatybilnych czastek , takich jak poli ( tlenek etylenu ) ( peg ) . niektore badania potwierdziły , ze to własnie swobodne czasteczki ctab były odpowiedzialne za zaobserwowane działania toksyczne , nie zas same nanoczastki złota . [SEP]
[CLS] przeprowadzono wiele badan in vitro , ktore wykazały , ze cytotoksycznosc ( czyli toksycznosc wzgledem komorek w danym organizmie ) nanoczastek złota silnie zalezy od ich wielkosci i stezenia . zmniejszenie wielkosci aunps B-nanoparticle skorelowane było z poszerzona dystrybucja tkankowa , podwyzszonym potencjałem do głebszej penetracji w okreslonych tkankach , bardziej efektownym przyswajaniem nanoczastek przez komorki oraz zwiekszonymi efektami toksycznymi . [SEP]
[CLS] w jednym z badan [SEP]
[CLS] wykazano , ze bardzo małe nanoczastki złota ( około 1 , 4 nm ) spowodowały martwice komorek na skutek stresu oksydacyjnego i zniszczen mitochondrialnych . w innym eksperymencie potwierdzono , ze dla niskich stezen ( 1 ppm ) aunps B-nanoparticle o rozmiarach 2 - 20 nm nie wystepowały efekty toksyczne w stosunku do linii komorkowej mysich makrofagow , podczas gdy stezenia wyzsze niz 10 ppm wywołały apoptoze komorek i zwiekszenie ekspresji genow pro - zapalnych . stres oksydacyjny zaobserwowano takze przy badaniach na komorkach fibroblastow mrc - 5 ludzkiego płuca ( pochodzacych od płodu ) poddanych działaniu 20 nm aunps B-nanoparticle , z jednoczesnym zmniejszeniem ekspresji genow kodujacych białka regulujace cykl komorkowy w fazie g2 / m oraz punkt kontrolny wrzeciona mitotycznego , a takze uszkodzeniem genow odpowiedzialnych za mechanizmy naprawy dna . [SEP]
[CLS] podsumowujac , czynniki odpowiedzialne za toksycznosc nanoczastek złota to ich wielkosc , kształt , powierzchnia ( ligandy oraz ładunek elektryczny ) , stopien agregacji , interakcje z czynnikami biologicznymi oraz typ i rodzaj uzytego medium hodowlanego do komorek . zauwazalnej toksycznosci mozna uniknac przede wszystkim poprzez metody modyfikacji powierzchni . [SEP]
[CLS] uogolniajac , nanoczastki złota sa duzo mniej toksyczne niz nanoczastki innych metali , a ich optymalizacja dla potencjalnych zastosowan moze byc w prosty sposob kontrolowana . [SEP]
[CLS] nanoczastki złota moga byc wykorzystywane do tworzenia wyjatkowych koniugatow . [SEP]
[CLS] ze wzgledu na rodzaj uzytego izotopu au ( radioaktywne 198 au i 199 au lub stabilny 197 au ) , koniugaty takie tworza radiofarmaceutyki lub chemoterapeutyki , choc opracowywane sa juz metody terapii synergicznej , w ktorej czasteczka aktywna biologicznie i znakowana radionuklidem moze byc jednoczesnie nosnikiem leku stosowanego w chemioterapii . [SEP]
[CLS] radiofarmaceutyki wykorzystujace znakowane nanoczastki moga byc klasyfikowane jako typ a lub b . w typie a radioizotop jest inkorporowany w biodegradowalnych nanoczastkach matryc organicznych . typ b pochodzi natomiast od bezposredniego pokrywania nieorganicznych nanomatryc radionuklidem - takie czastki moga byc powierzchniowo koniugowane z przeciwciałami , peptydami lub proteinami . [SEP]
[CLS] izotop złota 198 au ( tabela 1 ) wykazuje odpowiednie własciwosci do zastosowania w roli terapeutyku : optymalny okres połowicznego rozpadu , tj . 2 , 7 dnia , czy emisja krotkozasiegowych czastek β - penetrujacych miekkie tkanki na odcinku maksymalnie 3 , 8 mm ( połowa czastek przebywa jednak odległosc jedynie 0 , 38 mm ) . [SEP]
[CLS] produkty rozpadu opisywanego izotopu złota powstaja w ilosciach duzo nizszych od poziomu toksycznosci chemicznej . [SEP]
[CLS] wytwarzanie 198 au polega na bombardowaniu neutronami folii z naturalnego izotopu złota 197 au . jedna z udanych syntez radiofarmaceutyku znakowanego izotopem 198 au przeprowadzono poprzez redukcje h 198 aucl4 trimeryczna fosfina oparta na alaninie ( thpaltris ( hydroxymethyl ) phosphine - alanine ) w obecnosci biokompatybilnej matrycy z gumy arabskiej ( ga ) , tworzac ga - 198 aunps B-nanoparticle z bardzo wysoka wydajnoscia . [SEP]
[CLS] guma arabska jest ekstraktem roslinnym powszechnie stosowanym w przemysle spozywczym . [SEP]
[CLS] w innej z prac poprzez reakcje z arabinoksylanem ( bez dodatku innych reduktorow i stabilizatorow ) uzyskano wysoce stabilne 198 aunps B-nanoparticle , po podaniu doustnym głownie akumulowane w okreznicy . [SEP]
[CLS] z sukcesem przeprowadzono rowniez " zielona " synteze radioaktywnych nanoczastek funkcjonalizowanych mangiferyna , ktore wykazały wysokie powinowactwo do komorek pc3 nowotworu prostaty . [SEP]
[CLS] w badaniach in vivo udowodniono , ze nanoczastki te spowodowały pieciokrotne zmniejszenie guza w ciagu 3 tygodni od ich podania . [SEP]
[CLS] radioaktywne nanoczastki maja duzy potencjał jesli chodzi o zastosowanie w tworzeniu planu leczenia pacjenta . głownymi wyzwaniami obecnych badan sa skutecznosc w celowanym dostarczaniu radioaktywnych nanoczastek do nowotworu , opracowanie nowych metod znakowania promieniotworczego oraz koniugacja z ligandem ukierunkowujacym transport molekuły , pozadany profil bezpieczenstwa , a takze znaczace przeszkody handlowe i regulacyjne . [SEP]
[CLS] jednym z wazniejszych zastosowan stabilnych aunps B-nanoparticle jest ich wykorzystanie w roli nosnikow lekow . wykazano , ze własciwosci , takie jak rozmiar , ładunek elektryczny i chemia powierzchni nanoczastek maja wpływ zarowno na ich wychwyt do komorek , jak i pozniejsze działanie wewnatrzkomorkowe . [SEP]
[CLS] przy wydajnym dostarczaniu lekow nalezy wziac pod uwage takze nature oddziaływania lek - nanoczastka ( wiazania kowalencyjne lub niekowalencyjne ) oraz sposob uwalniania tego kompleksu do komorek . idealny byłby nosnik , ktorego trwałosc ograniczałaby sie czasowo do okna terapeutycznego . kolejnym czynnikiem , ktory nalezy brac pod uwage jest szybkosc penetracji nowotworu przez nanoczastki oraz specyfika miejsc docelowych . za głowna przeszkode , ktora musza pokonac aunps B-nanoparticle , uwaza sie bariery nabłonka i srodbłonka - uzyteczne moga stac sie w tym przypadku srodki zwiekszajace penetracje , takie jak metaloproteazy przeciwko błonom podstawnym oraz toksyny w stosunku do scisłych połaczen wewnatrzkomorkowych . [SEP]
[CLS] wazny jest takze czas cyrkulacji aunps B-nanoparticle we krwi , ktory według niektorych badaczy zalezy od rozmiaru nanoczastek . czas ten skorelowany jest z szybkoscia dotarcia leku do nowotworuz rosnacym czasem cyrkulacji rosnie tez szybkosc , z jaka chemoterapeutyk dociera do guza . [SEP]
[CLS] juz w 2008 roku huang i in . opisali w swojej pracy [SEP]
[CLS] dwie metody kierowania leku do nowotworu : w pierwszej nastepowała koniugacja nanoczastek złota z poli ( tlenkiem etylenu ) ( peg ) , w drugiej zas ze specyficznymi przeciwciałami , ktore wiaza sie z receptorami wystepujacymi na komorkach guza . peg zapobiega agregacji nanoczastek i wydłuza czas ich retencji we krwi . uzycie tego polimeru skutkuje pasywnym dostarczaniem leku do nowotworu , w przeciwienstwie do aktywnego mechanizmu z udziałem przeciwciał . po wychwycie przez komorki , aunps B-nanoparticle sa gromadzone w pecherzykach endo - lub lizosomalnych . w celu uwolnienia nanoczastek i wprowadzenia leku do cytoplazmy komorek nowotworowych , aunps B-nanoparticle musza byc modyfikowane poprzez koniugacje z peptydowymi sekwencjami przepuszczalnymi przez błony , co umozliwia pokonywanie pojedynczych warstw komorki . [SEP]
[CLS] nanoczastki złota były juz wielokrotnie wykorzystywane jako nosniki takich lekow , jak doksorubicyna , cisplatyna [SEP]
[CLS] czy docetaksel . zastosowanie ich w tej formie moze prowadzic do zmniejszenia efektu toksycznego w przypadku komorek prawidłowych przy jednoczesnie wzmocnionej efektywnosci terapii dla komorek nowotworowych , poszerzenia okna terapeutycznego dla danego leku czy zwiekszenia poziomu akumulacji leku w tkankach zmienionych chorobowo . omowione wczesniej własciwosci fizykochemiczne nanoczastek złota , a takze stosunkowo prosta modyfikacja ich powierzchni ( funkcjonalizacja ) , rozmiaru i kształtu , daja mozliwosc utworzenia wyjatkowo duzej liczby lekow opartych na aunps B-nanoparticle do terapii celowanych . [SEP]
[CLS] testy in vitro stanowia kluczowy element opieki klinicznej , wykorzystujac probki biologiczne , takie jak krew , mocz oraz pobrane tkanki pacjenta . [SEP]
[CLS] testy tego typu stosuje sie przede wszystkim do ustalenia lub potwierdzenia obecnosci roznych schorzen . [SEP]
[CLS] procedura pobierania probek jest zwykle nieinwazyjna i bezpieczna dla pacjenta , dzieki czemu mozliwe jest unikniecie wszelkiego rodzaju powikłan pozabiegowych , a wyniki badan sa relatywnie szybko dostepne . jest to szczegolnie wazne w przypadku wykrywania ciezkich i szybko postepujacych chorob . [SEP]
[CLS] ze wzgledu na wymienione wczesniej własciwosci nanoczastek złota , stanowia one doskonały surowiec do badan nad nowymi materiałami do detekcji chemicznej i biologicznej . wysoki stosunek powierzchni do objetosci oraz mozliwosc łatwej funkcjonalizacji powierzchni nanoczastek umozliwia dołaczenie do nich roznego rodzaju przetwornikow sygnału ( fluoroforow , dna czy enzymow ) , co z kolei pozwala na znaczace obnizenie limitow detekcji oraz jednoczesna analize wielu parametrow . dotychczasowe badania wykazały , ze istnieje mozliwosc zastosowania aunps B-nanoparticle w takich rodzajach diagnostyki in vitro , jak oznaczanie kolorymetryczne , elektrochemiczne , fluorescencyjne czy wykorzystujace powierzchniowo wzmocniona spektroskopie ramanowska ( sers ) , a takze wykorzystanie systemow opartych na nanoczastkach złota w badaniach w miejscu opieki nad pacjentem . [SEP]
[CLS] w dalszej czesci tej sekcji omowione zostana wybrane układy wykorzystujace aunps B-nanoparticle w roznych rodzajach testow in vitro . [SEP]
[CLS] jedna z ciekawszych metod diagnostycznych wykorzystujacych nanoczastki złota jest analiza biologicznego " kodu kreskowego " . [SEP]
[CLS] po raz pierwszy przedstawiona w pracy z 2003 roku , pokazała mozliwosc uzycia aunps B-nanoparticle funkcjonalizowanych przeciwciałami wiazacymi sie z celem molekularnym oraz dwuniciowym dna stanowiacymi barkod do detekcji swoistego antygenu sterczowego ( psa , markeru nowotworu prostaty ) z bardzo wysoka czułoscia . [SEP]
[CLS] w dalszych badaniach [SEP]
[CLS] udowodniono takze skutecznosc tej metody w jednoczesnej detekcji wielu markerow nowotworowych , oznaczajac psa , ludzka gonadotropine kosmowkowa ( marker nowotworu jader ) i α - fetoproteine ( marker raka watrobowokomorkowego ) rownoczesnie . w podejsciu tym stosuje sie dwie probki - mikroczastki magnetyczne połaczone z przeciwciałami monoklonalnymi , specyficznymi dla okreslonego epitopu danego celu molekularnego , oraz nanoczastki złota sfunkcjonalizowane oligonukleotydami ( stanowiacymi barkod ) i przeciwciałami rozpoznajacymi inny niz rozpoznawany przez mikroczastki magnetyczne epitop danego celu molekularnego . po połaczeniu celow molekularnych z obydwiema probkami otrzymywany jest kompleks , ktory nastepnie jest rozdzielany za pomoca pola magnetycznego i przemywany . nici analizowanego barkodu uwalnia sie za pomoca indukowanej przez dodatek ditiotreitolu wymiany ligandow i identyfikuje przy uzyciu metody skanometrycznej . zastosowanie tej koncepcji pozwala na detekcje wybranych markerow nowotworowych na poziomie femtomoli . [SEP]
[CLS] wiele badan nad zastosowaniem aunps B-nanoparticle dotyczy rowniez techniki sers . w tym rodzaju spektroskopii wykorzystuje sie zjawisko wzmocnienia ( az do 10 14 razy ) intensywnosci swiatła pochodzacego z rozproszenia ramanowskiego rozproszenia nieelastycznego ) i obserwuje sie je m . in . w układach zawierajacych nanoczastki metali szlachetnych . jednym z wyjasnien istnienia tego zjawiska jest efekt spr , opisany w podrozdziale 3 . juz w 2011 roku wang i in . [SEP]
[CLS] opisali metode detekcji krazacych komorek nowotworowych za pomoca metody sers wykorzystujacej nanoczastki złota z przyłaczonym reporterem ramanowskim ( czasteczka wrazliwa na obecnosc analitu ) i peptydem egf ( ang . epidermal growth factor - nabłonkowy czynnik wzrostu ) w roli ligandu celujacego oraz funkcjonalizowanych polimerami . po zbadaniu krwi obwodowej pochodzacej od 19 pacjentow z płaskonabłonkowym rakiem głowy i szyi skutecznie wykryto krazace komorki nowotworowe na poziomie od 1 do 720 komorek / ml krwi . [SEP]
[CLS] odrebna grupe zwiazkow opartych na aunps B-nanoparticle tworza sferyczne kwasy nukleinowe ( sna ) , zaprojektowane przez grupe badawcza pod kierownictwem chada mirkina z uniwersytetu northwestern . sa to trojwymiarowe struktury zbudowane zazwyczaj z nanoczastek złota w formie sferycznych powłok zwiazanych kowalencyjnie z gesto funkcjonalizowanymi i wysoce zorientowanymi kwasami nukleinowymi . [SEP]
[CLS] ich potencjalne zastosowanie obejmuje nie tylko diagnostyke , ale rowniez terapie ukierunkowana na regulacje genowa oraz immunoterapie . jednym z kluczowych osiagniec grupy mirkina było stworzenie tzw . " nanoflare " , dostepnych handlowo jako [UNK] , do detekcji wewnatrzkomorkowego mrna w zywych komorkach . zwiazki te sa zbudowane z nanoczastek złota koniugowa - nych z sna , ktorych sekwencja " rozpoznajaca " cel molekularny jest zhybrydyzowana do krotszych odcinkow zawierajacych fluorescencyjny reporter - " flare " , wygaszany w małej odległosci od nanoczastki . [SEP]
[CLS] w momencie połaczenia celu molekularnego z odpowiadajaca sekwencja sna " flara " zostaje przemieszczona i uwolniona , co pozwala na detekcje fluorescencji . [SEP]
[CLS] zastosowanie tych zwiazkow w połaczeniu z cytometria przepływowa moze słuzyc do fluorescencyjnej detekcji genetycznych markerow krazacych komorek nowotworowych we krwi pacjenta , umozliwiajac wykrycie na poziomie 100 zywych komorek nowotworowych / ml krwi . [SEP]
[CLS] wspomniane juz kilkukrotnie własciwosci nanoczastek złota wywołały ogromne zainteresowanie wykorzystaniem ich w technikach spect ( tomografii komputerowej emisji pojedynczego fotonu ) i ct ( tomografii komputerowej ) . [SEP]
[CLS] spect wykorzystuje promieniowanie gamma pochodzace od izotopu promieniotworczego w radiofarmaceutyku , wczesniej podanego pacjentowi . [SEP]
[CLS] zakres energii kwantow gamma mozliwych do detekcji przez tomograf spect to około 30 - 250 kev , zwykle jednak wykorzystuje sie energie od 100 do 200 kev . potencjalne zastosowanie w tym badaniu moga wiec miec radioaktywne nanoczastki złota . dwa izotopy złota , 198 au i 199 au , emituja promieniowanie gamma o energiach odpowiednich do zarejestrowania przez aparat spect , jednak energia kwantow wysyłanych przez 198 au ( 412 kev ) nie jest optymalna do tego rodzaju badania - tak wysoka wartosc powoduje problemy w kolimacji wiazki . natomiast energia gamma pochodzaca od 199 au ( 37 % , 159 kev i 22 % , 208 kev ) , jest bardzo bliska energii emitowanej przez najpopularniejszy radioizotop uzywany w spect i innych technikach medycyny nuklearnej , czyli 99m tc ( 140 kev ) . badania nad obrazowaniem z wykorzystaniem 199 au pozwoliłyby na oszacowanie indywidualnego wychwytu przez tkanki oraz okreslenie biodystrybucji i wydalania leku . tym samym , uzyskane dane umozliwiłyby obliczenie dokładnej dawki dla konkretnego pacjenta przed rozpoczeciem terapii stosujacej izotop 198 au . rozwoj jednoczesnej diagnostyki i terapii ( " teranostyki " ) moze zapewnic wyja [SEP]
[CLS] w jednym z badan nad wykorzystaniem nanoczastek złota w diagnostyce spect zhao i in . [SEP]
[CLS] zastosowali izotop 199 au , bezposrednio inkorporujac go na siatce krystalicznej stabilnych nanoczastek , osiagajac tym samym wysoka stabilnosc preparatu . poprzez funkcjonalizacje otrzymanych nanoczastek peptydem dapta , specyficznie wiazacym sie z receptorem chemokin ccr5 ( w tym przypadku uzytym do diagnostyki raka piersi ) , udało sie uzyskac wysokiej jakosci obrazy z poprawionym profilem biodystrybucji w stosunku do niemodyfikowanych nanoczastek . [SEP]
[CLS] tomografia komputerowa wykorzystuje roznice w zdolnosci pochłaniania kwantow rentgenowskich przez dane tkanki . [SEP]
[CLS] w badaniu tym zazwyczaj stosuje sie kontrasty majace na celu poprawienie jakosci uzyskiwanego obrazu . kwanty rentgenowskie sa emitowane z zewnetrznego zrodła , ustawionego dokładnie naprzeciw detektora . [SEP]
[CLS] ze wzgledu na wysoka zdolnosc pochłaniania promieniowania x przez złoto , aunps B-nanoparticle wydaja sie byc obiecujaca alternatywa dla srodkow kontrastowych do ct . liczba atomowa złota ( 79 ) jest duzo wieksza niz jodu , obecnie stosowanego kontrastu , dlatego au moze powodowac duzo silniejsze tłumienie promieniowania x . dodatkowo , bardzo mały rozmiar czasteczek jodu umozliwia jedynie krotki czas skanowania z powodu szybkiego usuwania kontrastu przez nerki . nanoczastki złota mozna natomiast tak modyfikowac , aby były one w stanie pokonac bariery biologiczne i pozostac w obszarze ograniczonym do przestrzeni wewnatrznaczyniowej na czas duzo dłuzszy . jedne z badan nad zastosowaniem aunps B-nanoparticle jako kontrastu krwi w rentgenowskiej tomografii komputerowej przeprowadzono w 2007 roku . nanoczastki złota pokryte peg ( o wymiarach 38 nm ) wstrzyknieto w dawce 493 mg au / kg m . c . do organizmu myszy , a nastepnie wykonano skanowanie za pomoca aparatu mikro - ct . zaobserwowano długotrwała poprawe kontrastu w naczyniach krwionosnych myszy - wzrost tłumienia promieniowania o 100 hu ( jednostki hounsfielda odnoszace sie do wspołczynnika pochłaniania [SEP]
[CLS] w badaniu z 2018 roku zaproponowano natomiast nanoczastki złota modyfikowane guma arabska w medium bedacym ciecza jonowa , mrowczanie glukozamonu , w roli kontrastu do ct . jako głowne zalety takiego koniugatu wskazano biokompatybilnosc i brak toksycznosci uzytych prekursorow ( gumy arabskiej i cieczy jonowej ) . [SEP]
[CLS] w badaniu z uzyciem fantomu udowodniono tez , ze stworzony zwiazek wykazywał lepsze działanie w stosunku do wybranego , komercyjnie stosowanego kontrastu . [SEP]
[CLS] radioterapia , polegajaca na naswietlaniu chorego narzadu zrodłem promieniowania x , jest wiodacym narzedziem terapeutycznym w walce z prawie połowa typow nowotworow . [SEP]
[CLS] lokalna kontrola guza jest osiagana poprzez zapewnienie wystarczajaco duzej dawki do zniszczenia objetosci nowotworu oraz zahamowania postepu i nawrotow choroby . [SEP]
[CLS] całkowita dawka terapeutyczna promieniowania rentgenowskiego zalezy od rodzaju guza i waha sie od kilkudziesieciu do około 100 gy . [SEP]
[CLS] najbardziej znaczace dla tego rodzaju terapii sa rozproszone fotony i kwanty x , fotoelektrony , elektrony komptonowskie , augera oraz fluorescencyjne . [SEP]
[CLS] energia kinetyczna wybitego przez foton elektronu w tkance odgrywa wazna rola w głebokosci penetracji przez elektron . [SEP]
[CLS] efekt fotoelektryczny zachodzi z prawdopodobienstwem ~ ( z / e ) 3 , gdzie e to energia padajacego kwantu x , a z jest liczba atomowa atomu , z ktorego wybijany jest elektron - dlatego własnie złoto ( z = 79 ) wykazuje wiekszy efekt oddziaływania z promieniowaniem rentgenowskim niz inne , czułe na promieniowanie pierwiastki , na przykład wegiel ( z = 6 ) , jod ( z = 53 ) , gadolin ( z = 64 ) czy platyna ( z = 78 ) . [SEP]
[CLS] polimery uzywane do pokrywania nanoczastek złota o potencjalnym zastosowaniu w teleradioterapii to głownie peg , polisacharydy , poloksaminy i poloksamery . wiekszosc dotychczasowych eksperymentow badajacych wrazliwosc złotych nanoczastek na promieniowanie wykonywana była na fantomach wodnych , z uzyciem aunps B-nanoparticle o rozmiarach 4 - 250 nm . [SEP]
[CLS] w ostatnich latach shi i in . przeprowadzili badanie na modelu komorkowym i mysim , sprawdzajac efekt podania nanoczastek złota pokrytych tiopronina na radioterapie nowotworu okreznicy . [SEP]
[CLS] w badaniu tym udowodniono , ze poprzez bezposrednie wstrzykniecie zwiazku do guza udało sie znaczaco opoznic jego wzrost - nastapił po 54 dniach w stosunku do 37 dni dla grupy kontrolnej . [SEP]
[CLS] terapia fototermiczna ( ptt ) jest rodzajem leczenia nowotworu , w ktorym wykorzystuje sie zjawisko hipertermii , czyli stan podwyzszonej temperatury ciała . [SEP]
[CLS] hipertermia jest niemal zawsze stosowana razem z innymi rodzajami leczenia , takimi jak chemio - i radioterapia . [SEP]
[CLS] sprawia ona , ze komorki nowotworowe staja sie bardziej wrazliwe na promieniowanie , czesto tez powoduje niszczenie tych komorek , ktore nie zostały uszkodzone poprzez napromienianie ( schemat działania ptt przedstawiono na ryc . 2 ) . [SEP]
[CLS] wzrost temperatury w okolicy guza zwieksza tempo zarowno endocytozy , jak i fagocytozy , ktore nastepnie moga nasilac wychwyt makroczasteczkowy oraz transport wewnatrzkomorkowy , wazne w dostarczaniu lekow . [SEP]
[CLS] przy ogrzewaniu guza do 43°c , zauwazono na poziomie naczyniowym dwukrotne podwyzszenie przepływu krwi przez chore tkanki , co ostatecznie skutkuje wzrostem wynaczynienia makroczasteczkowego . [SEP]
[CLS] selektywne dostarczanie ciepła do komorek nowotworowych uzyskuje sie głownie poprzez zastosowanie fotosensorow takich jak aunps B-nanoparticle , naswietlanych swiatłem nir , czyli bliskiej podczerwieni ( ze wzgledu na jego przenikalnosc i zdolnosc penetracji ) . [SEP]
[CLS] komorki rakowe sa nieodwracalnie uszkadzane przy hipertermii powodowanej temperatura z zakresu 41 - 47°c , trwajacej około 10 minut . hipertermia aktywuje mechanizmy przekazywania sygnałow prowadzace do smierci komorki na drodze apoptozy , a w przypadku zastosowania temperatur powyzej 50°c smierc komorek nastepuje głownie poprzez nekroze . [SEP]
[CLS] w ptt wykorzystuje sie zakres swiatła nir , co sprawia , ze nanoczastki złota ( ze wzgledu na wystepowanie spr ) , staja sie odpowiednim narzedziem do tego rodzaju leczenia . [SEP]
[CLS] przeprowadzono juz wiele badan ukazujacych synergistyczne działanie hipertermii z chemioterapia . [SEP]
[CLS] w jednym z takim badan [SEP]
[CLS] stworzono czuły na zmiany ph , dendrymerowy nano - koniugat do transportu doksorubicyny ( popularnie stosowany cytostatyk ) , w ktorym aunps B-nanoparticle były uwiezione wewnatrz dendrymerow . w trakcie leczenia zaobserwowano synergistyczny efekt pomiedzy ptt i chemioterapia . w innym eksperymencie zastosowano podejscie kombinacyjne oparte na nowotworowych komorkach macierzystych ( cscs ) , wykorzystujac w synergii rowniez te same rodzaje terapii . [SEP]
[CLS] nanorurki [SEP]
[CLS] złota [SEP]
[CLS] sprzegano z czwartorzedowa grupa amoniowa miedzy innymi poli ( chlorku diallilodimetyloamoniowego ) w celu zwiekszenia wychwytu aunrs przez komorki nowotworowe . nanorurki wykazały efekt fototermiczny , wybiorczo eliminujac cscs w linii komorkowej mcf - 7 raka piersi . platforma termochemioterapii została stworzona poprzez dodanie salinomycyny , inhibitora csc . w badaniu opisanym w roku 2019 zastosowano kolejna " zielona " synteza nanoczastek złota z uzyciem ekstraktu z kurkumy . udowodniono przeciwnowotworowe działanie preparatu po naswietleniu komorek mcf - 7 promieniowaniem laserowym , kierowane głownie przez mechanizm apoptozy . w jeszcze innym eksperymencie z tego samego roku wykazano natomiast efekt synergistyczny pomiedzy ptt i radioterapia , wykorzystujac puste w srodku nanosfery złota pokryte peg . zasugerowano rowniez mozliwosc zastosowania takiego zwiazku w teranostyce , łaczac dwa wymienione rodzaje terapii z diagnostyka ct . ryc . 2 . schemat działania terapii fototermicznej . [SEP]
[CLS] realizacja badan translacyjnych , czyli procesu przełozenia wynikow eksperymentow naukowych do zastosowan klinicznych , w przypadku nanomedycyny stanowi duze wyzwanie . [SEP]
[CLS] specyfika pracy z materiałami w tak małej skali wymaga od badaczy ciagłego doskonalenia stanu wiedzy na temat interakcji nanoczastek z układami biologicznymi oraz układem odpornosciowym człowieka . [SEP]
[CLS] dla naukowcow opracowujacych preparaty lecznicze jednym z kluczowych zadan jest scharakteryzowanie własciwosci farmakologicznych oraz biodystrybucji danego leku . w przypadku nanoczastek złota zaden z wymienionych powyzej problemow nie jest jeszcze dostatecznie dobrze poznany , stad stosunkowo niewielka liczba prowadzonych badan klinicznych w porownaniu z iloscia publikacji na temat aunps B-nanoparticle . [SEP]
[CLS] w 2018 roku singh i in . [SEP]
[CLS] stworzyli podsumowanie preparatow opierajacych sie na nanoczastkach złota , znajdujacych sie w badaniach klinicznych roznych faz . wsrod tych preparatow wyroznic nalezy miedzy innymi lek aurimune® ( cyt - 6091 ) opracowany przez cytimmune ( w obecnych badaniach w partnerstwie z astrazeneca ) . lek ten , w postaci nanoczastek złota pokrytych peg , działa jako nosnik dla rekombinowanego ludzkiego czynnika martwicy nowotworu ( rhtnf ) , dostarczajac go do obszaru zajetego przez nowotwor w celu zniszczenia jego naczyn krwionosnych . pierwsza faza badan klinicznych przyniosła bardzo dobre wyniki , pokazujac , ze dzieki zastosowaniu nanoczastek złota dawka rhtnf dostarczana pacjentom moze byc nawet trzykrotnie wyzsza niz dotychczas , bez dodatkowych efektow toksycznych . warto zaznaczyc , ze na pierwsza faze badan przedklinicznych czeka juz nastepny preparat cytimmune , cyt - 21625 , ktory oprocz rhtnf dostarczac ma do nowotworu rowniez lek cytostatyczny , paklitaksel . [SEP]
[CLS] innym przykładem leku opartego na aunps B-nanoparticle w badaniach klinicznych jest auroshells® , stworzony przez nanospectra . preparat ten dedykowany jest do zastosowania w ablacji laserowej guzow litych , a obecne badania kliniczne skupiaja sie na pacjentach z nowotworami prostaty - wczesniej technologie testowano rowniez w przypadkach guzow płuc , głowy i szyi . nanosfery stworzone z połaczenia złota i krzemionki i pokryte peg sa wprowadzane do ciała pacjenta poprzez wstrzykniecie , a nastepnie wzbudzane wiazka lasera z zakresu nir , co prowadzi do lokalnego wzrostu temperatury i niszczenia guza . ze wzgledu na brak zastosowania substancji aktywnych w tej metodzie , efekt toksyczny w stosunku do tkanek zdrowych jest skutecznie zminimalizowany . [SEP]
[CLS] w fazie badan klinicznych znajduje sie rowniez lek nu - 0129 , oparty na sferycznych kwasach nukleinowych przyłaczonych do powierzchni nanosfer złota . [SEP]
[CLS] skutecznosc i bezpieczenstwo preparatu badane sa obecnie w przypadku leczenia pacjentow z nawracajacym glejakiem wielopostaciowym oraz glejakomiesakiem . [SEP]
[CLS] jeszcze inne badania kliniczne przeprowadzono nad zastosowaniem nanoczastek złota jako nanosensorow w analizie oddechu pacjenta , w celu rozpoznawania chorob nowotworowych zoładka . poprzez zastosowanie takiego podejscia mozliwe byłoby nieinwazyjne rozroznienie raka zoładka od zwiazanych z nim zmian przednowotworowych . [SEP]
[CLS] nanoczastki złota ze wzgledu na swoje unikalne własnosci oraz duze mozliwosci modyfikacji ich rozmiarow i kształtow stanowia duza nadzieje dla terapii i diagnostyki nowotworowej . [SEP]
[CLS] moga byc potencjalnie uzyte zarowno w roli materiału bazowego , do tworzenia bardziej złozonych struktur z markerami biologicznymi i ligandami , jak i samodzielnego leku . [SEP]
[CLS] na uwage zasługuja przede wszystkim wystepowanie w aunps B-nanoparticle efektu powierzchniowego rezonansu plazmonowego , dzieki czemu nanoczastki złota stanowia obecnie jeden z najwazniejszych materiałow do badan nad hipertermia , a takze mozliwosc prowadzenia syntez bedacych czescia " zielonej " chemii . [SEP]
[CLS] obecnie wsrod problemow zwiazanych z opracowaniem preparatow medycznych bazujacych na nanoczastkach złota znajduja sie m . in . efektywne zredukowanie toksycznosci i poprawa biokompatybilnosci , stworzenie leku specyficznie wiazacego sie z wybranym nowotworem , w tym dalsze badania nad poszukiwaniem skutecznych biomarkerow , oraz opracowanie syntezy korzystnej z ekonomicznego punktu widzenia . [SEP]
[CLS] w przypadku preparatyki i badan materiałowych , najwieksze wyzwanie stanowi otrzymanie produktu homogenicznego . istnieje wiec koniecznosc prowadzenia dalszych badan , ktore pomoga w głebszym zrozumieniu własciwosci nanoczastek złota , ich biodystrybucji w organizmie oraz zachodzacych oddziaływaniach , a co za tym idzie opracowaniu efektywniejszych metod wykorzystania aunps B-nanoparticle jako lekow badz nosnikow lekow . [SEP]
[CLS] tabela 1 . dane dotyczace izotopu 198 au . [SEP]
[CLS] wkład emilii balcer został zrealizowany w ramach projektu nr powr . 03 . 02 . 00 - 00 - i009 / 17 - 00 ( program operacyjny wiedza edukacja rozwoj 2014 - 2020 wspołfinansowany ze srodkow europejskiego funduszu społecznego ) . [SEP]
[CLS] high - fidelity detection of targets with biosensors based on the au−s bond remains challenging due to the inevitable biothiol interference . [SEP]
[CLS] the emerging au−se bond - based biosensors have well resolved this concern owing to the higher stability of the au−se bond . [SEP]
[CLS] this feature highlights the developed au−se bond - based biosensors and discusses their design , preparation , application , current limitations , and potential future directions . [SEP]
[CLS] s elective and accurate detection of biomolecules in different biological samples holds tremendous promise for physiological and pathological investigations . [SEP]
[CLS] to achieve this goal , biosensors are indispensable to sense and visualize the targets . [SEP]
[CLS] compared with molecular sensors , nanoscale biosensors possess the characteristics of easy synthesis and modification , have high stability and signal strength , and are suitable for long - term circulation during in vivo applications . [SEP]
[CLS] therefore , over the past decades , researchers have designed and prepared a considerable number of nanoscale biosensors for tracing targets in biofluids , living cells B-material , tissues , and living bodies . [SEP]
[CLS] among which , gold B-material - based biosensors are the most widely recognized ones since the unique physicochemical properties and biocompatibilities B-property of gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) . [SEP]
[CLS] to construct versatile au - based biosensors , diverse recognition groups should be integrated onto the surface of aunps B-nanoparticle via either noncovalent or covalent strategies . [SEP]
[CLS] the noncovalent strategy mainly including van der waals ' force , hydrogen B-material bond , hydrophobic B-property interaction I-property , electrostatic adsorption , and π−π interaction . [SEP]
[CLS] on account of the complex biological environment possibly influencing the stability of the noncovalent bond constructed sensors , covalent bond - based sensors are more preferable . au−s bond - based biosensors have been the most studied ones during the past several decades , since the facile preparation of thiol decorated small / macro - molecules and easy formation of au−s bond . [SEP]
[CLS] it is obvious that au−s bond - based biosensors greatly facilitated the development of bioanalysis and nanomedicine fields . [SEP]
[CLS] though promising , recent studies revealed the stability of au− s bond - based biosensors could be severely affected by the abundant biothiols in living systems and cause false - positive signals . [SEP]
[CLS] therefore , precise detection of targets in biological systems with au−s bond - based sensors remains a challenge . [SEP]
[CLS] selenium B-material ( se ) , another chalcogen , is an essential micronutrient for the human body . [SEP]
[CLS] se is the critical component for many enzymes as it can replace several sulfur B-material - containing amino B-material acids I-material , including methionine B-material ( met ) , cysteine B-material ( cys ) , and cystine . [SEP]
[CLS] selenol could also form a stable covalent bond with au . [SEP]
[CLS] importantly , the bond energy of au−se bond is higher than that of au−s bond , indicating au−se bond - based biosensors may be more stable than the au−s bond constructed counterparts . [SEP]
[CLS] based on this property , our group developed the first au−se bond - based biosensor for highfidelity biological imaging in 2018 . [SEP]
[CLS] since then , a series of attractive biosensors were developed and have brought new opportunities for au - based biosensors . [SEP]
[CLS] in this feature , we will focus on the developed biosensors based on the au−se bond . [SEP]
[CLS] we will start from the detection of tumor B-material biomarkers B-property with au−se bond - based biosensors . [SEP]
[CLS] subsequently , imaging of signal molecules ' evolution with au−se bond - based biosensors will be introduced . [SEP]
[CLS] next , published : june 10 , 2020 feature pubs . acs . org / ac evaluating the therapeutic mechanisms of drugs with au−se biosensors and the novel freezing method for biosensor preparation will be highlighted . [SEP]
[CLS] finally , our thinking about the current limitations and potential future directions of these promising biosensors will be presented . [SEP]
[CLS] nanoprobes B-nanoparticle for selectively distinguishing cancer cells B-material from normal ones have received increasing research interest in recent years . [SEP]
[CLS] typically , cancer cells B-material metabolize differently from normal cells B-material , and the gene expression levels of cancer cells B-material are also different . [SEP]
[CLS] therefore , by precisely detecting diverse biomarkers B-property ( including diverse enzymes , genes , and other bioactive species ) , nanoprobes B-nanoparticle that could effectively identify cancer are of great significance for the diagnosis , treatment , and prognosis of cancer . [SEP]
[CLS] as talked about above , the most widely used au−s bond - based nanosensors B-nanoparticle suffer from biothiols interference . [SEP]
[CLS] given this , more stable au−se bond - based nanosensors B-nanoparticle came into being and have been proven to be better candidates for the detection of biomarkers B-property . [SEP]
[CLS] up until now , the available au−se bond - based biosensors mainly take enzymes and mrna as targets . [SEP]
[CLS] detecting enzymes [SEP]
[CLS] matrix metalloproteinases ( mmps ) are a large group of proteases , which play vital roles in diverse living cells B-material because they take part in cell B-material proliferation , migration , cell B-event adhesion I-event / dispersion , differentiation , and so on . [SEP]
[CLS] overexpression of mmps is also associated with inflammation , cancer cell B-material infiltration , metastasis B-event , and angiogenesis B-event . [SEP]
[CLS] therefore , selective detection of mmps enables efficient cancer diagnosis . [SEP]
[CLS] in 2018 , our group reported the design of au−se bond - based fluorescent B-property probe 1 for highfidelity tumor B-material biomarker B-property imaging in living cells B-material ( figure 1a ) . [SEP]
[CLS] in our design , fluorophore fitc - labeled selenol terminated mmp - 2 recognition peptides B-material were assembled onto the surface of 13 nm aunps B-nanoparticle . [SEP]
[CLS] since the similar chemical property of selenol and thiol B-material , the au−se probe 1 was successfully prepared by the traditional method for the au−s bond - based nanoprobe B-nanoparticle . [SEP]
[CLS] owing to the excellent fluorescence B-property quenching effect of aunps B-nanoparticle , the obtained nanoprobes B-nanoparticle have a low background fluorescence B-property . [SEP]
[CLS] after meeting the biomarker B-property mmp - 2 in cancer cells B-material , the cleavage of peptides B-material resulted in the release of the fluorescence B-property signal . [SEP]
[CLS] compared to the au−s bond constructed counterparts , negligible interference by biothiols was observed ( figure 1b ) . [SEP]
[CLS] importantly , the better stability of au−se probe 1 rendered a higher signal - to - noise ratio and lower detection limit . [SEP]
[CLS] this probe could serve as a reliable biosensor for tracing the real information in living cells B-material . [SEP]
[CLS] the expression levels of biomarkers B-property in different cell B-material lines are quite different . [SEP]
[CLS] probes for detecting one target are inefficient for identifying multiple cancer cells B-material from normal ones . [SEP]
[CLS] therefore , the simultaneous detection of multiple biomarkers B-property is helpful to further avoid misdiagnosis . [SEP]
[CLS] recently , an au−se bond - based two - color nanoprobe B-nanoparticle 2 was developed for simultaneous imaging of mmp - 2 and mmp - 7 ( figure 1c ) . [SEP]
[CLS] the probe possesses high selectivity and antibiothiol effect . [SEP]
[CLS] during ex vivo application , the elevated mmp - 2 and mmp - 7 levels in hepg2 and mcf - 7 cells B-material than hl - 7702 cells B-material were visualized ( figure 1d ) . [SEP]
[CLS] electrochemical sensors have the characteristics of rapid , sensitive , and easy integration and have been widely used for biological detection . [SEP]
[CLS] au electrodes are one of the most ideal electrodes for biological analysis owing to their good conductivity , biocompatibility B-property , and stability . [SEP]
[CLS] by choosing different functional molecules and electrochemically active components , au - based electronic sensors could be easily prepared for the efficient detection of targets in diverse biosamples . [SEP]
[CLS] the stability of the au electrode - functional molecules interface is also one of the critical factors detecting rnas . [SEP]
[CLS] in addition to proteins B-material , nucleic B-material acids I-material are also important biomarkers B-property for cancer diagnosis . [SEP]
[CLS] never - theless , the commercialized selenol functionalized nucleic B-material acid I-material is still unavailable . [SEP]
[CLS] therefore , a novel chemical strategy for the preparation of au−se bond - based nucleic B-material acid I-material probes is highly attractive . [SEP]
[CLS] very recently , our group prepared a kind of nanoflare based on the au−se bond ( probe 4 ) ( figure 2b ) . [SEP]
[CLS] the amino - modified recognition ssdna was linked with selenocystine via amidation B-event . [SEP]
[CLS] to attach the recognition sequences onto the aunps B-nanoparticle by forming a stable au−se bond , visible light irradiation was introduced to induce the cleavage of the diselenide bond . [SEP]
[CLS] the au−se nucleic probe could be obtained after further hybridizing the dye - labeled flare strands with ssdna . [SEP]
[CLS] after meeting the target vimentin mrna , the recognition ssdna could hybridize with the targets , resulting in the release of prequenched flare strands ( figure 2b ) . [SEP]
[CLS] the probe showed a good anti - gsh interference effect , and its application for the selective imaging of vimentin mrna in several breast cancer cell B-material lines was demonstrated . [SEP]
[CLS] visible light irradiation restricted the above method to prepare other kinds of biosensors . [SEP]
[CLS] more recently , our group developed a tcep mediated diselenide bond cleavage strategy to prepare selenol - terminated molecular beacon ( figure 2c ) . [SEP]
[CLS] the obtained product was further employed to prepare probe 5 . [SEP]
[CLS] probe 5 exhibited a similar detection performance to the au−s probe , but its anti gsh interference effect was significantly stronger ( figure 2d ) . [SEP]
[CLS] during imaging biomarker B-property mir - 221 in living cells B-material , probe 5 successfully avoided the biothiol interference , even in high concentrations of gsh pretreated normal cells B-material . [SEP]
[CLS] in living cells B-material , signaling transduction pathways are critical to maintain cell B-material homeostasis . [SEP]
[CLS] activation of abnormal signal transduction pathways usually cause cell B-material dysfunction and diverse diseases . [SEP]
[CLS] selective detection of the concentration of diverse biomolecules in cells B-material , tissues , and living bodies may facilitate the investigation of the molecular mechanisms of diverse bioevents . [SEP]
[CLS] however , real - time detection of signaling molecules in complex bioenvironments remains challenging , not only because the concentration of different biomolecules remains low but also many other species can influence the detection result . [SEP]
[CLS] therefore , probes with high sensitivity and specificity to different biomolecules are promising tools for investigating the relationships between these species and identifying potential signaling transduction pathways . [SEP]
[CLS] in 2018 , our group developed an au−se bond - based nanoprobe B-nanoparticle 6 for imaging caspase - 9 , an apoptosis B-event protein B-material , in cancer cells B-material , which showed a high anti - interference effect in the presence of biothiols and was demonstrated to reveal the real level of target in cells B-material . [SEP]
[CLS] inspired by this successful case , several multicolor biosensors were developed later for the detection of signal molecules ' evolution in living cells B-material . [SEP]
[CLS] the overexpressions of urokinase - type plasminogen activator ( upa ) and mmp - 9 are closely related to the invasion and metastasis B-event of cancer . [SEP]
[CLS] imaging of the expression levels of upa and mmp - 9 in cancer cells B-material may help to evaluate the tumor B-material development status and metastasis B-event risks . [SEP]
[CLS] simultaneously , investigating the upstream and downstream relationships between the proteins B-material may further inspire novel strategies for metastasis B-event prevention and cancer treatment . [SEP]
[CLS] by grafting two dye modified selenol - terminated recognition peptides B-material onto aunps B-nanoparticle , our group developed a fluorescent B-property nanoprobe B-nanoparticle 7 for investigating the relationships between upa and mmp - 9 ( figure 3a ) . [SEP]
[CLS] the probe was employed to visualize the realtime expression levels of upa and mmp - 9 in lps treated mcf - 7 cells B-material . [SEP]
[CLS] after incubation B-technique with au−se probe , the real - time fluorescence B-property signal changes for upa and mmp - 9 were captured , and the signal for upa appears before mmp - 9 , indicating upa is the upstream signaling molecule of mmp - 9 . [SEP]
[CLS] however , au−s bond constructed probes suffer from biothiol interference and exhibited obvious signal distortion . [SEP]
[CLS] therefore , au−se bond - based nanosensors B-nanoparticle are ideal candidates for evaluating the upstream and downstream relationships between different biomolecules . [SEP]
[CLS] later , visualization of the apoptosis B-event cascade was also realized with an au−se bond - based fluorescent B-property probes 8 , which revealed caspase 8 and caspase 9 , which are the upstream signaling molecules of apoptosis B-event protein B-material caspase 3 . [SEP]
[CLS] cathepsin b is a critical cysteine B-material proteinase in living cells B-material , which mainly localizes in lysosomes . [SEP]
[CLS] under stimulations , lysosomes may become permeable to release cathepsin b into the cytoplasm . [SEP]
[CLS] the released enzyme could serve as the initiator to induce cell B-material apoptosis B-event , even in apoptosis resistant cancer cells B-material . [SEP]
[CLS] recently , our group prepared a dual - color au−se bond - based fluorescent B-property nanoprobe B-nanoparticle 9 , for visualizing this apoptosis B-event process ( figure 3b ) . [SEP]
[CLS] two fluorophore modified selenol - terminated peptides B-material were assembled onto the surface of aunps B-nanoparticle to construct the probe . [SEP]
[CLS] ■ evaluating the therapeutic mechanism of drugs in addition to the above - mentioned applications , the au−se probe was also employed to investigate the therapeutic mechanism of chemotherapeutic drugs [SEP]
[CLS] it is demonstrated that autophagy may survive cancer cells B-material from different treatments including chemotherapy . autophagy inhibition has been proved to be a potential strategy for enhancing the therapy efficiency of diverse cancer treatments . [SEP]
[CLS] however , there are also some drugs inducing apoptosis B-event by excessive autophagy activation . [SEP]
[CLS] therefore , autophagy inhibition might compromise the therapeutic effect of these drugs . [SEP]
[CLS] very recently , we devised an au−se probe 10 for simultaneous imaging of autophagy biomarker B-property atg4b and apoptosis B-event biomarker B-property caspase - 3 . [SEP]
[CLS] instead of using traditional sodium B-material dodecyl sulfate ( sds ) - assisted probe preparation strategy , this work explored a facile freezing method ( figure 4a ) . [SEP]
[CLS] by simply mixing aunps B-nanoparticle with selenol - terminated peptides B-material and storing at −20 °c for 1 h , the peptides B-material could be efficiently assembled onto aunps B-nanoparticle by forming a stable au−se bond . [SEP]
[CLS] the underlying mechanism of this preparation method is that the freezing process induces the precipitation of peptides B-material , and the elevated local concentration of peptides B-material and aunps B-nanoparticle facilitated the connection of selenol with aunps B-nanoparticle , thus the peptide B-material density of the probe prepared by the freezing method was higher than that by traditional sds - assisted strategy ( figure 4b ) . [SEP]
[CLS] this surfactant - free strategy not only improved the utilization efficiency of peptides B-material but also ensured the biocompatibility B-property of the probes . [SEP]
[CLS] with probe 10 , the real - time expression of atg4b and caspase - 3 in chemotherapeutic drug curcumin and 7 - ethyl - 10 - hydroxycamptothecin ( sn - 38 ) treated hepg2 cells B-material were effectively visualized . [SEP]
[CLS] in the real - time confocal fluorescent B-property imaging experiment , this probe successfully revealed curcumin induces apoptosis B-event by triggering excessive apoptosis B-event , while sn - 38 induces apoptosis B-event with an apoptosis - independent manner ( figure 4c , d ) . [SEP]
[CLS] in summary , au−se bond - based biosensors have emerged as the paragons for resolving the biothiol interference of au - based biosensors . [SEP]
[CLS] ever since the first example reported in 2018 , a series of biosensors based on au−se bond have been devised for high - fidelity detection of biomolecules in biological systems . [SEP]
[CLS] although promising , research is still in the infancy stage and there is still room for further development of related biosensors [SEP]
[CLS] in regards to the limitations , most of the developed biosensors are fluorescent B-property probes , and almost all the probes use enzyme as targets . [SEP]
[CLS] though a au−se bond - based nanoflare has been developed for intracellular mrna imaging very recently , the relative complex synthesis procedure and photoinduced cleavage of diselenide bond restricted the further exploration of other types of the au−se - dna probe . [SEP]
[CLS] tcep mediated cleavage of the diselenide bond seems to be a more practical strategy for the design of other au−se - dna probes , but the universality of this method still needs to be further verified . [SEP]
[CLS] the freezing method is fascinating for the rapid preparation of high - performance au - based biosensors , it ' s potential also remains to be explored on other types of biosensors , and the mechanism remains to be further cleared . [SEP]
[CLS] the au−se bond - based electrochemical probe was also synthesized ; however , at present , only the application of detection biomarker B-property mmp - 2 in biological fluids was demonstrated . [SEP]
[CLS] the highly stable au−se bond should also be useful for constructing therapeutic and theranostic probes , but the related investigation is still absent . [SEP]
[CLS] compared to in vitro conditions , the in vivo environments are more complicated , and there may be more potential barriers B-property to au−se probes used in vivo . [SEP]
[CLS] potential future directions include ( 1 ) developing more versatile au−se bond - based biosensors . [SEP]
[CLS] au possesses numerous attractive features , and au−se bond - based colorimetric probes , raman probes , photoacoustic probes , ratiometric probes as well as multimodal probes could be devised with diverse feasible synthetic strategies in the following studies ; ( 2 ) au−se - dna probes are ideal platforms for bioanalysis , and it is essential to develop more methods to prepare selenolfunctional nucleic B-material acids I-material , such as dnazyme , aptamer , and so on . [SEP]
[CLS] ( 3 ) the potential therapeutic application of the probes should be investigated . [SEP]
[CLS] by connecting different bioactive molecules , the au−se bond - based therapeutic / theranostic probes should be promising nanomedicine for chemotherapy , phototherapy , radiotherapy , and immunotherapy . [SEP]
[CLS] ( 4 ) investigating the bioactivity / toxicity B-property of au−se bond - based biosensors systematically and further evaluating their in vivo distribution and pharmacokinetics pave the road for future clinical translation . [SEP]
[CLS] ( a , a ) design of au−se probe 1 and the counterpart au−s probe and tem images of au−se ( b ) and au−s ( c ) probes . [SEP]
[CLS] ( b ) flow B-technique cytometry I-technique imaging of mmp - 2 in hepg2 cells B-material with au−se probe 1 ( a ) and the counterpart au−s ( b ) probes . [SEP]
[CLS] parts c and d are the fluorescence B-property intensity distribution of cells B-material in parts a and b ) . [SEP]
[CLS] ( c ) responsive mechanism of au−se probe 2 for simultaneous mmp - 2 and mmp - 7 imaging . [SEP]
[CLS] ( d ) confocal imaging of mmp - 2 and mmp - 7 in different cells B-material with au−se probe 2 , scale bars = 25 μm . [SEP]
[CLS] adapted with permission from ref 83 . [SEP]
[CLS] copyright 2019 science china press and springer - verlag gmbh germany . [SEP]
[CLS] ( a ) schematic illustration of the structure and performance of au−se electrode 3 and the counterpart au−s electrode . [SEP]
[CLS] ( b ) schematic illustration of the preparation and response mechanism of au−se probe 4 . [SEP]
[CLS] adapted with permission from ref 96 . [SEP]
[CLS] copyright 2020 royal society of chemistry . [SEP]
[CLS] ( c , a ) schematic illustration of the preparation of selenol - terminated molecular beacon , ( b ) responsive mechanisms of probe 5 and the related au−s probe in the gsh - containing environments . [SEP]
[CLS] ( d , a ) fluorescence B-property responses of probe 5 , and the related au−s probe toward different targets and ( b ) time - dependent fluorescence B-property background signal of probe 5 and the related au−s probe in the presence of gsh ( 5 mm ) . [SEP]
[CLS] ( a ) schematic illustration of the response mechanism of au−se probe 7 . [SEP]
[CLS] adapted with permission from ref 103 . [SEP]
[CLS] copyright 2019 royal society of chemistry . [SEP]
[CLS] ( b ) schematic illustration of the response mechanism of au−se probe 9 . [SEP]
[CLS] ( c ) real - time monitoring of the mcf - 7 cells B-material with the nanoprobes B-nanoparticle after resveratrol addition : ( a and b ) the probe 9 treated group and ( c and d ) the au−s nanoprobe B-nanoparticle group . [SEP]
[CLS] red fluorescence B-property of 5tamra for cathepsin b ( a , c ) and green fluorescence B-property of fitc for caspase - 3 ( b , d ) . [SEP]
[CLS] ( d ) normalized fluorescence B-property intensities of part c . [SEP]
[CLS] adapted with permission from ref 105 . [SEP]
[CLS] copyright 2019 elsevier [SEP]
[CLS] probe 9 releases the related fluorescence B-property signals specifically after the peptides B-material were cleaved by cathepsin b / caspase 3 . [SEP]
[CLS] in the resveratrol treated mcf - 7 cells B-material , probe 9 successfully visualized the sequential activation of cathepsin b and apoptosis B-event protein B-material caspase 3 . [SEP]
[CLS] however , the biothiols caused signal distortion resulted the au−s bond - based probe fail to detect the signal difference effectively ( figure 3c , d ) . [SEP]
[CLS] all the results proved the feasibility of high - fidelity tracing intracellular biomolecules with au−se bond - based biosensors . [SEP]
[CLS] ( a ) schematic illustration of the preparation and response mechanism of au−se probe 10 by the freezing method . [SEP]
[CLS] ( b ) comparison of the performance of au−se probe 10 prepared by the traditional method and the freezing method : ( a−c ) tem morphology characterization and ( d ) surface zeta B-property potential I-property of aunps B-nanoparticle and probes prepared by the traditional and freezing methods , ( e ) peptide B-material density on per aunps B-nanoparticle of probes . [SEP]
[CLS] real - time imaging the intracellular expression of atg4b and caspase - 3 in hepg2 cells B-material treated with curcumin ( c ) and sn - 38 ( d ) with probe 10 . [SEP]
[CLS] college of chemistry , chemical engineering and materials science , key laboratory of molecular and nano probes , ministry of education , collaborative innovation center of functionalized probes for chemical imaging in universities of shandong , institute of molecular and nano science , shandong normal university , jinan 250014 , p . r . china wei pan − college of chemistry , chemical engineering and materials science , key laboratory of molecular and nano probes , ministry of education , collaborative innovation center of functionalized probes for chemical imaging in universities of shandong , institute of molecular and nano science , shandong normal university , jinan 250014 , p . r . china ; orcid . org / 0000 - 0002 - 2281 - 8749 [SEP]
[CLS] complete contact information is available at : https : / / pubs . acs . org / 10 . 1021 / acs . analchem . 0c01624 [SEP]
[CLS] biographies peng gao obtained his b . s . degree in chemistry from nanchang university in 2017 . [SEP]
[CLS] he is now pursuing a m . s . degree at shandong normal university under the supervision of prof . bo tang . [SEP]
[CLS] his research focuses on the development of nanoscale biosensors for tumor B-technique imaging I-technique and therapy . [SEP]
[CLS] yuanyuan chen obtained her b . s . degree in chemistry from shandong normal university in 2015 . [SEP]
[CLS] she is now pursuing her ph . d . studies with prof . bo tang . [SEP]
[CLS] her current research interest is design and synthesis of nanoscale biosensors for tumor B-material radiotherapy . [SEP]
[CLS] wei pan received his ph . d . in analytical chemistry from shandong normal university in 2014 . [SEP]
[CLS] currently , he is an associate professor in the college of chemistry , chemical engineering and materials science at shandong normal university . [SEP]
[CLS] his research interests focus on the design and application of fluorescent B-property nanoprobes B-nanoparticle for cancer imaging and therapy . [SEP]
[CLS] na li received her b . s . in 2004 and ph . d . in 2009 from shandong university . [SEP]
[CLS] after a postdoctoral fellowship at mcmaster university , she joined shandong normal university in 2010 . [SEP]
[CLS] she became an associate professor in 2012 and was promoted to a full professor in 2014 . [SEP]
[CLS] her research interests include the design and synthesis of functional nanoprobes B-nanoparticle for detection , imaging , and therapy . [SEP]
[CLS] bo tang obtained his ph . d . in analytical chemistry in 1994 from nankai university . [SEP]
[CLS] then he joined the college of chemistry , chemical engineering and materials science as a full professor at shandong normal university . [SEP]
[CLS] his current research interests include development of molecular and nanoprobes B-nanoparticle for analytical and biomedical applications , solar energy chemical transformation and storage , and clean synthesis of chemicals . [SEP]
[CLS] a wide range of analytical techniques in bioanalysis relies on surface - based biomolecular detection , which requires the confinement of probes onto a heterogeneous surface to react with targets . [SEP]
[CLS] probe arrangement on the interface is critical for target recognition and determines assay performance . [SEP]
[CLS] much effort has been devoted to screen the optimized probe arrangement according to experimental tests . [SEP]
[CLS] such a data - driven posteriori pattern faces low efficiency , ambiguous orientation , and possible deviated tested ranges from the best case . [SEP]
[CLS] herein , we demonstrate that a model can effectively guide probe arrangement onto the interface to facilitate probe−target recognition , embodied by the assay of human telomerase activity with dna - conjugated gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) . [SEP]
[CLS] both theoretical calculation and experimental results indicate that telomerase activity is maximized on the aunp B-nanoparticle surface under guidance of the model . [SEP]
[CLS] the detection limit is at least 1 order of magnitude lower than that of aunp B-nanoparticle bearing densely packed dna , comparable to that of the telomeric repeat amplification protocol ( trap ) . [SEP]
[CLS] the model - guided interface probe arrangement is proved to be highly useful in regulating interface recognition and may offer a new paradigm to promote surface - based biomolecular detection . [SEP]
[CLS] s urface - based biomolecular detection is extensively adopted and plays a vital role in bioanalysis , which confines the probe onto the heterogeneous surface to react with targets . [SEP]
[CLS] the probe arrangement on the interface has a crucial impact on the probe−target recognition and detection performance . [SEP]
[CLS] to obtain a proper probe arrangement , much effort has been devoted to test assay responses under an artificially chosen range of probe densities , 3−5 conformations , and surface chemistries [SEP]
[CLS] however , those methods rely on experiments to screen an optimized probe arrangement and have a data - driven posteriori pattern . [SEP]
[CLS] the limitations are low efficiency , ambiguous orientation , and possible deviated tested ranges from the best case . [SEP]
[CLS] alternatively , if a model can be established by integrating the surface , probe , and target , probes can be arranged in a more effective and oriented way under guidance of the model , so as to promote surface - based biomolecular detection . [SEP]
[CLS] herein , we demonstrate that a model can efficiently guide probe arrangement on the interface for sensitive detection , embodied by an assay of human telomerase activity with dnaconjugated gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) . [SEP]
[CLS] dna - conjugated aunps B-nanoparticle constitute an important part in surface - based biomolecular detection , finding powerful applications in major analytical techniques including a colorimetric / fluorescent B-property / electrochemical / scanometric assay and bioimaging . [SEP]
[CLS] human telomerase is a ribonucleoprotein comprised of telomerase rna and telomerase reverse transcriptase , catalyzing the addition of telomeric repeats ( ttaggg ) n onto the 3 ′ - end of telomere . [SEP]
[CLS] telomerase activity is overexpressed in most types of cancer cells B-material but not in normal somatic cells B-material . [SEP]
[CLS] besides the biological importance , it is chosen as a target since the large size and functional sites make its interaction with probes more complex , necessitating a proper probe arrangement on the interface . [SEP]
[CLS] telomerase activity was colorimetric detected by dna−aunp conjugates . [SEP]
[CLS] the improperly arranged , densely packed dnas on the aunp B-nanoparticle surface however impair their recognition to telomerase . [SEP]
[CLS] this in turn results in a low proportion of elongated dna ( merely one percent ) that undermines the sensitivity . [SEP]
[CLS] therefore , this system serves as a proof of our concept . [SEP]
[CLS] our principle to detect telomerase activity is illustrated in figure 1 . [SEP]
[CLS] telomerase binding substrates ( ts ) are conjugated onto aunps B-nanoparticle via the 5 ′ - end and undergo 3 ′ - end elongation under the action of telomerase . [SEP]
[CLS] the sequence near 5 ′ - end ( depicted in italic letters ) is designed to be complementary to telomeric repeats , inducing a hairpin structure folding on aunp B-nanoparticle surface . [SEP]
[CLS] the tertiary structure can enhance stability of aunps B-nanoparticle at high salt B-material concentration ; thus , aunps B-nanoparticle remain dispersed to stay red . [SEP]
[CLS] however , the ts would not be elongated with deactivated telomerase . [SEP]
[CLS] aunps B-nanoparticle cannot be stabilized and form aggregates to induce a color change to blue . [SEP]
[CLS] first , we established a model between aunp B-nanoparticle - confined dna and telomerase . [SEP]
[CLS] the synthesized aunps B-nanoparticle are uniform in size with an average radius of 7 . 1 nm ( figure s - 1 ) . [SEP]
[CLS] telomerase is recently identified to function as a dimer , and the distance between the two catalytic pockets containing rna templates is approximately 18 . 5 nm . [SEP]
[CLS] molecular graphics of telomerase dimer are reconstructed by the ucsf chimera package and em density map emd - 2310 ( figure 2a , inset ) . [SEP]
[CLS] during the binding , telomerase is actually separated from the aunp B-nanoparticle surface by a dna segment from the " t " base close to the particle surface to the " a " base next to the telomeric domain that inserts into the catalytic pockets ( figure 2a ) . [SEP]
[CLS] assuming that dna adopts an extended conformation , the length of the dna segment is calculated to be 13 . 3 nm by the size of each base in single - stranded dna ( dna segment and length calculation are detailed in figure s - 2 ) . [SEP]
[CLS] to maximize the catalytic activity of telomerase , the dna arrangement on the aunp B-nanoparticle surface should satisfy that the two neighboring dnas interact just right with the two catalytic sites ( marked with red dots ) . [SEP]
[CLS] the distance between the neighboring incipient telomeric bases housed in the catalytic pocket to hybridize with telomerase rna is thus anticipated to be 18 . 5 nm . [SEP]
[CLS] the optimal distance between neighboring dna strands is calculated to be 6 . 4 nm according to the sector formula . [SEP]
[CLS] next , we calculated the dna arrangement that facilitated telomerase binding . [SEP]
[CLS] each dna is assumed to occupy a square area , and the strand is considered to be located at the center ( figure 2b ) . [SEP]
[CLS] since the distance between neighboring dna strands is 6 . 4 nm , each dna holds an area of 41 . 0 nm 2 . [SEP]
[CLS] besides , the surface area of aunp B-nanoparticle is calculated to be 633 . 1 nm 2 . [SEP]
[CLS] since conjugated dna covers the aunp B-nanoparticle surface , the number of dna on each nanoparticle B-nanoparticle is calculated to be 15 . 4 by dividing the aunp B-nanoparticle surface area by the dna - occupied area . [SEP]
[CLS] this means that the optimal dna surface density is between 15 and 16 strands per particle . [SEP]
[CLS] because the dna surface density derived from the model simulation is relatively low and the dna is presumed to stay extended on the aunp B-nanoparticle surface , consecutive adenines were chosen to conjugate and arrange ts onto aunp B-nanoparticle . [SEP]
[CLS] this is because the dna anchored by consecutive adenines is spatially distributed and adopts an extended and upright conformation , as a result of the occupied adenines on aunp B-nanoparticle . [SEP]
[CLS] moreover , the dna surface density can be adjusted by varying the length of consecutive adenines . [SEP]
[CLS] ts were modified onto the aunp B-nanoparticle surface via the 10 , 20 , and 30 consecutive adenines at the 5 ′ end , obtaining a10 - ts - aunp B-nanoparticle , a20 - ts - aunp B-nanoparticle , and a30 - ts - aunp B-nanoparticle conjugates , respectively . [SEP]
[CLS] dynamic B-technique light I-technique scattering I-technique ( dls ) indicates that dnas tend to adopt an extended conformation on the aunp B-nanoparticle surface ( figure s - 3 ) . [SEP]
[CLS] the dna densities on the aunp B-nanoparticle surface were determined by the dtt - displacement method ( figure s - 4 ) . [SEP]
[CLS] it is revealed that 19 , 16 , and 11 dna strands per aunp B-nanoparticle are obtained for the three types of conjugates . [SEP]
[CLS] their stabilities demonstrate an order of a20 - ts - aunp B-nanoparticle > a10 - ts - aunp B-nanoparticle > a30 - ts - aunp B-nanoparticle , roughly consistent with the order of conjugated mononucleotide quantities on each nanoparticle B-nanoparticle ( figure s - 5 ) . [SEP]
[CLS] the dna surface density of a20 - ts - aunp B-nanoparticle conjugate coincides with the theoretical model . [SEP]
[CLS] note that the dna surface density could be regulated until close to theoretical value by precisely altering the length of consecutive adenines if the coincidence did not occur . [SEP]
[CLS] a20 - ts - aunp B-nanoparticle was complexed with telomerase from hela extracts equivalent to cell B-material concentrations of 0 to 200 cells B-material / μl , and time - dependent absorption spectra were recorded . [SEP]
[CLS] the absorption ratio ( a700 / a520 ) is used to evaluate the aggregation of aunps B-nanoparticle . [SEP]
[CLS] it is observed that aunps B-nanoparticle aggregate slower and tend to stay dispersed as telomerase activity rises ( figure 3a ) . [SEP]
[CLS] for comparison , control experiments were tested under otherwise identical conditions but utilizing a10 - ts - aunp B-nanoparticle and a30 - ts - aunp B-nanoparticle conjugates . [SEP]
[CLS] it is found that their aggregation kinetics are similar but differ from that of a20 - ts - aunp B-nanoparticle . [SEP]
[CLS] the discrepancy between 0 and 2 cells B-material / μl is much smaller than that of a20 - ts - aunp B-nanoparticle ( figure 3b , c ) . [SEP]
[CLS] models were simulated on the basis of the actual dna arrangement . [SEP]
[CLS] ( calculations were detailed in the supporting information . [SEP]
[CLS] ) for a20 - ts - aunp B-nanoparticle , the distance between neighboring incipient telomeric bases is 18 . 1 nm ( figure 3d ) , matching well with the distance between two catalytic pockets in a telomerase dimer . [SEP]
[CLS] therefore , one dimer could simultaneously elongate two individual dna , maximizing telomerase ' s catalytic activity . [SEP]
[CLS] however , for a10 - ts - aunp B-nanoparticle and a30 - ts - aunp B-nanoparticle , the distances are 16 . 6 and 21 . 8 nm , respectively ( figure 3e , f ) . [SEP]
[CLS] the dna arrangement is either too dense or too sparse to insert into both catalytic pockets of telomerase , and thus , one dimer could only elongate one dna . [SEP]
[CLS] the initial kinetic curves within 20 min were analyzed so that dna substrates were sufficient , more objective to compare telomerase catalysis on dna . [SEP]
[CLS] fitting of plots for three conjugates , complexed with telomerase from 20 cells B-material / μl hela extracts , demonstrates good linear relationships ( figure 4a ) . [SEP]
[CLS] slopes of variation in ratio versus time provide exact aggregation rate constants ( k ) ( specified in figure 4a ) . [SEP]
[CLS] k is inversely proportional to the stabilities of nanoparticles B-nanoparticle ( w ) according to colloids ' aggregation kinetics ( eq 1 ) . [SEP]
[CLS] here , k is the boltzmann constant , t is absolute temperature , η is the viscosity B-property of dispersion medium , and w is the stability ratio indicating the stability of colloids . [SEP]
[CLS] nanoparticle B-nanoparticle aggregation is caused by salt B-material - screening of surface charges ; thus , their stability depends on the increased double layer energy . [SEP]
[CLS] the double layer energy ( w ) for spherical particles is formulated as eq 2 . [SEP]
[CLS] π σ κ ε ε [SEP]
[CLS] ( 2 ) a20 - ts - aunp B-nanoparticle was then complexed with telomerase from hela extracts equivalent to various cell B-material concentrations . [SEP]
[CLS] the absorption ratio increases slower along with the increasing cell B-material concentration , indicating retarded aggregation of aunps B-nanoparticle ( figure 5a ) . [SEP]
[CLS] this is because the proportion of elongated ts that could stabilize aunp B-nanoparticle gradually scales up as telomerase activity rises . [SEP]
[CLS] kinetic curves under the action of different telomerase activities , even with low concentration of hela cells B-material , are elegantly distinguished from each other . [SEP]
[CLS] quantification was achieved by plotting the absorption ratio at the final time point versus hela cell B-material concentration . [SEP]
[CLS] the absorption ratio drops dramatically along with cell B-material concentration from 0 to 50 cells / μl and gradually reaches saturation until 500 cells B-material / μl ( figure 5b ) . [SEP]
[CLS] the ratio shows a linear range toward cell B-material concentration of 0 to 50 cells B-material / μl . for the assay of 2 , 20 , and 50 cells B-material / μl , the relative standard deviations conducted in three independent experiments are 4 . 2 % , 4 . 1 % , and 4 . 3 % within 1 day and are 6 . 2 % , 5 . 3 % , and 4 . 9 % on three different days , showing a satisfactory precision . [SEP]
[CLS] for comparison , pure telomerase was meanwhile tested ( figure s - 13 ) . [SEP]
[CLS] by correlating cell B-material concentration and telomerase concentration with the same absorption ratio , it is estimated that telomerase activity in a single hela cell B-material is around 3 × 10 −11 iu , which is consistent with previous reports . [SEP]
[CLS] the directly measured detection limit is 0 . 2 cell B-material / μl , equivalent to telomerase activity extracted from 10 hela cells B-material since the total reaction volume is 50 μl . [SEP]
[CLS] the detection limit is lower than that of previous colorimetric methods and is improved by at least 1 order of magnitude in comparison to ts - aunp B-nanoparticle conjugates with a densely packed dna layer ( figures s - 14 and s - 15 ) . [SEP]
[CLS] the sensitivity is comparable to that of the telomeric repeat amplification protocol ( trap ) and biobarcode assay , but the procedure is more simplified , without resorting to polymerase chain reaction ( pcr ) and repeated dna capture / release . [SEP]
[CLS] the impressive sensitivity is primarily attributed to a dna interface arrangement that hit both catalytic sites of telomerase dimer . [SEP]
[CLS] telomerase activity was visually observed by the aid of aunp B-nanoparticle ' s size - dependent color . [SEP]
[CLS] along with the increasing amount of telomerase , the color of a20 - ts - aunp B-nanoparticle gradually turns from blue - gray to purple and then to red ( figure 5c ) . [SEP]
[CLS] this is because more ts are elongated as telomerase activity rises , which better protects aunp B-nanoparticle from salt - induced aggregation . [SEP]
[CLS] the conjugates thus show a color change from aggregated aunps B-nanoparticle to dispersed aunps B-nanoparticle . [SEP]
[CLS] the visual discrimination of active telomerase from deactivated telomerase was further achieved . [SEP]
[CLS] the color of the solution remains red when a20 - ts - aunp B-nanoparticle is complexed with untreated hela extracts but turns to purple when complexed with heat - deactivated or inhibitor ( 3 ′ - azido - 3 ′ - deoxythymidine , azt ) - treated extracts ( figure 5d ) . [SEP]
[CLS] in summary , we have demonstrated a model - guided interface probe arrangement for sensitive target detection . [SEP]
[CLS] integrating the interface , probe , and target , a model that facilitates target recognition by surface - confined probes was rationally designed . [SEP]
[CLS] under the guidance of this model , probes were properly arranged on the surface to effectively bind target . [SEP]
[CLS] the concept is embodied by the assay of human telomerase activity with dna - conjugated aunps B-nanoparticle . [SEP]
[CLS] as a result of the maximized catalytic activity of telomerase on the aunp B-nanoparticle surface , the sensitivity is greatly improved . [SEP]
[CLS] model - guided interface probe arrangement avoids a data - driven posteriori pattern , offering an effective and oriented way to pack probes onto the interface . [SEP]
[CLS] this strategy can be adapted to direct other concrete surface - based bioassays B-technique and may offer a new paradigm to promote surface - based biomolecular detection . [SEP]
[CLS] the supporting information is available free of charge on the acs publications website at doi : 10 . 1021 / acs . analchem . 6b02972 . [SEP]
[CLS] experimental details , dna sequences ( table s - * fax : + 86 531 88564464 . [SEP]
[CLS] e - mail : wjiang @ sdu . edu . cn . [SEP]
[CLS] the authors declare no competing financial interest . [SEP]
[CLS] ■ acknowledgments [SEP]
[CLS] moreover , aggregation kinetic curves demonstrate elegant distinctions toward different telomerase activities . [SEP]
[CLS] transmission B-technique electron I-technique microscopy I-technique ( tem ) images verify the aggregation of aunps B-nanoparticle in the absence of telomerase and the dispersion of aunps B-nanoparticle complexed with telomerase ( figure s - 6 ) . [SEP]
[CLS] gel B-technique electrophoresis I-technique confirmed that the dna has indeed been enlongated on the aunp B-nanoparticle surface ( figure s - 7 ) , to stabilize colloids from saltinduced aggregation . [SEP]
[CLS] 1 . principle of colorimetric detection of telomerase activity by dna−aunp conjugates . [SEP]
[CLS] 2 . an ideal model of dna−aunp conjugates for interaction with telomerase ( a ) and the derived dna arrangement on aunp B-nanoparticle surface ( b ) . [SEP]
[CLS] 3 . time - dependent changes of absorption ratio ( a700 / a520 ) for a20 - ts - aunp B-nanoparticle , a10 - ts - aunp B-nanoparticle , and a30 - ts - aunp B-nanoparticle conjugates complexed with telomerase ( a−c ) and their interaction models ( d−f ) [SEP]
[CLS] 4 . linear fitting of aggregation kinetic curves ( a ) and histogram of square root of inverse aggregation rate constants ( b ) for three types of ts - aunp B-nanoparticle conjugates . [SEP]
[CLS] 5 . time - dependent changes of absorption ratio ( a700 / a520 ) for a20 - ts - aunp B-nanoparticle complexed with telomerase from hela extracts equivalent to cell B-material concentrations from 0 to 500 cells B-material / μl ( a ) , dynamic detection scope and linear range ( b ) , and visual observation of telomerase activity ( c and d ) . [SEP]
[CLS] 1 ) , characterization of synthesized aunps B-nanoparticle ( figure s - 1 ) , detailed dna sequence and length calculation ( figure s - 2 ) , dls characterization ( figure s - 3 ) , quantification of dna surface density for consecutive adenine - ts - aunp B-nanoparticle ( figure s - 4 ) , stability comparison of three consecutive adenine - ts - aunp B-nanoparticle conjugates ( figure s - 5 ) , tem characterization of the sensing system ( figure s - 6 ) , gel B-technique electrophoresis I-technique confirmation of dna elongation on aunp B-nanoparticle surface ( figure s - 7 ) , proof - of - concept validation with 20 nm aunp B-nanoparticle ( figures s - 8 to s - 12 ) , detection of pure telomerase ( figure s - 13 ) , quantification of dna surface density for thiolated ts - aunp B-nanoparticle conjugates ( figure s - 14 ) , sensitivity comparison between a20 - ts aunp B-nanoparticle and sh - ts - aunp B-nanoparticle conjugates ( figure s - 15 ) , and supplementary calculations ( tables s - 2 to s - 5 ) and discussion ( pdf ) [SEP]
[CLS] the detection of nucleic B-material acid I-material is critical for clinic diagnostics . [SEP]
[CLS] single - nanoparticle inductively coupled plasma mass spectrometry ( sp - icpms ) has demonstrated unique advantages for nucleic B-material acid I-material detection . [SEP]
[CLS] here we report the development of a novel sp - icpms dna assay based on a target - induced hybridization chain reaction to achieve controlled spherical nucleic B-material acid I-material assembly . [SEP]
[CLS] the assembly process generated large gold B-nanoparticle nanoparticle I-nanoparticle aggregates , and the number of aggregates could be counted by sp - icpms , which was closely correlated to the concentration of the target dna . [SEP]
[CLS] this simple homogeneous assay could analyze dna within the range of 5 fm to 10 pm with excellent selectivity and applicability for real sample analysis . [SEP]
[CLS] it is envisioned that the developed approach might create a useful sp - icpms platform for biomolecule detection . [SEP]
[CLS] i nductively coupled plasma mass spectrometry ( icpms ) has demonstrated unique advantages for nucleic B-material acid I-material detection and related clinic diagnostics with absolute quantification and high - throughput analysis . [SEP]
[CLS] lanthanide B-material elements and different kinds of nanoparticles B-nanoparticle have been utilized as element tags for nucleic B-material acid I-material detection with the icpms method . [SEP]
[CLS] the simultaneous quantitative detection of multiple dna targets or proteases by the icpms method with different lanthanide B-material elements as labels has demonstrated an excellent ability for multiplexing analysis . [SEP]
[CLS] the gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) have also been widely used as tags for icpms measurement due to their unique advantages of a high signal - to - background ratio , ease of preparation and modification , as well as excellent biocompatibility B-property . [SEP]
[CLS] several nucleic B-material acid I-material amplification methods such as rolling circle amplification ( rca ) , duplex - specific nuclease - mediated amplification , and ligation - mediated amplification have been combined with icpms to achieve nucleic B-material acid I-material detection . [SEP]
[CLS] these strategies significantly improve the detection sensitivity . [SEP]
[CLS] nevertheless , dissolving nanoparticles B-nanoparticle was required , and signals were obtained from a homogeneous solution . [SEP]
[CLS] single - nanoparticle icpms ( sp - icpms ) is an operating mode specifically developed to detect and count single nanoparticles B-nanoparticle . [SEP]
[CLS] the frequency of pulses generated by single nanoparticle B-nanoparticle is a function of the nanoparticle B-nanoparticle concentration , and the peak intensity reflects the particle size distribution . [SEP]
[CLS] hence utilizing nanoparticles B-nanoparticle as a tag and counting their numbers presents a useful platform for icpms - based biosensors . [SEP]
[CLS] zhang et al . developed a onestep homogeneous sp - icpms assay for sensitive dna detection without signal amplification , where the hybridization of dna resulted in large aunp B-nanoparticle aggregates . [SEP]
[CLS] multiplex dna detection has also been realized by the sp - icpms method with different nanoparticles B-nanoparticle as labels . [SEP]
[CLS] sp - icpms provides a simple , rapid , and accurate tool for dna detection ; nevertheless , some challenges still remain . [SEP]
[CLS] the small nanoparticle B-nanoparticle generates a relatively low signal - to - background ratio because of the limited atoms B-material , and in the dna hybridization - based assembly of large aunp B-nanoparticle aggregates method , one large aggregate required hundreds of target dna molecules for assembly , limiting the sensitivity of the assay . [SEP]
[CLS] further improvements in assay sensitivity may be useful for the analysis of low - abundance dna samples . [SEP]
[CLS] herein , in this work , we reported an ultrasensitive homogeneous sp - icpms dna assay utilizing the hybridization chain reaction ( hcr ) to mediate aunp B-nanoparticle assembly . [SEP]
[CLS] the large aunp B-nanoparticle aggregates provide a high signal - to - background ratio for single - particle counting . [SEP]
[CLS] as illustrated in scheme 1 , the dna target was able to trigger the hcr with two hairpin probes h1 and h2 to form long dsdna polymer B-material strands . [SEP]
[CLS] detailed information on all of the dna probes is listed in table s1 . [SEP]
[CLS] the ultrastable spherical nucleic B-material acids I-material ( usnas ) were prepared by assembling a thiolated dna probe on the surface of aunps B-nanoparticle and then hybridizing with a linker ( l - dna ) . [SEP]
[CLS] because the hairpin probe h1 was designed with a tail sequence complementary to the l - dna , hundreds of aunps B-nanoparticle were assembled to form one large aggregate via the hcr product . [SEP]
[CLS] to this end , a much larger pulse signal of au 197 could be obtained in contrast with the dispersed single aunp B-nanoparticle . [SEP]
[CLS] the sp - icpms was then performed to detect and count the number of aggregates , which was correlated to target the dna concentration . [SEP]
[CLS] details of operating parameters for sp - icpms are listed in table s2 . [SEP]
[CLS] the hcr - mediated usna assembly provides more control of the distances between each single aunp B-nanoparticle for aggregation induced by dna target hybridization . [SEP]
[CLS] moreover , in our approach , one dna target molecule was able to induce one large aunp B-nanoparticle aggregate , corresponding to one strong pulse signal of au 197 , enabling ultrasensitive dna detection . [SEP]
[CLS] the spherical nucleic B-material acid I-material ( sna ) generally consisted of a aunp B-nanoparticle core B-material and an oligonucleotide shell B-material , which provided excellent stability for aunps B-nanoparticle in the sample matrix . [SEP]
[CLS] three factors were taken into account for the construction of our usnas . one was the high salt B-material concentration , which is important for both the hcr and the hybridization process to overcome electrostatic repulsion induced by phosphate backbones . [SEP]
[CLS] the dna density and conformation could also affect the hybridization efficiency . [SEP]
[CLS] hence we constructed ultrastable spherical nucleic B-material acids I-material ( usnas ) utilizing double dna layers . [SEP]
[CLS] first , the thiolated dna probe was anchored on the inner surface of aunps B-nanoparticle with high density to provide high stability in high salt B-material solutions . [SEP]
[CLS] then , l - dna was hybridized with thiolated dna probe to form a double - strand structure with low dna density . [SEP]
[CLS] because the complementary region of l - dna was pushed away from the aunp B-nanoparticle surface by the rigid dsdna structure , a high hybridization efficiency with hcr products could be achieved . [SEP]
[CLS] the usnas showed stronger absorbance than dna @ aunp B-nanoparticle at 260 nm , indicating the formation of a dsdna structure . [SEP]
[CLS] the dynamic B-technique light I-technique scattering I-technique ( dls ) and zeta B-property potential I-property have also been utilized to characterize usnas ( figures s2 and s3 ) . [SEP]
[CLS] the uplc - ms / ms experiments were then performed to estimate the dna density ; details of the calculation process are described in figure s4 . [SEP]
[CLS] the thiolated dna number per aunp B-nanoparticle was determined to be 158 ± 5 ; the high loading capacity was comparable to the previously reported value and could provide excellent stability of aunps B-nanoparticle in salt B-material solution . [SEP]
[CLS] the number of l - dna linkers per aunp B-nanoparticle was determined to be 14 ± 2 , which verified a low - density nucleic B-material acid I-material structure at the outer layer of the usnas ( figure s4 ) . [SEP]
[CLS] the prepared usnas demonstrated excellent stability in high salt B-material solutions compared with different assembly approaches such as peg and pvp modification ( figure s5 ) . [SEP]
[CLS] the hcr was a well - developed enzyme - free amplification approach , where two metastable hairpin probes h1 and h2 were triggered by an initiator dna sequence . [SEP]
[CLS] herein the design of h1 and h2 for target dna was based on the unafold web server , and the process was validated by agarose B-technique gel I-technique electrophoresis I-technique . [SEP]
[CLS] the hcr process and assembly of large usna aggregates mediated by hcr products have been verified by the uv absorption spectrum , gel B-technique electrophoresis I-technique , and dls measurements , as shown in figure 1 . [SEP]
[CLS] in the presence of target dna , bright ladder - like bands appeared , and old band disappeared ( lane 5 ) , indicating that long - nicked hcr dna polymers B-material products were formed . [SEP]
[CLS] on the contrary , hairpin probes h1 and h2 maintained stability without target dna ( lane 4 ) . [SEP]
[CLS] these results demonstrated the success of the hcr assembly . [SEP]
[CLS] it is worth noting that the ladder - like bands in lane 5 indicate that the hcr reaction generated many differentlength dsdna products . [SEP]
[CLS] this result is consistent with other work involved with hcr amplification . [SEP]
[CLS] because of the mechanism of hcr , the products are different - length dsdnas , and usnas will have different sizes . [SEP]
[CLS] however , the dsdna products could be long enough , and the usnas would be able to generate a significantly strong signal compared with that of a single aunp B-nanoparticle . [SEP]
[CLS] therefore , counting of usnas by the sp - icpms approach will not be affected by the extent of the hcr and the size of the usnas . [SEP]
[CLS] the significant decrease in the absorbance at 520 nm after incubating B-technique target dna with h1 , h2 , and usnas indicated the assembly of usna aggregates ( figure 1b ) , and the results were confirmed by dls measurements ( figure 1c ) . [SEP]
[CLS] the successful assembly of usna aggregates was confirmed by gel B-technique electrophoresis I-technique , as shown in lane 1 of figure 1d . [SEP]
[CLS] the unique advantages of the hcr - mediated usnas assembly have been demonstrated by tem images and sp - icpms results , as shown in figure 2 . [SEP]
[CLS] after incubating B-technique 10 pm target dna with h1 , h2 , and usnas , the dispersed usnas were clustered together ( figure 2a , b ) . [SEP]
[CLS] this result demonstrated the assembly of usnas with the long dsdna formed by the dna - triggered hcr . [SEP]
[CLS] it is worth noting that the usna aggregate showed uniform distances from each single usna . [SEP]
[CLS] compared with previous aunp B-nanoparticle aggregation methods based on complementary dna hybridization , the proposed approach provided a more controllable assembly process with less dense packing . [SEP]
[CLS] a much stronger pulse signal of au 197 was observed for the single usna aggregate compared with dispersed single usnas ( intensity count > 50 vs < 6 ) . [SEP]
[CLS] hence the threshold of the intensity count was set as 50 to enable high signal - to - background ratio detection and single - particle counting ( figure 2c , d ) . [SEP]
[CLS] then , the experimental parameters were investigated and optimized . [SEP]
[CLS] because numerous extra - dispersed usnas were in the detection solutions , sample dilution was necessary to reduce the dispersed usna signal relative to the aggregate . [SEP]
[CLS] in addition , an appropriate sample dilution was helpful for reducing the occurrence of aggregate analytical coincidence ( i . e . , two or more aggregates entering the plasma at once ) . [SEP]
[CLS] as shown in figure s6 , the optimal dilution factor was set as 10 000× , which reduced the dispersed signal to far below the aggregate and provided maximum counts . [SEP]
[CLS] four different buffer solutions were investigated to see how the ionic strength affected the analytical performance , and 5 mm tris - hcl / 50 mgcl 2 was chosen as the buffer system ( figure s7 ) . [SEP]
[CLS] other parameters such as the concentrations of hairpin probes h1 and h2 and the usna concentration have also been optimized ( figures s8 and s9 ) . [SEP]
[CLS] under the optimized conditions , the ability of the proposed hcr - mediated usna assembly approach for quantitative dna detection has been investigated . [SEP]
[CLS] a series of solutions containing different concentrations of target dna were tested by the assay . [SEP]
[CLS] as shown in figure 3 , the threshold was set at 50 intensity counts , and the frequencies increased with increasing concentrations of target dna , exhibiting a good linear relationship with the natural logarithm of target dna concentrations from 5 fm to 10 pm . [SEP]
[CLS] the limit of detection ( lod ) was calculated to be 3 fm on the basis of three times the standard deviation of blank values . [SEP]
[CLS] these results revealed that the developed strategy offers improved sensitivity compared with the previous hybridization - based sp - icpms method for dna detection . [SEP]
[CLS] the method also demonstrated better or comparable sensitivity as compared with icpms combined with enzyme - mediated nucleic B-material acid I-material amplification assays . [SEP]
[CLS] the low lod of the method demonstrated an excellent ability for low - level nucleic B-material acid I-material detection in real samples . [SEP]
[CLS] the developed sp - icpms assay was very specific for the detection of target dna ( figure s10 ) . [SEP]
[CLS] the detection of noncomplementary and single - base mismatched dna sequences merely generated significant au 197 intensity of the pulse signal . [SEP]
[CLS] this result revealed that the proposed strategy has excellent selectivity for specific dna sequence detection . [SEP]
[CLS] the developed method also demonstrated decent reproducibility from repeated trials of samples with the same concentration ( figure s11 ) . [SEP]
[CLS] to evaluate the performance of the developed sp - icpms assay in complex biological media , the detection of target dna in 10 % human serum samples was then performed . [SEP]
[CLS] serum samples spiked with different concentrations of target dna were measured , and satisfactory recoveries were obtained ( table s3 ) , indicating the potential of the developed strategy for real sample analysis . [SEP]
[CLS] in summary , a novel sp - icpms dna assay has been developed for ultrasensitive dna detection . [SEP]
[CLS] the strategy relies on hcr - meditated usna assembly to generate controllable large aunp B-nanoparticle aggregates and significant au 197 counts as compared with the background of a single dispersed aunp B-nanoparticle . [SEP]
[CLS] the hcr process successfully transferred one target dna to one large usna aggregate , favoring ultrasensitive dna detection by the sp - icpms counting approach . [SEP]
[CLS] moreover , a homogeneous format can be performed without the need to remove excess aunps B-nanoparticle , providing convenience and robustness for the assay . [SEP]
[CLS] the developed method demonstrated its applicability for the analysis of nucleic B-material acid I-material in a complex biological medium . [SEP]
[CLS] by virtue of these advantages , the proposed strategy provides a new paradigm for the design of sp - icpmsbased biosensors and might hold great potential for dna detection in biological samples . [SEP]
[CLS] the supporting information is available free of charge at https : / / pubs . acs . org / doi / 10 . 1021 / acs . analchem . 9b05741 . [SEP]
[CLS] illustration of the sp - icpms dna assay based on the hcr - mediated ultrastable spherical nucleic B-material acid I-material assembly [SEP]
[CLS] 2 . tem images of usnas ( a ) in the absence and ( b ) in the presence of 10 pm target dna of the assay . [SEP]
[CLS] sp - icpms profile ( c ) without or ( d ) with the 10 pm target of the assay . [SEP]
[CLS] the signal of au 197 was recorded per 5 ms and acquired in 150 s . [SEP]
[CLS] ( a−h ) sp - icpms profile spectra of 5 fm , 10 fm , 50 fm , 100 fm , 500 fm , 1 pm , 5 pm , and 10 pm target dna of the assay . [SEP]
[CLS] ( i ) relationship between the frequencies and the concentrations of the target from 5 fm to 10 pm . [SEP]
[CLS] the dwell time was 5 ms , and the duration was 150 s . [SEP]
[CLS] error bars represent the standard deviations of the results from three independent experiments . [SEP]
[CLS] double - stranded dna - grafted nanoparticles B-nanoparticle ( dsdna−nps ) exhibit a unique dispersion behavior under high - salt B-material conditions depending on the pairing status of their outermost base pairs ( pairing or unpairing ) . [SEP]
[CLS] the dsdna−nps having complementary ( i . e . , pairing ) outermost base pairs spontaneously aggregate under high - salt B-material conditions , but not when their outermost base pairs are mismatched ( unpairing ) . [SEP]
[CLS] in this study , we used colloidal probe atomic B-technique force I-technique microscopy I-technique to examine how the outermost base pairs affect the interaction between the dsdna - grafted layers ( dsdna layers ) . [SEP]
[CLS] to precisely assess the subtle structural differences in the dsdna layers , we developed a method for the formation of a homogenous dsdna layer on gold B-material surfaces using hairpinshaped dnas . [SEP]
[CLS] homogenous dsdna layers having complementary ( g−c ) or mismatched ( c−c ) outermost base pairs were grafted onto the surfaces of colloidal probes and gold B-material substrates . [SEP]
[CLS] force−distance curves measured in an aqueous medium under high - salt B-material conditions revealed that the surface forces between the dsdna layers were bilateral in nature and were governed by the outermost base pairs . [SEP]
[CLS] between complementary outermost dsdna layers , the surface force changed from repulsive to attractive with an increase in the nacl concentration ( 10−1000 mm ) . [SEP]
[CLS] the attraction observed under high - salt B-material conditions was attributed to the site - specific interaction proceeded only between complementary dsdna terminals , the so - called blunt - end stacking . [SEP]
[CLS] in fact , between mismatched outermost dsdna layers , the repulsive force was mostly dominant within the same nacl concentration range . [SEP]
[CLS] our results clearly revealed that the pairing status of the outermost base pairs has significant implications for the surface forces between dsdna layers , leading to the unique dispersion behavior of dsdna−nps . [SEP]
[CLS] the properties of dna strands immobilized at a solid−liquid interface have received considerable attention in fundamental and applied research because they play key roles in various dna - related functional devices , including biosensors . [SEP]
[CLS] the formation of dna layers on solid surfaces often results in restricted flexibility of the conformation of dna strands because of crowding molecular packing ( steric hindrance ) and an aligned configuration of dna on the surface . [SEP]
[CLS] in addition , the local ionic environment ( ionic strength and composition ) surrounding the dna layer affects the physicochemical characteristics of the surface - immobilized dna strands , yielding characteristics very different from those of free dna in solution . [SEP]
[CLS] for example , it has been reported that singlestranded dna ( ssdna ) that is densely immobilized on gold B-nanoparticle nanoparticles I-nanoparticle ( gnps ) [ spherical nucleic B-material acids I-material ( snas ) ] shows a higher affinity for complementary dna than the same ssdna that is free in solution . [SEP]
[CLS] moreover , the melting transition of dna duplexes formed on the sna surface or cross - linked between the snas occurs at a higher and narrower temperature range compared with that of free dna duplexes of the same sequence . [SEP]
[CLS] more than 10 years ago , we identified a unique dispersion behavior of dna - immobilized nps B-nanoparticle , that is , the dispersion state of double - stranded dna - grafted nanoparticles B-nanoparticle ( dsdna−nps ) under high - salt B-material conditions is governed exclusively by the pairing status of the outermost base pairs ( base pairing or unpairing ) . [SEP]
[CLS] the dispersion of dsdna−nps having complementary ( i . e . , pairing ) outermost base pairs shows a non - cross - linking aggregation response under high - salt B-material conditions , but the dispersion of dsdna−nps having mismatched ( i . e . , unpairing ) outermost base pairs does not . [SEP]
[CLS] interestingly , this behavior is generally observed irrespective of the material B-material ( gold B-material and polymers B-material ) , size , and shape ( sphere , rod , triangle ) of the particle core B-material , and it is also independent of the base sequence of the inner region of dsdna - grafted layers ( dsdna layers ) . [SEP]
[CLS] these observations reveal that a single - base pairing or unpairing at the outermost surface of the dsdna layer on nps B-nanoparticle dominates the aggregation or dispersion of dsdna−nps , although the underlying mechanism of this phenomenon is still unclear . [SEP]
[CLS] we consider that the unique dispersion behavior of dsdna−nps is caused by an unidentified but outermost surface - specific interaction between the dsdna−nps . [SEP]
[CLS] atomic B-technique force I-technique microscopy I-technique ( afm ) is a powerful tool to examine not only the topology but also the interaction between surface - immobilized molecules such as proteins B-material , peptides B-material , and nucleic B-material acids I-material under various conditions . [SEP]
[CLS] by using afm probes with a spherical end of defined radius ( so - called colloidal probes ) , the surface force at work between two surfaces can be measured quantitatively . [SEP]
[CLS] in this study , we have employed the colloidal probe afm ( cp - afm ) technique to examine the interaction between the dsdna layers . [SEP]
[CLS] one of the major issues in precisely assessing the effect of subtle structural differences on the surface force using this technique is the structural homogeneity of the surface layers . [SEP]
[CLS] the dsdna layer is usually prepared via the hybridization of ssdna strands with surface - immobilized ssdna strands . [SEP]
[CLS] in general , the hybridization efficiency at the solid−liquid interface is lower than that of the solution phase , with some amount of surface - immobilized ssdna molecules remaining unhybridized , that is , the resultant dsdna layer has a heterogeneous structure . [SEP]
[CLS] the process of reducing the residual ssdna by enhancing the hybridization efficiency on the substrate through the optimization of various factors including the surfaceimmobilized ssdna density , hybridization temperature , and buffer composition is quite labor - intensive . [SEP]
[CLS] nevertheless , a perfect dsdna layer cannot be guaranteed even after the optimization . [SEP]
[CLS] in the present study , we developed a simple alternative method for the preparation of " homogenous " ( ssdna - free ) dsdna layers on a gold B-material surface : the hairpin dna ( hpdna ) anchoring method . [SEP]
[CLS] first , we applied this method to the dispersion of gnps and confirmed that the outermost base pairing - specific dispersion behavior was similar to that previously observed for dsdna−nps prepared by the surface hybridization procedure . [SEP]
[CLS] then , we adopted this method to prepare homogenous dsdna layers on the surfaces of colloidal probes and gold B-material substrates and measured the force−distance curves between the dsdna layers using the cp - afm technique . [SEP]
[CLS] from the analyses of force−distance curves , we examined the correlation between the dispersion behavior of the dsdna−nps and the surface forces at work between the dsdna layers under corresponding conditions . [SEP]
[CLS] finally , we discussed the potential mechanism underlying the outermost base pairing - specificity of the surface forces observed for dsdna layers . [SEP]
[CLS] 2 . 1 . evaluation of the colloidal dispersion behavior of hpdna−gnps . [SEP]
[CLS] the hpdna−gnp stock solution was mixed with sodium B-material phosphate buffer ( 10 mm , ph 7 . 4 ) containing various concentrations of nacl . [SEP]
[CLS] after incubation B-technique at 25 °c for 15 min , the mixture was photographed using a digital camera ( camedia c - 5050zoom ; olympus , tokyo , japan ) . [SEP]
[CLS] ultraviolet−visible ( uv−vis ) absorption spectra measurements were performed on a uv - 2550 spectrophotometer equipped with a temperature control unit ( shimadzu , kyoto , japan ) at 25 °c . [SEP]
[CLS] 2 . 2 . force−distance curve measurements between two dsdna layers using colloidal probe afm . [SEP]
[CLS] for the force− distance curve measurements , we used a commercial afm system equipped with a liquid cell B-material ( mfp - 3d ; oxford instruments , santa barbara , ca ) . [SEP]
[CLS] the spring constant of each colloidal probe was determined by monitoring the thermal fluctuations . [SEP]
[CLS] the colloidal probe was made to approach the surface until the detection of a 200 pn repulsion with a speed of 200 nm • s −1 . [SEP]
[CLS] all force curve measurements were performed in sodium B-material phosphate buffer ( 10 mm , ph 7 . 4 ) containing various concentrations of nacl ( 10−1000 mm ) at room temperature . [SEP]
[CLS] to avoid the sample−sample variation , the same set of colloidal probe−gold substrate was used with different nacl concentrations through the measurements for a specific surface combination , for example , c - dna - to - c - dna layers . [SEP]
[CLS] we measured 20 single - force curves , each at five different positions , for each nacl concentration . [SEP]
[CLS] 2 . 3 . force−distance curve data processing . [SEP]
[CLS] each single force−distance curve was smoothed with a moving average of 10 sequential data points to reduce noise . [SEP]
[CLS] we shifted the curves to set the minimum distance data to zero for simplicity . [SEP]
[CLS] a representative single force−distance curve was picked up for each nacl concentration to show the characteristics of the approaching events ( figure 4a , c ) . [SEP]
[CLS] a histogram of the maximum attractive force and the corresponding distance was deduced from the single force−distance curves for c - dna layers ( figure 5a , b ) , whereas forces at a distance of 3 . 0 nm were used for m - dna layers ( figure 5c ) . [SEP]
[CLS] the averaged force−distance curves in figure 4b , d were produced by binning the data points in 0 . 1 nm bins and subsequent averaging of the data in each bin . [SEP]
[CLS] because of the scattered jump - in events in figure 4a , the peak depth and position in figure 4b do not correspond to the average of the maximum attractive force seen in figure 5a . [SEP]
[CLS] 3 . 1 . dsdna−gnps prepared using the hpdna - anchoring method . [SEP]
[CLS] an outline of the hpdna - anchoring method is illustrated in scheme 1 . [SEP]
[CLS] an oligo dna strand with a self - complementary sequence spontaneously folds into a stable hairpin structure consisting of stem and loop moieties . [SEP]
[CLS] we introduced an anchor unit containing a thiol B-material group into the loop moiety to immobilize the hpdna strands on a gold B-material surface in a site - specific manner with au−s bonding . [SEP]
[CLS] the surface - immobilized hpdna strands formed the outer layer consisting solely of stem moieties ( dsdna layer ) . [SEP]
[CLS] our hpdnaanchoring method is more facile and advantageous than the scheme 1 . [SEP]
[CLS] outline of the hpdna - anchoring method a a hairpin structure - folded dna strands were anchored on the gold B-material surface in a site ( loop moiety ) - specific manner through au−s bond formation , resulting in the formation of ssdna - free outer layers consisting solely of stem moieties , that is , homogenous dsdna layers . [SEP]
[CLS] surface - hybridization method because this technique guarantees a homogenous ( ssdna - free ) surface structure of dsdna layers on gold B-material surfaces without complicated optimization of hybridization conditions . [SEP]
[CLS] the base sequences of 25 - mer oligo dna strands ( hpdna - sh ) used in this study are depicted in figure 1 . [SEP]
[CLS] the base sequences of hpdna - sh with a complementary / mismatched terminal base pair ( c - dna - sh , m - dna - sh ) were designed to fold into a hairpin structure using an mfold dna web - based program . [SEP]
[CLS] upon folding , each oligo dna forms the 5 - mer loop of t ( t5 loop ) with a hairpin stem . [SEP]
[CLS] c - dna - sh has a complementary stem terminal ( g−c pair ) , and m - dna - sh has a single - base mismatch at the stem terminal ( c−c pair ) . [SEP]
[CLS] the stability of these hairpin structures was guaranteed by melting temperature ( t m ) measurements ( figure s1 ) . [SEP]
[CLS] for anchoring of hpdna strands on gnps , a thiol B-material group was introduced through an alkyl spacer B-material unit ( x : dt - sh ; figure 1 ) at the fifth position of the thymine base of the t5 loop center . [SEP]
[CLS] through au−s bonding , the hpdna - sh strands were sitespecifically anchored on the gold B-material surface , forming a homogenous dsdna layer as illustrated in scheme 1 . [SEP]
[CLS] details of the synthesis and characterizations of hpdna - sh are described in the supporting information . [SEP]
[CLS] two types of hpdna - functionalized gnps ( hydrodynamic diameter of gnp core B-material = 41 . 0 nm ) , that is , c - dna−gnps and m - dna−gnps , were prepared using the hpdna - anchoring method . [SEP]
[CLS] both dispersions were stable in the presence of 50 mm nacl and showed a clear red color . [SEP]
[CLS] the hydrodynamic diameters of c - dna−gnps and m - dna−gnps estimated using dynamic B-technique light I-technique scattering I-technique measurements were 46 . 4 and 45 . 6 nm , respectively . [SEP]
[CLS] the site - specific immobilization of the hpdna - sh strands on the gnps was confirmed by the incubation B-technique of bare gnps with anchor - free hpdna ( c - hpdna , m - hpdna ) and subsequent addition of 50 mm nacl , which resulted in the immediate aggregation of the gnps ( figure s5 ) . [SEP]
[CLS] the immediate aggregation revealed , in turn , that the hpdna - sh strands were anchored specifically on the gnps with au−s bonding . [SEP]
[CLS] moreover , the anchoring of hpdna - sh strands via au−s bonding guarantees the formation of a layer with an exposed stem terminal ( dsdna layer ) as the outermost structure , as we expected in scheme 1 . [SEP]
[CLS] the number of hpdna - sh strands immobilized on the gnps was evaluated using the dithiothreitol ( dtt ) displacement assay . [SEP]
[CLS] the average number of c - dna - sh strands immobilized on the gnp surface was 309 ± 17 strands per particle ( surface area of c - dna - sh of 16 . 9 ± 0 . 8 nm 2 ) , whereas that of m - dna - sh strands was 313 ± 4 strands per particle ( surface area of m - dna - sh of 16 . 8 ± 0 . 2 nm 2 ) . [SEP]
[CLS] the difference between the two was sufficiently small , indicating that the stem terminal structure of hpdna - sh had almost no effect on the efficiency of immobilization on the gold B-material surface . [SEP]
[CLS] the above - mentioned results clearly demonstrate that the hpdna - anchoring method is a powerful means of preparing a dsdna layer on gold B-material surfaces with an ssdna - free , stem terminal base - pair - independent , and well - controlled dna strand configuration . [SEP]
[CLS] behavior of hpdna - functionalized gnps . [SEP]
[CLS] the colloidal dispersion behavior of the hpdna - functionalized gnps exhibited a strong dependence on their outermost base pairs . [SEP]
[CLS] as shown in figure 2a , the dispersion of c - dna−gnps showed a distinctive color change from red to light purple when the concentration of nacl in the dispersion was above 250 mm , indicating that the c - dna− gnps aggregated in a non - cross - linking manner . [SEP]
[CLS] the uv−vis absorption spectra also qualitatively supported the naclinduced non - cross - linking aggregation of c - dna−gnps ( figure 2b , c ) . [SEP]
[CLS] by contrast , the m - dna−gnps showed an extremely high colloidal dispersion stability with no visible change in color ( figure 3a ) and no significant change in uv− vis absorption spectra over the entire nacl concentration range examined up to 1 m ( figure 3b , c ) . [SEP]
[CLS] it should be emphasized that the difference between c - dna−gnps and m - dna− gnps is only the outermost base pair ( complementary or mismatched ) because the other factors such as remaining unhybridized ssdnas or unfavorably aligned dnas were completely excluded with the hpdna - anchoring method . [SEP]
[CLS] the result , therefore , strongly suggests that the terminal difference of the surface - tethered dsdna evokes a large deviation in the physicochemical properties of the hpdna - functionalized gnp surface , leading to the obvious difference in the colloidal dispersion behavior observed . [SEP]
[CLS] in addition , the clear contrast in the colloidal dispersion behavior observed for hpdnafunctionalized gnps is consistent with our previous observations on dsdna - grafted nps B-nanoparticle prepared using surface hybridization , where unhybridized ssdnas might remain on the surface of the nps B-nanoparticle . [SEP]
[CLS] one possible explanation for the above - described difference in the dispersion behavior of hpdna - functionalized gnps is that these particles may have a different surface charge . [SEP]
[CLS] the a small dip related to the jump - in event was also observed on the curves for nacl concentrations lower than 100 mm , although the surface force was mostly repulsive . [SEP]
[CLS] as will be mentioned later , the jump - in event takes place in a stochastic manner , resulting in a scattered depth ( force strength ) and position ( distance ) of the valley on the curves . [SEP]
[CLS] the dependence of the force−distance curve on the nacl concentration can be discussed more qualitatively based on the averaged curves in figure 4b , where the increase in the attractive interaction with the nacl concentration is also observed and the attractive force becomes obvious at a distance of approximately 8 nm . [SEP]
[CLS] one important point that should be noted from the series of force−distance curves is that the increase in the nacl concentration facilitates the attractive interaction between the c - dna layers , rather than reducing the repulsive interaction . [SEP]
[CLS] when approaching the c - dna layers at a nacl concentration above 250 mm , the attractive interaction begins at a distance of 8 nm ( figure 4b ) and leads to a jump - in event at a distance of 1−5 nm ( figure 4a ) . [SEP]
[CLS] this means that the attractive force observed here cannot be assigned to van der waals force , which usually shows no significant dependence on the salt B-material concentration . [SEP]
[CLS] on the basis of our observation that the attractive force was strongly dependent on the nacl concentration , we consider that the hydrophobic B-property interaction I-property between the c - dna layers facilitated by nacl is the most probable origin of the attraction . [SEP]
[CLS] a further discussion of the hydrophobic B-property interaction I-property , that is , the molecular mechanism of the attraction between the c - dna layers facilitated by nacl , will be made in a later section . [SEP]
[CLS] 5 shows the histograms for ( a ) the maximum attraction force ( the lowest force value on the curve ) observed above 100 mm nacl and ( b ) the corresponding distance where the maximum attraction was achieved . [SEP]
[CLS] upon increasing the nacl concentration from 100 mm to 1 m , the distribution of the maximum attraction force became broad . [SEP]
[CLS] the distribution of the corresponding distance was similarly scattered within this nacl concentration range . [SEP]
[CLS] the broad distribution of the attraction strength and distance indicates that the attractive interaction between the c - dna layers varies from event to event , that is , the jump - in event takes place in a stochastic manner . [SEP]
[CLS] 3 . 3 . 2 . force−distance dependence between m - dna layers . [SEP]
[CLS] the force−distance curves observed for the approaching m - dna layers are shown in figure 4c , d for single and averaged curves ( more than 100 curves ) . [SEP]
[CLS] 5c shows the histogram of the force observed at a distance of 3 nm , revealing a higher reproducibility of the force−distance curves in figure 4c , compared with that for c - dna layers ( figure 4a ) . [SEP]
[CLS] unlike in the case of the c - dna layers , the force existing between the m - dna layers was simply repulsive with an exponential - type decay with distance , except at a nacl concentration of 1 m . [SEP]
[CLS] notably , the repulsion force starts as it approaches to within a distance of approximately 8 nm , and the force−distance curves showed no significant dependence on the nacl concentration within the range of 50−500 mm ( figure 4d ) . [SEP]
[CLS] the observed distance of 8 nm is far beyond the distance range of the electrostatic force , which has been evaluated as 1 . 2 nm for a nacl concentration of 10 mm in sodium B-material phosphate buffer ( 10 mm , ph 7 . 4 ) , deduced as the debye length . [SEP]
[CLS] the small deviation observed for 10 mm nacl was reproducible , but the reason for the deviation is not clear at the moment . [SEP]
[CLS] only at 1 m of nacl , we observed a weak attractive interaction on the single force−distance curves , but we rarely observed an attractive force higher than 50 pn ( figure s7 ) . [SEP]
[CLS] on the averaged curve , we observed an attraction of approximately 20 pn at 1 m nacl ( figure 4d ) , which was far below that for the c - dna layers , revealing that the attractive interaction present between the m - dna layers was not significant even at higher nacl concentrations . [SEP]
[CLS] nevertheless , the existence of a weak attractive interaction between the m - dna layers was revealed by the force−distance curve measurements , and such an interaction cannot be deduced from the dispersion behavior of m - dna−gnps . [SEP]
[CLS] the onset of the repulsive force observed on the averaged curves in figure 4d was approximately 8 nm for 50−500 mm nacl , whereas it was approximately 1 nm for 1 m nacl . [SEP]
[CLS] at the same time , the repulsive force increased slowly for 50−500 mm nacl , compared with the steep increase observed for 1 m nacl . [SEP]
[CLS] it seems reasonable to attribute this steep increase to the soft and direct contact of the m - dna layers . [SEP]
[CLS] the differences in the force−distance curves suggested that the properties of the m - dna layers and the surrounding solvent environments were drastically changed at a nacl concentration of 1 m . [SEP]
[CLS] 3 . 4 . mechanism of the outermost base pair - specific surface forces between dsdna layers . [SEP]
[CLS] the average values of the maximum attraction force for the c - dna layers and the force observed at a distance of 3 nm for the m - dna - layers were plotted as a function of the nacl concentration , together with the absorption ratio ( a 520 / a 600 ) of the corresponding hpdna - functionalized gnp dispersions ( figure 6 ) . [SEP]
[CLS] the nacl concentration dependence of each force agrees well with that of the dispersion / aggregation behavior of dsdna - functionalized gnps , showing the significant correlation between the surface force and the dispersion behavior . [SEP]
[CLS] aggregation of hpdnafunctionalized gnps occurred ( a 520 / a 600 value was decreased ) when the attractive force between the corresponding dsdna layers reached above 89 pn ( 9 . 9 n • m −1 ) . [SEP]
[CLS] the weak attractive force observed for the c - dna layers at 100 mm nacl ( ca . 45 pn , 5 . 0 n • m −1 ) and the m - dna layers at 1 m nacl ( ca . 15 pn , 1 . 7 n • m −1 ) had no significant effect on the dispersion state of hpdna - functionalized gnps , suggesting that the strength of the attraction force was too weak to maintain contact between gnps against their brownian motion . [SEP]
[CLS] notably , a large attractive force was observed only for c - dna layers with higher nacl concentrations . [SEP]
[CLS] this was attributed to a terminal - specific attraction between complementary dsdna terminals facilitated by nacl . [SEP]
[CLS] in general , an increase in the salt B-material concentration reduces the water B-material activity , leading to a dehydration of polyelectrolytes including dna . [SEP]
[CLS] we observed a characteristic change in the circular dichroism ( cd ) spectra of hpdna solutions with an increase in the nacl concentration from 10 mm to 1 m ( figure s8 ) , which could be assigned to the gradual conformational change of the stem moiety ( b - form to c - form ) by reducing the degree of hydration . [SEP]
[CLS] correlating the strong dependence of the attractive interaction between the c - dna layers on the nacl concentration ( figure 6 ) with the reduction in the degree of hydration suggested by the cd spectra , we conclude that a hydrophobic B-property interaction I-property is the most probable origin of the attraction . [SEP]
[CLS] we consider that the terminal - specific hydrophobic B-property interaction I-property is the end - to - end attraction of dsdna strands with complementary terminals , the so - called blunt - end stack - ing . [SEP]
[CLS] this consideration was experimentally supported by the force−distance curve measurements taken for the heterogenic surface combination of an m - dna layer and a c - dna layer . [SEP]
[CLS] in this measurement , no significant attraction appears even at 1 m nacl ( figure s9 ) , revealing that the attraction functions selectively between dsdna strands with complementary terminals , that is , the nacl - facilitated attractive interaction takes place only between the c - dna layers . [SEP]
[CLS] the appearance of the blunt - end stacking between the dna strands has typically been reported at locally concentrated dnas such as the dna tile , dna bundle , dna liquid crystal , and dna origami . [SEP]
[CLS] pollack and co - workers reported that divalent salts B-material such as mgcl 2 ( above 20 mm ) facilitate the blunt - end stacking between complementary dsdna strands even in a semidilute solution ( [UNK] mm ) . [SEP]
[CLS] they also revealed that monovalent salts B-material including nacl have no ability to promote the blunt - end stacking even at high concentrations ( [UNK] mm ) . [SEP]
[CLS] by contrast , our present results revealed that the concentration of nacl above 250 mm promotes blunt - end attraction between dsdna layers with complementary terminals ( figure 4a , b ) . [SEP]
[CLS] this might be due to the surface - anchoring of the dsdna strands . [SEP]
[CLS] the diffusional motion of the dsdna strands is strongly restricted by anchoring to the gold B-material surface , and the resultant configuration promotes terminal−terminal interaction between the dsdna layers on approaching . [SEP]
[CLS] in addition , the reduction in the number of water B-material molecules surrounding dsdna with an increase in nacl concentration enables dsdna terminals to approach more closely . [SEP]
[CLS] the terminal closure promotes attractive interactions between complementary dsdna terminals through the blunt - end stacking . [SEP]
[CLS] on the other hand , dsdna strands with mismatched terminals cannot attract each other even by approaching closely because of their lack of hydrophobic B-property terminal base pairs . [SEP]
[CLS] in addition , the number of potential binding sites of water B-material molecules to the unpairing terminal nucleobases is larger than that of pairing ones ( figure s10 ) . [SEP]
[CLS] this leads to the persistence of tightly bound water B-material molecules surrounding the mismatched dsdna terminals even at a higher nacl concentration , resulting in the persistent repulsion force between the m - dna layers observed in figure 6 up to a salt B-material concentration of 500 mm . the working distance of the repulsion observed in figure 4c , d was larger than twice the thickness of the dsdna layer ( estimated to be approximately 7 nm ) , supporting the notion that environmental factors such as water B-material configuration are important for the repulsion interaction of the mismatched dsdna terminals . [SEP]
[CLS] when the number of water B-material molecules binding to the terminal moiety is reduced at 1 m nacl , the m - dna layers can approach more closely together , but the attractive interaction is too weak to form a stable adhesion ( colloidal aggregation ) , as indicated by the significantly small attraction observed in figure 6 . [SEP]
[CLS] we explored the mechanism underlying the outermost base pairing dependence of the dispersion behavior of dsdna−nps . [SEP]
[CLS] site - specific immobilization of the hpdna strands enabled us to prepare homogenous dsdna layers on gold B-material surfaces with complementary or mismatched outermost base pairs . [SEP]
[CLS] the force−distance curve analyses between the dsdna layers using the cp - afm technique showed that the outermost complementary ( mismatched ) dsdna layers attract ( repulse ) each other at a higher nacl concentration . [SEP]
[CLS] these results agree well with the outermost base pairing dependence of the colloidal dispersion behavior of dsdna−nps , indicating that the outermost base - pairing - specific interaction between the dsdna−nps dominates their dispersion behavior at a higher nacl concentration . [SEP]
[CLS] we concluded that the nacl - facilitated end - to - end hydrophobic B-property interaction I-property ( blunt - end stacking ) between complementary dsdna terminals caused the attraction between the c - dna layers , whereas the repulsive interaction observed between the m - dna layers was mainly attributable to a repulsive hydration force . [SEP]
[CLS] although the number of dsdna pairs involved in the surface force is not clear at this moment , this is the first direct observation of the terminal - specific interaction between dsdna layers in terms of force , to the best of our knowledge . [SEP]
[CLS] we believe that our findings will contribute not only to designing the dsdna interface - based molecular systems including single - nucleotide polymorphism ( snp ) genotyping devices but also to understanding the molecular basis of dna−dna interactions in biology . [SEP]
[CLS] in fact , the concentration of monovalent cations B-material ( sodium B-material and potassium B-material ions B-material ) in the nucleoplasm is reported to be approximately 500 mm . [SEP]
[CLS] further studies along these lines are currently under way . [SEP]
[CLS] the supporting information is available free of charge on the acs publications website doi : 10 . 1021 / acs . langmuir . 6b03470 . [SEP]
[CLS] experimental details , melting curves , and melting temperature of hpdnas , synthesis of thiol B-material groupincorporated hairpin dnas ( hpdna - shs ) , colloidal dispersion stability of hpdna / gnp mixtures , zeta B-property potentials I-property of c - dna - gnps and m - dna - gnps at various nacl concentrations , histogram of minimum force value observed on approaching m - dna layers , cd spectra of hpdna , force−distance dependence observed for the approaching m - dna layer and c - dna layer , and the potential binding site of water B-material molecules to the pairing / unpairing terminal base pairs ( pdf ) [SEP]
[CLS] ( a ) base sequences and folded structures of hpdna - sh . [SEP]
[CLS] self - complementary regions in hpdna - sh sequences are shown as underlined text . [SEP]
[CLS] upon folding , each hpdna - sh forms a 5 mer loop of t with a hairpin stem . [SEP]
[CLS] ( b ) chemical structure of the anchor unit ( x : dt - sh ) . [SEP]
[CLS] a thiol B-material group acts as an anchor for the gold B-material surface . [SEP]
[CLS] photographs ( a ) , uv−vis spectra ( b ) , and the ratio of absorption observed at 520 and 600 nm ( a 520 / a 600 ) ( c ) for the c - dna−gnp dispersions in sodium B-material phosphate buffer ( 10 mm , ph 7 . 4 ) containing various concentrations of nacl at 25 °c . [SEP]
[CLS] above 250 mm nacl , the c - dna−gnp dispersion showed a distinctive change in color and uv−vis spectrum , revealing the aggregation . [SEP]
[CLS] photographs ( a ) , uv−vis spectra ( b ) , and the ratio of absorption observed at 520 and 600 nm ( a 520 / a 600 ) ( c ) for the m - dna−gnp dispersions in sodium B-material phosphate buffer ( 10 mm , ph 7 . 4 ) containing various concentrations of nacl at 25 °c . [SEP]
[CLS] unlike c - dna− gnp dispersion , the m - dna−gnp dispersion showed no significant change in either the color or the uv−vis spectrum even in high nacl concentrations . [SEP]
[CLS] representative single force−distance curves ( a ) and averaged curves ( b ) obtained for the approaching c - dna layers in sodium B-material phosphate buffer ( 10 mm , ph 7 . 4 ) containing various concentrations of nacl . [SEP]
[CLS] straight slopes in the curves in ( a ) correspond to jump - in events , that is , the probe was pulled to the surface with a strong attractive force . [SEP]
[CLS] the deviation in the peak shape between single and averaged curves was due to the scattered distribution of maximum attraction distances , as plotted in figure 5 ( b ) . [SEP]
[CLS] representative single force−distance curves ( c ) and averaged curves ( d ) obtained for approaching m - dna layers . [SEP]
[CLS] histograms for ( a ) the minimum force value ( maximum attraction force ) for c - dna layers within a distance of 20 nm and ( b ) the distance of maximum attraction point for c - dna layers . [SEP]
[CLS] the average maximum attraction forces were 18 , 21 , 45 , 90 , 181 , and 224 pn for nacl concentrations of 10 , 50 , 100 , 250 , 500 , and 1000 mm , respectively , and forces less than 50 pn could be attributed to a fluctuation error , as evidenced by the distance distribution beyond 10 nm in ( b ) . [SEP]
[CLS] ( c ) force observed at a distance of 3 nm for m - dna layers . [SEP]
[CLS] the standard deviation for the distribution was less than 25 pn for all nacl concentrations , which could be attributed to measurement fluctuations . [SEP]
[CLS] correlation between the dispersion behavior of hpdna− gnps and the surface force of dsdna layers at various nacl concentrations . [SEP]
[CLS] the dispersion behavior of hpdna−gnps as expressed by the absorption ratio ( a 520 / a 600 ) was plotted as a function of nacl concentration . [SEP]
[CLS] the maximum attraction force observed within a distance of 20 nm was plotted for c - dna layers , whereas the force observed at a distance of 3 nm was plotted for m - dna layers . [SEP]
[CLS] enzymes are important biomarkers B-property for molecular diagnostics and targets for the action of drugs . [SEP]
[CLS] in turn , inorganic B-nanoparticle nanoparticles I-nanoparticle ( nps B-nanoparticle ) are of interest as materials for biological assays , biosensors , cellular and in vivo imaging probes , and vectors for drug delivery and theranostics . [SEP]
[CLS] so how does an enzyme interact with a np B-nanoparticle , and what are the outcomes of multivalent conjugation of its substrate to a np B-nanoparticle ? [SEP]
[CLS] this invited feature article addresses the current state of the art in answering this question . [SEP]
[CLS] using gold B-nanoparticle nanoparticles I-nanoparticle ( au nps B-nanoparticle ) and semiconductor quantum B-nanoparticle dots I-nanoparticle ( qds ) as illustrative materials , we discuss aspects of enzyme structure−function and the properties of np B-nanoparticle interfaces and surface chemistry that determine enzyme−np interactions . [SEP]
[CLS] these aspects render the substrate - on - np B-nanoparticle configurations far more complex and heterogeneous than the conventional turnover of discrete substrate molecules in bulk solution . [SEP]
[CLS] special attention is also given to the limitations of a standard kinetic analysis of the enzymatic turnover of these configurations , the need for a well - defined model of turnover , and whether a " hopping " model can account for behaviors such as the apparent acceleration of enzyme B-property activity I-property . [SEP]
[CLS] a detailed and predictive understanding of how enzymes turn over multivalent np B-nanoparticle - substrate conjugates will require a convergence of many concepts and tools from biochemistry , materials , and interface science . [SEP]
[CLS] in turn , this understanding will help to enable rational , optimized , and value - added designs of np B-nanoparticle bioconjugates for biomedical and clinical applications . [SEP]
[CLS] nanoparticles B-nanoparticle ( nps B-nanoparticle ) are a diverse group of colloidal materials with dimensions between ca . 1 and 100 nm and span a wide range of properties and applications . [SEP]
[CLS] the properties of inorganic nps B-nanoparticle are often different or enhanced versus those of the analogous bulk materials . [SEP]
[CLS] for example , gold B-material and other metallic nps B-nanoparticle exhibit strong plasmonic resonances , semiconductor quantum B-nanoparticle dots I-nanoparticle are brightly photoluminescent B-property , and some metal B-material oxides I-material and metal alloys evolve unique magnetic B-property properties as nps B-nanoparticle . [SEP]
[CLS] more general advantages of these materials include the combination of molecule - like diffusion with a surface area that can be functionalized , as well as large surface area - tovolume ratios . [SEP]
[CLS] a focus of research for many inorganic nps B-nanoparticle has been the utilization of their properties to address challenges in biology and medicine . [SEP]
[CLS] examples include but are not limited to cellular and tissue imaging , biosensing , drug delivery , and theranostics . [SEP]
[CLS] there is still much that needs to be learned before these ambitions can be fully realized , not the least of which is how interfacial chemistry controls the interactions between inorganic nps B-nanoparticle and biological molecules . [SEP]
[CLS] interfacial chemistry is the principal determinant of the interactions between inorganic nps B-nanoparticle and biomolecules , and thus the optimization of surface chemistry , such as ligand and polymer B-material coatings B-material , has been a significant endeavor toward biomedical applications . [SEP]
[CLS] ( here , the term ligand refers to a molecule that binds to the inorganic surface of a np B-nanoparticle . [SEP]
[CLS] ) the selection of surface chemistry is not trivial as it ideally addresses several competing requirements : colloidal stability , hydrodynamic size , nonspecific interactions , and methods for bioconjugation . [SEP]
[CLS] substantial effort has therefore gone into the development of coatings B-material that tick these boxes and others depending on the details of the application . [SEP]
[CLS] there is a sometimes - underappreciated correlation between the scope of applications for a np B-nanoparticle material B-material and the ease , reliability , and overall development of its surface chemistry . [SEP]
[CLS] a recurring theme for the surface chemistry of many np B-nanoparticle materials is control over noncovalent interactions with biomolecules . [SEP]
[CLS] the most common context is nonspecific adsorption or biofouling , where proteins B-material and other biomacromolecules tend to be the chief offenders but molecules of all sizes and types have the potential to adsorb . [SEP]
[CLS] the functionalization of nps B-nanoparticle with poly ( ethylene glycol ) ( peg ) and zwitterionic surface chemistries are common strategies for the prevention of nonspecific adsorption , albeit that this goal is not easy to achieve . [SEP]
[CLS] a case in point is that the protein B-material corona I-material that tends to form around nps B-nanoparticle in biological fluids ( e . g . , blood ) and in physiological environments remains a significant challenge in the translation of nps B-nanoparticle from laboratory development to clinical application . [SEP]
[CLS] in other contexts , the goal may be an engineered interaction between a np B-nanoparticle and a particular biomolecule of interest , for example , the cationic B-material functionalization of a np B-nanoparticle as a vector for gene delivery . [SEP]
[CLS] whether in the context of this protein B-material corona I-material , engineered interactions , or adsorption in general , the noncovalent interactions of biomolecules with colloidal nps B-nanoparticle range from low affinity , dynamic , and reversible to high affinity , static , and irreversible on the time scale of experiments , with a corresponding range of effects on biological activity and identity within a system . [SEP]
[CLS] enzymes are especially interesting from the perspective of noncovalent interactions with colloidal nps B-nanoparticle . these catalytic proteins B-material regulate the rates of myriad biochemical processes and are some of the most important molecular machines of life . [SEP]
[CLS] both the concentration and the state of an enzyme are important in determining its biological activity : the former is largely controlled via expression levels , and the latter is controlled through various mechanisms , including the proteolytic activation of proenzymes , the covalent modification of enzymes ( e . g . , phosphorylation ) , changes in the oligomerization state , and binding with small - molecule effectors and endogenous inhibitors . [SEP]
[CLS] enzymes are also important biomarkers B-property for disease diagnostics ( e . g . , molecular assays and biosensors ) and as targets for the action of drugs . [SEP]
[CLS] these two areas of interest are shared with nps B-nanoparticle , so it follows that nps B-nanoparticle are materials of interest for the detection of enzymes and their activity , and as potential vectors for the delivery of drugs that inhibit enzymes or have their release actuated by enzymes . [SEP]
[CLS] whereas the study of the adsorption of abundant serum proteins B-material on a np B-nanoparticle is mainly concerned with the downstream fate and function of the np B-nanoparticle , the case of adsorption or other interactions between an enzyme and a np B-nanoparticle necessitates concern about the fate and function of both the np B-nanoparticle and enzyme . [SEP]
[CLS] what are the effects of a colloidal inorganic np B-nanoparticle and its interfacial chemistry on the activity B-property of I-property an I-property enzyme I-property ? [SEP]
[CLS] is it possible to control and design these effects to be advantageous in applications ? [SEP]
[CLS] here , we review the interactions between enzymes and colloidal inorganic nps B-nanoparticle conjugated with their substrates . [SEP]
[CLS] that is , substrate - on - np B-nanoparticle configurations with enzyme in bulk solution . [SEP]
[CLS] these configurations are very useful for leveraging the favorable properties of nps B-nanoparticle for the bioanalysis of enzymes . [SEP]
[CLS] the converse configuration , which is enzyme - on - np B-nanoparticle with a substrate in bulk solution , has been the topic of a greater number of fundamental studies aimed at elucidating the interactions between enzymes and nps B-nanoparticle , including effects on catalytic activity and stability . [SEP]
[CLS] many of these studies have been highlighted in recent reviews . [SEP]
[CLS] the emphasis on how the enzyme - on - np B-nanoparticle configuration impacts catalysis is natural given that bioprocessing and manufacturing are prospective applications of these materials . [SEP]
[CLS] although studies with substrate - on - np B-nanoparticle configurations are also numerous , most of these studies have been in the aforementioned analytical or biomedical diagnostic context : assays , sensors , and imaging probes for enzymes of biological or clinical importance . [SEP]
[CLS] there is also interest in using np B-nanoparticle - substrate conjugates as vectors for enzyme - mediated drug delivery . [SEP]
[CLS] the emphasis of study has therefore been function ; however , the optimization of the foregoing applications and the realization of new opportunities for substrate - on - np B-nanoparticle configurations will require a detailed understanding of their interfacial chemistry , their interactions with enzymes , and the corresponding effects on substrate turnover . [SEP]
[CLS] the following sections of this article address progress and challenges toward a detailed understanding of enzymatic catalysis associated with substrate - on - np B-nanoparticle configurations . [SEP]
[CLS] the scope is limited to " hard " colloidal inorganic nps B-nanoparticle , with gold B-nanoparticle nanoparticles I-nanoparticle ( au nps B-nanoparticle ) and semiconductor quantum B-nanoparticle dots I-nanoparticle ( qds ) featured prominently . [SEP]
[CLS] " soft " organic nps B-nanoparticle , such as those based on lipids B-material and polymers B-material , are also of interest for applications of substrate - on - np B-nanoparticle configurations ; however , we exclude these materials from discussion because of their markedly different chemistries and morphologies versus those of inorganic nps B-nanoparticle . [SEP]
[CLS] the prominence of au nps B-nanoparticle and qds reflects their frequent use in fundamental studies of enzyme B-property activity I-property toward substrate - on - np B-nanoparticle configurations , which follows from the reaction tracking modalities available to these materials , their well - established chemistry , and their overall breadth of prospective applications in biology and medicine . [SEP]
[CLS] we begin by first reviewing important aspects of enzyme structure−function and important features of the colloidal inorganic np B-nanoparticle interface . [SEP]
[CLS] a rudimentary understanding of both np B-nanoparticle and enzyme chemistry is assumed , and both sections are selective in their content rather than exhaustive . [SEP]
[CLS] we then summarize methods for tracking enzyme−substrate reaction progress and address the use of the michaelis−menten formalism for the analysis of kinetic parameters , as well as alternative conceptual models and caveats for the interpretation of experimental data from substrate - on - np B-nanoparticle configurations . [SEP]
[CLS] these sections are followed by a review of the recent literature , organized by the concepts of adsorption , steric effects , interfacial environment , and the acceleration of substrate turnover . [SEP]
[CLS] a putative hopping model of enzyme B-property activity I-property with np B-nanoparticle - substrate conjugates is then critically discussed . [SEP]
[CLS] to close , we offer a perspective on how this research will move forward and propose future opportunities for impact on applications in biology and medicine . [SEP]
[CLS] as figure 1 illustrates , the successful development of predictive models for the activity B-property of I-property enzymes I-property toward npsubstrate conjugates will be an important advance toward these applications and a remarkable convergence of research at the intersection of biochemistry , materials science , and fundamental interface science . [SEP]
[CLS] an enzyme potentially interacts with both components of a substrate - on - np B-nanoparticle configuration . [SEP]
[CLS] it is therefore useful to discuss the structural features of an enzyme that may contribute to substrate−enzyme and np−enzyme B-nanoparticle interactions , which may occur in parallel . [SEP]
[CLS] the active site of an enzyme is its most famous structural feature . [SEP]
[CLS] introductory texts generally conceptualize the remarkably selective and efficient catalysis of enzymes through descriptions of the active site , first in terms of fischer ' s " lockand - key " model and then as the current " hand - in - glove " or induced - fit model . [SEP]
[CLS] however , the active site is not necessarily the most important structural feature of an enzyme in the context of interactions with nps B-nanoparticle . [SEP]
[CLS] the active site comprises a relatively small percentage of the total number of amino B-material acid I-material residues of an enzyme , and the number of residues that directly engage in the catalytic mechanism is typically even smaller . [SEP]
[CLS] serine B-material proteases , for example , are well known for their catalytic triads of aspartate , histidine B-material , and serine B-material residues in the active site . [SEP]
[CLS] chymotrypsin is a canonical example of a serine B-material protease and has [UNK] total amino B-material acid I-material residues , but only [UNK] of these residues form the main ( s1 ) substrate binding pocket , inclusive of the triad , and an additional 8 residues form 2 loops near the binding pocket that also play a role in determining substrate specificity . [SEP]
[CLS] in addition to representing a small fraction of the total amino B-material acid I-material residues of an enzyme , the location of the active site varies between enzymes , with some active sites at the enzyme surface and others within pockets and clefts , accessible via one or more channels of up to 2 nm or more in length . [SEP]
[CLS] given all of the above , enzyme structure other than the active site is generally expected to predominate interactions with nps B-nanoparticle and other interfaces . [SEP]
[CLS] indeed , the nonactive site structure of some enzymes evolved for the very purpose of interactions with surfaces . [SEP]
[CLS] structure away from the active site is not necessarily structure that is independent of the active site because some enzymes have allosteric sites or exosites . [SEP]
[CLS] both terms refer to binding interactions at sites topologically distinct from the active site but with effects that propagate to the active site and affect the substrate turnover and specificity . [SEP]
[CLS] these binding interactions may , for example , refer to the docking of small - molecule ligands or macromolecular ligands , dimerization of the enzyme , or association with a membrane . [SEP]
[CLS] to illustrate structure away from the active site and highlight that enzymes have their own surface chemistry , figure 2 shows simple ribbon and surface representations of four serine B-material proteases : chymotrypsin , trypsin , thrombin , and plasmin . [SEP]
[CLS] select structural features and properties are highlighted . [SEP]
[CLS] chymotrypsin and trypsin have orthogonal substrate specificities but have largely analogous structures except for small , but important , differences between the main binding pocket and loops . [SEP]
[CLS] trypsin , thrombin , and plasmin share some substrate specificity in vitro but have very different structures away from the active site . [SEP]
[CLS] for trypsin , few structural features away from its active site are noteworthy ; however , thrombin has two exosites and is perhaps the bestcharacterized example of such an enzyme . [SEP]
[CLS] plasmin has multiple kringle domains ( large disulfide - stabilized multiloop structures ) . [SEP]
[CLS] the exosites of thrombin and the kringle domains of plasmin ( ogen ) both bind with various regulators of their activity ( or activation ) . [SEP]
[CLS] given the above , it is reasonably hypothesized that the similar structures of trypsin and chymotrypsin will lead to similar interactions with nps B-nanoparticle despite their different substrate specificities . [SEP]
[CLS] a second hypothesis is that the distinct structural features of thrombin and plasmin will lead to quite different interactions with a np B-nanoparticle versus trypsin , despite the ability of all three of these serine B-material proteases to hydrolyze a common substrate by a common mechanism . [SEP]
[CLS] as with other proteins B-material , van der waals , electrostatic , hydrogen B-material bonding , and hydrophobic B-property interactions I-property , or some combination thereof , are the likely driving forces of an interaction between an enzyme and a np B-nanoparticle . [SEP]
[CLS] both the surface chemistry of the np B-nanoparticle and its size play a role , the latter determining the surface area and curvature ( if the np B-nanoparticle is approximately spherical ) . [SEP]
[CLS] for a given molar concentration of nps B-nanoparticle , more surface area offers more adsorption sites . [SEP]
[CLS] curvature is important because the interactions that lead to enzyme adsorption strongly depend on the distance and contact area of interaction , which must increase and decrease , respectively , as the surface of the np B-nanoparticle falls away from a globular enzyme more quickly with a smaller diameter . [SEP]
[CLS] for an enzyme , properties such as its molecular weight , isoelectric point ( pi ) , and solubility B-property may be useful predictors of interactions and their magnitude but may also be oversimplifications because these properties treat the enzyme structure as homogeneous . [SEP]
[CLS] in general , higher molecular weight correlates with more contact area for van der waals interactions , pi indicates net charge toward electrostatic interactions , and lower solubility B-property may portend interactions that decrease the solvated surface area of an enzyme . [SEP]
[CLS] what is obscured by these three parameters is the potential for orientation and localization of these interactions to distinct structural features of an enzyme , for example , subunits , domains , and the aforementioned allosteric sites and exosites . [SEP]
[CLS] as a thought experiment , imagine two enzymes with similar pi values : the first enzyme has cationic B-material and anionic residues distributed uniformly over its surface , and the second enzyme has anionic residues uniformly distributed over its surface but has its cationic B-material residues concentrated on only one face . [SEP]
[CLS] the second enzyme will have much stronger electrostatic interactions with an anionic np B-nanoparticle , which is not predicted by the similar pi values . [SEP]
[CLS] trypsin and thrombin are two enzymes thought to exhibit this type of difference in their interactions with anionic nps B-nanoparticle because of the two cationic B-material exosites of thrombin . [SEP]
[CLS] analogously , enzymes with a scattered versus localized distribution of hydrophobic B-property residues are anticipated to have different interactions with a np B-nanoparticle interface . [SEP]
[CLS] another aspect related to enzyme structure that warrants mentioning is steric hindrance . [SEP]
[CLS] the context here is not the potential role of steric effects in the active site determining enzyme−substrate specificity but rather the physical size of enzymes and nps B-nanoparticle relative to one another and relative to the substrate . [SEP]
[CLS] the enzymes discussed in this article range in size from 23 to 128 kda or , if modeled as ellipsoids , have approximate dimensions of 2 nm × 2 nm × 2 . 5 nm up to 2 . 5 nm × 2 . 5 nm × 4 nm . [SEP]
[CLS] the nps B-nanoparticle are approximately spherical with diameters that range from [UNK] to [UNK] nm . [SEP]
[CLS] it follows from these relative sizes that there is the potential for steric hindrance between the enzyme and np B-nanoparticle that limits access to the substrate conjugated to a np B-nanoparticle , particularly if the length of the substrate is much shorter than the radii of the np B-nanoparticle and enzyme , or if the active site of the enzyme is located within a deep cleft . [SEP]
[CLS] likewise , there is the potential for steric hindrance that impacts the ability of van der waals , electrostatic , and hydrophobic B-property interactions I-property to occur between specific regions of an enzyme and a np B-nanoparticle . [SEP]
[CLS] the takeaway message is that enzymes have size , surface chemistry , and caveats with respect to assumptions of homogeneity . [SEP]
[CLS] enzymes , like other proteins B-material , also exhibit features of colloidal behavior . [SEP]
[CLS] as discussed next , nps B-nanoparticle are conceptually analogous in these respects . [SEP]
[CLS] indeed , the analogy of functionalized nps B-nanoparticle as protein B-material mimics has been proposed 56 because of the size and behavioral similarities . [SEP]
[CLS] ribbon and surface diagrams for the comparison of selected structural features between four members of the serine B-material protease family : chymotrypsin , trypsin , thrombin , and plasmin . [SEP]
[CLS] the ser - his - asp catalytic triads are colored red , the s1 substrate binding pockets are colored green , and select loops are colored yellow and orange . [SEP]
[CLS] the loops for chymotrypsin and trypsin are the l1 and l2 loops , which play a role in determining substrate specificity . [SEP]
[CLS] for thrombin , the loops are the 60 - loop and the γ - loop , which contribute to substrate binding . [SEP]
[CLS] loop - 5 is highlighted for plasmin and is most analogous to the 60 - loop for thrombin . [SEP]
[CLS] in addition , important residues for exosites i and ii are highlighted in blue and cyan for thrombin , and the five kringle domains are highlighted in various shades of blue for plasmin . [SEP]
[CLS] surfaces that are colored according to hydrophobicity B-property and electrostatic potential are also shown . [SEP]
[CLS] colloidal inorganic nps B-nanoparticle [SEP]
[CLS] it is sometimes overlooked that the name of a np B-nanoparticle material B-material is , at best , a partial and ambiguous description . [SEP]
[CLS] the name defines almost nothing about the number or arrangement of atoms B-material , whereas for a small molecule such as aspirin ( acetylsalicylic acid ) the name defines an exact number and arrangement of atoms B-material of specific types . [SEP]
[CLS] for example , the " au np B-nanoparticle " nomenclature indicates that gold B-material atoms I-material are bonded in sufficient number to form a particle with dimensions between ca . 2 and 100 nm . [SEP]
[CLS] a variant such as " gold B-material nanorod B-nanoparticle " indicates something about the shape . [SEP]
[CLS] " cdse / zns quantum B-nanoparticle dot I-nanoparticle " is more descriptive , indicating composition , a core / shell structure , and a size where quantum confinement effects are manifest ( ca . 2−10 nm ) , but is nevertheless ambiguous with respect to the precise number and arrangement of atoms B-material . [SEP]
[CLS] whereas a pure sample of aspirin is homogeneous , an arguably pure sample of nps B-nanoparticle is inherently heterogeneous , starting but not ending with distributions of particle sizes and shapes ( i . e . , polydispersity ) . [SEP]
[CLS] it is likely that the heterogeneity in size and shape propagates to heterogeneity with respect to the surface area , facets , and surface structure of each individual nanocrystal and then further propagates to the organic functionalization of each nanocrystal surface and the interface between the np B-nanoparticle and the solvent . [SEP]
[CLS] au nps B-nanoparticle and qds are highlighted throughout this review as two exemplars of colloidal inorganic nanocrystals and are the materials with which studies relevant to our topic have been most frequently undertaken . [SEP]
[CLS] some particular features of the structures of au nps B-nanoparticle and qds are summarized in the following subsections before we return to a more general discussion of inorganic np B-nanoparticle interfaces . [SEP]
[CLS] several of the features highlighted with au nps B-nanoparticle and qds are also relevant to other inorganic np B-nanoparticle materials , including but not limited to nongold metallic nps B-nanoparticle , lanthanide - based upconversion nps B-nanoparticle , and metal B-material oxide I-material and alloyed magnetic B-property nps B-nanoparticle . [SEP]
[CLS] more detailed information about np B-nanoparticle interfaces can be found in recent reviews . [SEP]
[CLS] 3 illustrates some of the main points in the next subsections , including the general design and interfacial features of an inorganic np B-nanoparticle functionalized with a ligand coating B-material , cartoons of spherical au nps B-nanoparticle and qds , example depictions of how real crystals may be faceted , and examples of ligand structures for tuning the surface chemistry of these two types of nps B-nanoparticle . [SEP]
[CLS] au np B-nanoparticle interface [SEP]
[CLS] au nps B-nanoparticle , analogous to bulk gold B-material crystals , typically have a face - centered cubic ( fcc ) lattice structure . [SEP]
[CLS] although typically approximated as spherical , crystalline au nps B-nanoparticle tend to be polyhedra in reality , whether platonic , archimedean , or catalan . [SEP]
[CLS] crystal twinning is not uncommon and leads to some of these shapes . [SEP]
[CLS] the precise shape of the np B-nanoparticle determines the relative proportions of facets that are { 111 } and { 100 } . for example , an icosahedron has exclusively { 111 } facets , whereas a cuboctahedron is a mixture of eight { 111 } facets and six { 100 } facets , albeit that surface reconstructions are expected . [SEP]
[CLS] common au np B-nanoparticle surface chemistries include citrate ligands , polymers B-material , and discrete thiol ( ate ) ligands . [SEP]
[CLS] citrate can be convenient to retain from synthesis but is often a poor or mediocre ligand in many applications because of its weak binding and tenuous electrostatic colloidal stabilization of the au np B-nanoparticle . thiol ( ate ) ligands with distal hydrophilic B-property groups tend to be the most common and effective strategy in applications that require control of surface chemistry . these ligands include both small molecules and biomacromolecules such as oligonucleotides terminated with a thiol B-material linker . [SEP]
[CLS] as an example of the former , the rotello group has developed an impressive library of smallmolecule ligands for au nps B-nanoparticle with tunable distal functionality that has been shown to modulate protein−np interactions . [SEP]
[CLS] pioneered by the mirkin group , the multivalent functionalization of citrate - stabilized au nps B-nanoparticle with oligonucleotides has been the foundation of several bionanotechnological advances and the foundation of a proposed class of materials called spherical nucleic B-material acids I-material . [SEP]
[CLS] thiols B-material are well known for their strong binding to metallic gold B-material ; however , the precise modes of binding remain under scrutiny . [SEP]
[CLS] the putative binding modes between thiol B-material ligands and au nps B-nanoparticle are inferred from studies on bulk planar gold B-material interfaces and with atomically precise gold B-material nanoclusters . [SEP]
[CLS] the original model of thiols B-material binding at 3 - fold hollow sites on an unreconstructed planar gold B-material { 111 } surface to form √3 × √3r 30°lattices and c ( 4 × 2 ) superlattices has been challenged by the observation of monomeric rs−au ad −sr and dimeric rs− ( au ad −sr ) 2 " staple " binding motifs and au−sr−au bridge - site binding motifs , largely but not exclusively with nanoclusters . [SEP]
[CLS] experiments suggest that staples are associated with adatoms and are preferred on { 111 } facets , whereas bridge binding may be preferred on { 100 } facets . [SEP]
[CLS] the process of adsorption of thiol B-material ligands on gold B-material surfaces can also induce surface reconstructions such that the inorganic component of the interface is not necessarily static . [SEP]
[CLS] the complete implications for au nps B-nanoparticle remain to be determined , but it is reasonably anticipated that there will be a size - and facetdependent distribution of thiol−gold binding motifs . [SEP]
[CLS] qd interface . [SEP]
[CLS] like au nps B-nanoparticle , qds are typically approximated as spherical but are also polyhedra , with the numbers and types of facets determined during nanocrystal synthesis . [SEP]
[CLS] cdse is the prototypical qd material B-material and can adopt both zinc B-material blende and wurtzite crystal structures . [SEP]
[CLS] zinc blende qds tend to be tetrahedral or faceted in a way that approximates a sphere , with the possibility of being isotropic and displaying only one type of facet , whereas wurtzite qds tend to be slightly elongated along their c axis and are multifaceted . [SEP]
[CLS] ultimately , the shape of the qd nanocrystal determines the facets displayed , where these facets can be cationic B-material and terminated with cd 2 + ions B-material with coordination number 2 or 3 , anionic and terminated with se 2− ions B-material , or neutral and terminated with both cd 2 + and se 2− ions B-material , albeit that surface reconstructions are expected . [SEP]
[CLS] although the foregoing is written in the context of cdse qds , the general concepts extend to many common core B-material qd materials , including other ii−vi semiconductors such as cds and zns , iii−v semiconductors such as inp , iv−vi semiconductors such as pbs , and epitaxial core / shell qd structures such as cdse / zns . [SEP]
[CLS] hydrophilic B-property qds are typically functionalized with one of three general chemistries : discrete ligands that bind to the inorganic nanocrystal , polymers B-material with pendant groups that bind to the inorganic nanocrystal , and amphiphilic B-property polymers B-material that wrap around the nanocrystals and retain hydrophobic B-property ligands from synthesis . [SEP]
[CLS] for studies of enzyme B-property activity I-property toward qdsubstrate conjugates , discrete ligands based on anchoring thiol ( ate ) groups and distal hydrophilic B-property groups have been the most common type of surface functionalization . [SEP]
[CLS] 3 shows several examples of thiol B-material ligands , including a family of ligands based on dihydrolipoic acid ( dhla ) and a subset of dhla - peg ligands with various distal functional groups . [SEP]
[CLS] many of these ligands are from or are inspired by the medintz and mattoussi groups . [SEP]
[CLS] the thiol ( ate ) groups of ligands preferentially bind to cationic B-material sites on the nanocrystal surface , and therefore different densities and displays of ligands are anticipated between facets . [SEP]
[CLS] in principle , the same ligands can be used to functionalize au nps B-nanoparticle and qds ; however , the coordinate bonding interactions between thiol ( ate ) ligands and cationic B-material sites on the qd surface are weaker than the corresponding bonding interactions with au nps B-nanoparticle . [SEP]
[CLS] this fact accounts , in part , for the predominance of monothiol ligands with au nps B-nanoparticle and dithiol ligands based on dhla with qds . [SEP]
[CLS] interfacial heterogeneity . [SEP]
[CLS] it emerges from the previous subsections that nanocrystals are not only heterogeneous in size and shape but also potentially heterogeneous as individual particles because of their different facets . [SEP]
[CLS] differences in inorganic structure between facets are anticipated to propagate to differences in organic ligand displays between facets , with the ligand numbers and densities being two such examples . [SEP]
[CLS] these differences may have downstream effects as , for example , density contributes to the frequency of gauche defects , as does the radius of curvature of a np B-nanoparticle . [SEP]
[CLS] ligands fan out as the radius of curvature decreases and thus more space is available to accommodate gauche defects . [SEP]
[CLS] with the idea that facets are locally flat faces of a nanocrystal , the concept of the radius of curvature translates into the total numbers and size of each facet and breaks in ligand coverage at edges and vertices between facets , which accommodate a large amount of structural disorder . [SEP]
[CLS] the structural disorder is generally expected to decrease as the facets grow larger and with features of a ligand that promote better packing ( e . g . , longer alkyl chain lengths anticipated to add to the differences between facets , perhaps to the point that no two facets in an ensemble of nps B-nanoparticle are truly equivalent . [SEP]
[CLS] even more heterogeneity may be encountered in the special case of core / shell nps B-nanoparticle because the epitaxial growth of a uniform shell B-material is not trivial . [SEP]
[CLS] the possibility of uneven or incomplete coverage with shell B-material material B-material , which has been observed with core / shell qds , brings with it the possibility that different facets have different chemical compositions . [SEP]
[CLS] likewise , if ligand exchange is used to apply the final organic coating B-material to a np B-nanoparticle , then this process is not necessarily 100 % efficient or uniform between different facets and is another potential source of heterogeneity in chemical composition between facets . [SEP]
[CLS] yet another source of interfacial heterogeneity is bioconjugation , which , for example , is required to attach an enzyme substrate to a np B-nanoparticle . [SEP]
[CLS] the concepts and challenges of np B-nanoparticle bioconjugation have been reviewed in detail elsewhere . [SEP]
[CLS] for many chemistries , both the np B-nanoparticle and the biomolecule of interest have multiple potential points of attachment , which generally results in a population of np B-nanoparticle - bioconjugates with broad distributions in the number of biomolecules per np B-nanoparticle and the orientations of those biomolecules . [SEP]
[CLS] the tendency of hydrolysis to compete with many popular bioconjugation reactions exacerbates the challenge and adds potentially significant batch - to - batch variation . [SEP]
[CLS] although some chemistries provide much better control and reproducibility than others ( e . g . , the self - assembly of polyhistidine - tagged and thiol - terminated biomolecules to qds and au nps B-nanoparticle , respectively ) , there are few , if any , chemistries that enable the conjugation of biomolecules to qds and au nps B-nanoparticle in a manner that is homogeneous in number and attachment point per np B-nanoparticle . [SEP]
[CLS] there is no all - in - one characterization method for the np B-nanoparticle interface . [SEP]
[CLS] instead , an array of methods must be utilized and cross - referenced , often with great attention to detail to correctly identify and interpret features in the data . [SEP]
[CLS] consequently , many of the above concepts are not yet directly characterized or fully understood but rather are inferred from an accumulation of experiments and observations , or extrapolated from cluster molecules or related bulk materials . [SEP]
[CLS] sources of interfacial variability [SEP]
[CLS] to this point , fundamental materials chemistry has been discussed as a root cause of heterogeneity . [SEP]
[CLS] a related challenge is the variability between preparations of nps B-nanoparticle . [SEP]
[CLS] although batch - to - batch variation is also rooted in chemistry , it is not just an outcome of the nominal identity of the np B-nanoparticle material B-material but also an outcome of the detailed route by which the np B-nanoparticle material B-material was attained . [SEP]
[CLS] synthesis factors that are expected to affect the details of a final inorganic np B-nanoparticle include the selection of precursors , solvents , and ligands ; impurities B-property ; reagent stoichiometry ; mixing / stirring efficiency ; and temperature , pressure , and ph , among other conditions . [SEP]
[CLS] for example , the practical challenge of precisely regulating high temperatures and reagent impurities B-property in the early solvothermal synthesis of qds was ( and remains ) a considerable source of variability , as are the conditions of shell B-material growth . [SEP]
[CLS] in addition , aqueous and solvothermal methods of synthesis for compositionally identical qd materials ( e . g . , cdte , cds ) do not tend to yield functionally equivalent materials . [SEP]
[CLS] the above arguments regarding reactants and reaction conditions also extend to surface functionalization methods such as ligand exchange . [SEP]
[CLS] given that ligand adsorption may induce surface reconstruction , it is not necessarily the case that the initial inorganic surface is immutable during ligand exchange . [SEP]
[CLS] it is thus possible that the combination of methods for np B-nanoparticle synthesis and ligand exchange determines the density , orientation , and other details of the final ligand - functionalized np B-nanoparticle interface . [SEP]
[CLS] in addition to all of the above , the aging of inorganic np B-nanoparticle materials is another potential source of heterogeneity . [SEP]
[CLS] for example , some np B-nanoparticle materials , including qds , are susceptible to oxidation and the formation of an oxide B-material layer at their interface . [SEP]
[CLS] corrosion , etching , and leaching are general degradation pathways for most metal - based nps B-nanoparticle , with the rates of degradation being dependent on the materials and their passivation and conditions such as temperature and ph . as an example , silver B-material nps B-nanoparticle are notorious for their poor chemical stability , which stands in contrast to the comparatively good stability of au nps B-nanoparticle . [SEP]
[CLS] ligands are also a potential source of aging because noncovalent binding makes their off - rate and desorption equilibrium important once a functionalized np B-nanoparticle is purified of excess ligand . [SEP]
[CLS] the off - rate and position of a new equilibrium between free and bound ligand will depend upon the dilution , the strength of the ligand−np binding interaction , and the ph or other conditions that affect that interaction . [SEP]
[CLS] the foregoing discussion also extends to conjugated biomolecules , which may desorb if bound noncovalently ( including covalent bonding to noncovalently bound np B-nanoparticle ligands ) and denature or otherwise degrade over time . [SEP]
[CLS] irrespective of material B-material and mechanism , the rapid , severe degradation of a np B-nanoparticle material B-material is perhaps a minimal source of variability because it is readily detected . [SEP]
[CLS] it is slower degradation over days , weeks , or months with minimal outward symptoms that may be more problematic over a series of experiments . [SEP]
[CLS] overall , the variability between the final np B-nanoparticle materials produced by different methods and between batches produced by the same nominal method is an obstacle to a more detailed understanding of how np B-nanoparticle surface chemistry affects downstream applications . [SEP]
[CLS] thorough , detailed reporting of methods is essential for published reports , and guidelines for the detailed characterization of nps B-nanoparticle have been proposed to help compare and reconcile observations between different studies and batches of materials ; however , the undertaking is nontrivial , and most studies tick only some of the boxes . [SEP]
[CLS] interfacial environment . [SEP]
[CLS] the full interfacial environment of a np B-nanoparticle comprises not only the inorganic nanocrystal surface and its organic ligands but also conjugated biomolecules ( e . g . , peptides B-material and other enzyme substrates ) and local solvent and solute molecules . [SEP]
[CLS] the local volume of solvent generally differs from that of bulk solution ( i . e . , far from the np B-nanoparticle ) as there is a reorganization of solvent within [UNK] nm of a np B-nanoparticle interface . [SEP]
[CLS] this reorganization varies between anionic and cationic B-material , zwitterionic , and hydrophilic B-material neutral coatings B-material ( e . g . , peg ) on a np B-nanoparticle , and indeed , the details of hydration and hydrogen B-material bonding are principal factors in the nonfouling character of peg and zwitterionic coatings B-material . [SEP]
[CLS] charge at the np B-nanoparticle interface also brings about the wellknown electrical double layer , resulting in a different local ionic strength at the np B-nanoparticle interface . [SEP]
[CLS] the debye length ranges from less than 1 nm to several nanometers depending on the bulk ionic strength . [SEP]
[CLS] other important considerations at the np B-nanoparticle interface arise from multivalency , which refers to the numbers of ligands and conjugated biomacromolecules per np B-nanoparticle . [SEP]
[CLS] nearest - neighbor interactions occur with some similarity to bulk interfaces but also with potential differences from the radius of curvature or faceting of a np B-nanoparticle . [SEP]
[CLS] as an example of interactions between ligands , the carboxyl B-material group I-material pk a of monothioalkyl acid ligands is elevated on au nps B-nanoparticle and qds because of hydrogen B-material bonding between adjacent ligand molecules , mediated in part by the radius of curvature of the np B-nanoparticle . [SEP]
[CLS] the same effect does not appear to be observed with dihydrolipoic acid ligands , presumably because of different organization and density imposed by their dithiol anchoring group . [SEP]
[CLS] a high local concentration of conjugated biomolecules can also induce concentration - dependent processes to occur efficiently at the interface of a np B-nanoparticle even when inefficient in bulk solution . [SEP]
[CLS] to illustrate , imagine a np B-nanoparticle of diameter d , conjugated with an average of n peptides B-material per np B-nanoparticle , where a distance of 10 nm from the np B-nanoparticle surface is ( arbitrarily ) defined as the local environment . [SEP]
[CLS] 4 plots the concentration of peptide B-material within this volume depending on the value of n and the diameter of the np B-nanoparticle . [SEP]
[CLS] the local concentration varies by 3 orders of magnitude from [UNK] μm to [UNK] mm . [SEP]
[CLS] one example of a demonstration of this effect is the self - quenching of fluorescence B-property from qd - conjugated , dye - labeled peptides B-material because of dimerization of the dyes between neighboring peptides B-material . [SEP]
[CLS] no such behavior was observed without the localization of multiple copies of the peptide B-material to a qd . [SEP]
[CLS] this result also suggests the potential for dimerization or other density - induced interactions between conjugated biomolecules themselves . [SEP]
[CLS] note that there is a distinction between the concepts of a high local concentration and high avidity for a np B-nanoparticle . [SEP]
[CLS] in our context , effects from a high local concentration are associated with an increased probability of productive encounters between an enzyme and substrate at a np B-nanoparticle interface , whereas effects from avidity are associated with multiple , concurrent binding interactions ( e . g . , multiple biomolecules conjugated to the same np B-nanoparticle are bound to multiple receptors on a cell B-material membrane ) . [SEP]
[CLS] a single enzyme with a single binding site does not experience avidity with respect to the np B-nanoparticle because it can bind only to a single substrate molecule at any given time . [SEP]
[CLS] another effect from the multivalent conjugation of biomacromolecules to a np B-nanoparticle is a reduction in their degrees of freedom versus the bulk solution because of their anchoring and interactions ( e . g . , steric , electrostatic ) with the surface of the np B-nanoparticle and between one another , which impacts the range and dynamics of conformations adopted . [SEP]
[CLS] for example , oligonucleotides and peptides B-material adopt an average conformation that is more upright as their number per np B-nanoparticle increases . [SEP]
[CLS] moreover , the radius of curvature of a np B-nanoparticle affects the maximum densities of conjugated biomacromolecules that can be achieved , with smaller - diameter nps B-nanoparticle supporting a smaller number but higher density because of the greater deflection angle between nearest neighbors . [SEP]
[CLS] the density of biomacromolecules on a np B-nanoparticle can therefore be greater than on a bulk interface . [SEP]
[CLS] the next section will review how measurements of the enzymatic turnover of np B-nanoparticle - substrate conjugates are made , and , in part , will highlight how conventional analyses overlook or homogenize much of the detail and heterogeneity discussed in this section . [SEP]
[CLS] assay methods [SEP]
[CLS] the most common methods for tracking enzyme B-property activity I-property toward np B-nanoparticle - substrate conjugates are based on fluorescence B-property . [SEP]
[CLS] these methods capitalize on the inherent photoluminescence B-property ( pl ) properties of qds and the fluorescence B-property quenching abilities of au nps B-nanoparticle . [SEP]
[CLS] fluorescence B-property methods are very useful for tracking enzyme B-property activity I-property because they are sensitive , multicolor , and , in the cases of forster resonance energy transfer ( fret ) and electron transfer quenching , enable real - time tracking without washing or developing steps . [SEP]
[CLS] real - time tracking is also advantageous because it allows the enzyme−substrate reaction to be followed with high temporal resolution . [SEP]
[CLS] suitable calibration also enables quantitative measurements . [SEP]
[CLS] these methods are robust but nevertheless work best when care is taken to minimize artifacts from strong light scattering , inner filter effects , and trivial radiative energy transfer . [SEP]
[CLS] the desired data from tracking is a measure of the substrate or product as a function of timea partial or full reaction progress curve . [SEP]
[CLS] the substrate selected for an assay must concurrently satisfy the criteria mandated by the specificity of the enzyme and support a mechanism of signaling upon its turnover to product . [SEP]
[CLS] ( see the si for more discussion . [SEP]
[CLS] ) dye - labeled substrates are a common strategy for tracking hydrolase activity with au nps B-nanoparticle and qds . [SEP]
[CLS] for example , dye - labeled peptides B-material are useful for tracking protease activity . [SEP]
[CLS] the dye initially quenches the qd pl emission intensity via fret , and peptide B-material hydrolysis is tracked through the recovery of qd pl as the dye diffuses beyond the range of energy transfer . [SEP]
[CLS] if this dye is fluorescent B-property , then the ratio of dye and qd pl emission intensities is another useful metric for tracking . [SEP]
[CLS] an analogous format substitutes an au np B-nanoparticle for the qd , where the fluorescent B-property dye label on a conjugated peptide B-material is quenched via energy transfer to the au np B-nanoparticle and the recovery of dye fluorescence B-property emission intensity tracks with the hydrolysis of the peptide B-material . [SEP]
[CLS] these two formats are equally applicable to nucleases with the substitution of an oligonucleotide for the peptide B-material and , in principle , to all types of hydrolases and their macromolecular substrates . [SEP]
[CLS] tracking of transferase activity is also possible using a similar assay format . [SEP]
[CLS] the distinction for transferases is that the enzyme does not cleave the fluorescent B-property dye from the np B-nanoparticle - peptide substrate conjugate but rather modifies a specific peptide B-material residue with the dye , [SEP]
[CLS] either directly or indirectly [SEP]
[CLS] direct modification is usually preferable for real - time kinetic measurements . [SEP]
[CLS] 5 summarizes the foregoing energy - transfer - based assay formats , which may also prove useful for tracking ligase and lyase activity . [SEP]
[CLS] analogous in concept to the above , there are some cases where the product itself quenches the qd pl emission via fret or electron transfer but the substrate does not ( or vice versa ) . [SEP]
[CLS] this format requires that the substrate changes color upon conversion to product in order to modify the spectral overlap parameter required for fret or that it changes its redox properties in order to modulate quenching by electron transfer . [SEP]
[CLS] these configurations tend to be most useful with oxidoreductases . [SEP]
[CLS] if fret and electron - transfer quenching are not practical , then the tracking of enzymatic activity may , in principle , be achieved through chromogenic and fluorogenic small molecules conjugated to nps B-nanoparticle as substrates for an enzyme of interest . [SEP]
[CLS] here , the signal for tracking enzyme B-property activity I-property is independent of the au np B-nanoparticle or qd . [SEP]
[CLS] these substrates are widely utilized for general assays of I-technique enzyme I-technique activity I-property and have frequently proven useful with enzyme - on - np B-nanoparticle configurations ( for example , refs 129−131 ) but have not been common with substrate - on - np B-nanoparticle configurations . [SEP]
[CLS] there is no fundamental limitation for the latter as resolving changes in color or fluorescence B-property intensity versus a background of au nps B-nanoparticle or qds has been feasible with enzyme - on - np B-nanoparticle configurations . [SEP]
[CLS] it is simply that there is little precedent for this assay format because the use of a chromogen or fluorogen in parallel with a au np B-nanoparticle or qd is redundant from the standpoint of detection . [SEP]
[CLS] nevertheless , the format may prove useful as the scope of fundamental studies on substrate - on - np B-nanoparticle configurations expands to more enzymes with nonmacromolecular substrates . [SEP]
[CLS] returning to fret assay formats , a potential challenge in assaying hydrolase activity toward substrate - on - np B-nanoparticle configurations is the nonspecific adsorption of product on the np B-nanoparticle , typically driven by electrostatic or hydrophobic B-property interactions I-property . [SEP]
[CLS] the culprit may be the substrate itself or the dye label that engages in energy transfer . [SEP]
[CLS] the result is that the signal for tracking substrate never goes to zero energy transfer efficiency , which is otherwise expected for complete conversion to product . [SEP]
[CLS] calibration methods to help account for adsorption have been proposed and are discussed in the next subsection , but nonspecifically adsorbed product fragments remain difficult to distinguish from a subpopulation of putative substrates that are inactive or inaccessible . [SEP]
[CLS] another challenge in assays is drift , which may take the form of drift in instrument response , drift in the chemistry of the npsubstrate system ( e . g . , photobleaching or other nonenzymatic degradation causing a slow change in the measured observable ) , and drift in the activity B-property of I-property the I-property enzyme I-property ( e . g . , denaturation or other degradation over time ) . [SEP]
[CLS] it is important that each of these forms of drift is evaluated and addressed through control experiments . [SEP]
[CLS] a third and occasional challenge in assays is the measurement of a np B-nanoparticle - free , substrate - only control experiment . [SEP]
[CLS] either an altogether different method must be used ( e . g . , a separation method ) or a fluorescent B-property dye or molecular quencher is introduced to replace the qd or au np B-nanoparticle [SEP]
[CLS] although there are good examples of both approaches , neither is trivial . [SEP]
[CLS] for example , in our hands , the former approach has been laborious and required a honed technique to avoid poor precision , and the latter strategy has been less reliable with peptide B-material substrates than with oligonucleotide substrates because of a greater propensity for dye−dye interactions with the dual labeling of a peptide B-material . [SEP]
[CLS] one must also consider the possibility that interchanging between np B-nanoparticle and dye alters a property of the substrate ( e . g . , average conformation ) that convolves with the desired loss of effects from the np B-nanoparticle to alter the apparent enzymatic activity . [SEP]
[CLS] the hypothetical use of small - molecule chromogenic or fluorogenic substrates avoids these potential challenges . [SEP]
[CLS] progress curves and michaelis−menten analysis . [SEP]
[CLS] the data obtained from assays are full or partial reaction progress curves that represent the rate of conversion of substrate to product . [SEP]
[CLS] the raw data is typically in the form of fluorescence B-property intensity versus time . [SEP]
[CLS] obtaining a turnover rate in standard units ( m s −1 ) requires a calibration to convert the progress curves to a molar quantity versus time , as detailed in the next subsection . [SEP]
[CLS] the reflexive kinetic analysis tends to be the michaelis−menten ( mm ) model for extracting the parameters k m and k cat , the michaelis constant and turnover number , respectively , and their ratio , k cat / k m , which is the specificity constant . [SEP]
[CLS] the appeal is that the mm parameters are the familiar and basic quantitative language of enzymology ; however , as elaborated on below , the model tries to standardize the results from configurations that are nonstandard and assumes homogeneity . [SEP]
[CLS] the formalism thus has deficiencies for np B-nanoparticle - substrate conjugates . [SEP]
[CLS] two methods are principally used to arrive at values for the mm parameters from progress curves : calculation from initial rates , per eq 1 , or the fitting of full progress curves with the integrated mm equation , per eq 2 . [SEP]
[CLS] the terms in these equations are the reaction velocity , v , and the concentrations of substrate and product , [ s ] and [ p ] , as a function of time , t , where the naught subscript denotes an initial value and the t subscript denotes a value at an arbitrary time point . [SEP]
[CLS] equation 3 is the closed - form solution of eq 2 , where w is the lambert function . [SEP]
[CLS] i k j j j j j y [SEP]
[CLS] the mm model in eqs 1−3 has several implicit assumptions and guidelines : a large excess of substrate in order to achieve the briggs−haldane or steady - state approximation of d [ es ] / dt = 0 , where es is the enzyme−substrate complex ; the free ligand condition of [ e ] 0 [UNK] k m ; and [ s ] 0 [UNK] 3k m to determine k m and k cat separately rather than as only the specificity constant . [SEP]
[CLS] moreover , the enzyme must be stable over the period of measurement , the reaction must be irreversible and without product inhibition , and progress curves for a series of enzyme concentrations should pass selwyn ' s test and follow a single trajectory when plotted in enzyme time . [SEP]
[CLS] for eq 1 , initial rates can be obtained from tracking over a time window that corresponds to the initial turnover of substrate ( typically a few percent consumption or less ) or from the mathematical fitting of full progress curves . [SEP]
[CLS] in the latter case , the equation for fitting can be empirical and need not be the integrated mm equation , but eq 1 and eqs 2 and 3 should ultimately yield the same values for k m and k cat in the absence of deviations from mm behavior . [SEP]
[CLS] even if the initial rates will be the basis of quantitative analysis , we recommend the measurement of full progress curves . [SEP]
[CLS] almost every reaction progress curve looks the same in its initial stages : approximate linearity at short times and negligible substrate consumption followed by the introduction of modest curvature at somewhat longer times as the substrate begins to be depleted . [SEP]
[CLS] substantial consumption of substrate is needed to test the validity of a kinetic model and resolve mechanism differences . [SEP]
[CLS] 6a shows an example of how the measurement of only the initial portion of the progress curves would completely obscure two different kinetic profiles . [SEP]
[CLS] this mock data is inspired by a real example ( vide infra ) where the differences between the initial and full progress curves revealed key differences in the models of substrate turnover with a np B-nanoparticle . [SEP]
[CLS] of course , it is obvious in this example that a naıve assumption of the mm model is inappropriate and that a full progress curve is useful . there are also more subtle examples , including our finding that the turnover of multivalent qd - peptide B-material substrate conjugates by a protease did not strictly follow the mm model . [SEP]
[CLS] 6b shows some of this data , where the deviations are the poor fit of the modeled mm progress curve and the lack of a single trajectory when the experimental progress curves are plotted in enzyme time . [SEP]
[CLS] in addition to the fit of the data , it is also important to consider the congruity between the assumptions of the mm model and the conditions of the experiment . [SEP]
[CLS] the mm formalism sometimes presents challenges for substrate - on - np B-nanoparticle configurations because nps B-nanoparticle are typically used at submicromolar concentrations and thus a large excess of substrate over enzyme may be impractical to achieve . [SEP]
[CLS] although there is a reformulation of the mm formalism for excess enzyme , the situation is complicated by the potential for concurrent turnover of multiple substrates on a np B-nanoparticle by multiple enzymes . [SEP]
[CLS] an alternative approach that has been adopted with a small excess of substrate is the determination of the specificity constant , k cat / k m , as a single value with the subsequent estimate of k m via the assumption that k cat is unchanged from assays with substrates but not with nps B-nanoparticle . [SEP]
[CLS] beyond technical challenges , there is a more fundamental question to answer with respect to the applicability of the mm model : what does [ s ] represent for a substrate - on - np B-nanoparticle configuration ? [SEP]
[CLS] the number of diffusing entities in an x m solution of np B-nanoparticle - [ substrate ] n conjugates ( i . e . , n substrates conjugated per np B-nanoparticle ) is less than in an equal volume of nx m solution of substrate alone , but the number of substrates that can be converted to product is greater for x m np B-nanoparticle - [ substrate ] n conjugates than for x m substrate alone ( notwithstanding the frequent challenge of measuring x accurately for samples of nps B-nanoparticle ) . [SEP]
[CLS] the choice of [ s ] = x m or [ s ] = nx m becomes important for quantitative comparisons of k m and k cat between different studies and for comparisons between assays with np B-nanoparticle - [ substrate ] n and substrate only , where the potential factor of n difference in these parameters needs to be explicit lest it affect the conclusions drawn . [SEP]
[CLS] as will be detailed in the next section , the proposed model of turnover for np B-nanoparticle - [ substrate ] n conjugates may also influence the choice of [ s ] = x m or [ s ] = nx m . there is a proposed hopping model where the conjugate undergoes singlestep conversion from np B-nanoparticle - [ substrate ] n to np B-nanoparticle - [ product ] n in an encounter with enzyme . [SEP]
[CLS] if this model is accepted , then the logical interpretation is that [ s ] = x m and each value of n for the conjugate represents a unique substrate that , in principle , has its own values for k m and k cat . [SEP]
[CLS] in any case , derived values for k m and k cat are best labeled as " apparent " but have utility for comparison between different multivalent np B-nanoparticle - substrate conjugates . [SEP]
[CLS] given the above , an mm analysis should not be automatic . [SEP]
[CLS] its use should be accompanied by clear statements of assumptions and an evaluation of how a change in assumptions would ( if at all ) impact the conclusions drawn . [SEP]
[CLS] a more general approach to kinetic analysis is stepping back to collision theory . [SEP]
[CLS] there are interpretations of enhancements of enzyme B-property activity I-property associated with np B-nanoparticle - substrate conjugates that point to parameters such as the collision cross - section and steric factor , and these parameters may be sufficient if single - step conversion of np B-nanoparticle - [ substrate ] n to np B-nanoparticle - [ product ] n is accepted . [SEP]
[CLS] if not accepted , modifications to collision theory should take into account that multiple productive collisions are needed to convert all n substrates per np B-nanoparticle into product . [SEP]
[CLS] more detailed models may also seek to tease apart potential enzyme−substrate and enzyme−np interactions and allow for distributions of values for model parameters that arise from nontrivial polydispersity or other heterogeneity of a np B-nanoparticle sample . [SEP]
[CLS] a more detailed look at the mathematics for models based on collision theory can be found elsewhere , and we will later address conceptual factors for these models in the context of the capacity for np B-nanoparticle - substrate conjugates to accelerate enzymatic activity . [SEP]
[CLS] model matters . [SEP]
[CLS] with the questionable applicability of a simple mm model , other models are needed for the interpretation of progress curve data . [SEP]
[CLS] as noted earlier , a hopping model has been proposed for the enzymatic turnover of npsubstrate conjugates . [SEP]
[CLS] in this model , all of the substrate conjugated to an individual np B-nanoparticle is hydrolyzed in a single encounter with a single enzyme . [SEP]
[CLS] the putative rate - limiting step is the diffusion of the enzyme between nps B-nanoparticle . [SEP]
[CLS] the opposite extreme is a model where only one of the conjugated substrates is hydrolyzed per encounter between an enzyme and the np B-nanoparticle , which we call the " colliding " model . [SEP]
[CLS] for this model , the putative rate - limiting step is enzyme−substrate binding . [SEP]
[CLS] 7 illustrates the difference between these two models in terms of the time - dependent distribution of the number of substrates per qd , assuming simple poisson statistics . [SEP]
[CLS] the colliding model , which invokes a series of discrete enzyme−substrate interactions , is conceptually most similar to the conventional mm model , but the hopping model is the most congruent mathematically . [SEP]
[CLS] the following paragraphs will illustrate how the results of a quantitative analysis of experimental data can depend on the model with which the data is interpreted . [SEP]
[CLS] as a thought experiment , consider an assay for protease activity with qd - [ substrate−dye ] n conjugates and the use of fret for tracking substrate hydrolysis . [SEP]
[CLS] 8a shows hypothetical progress curves for this experiment , which are initially obtained as the qd and dye pl emission intensities versus time . [SEP]
[CLS] the dye / qd pl intensity ratio versus time is calculated from this data and also plotted . [SEP]
[CLS] the data is modeled to be ideal with respect to fret theory and free of noise , drift , and the nonspecific adsorption of product . [SEP]
[CLS] consequently , the progress curves for qd pl intensity versus time and the pl ratio versus time will both yield equivalent results from a full analysis , albeit the latter is thought to be a more robust metric for real experiments . [SEP]
[CLS] to parametrize the thought experiment , we adopt an initial qd - [ substrate−dye ] n conjugate with a fret efficiency of e = 0 . 75 at n = 10 . [SEP]
[CLS] first , we consider a colliding model . [SEP]
[CLS] the calibration for this model is a series of samples of qd - [ substrate ] n conjugates ranging from n = 0 to 10 . [SEP]
[CLS] 8b shows the model calibration data . [SEP]
[CLS] the trend for the qd pl intensity versus n is a hyperbolic function typical of fret , and the trend for the dye / qd pl ratio is linear . [SEP]
[CLS] the mock data assumes equal quantum yields for the qd and dye , but the trend in the pl ratio would be linear regardless of the ratio of quantum yields . [SEP]
[CLS] the calibration data in figure 8b converts the raw data to the stoichiometric progress curve in figure 8c . [SEP]
[CLS] the vertical axis of the progress curves in figure 8c includes scales for the ensemble average number of peptides B-material per qd , n , which is the logical metric for the colliding model , and for the mole fraction of substrate , calculated as χ substrate = n / 10 for comparison to the hopping model . [SEP]
[CLS] the calibration for a hopping model is a series of samples with a mixture of qd - [ substrate ] 10 and qd - [ substrate ] 0 conjugates , with no intermediate B-property values of n , where χ substrate is the mole fraction of n = 10 conjugates . [SEP]
[CLS] ( in real experiments , qd - [ product ] 10 conjugates would be preferred over qd - [ substrate ] 0 conjugates to account for nonspecific adsorption . [SEP]
[CLS] ) the trend in the qd pl intensity is linear as a function of χ substrate , and the trend in the pl ratio is hyperbolic . [SEP]
[CLS] the hopping model calibration data in figure 8b converts the raw data to the stoichiometric progress curve in figure 8c . [SEP]
[CLS] note that the progress curves for the hopping and colliding models are not superimposed . [SEP]
[CLS] rather , the assumption of a colliding model translates into an apparent faster rate of turnover . [SEP]
[CLS] the difference arises from the requisite differences in calibration for the hopping and colliding models . [SEP]
[CLS] the n and χ parameters are indeed comparable between the models because each corresponds to the same number of freely diffusing , hydrolyzed - product peptide B-material fragments ( despite representing a different makeup of the np B-nanoparticle - substrate conjugates ) . [SEP]
[CLS] a quirk is that the final progress curves for the two models become more similar as the fret efficiency in the initial conjugate decreases and as nonspecific adsorption increases . [SEP]
[CLS] full details on the analysis of this thought experiment can be found in the si . [SEP]
[CLS] of course , there are prospective models that lie between the extremes of hopping and colliding . [SEP]
[CLS] the degree to which the real enzymatic turnover of np B-nanoparticle - [ substrate ] n conjugates approximates hopping or colliding may depend on the size of the np B-nanoparticle and the value of n . [SEP]
[CLS] moreover , if we define pseudohopping as the turnover of a large number of substrates per np B-nanoparticle per encounter with enzyme and define pseudocolliding as the turnover of a small number of substrates per np B-nanoparticle per encounter with enzyme , then we must consider the possibilities of each of these models in isolation and as a mixed model that transitions from pseudohopping to pseudocolliding as the reaction progresses . [SEP]
[CLS] although the mathematics for these models can be devised , experiments must first establish and parametrize the model that is most appropriate . [SEP]
[CLS] it is for this reason that we have recently stepped back from assigning a model to our data for the purposes of extracting apparent values of k m and k cat . [SEP]
[CLS] semiquantitative analysis remains possible without the selection of a colliding , hopping , or other model by comparing the nonstoichiometric progress curves with the qd pl intensity or pl ratio plotted versus time . [SEP]
[CLS] the caveat is that the properties of interest of the conjugate must be varied without substantially changing the fret efficiency of the initial conjugate , for example , by using a fixed amount of the dye - labeled substrate and varying only the surface ligands or identity and number of additional unlabeled substrates or nonsubstrate biomolecules . [SEP]
[CLS] in real systems , the fret efficiency generally changes slightly as the system changes , so small differences in progress curves must be interpreted cautiously . [SEP]
[CLS] nevertheless , much can still be learned about how changes in surface chemistry and other properties impact the turnover of np B-nanoparticle - substrate conjugates . [SEP]
[CLS] overview . [SEP]
[CLS] the previous sections of this article introduced concepts of enzymatic activity and interactions with npsubstrate conjugates apart from actual examples of experimental studies . [SEP]
[CLS] we now add more substance to the discussion through a review of recent studies that address this research question . [SEP]
[CLS] 1 summarizes several fundamental studies of the enzymecatalyzed turnover of np B-nanoparticle - substrate conjugates . [SEP]
[CLS] ( we apologize to the authors of any studies that we have inadvertently overlooked . [SEP]
[CLS] ) the results of these studies are distilled into prevailing factors that affect the rate of np B-nanoparticle - substrate conjugate turnover , each with its own subsection . [SEP]
[CLS] the final subsections address the recurring observation that the multivalent conjugation of substrate to a np B-nanoparticle appears to accelerate enzyme B-property activity I-property and assess the current state of the hypothesis that a hopping model accounts for this acceleration . [SEP]
[CLS] a pair of studies that we have completed indicate an important role for the adsorption of enzyme on a np B-nanoparticle in determining the kinetics of substrate turnover . [SEP]
[CLS] the proteases trypsin , thrombin , and plasmin were used as model enzymes , and glutathione ( gsh , 9 in figure 3 ) , cysteine B-material ( 7 ) , 3mercaptopropionic acid ( mpa , 6 ) , dihydrolipoic acid ( dhla , 8 ) , dhla - peg ( 12 ) , and dhla - sulfobetaine ( dhla - sb , 10 ) were used as surface ligands to functionalize cdses / zns and cdse / cds / zns qds . [SEP]
[CLS] the cumulative results of these studies can be summarized through five key experiments , as described below . [SEP]
[CLS] first , different surface ligand−protease pairings yielded different rates of turnover for x - qd - [ peptide substrate ] n conjugates and different shapes of progress curves , where x was one of the cysteine B-material , dhla , gsh , or mpa surface ligands and the proteases were trypsin and thrombin . [SEP]
[CLS] the progress curves were measured via fret with a distal dye label on the peptide B-material substrate . [SEP]
[CLS] there was a general correlation between faster turnover and progress curves that were one - phase and convergent to a common end point for different protease concentrations , and between slower turnover and progress curves that were two - phase and nonconvergent for different protease concentrations . [SEP]
[CLS] trypsin exhibited one - phase / convergent progress curves regardless of x , whereas thrombin exhibited two - phase / nonconvergent progress curves . [SEP]
[CLS] even with the different shapes of the progress curve , the same qualitative trend in the rate of substrate turnover with x was observed with both trypsin and thrombin . [SEP]
[CLS] subsequent experiments , shown in part in figure 9a , also revealed two - phase / nonconvergent progress curves for plasmin with x = dhla and gsh . [SEP]
[CLS] the interpretation of the two - phase / nonconvergent progress curves was that thrombin or plasmin rapidly associated with x - qd - [ peptide substrate ] n conjugates , leading to an initial phase of efficient substrate hydrolysis that then transitioned to a second phase where the strong adsorption of protease on x - qd hindered diffusion to a new conjugate , causing a marked decrease in the ensemble rate of hydrolysis . [SEP]
[CLS] accordingly , the interpretation of the one - phase / convergent progress curves was that , to a first approximation , trypsin was free to diffuse from conjugate to conjugate because of weak ( if any ) adsorption on the x - qd . [SEP]
[CLS] a second set of experiments investigated the effect of x 2 - qd on progress curves when mixed with x 1 - qd - [ substrate ] n conjugates , where x 1 and x 2 are two different ligands . [SEP]
[CLS] the added x 2 - qd had the strongest inhibitory effect with the x 2 = mpa and dhla ligand coatings B-material that were associated with the slowest turnover of substrate on cdses / zns qds by thrombin and trypsin . [SEP]
[CLS] in turn , the x 2 = gsh and cysteine ligand coatings B-material that were associated with faster turnover had the smallest inhibitory effect . [SEP]
[CLS] thrombin was much more sensitive to x 2 - qd than was trypsin , with the latter exhibiting almost no response to x 2 - qd with x 2 = gsh and cysteine B-material . [SEP]
[CLS] these trends were fully consistent with expectations from the first set of experiments , further supporting the potential effect of the qd interface as an inhibitor of protease activity , putatively via strong adsorption and the loss of diffusion between conjugates . [SEP]
[CLS] two additional experiments further addressed surface effects , this time from the standpoint of surface passivation to reduce protease adsorption . [SEP]
[CLS] 9b shows an example in which loading of the qd with an increasing number , m , of other biomacromolecules yielded an overall trend of increasing rate ( one - phase progress curves ) or extent ( two - phase progress curves ) of the substrate turnover . [SEP]
[CLS] these conjugates were of the form x - qd - [ peptide substrate ] n - [ y ] m , where y was an additional substrate peptide B-material , nonsubstrate peptide B-material , protein B-material , or discrete peg molecules [SEP]
[CLS] the trend applied to trypsin , thrombin , and plasmin , albeit that the biggest effects were observed with plasmin , consistent with it being most prone to adsorption . [SEP]
[CLS] in addition , it was found that a change from x = dhla or gsh to x = dhla - sb or dhla - peg resulted in a concomitant change from two - phase / nonconvergent progress curves to one - phase / convergent progress curves for plasmin activity . [SEP]
[CLS] this result , shown in figure 9c , was consistent with the well - characterized ability of peg and zwitterionic coatings B-material on nps B-nanoparticle to resist nonspecific protein B-material adsorption . [SEP]
[CLS] overall , the two experiments demonstrated that the tuning of surface chemistry can mitigate inhibitory adsorption and recover protease activity . [SEP]
[CLS] the fifth and final set of experiments was runs of agarose B-technique gel I-technique electrophoresis I-technique , which linked all of the foregoing inferences of adsorption from progress curves to its direct observation . [SEP]
[CLS] examples of gels are shown in figure 9d . the adsorption of protease on qds was observed in several ways : band streaking , band mobility B-property shifts , the formation of multiple discrete bands of different mobility B-property , and a complete loss of mobility B-property . [SEP]
[CLS] the order of these observations approximately trended with increasing affinity between the qd interface and protein B-material ( i . e . , lower k d ) and less dynamic interactions ( i . e . , slower exchange between adsorbed and unbound states ) . [SEP]
[CLS] importantly , the gel - derived trends in protease adsorption on the qds correlated with trends in progress curves and substrate turnover between surface ligands , x , between the three proteases , and with increased surface passivation from added biomacromolecules , y . the caveat was that the protease concentrations at which adsorption was observed on a gel were higher than the concentrations used to measure progress curves , but there was a correlation in trends nonetheless . [SEP]
[CLS] a study by dı az et al . drew conclusions similar to the above . [SEP]
[CLS] the activities of elastase and collagenase toward cdse / cdzns / zns qds functionalized with a zwitterionic ligand ( dhla - cl4 ) and conjugated dye - labeled peptide B-material substrate were compared . [SEP]
[CLS] fret was again used to measure progress curves . [SEP]
[CLS] differences between the progress curves for elastase and collagenase , albeit less than the differences we observed between plasmin and thrombin , were attributed to a much greater tendency for collagenase to adsorb on the qds , which was likewise observed as mobility B-property shifts for the qds in agarose B-technique gel I-technique electrophoresis I-technique . [SEP]
[CLS] greater passivation of the qds through conjugation of a greater number of substrate peptides B-material also increased the rate of substrate turnover . [SEP]
[CLS] the overarching conclusion from the above is that the minimization of enzyme adsorption on a np B-nanoparticle is likely to maximize the rate of substrate turnover . [SEP]
[CLS] that said , it remains an open question as to whether carefully optimized weak and dynamic adsorption can be more favorable than the complete and total elimination of adsorption . [SEP]
[CLS] for example , in cases where steric hindrance ( vide infra ) is equal between different surface ligands on a qd , dı az et al . have suggested that a weak affinity between a protease and np B-nanoparticle may increase the residence time of the protease at the np B-nanoparticle , potentially facilitating the turnover of conjugated substrate . [SEP]
[CLS] the affinity between rnase and au npoligonucleotide conjugates has also been reported to accelerate nuclease activity . [SEP]
[CLS] the means by which weak adsorption might accelerate enzyme B-property activity I-property are discussed later . [SEP]
[CLS] steric hindrance [SEP]
[CLS] in one of our studies , we saw a putative effect of steric hindrance between x = dhla - sb and dhla - peg for x - qd - [ peptide substrate ] n conjugates . [SEP]
[CLS] although both ligands engendered one - phase / convergent progress curves with plasmin ( cf . two - phase / nonconvergent progress curves with x = gsh , dhla ) , a much slower rate of substrate turnover was observed for dhla - peg . [SEP]
[CLS] both the dhla - sb and dhla - peg coatings B-material resisted adsorption , so the difference in rate was attributed to the much larger size of the dhla - peg ligand . [SEP]
[CLS] a significant portion of the length of the substrate was within the peg layer around the qd , representing a potential hindrance for binding by plasmin . [SEP]
[CLS] the above conclusion was in agreement with a contemporary study by dı az et al . that assessed the turnover of x - qd - [ peptide substrate ] n conjugates by trypsin , where x was a series of dhla - peg - r ligands ( figure 3c , 12 ) and r was a variable functional B-material group I-material at the distal terminus of the peg chain : amine B-material , acetyl , methoxy , hydroxyl , carboxyl B-material , or a zwitterionic group ( denoted as cl4 ) . [SEP]
[CLS] a dhla - cl4 ligand without peg was also evaluated as a control . [SEP]
[CLS] substrate turnover was fastest on the dhla - cl4 - qds , consistent with steric hindrance from the peg chains limiting access to the peptide B-material substrate by trypsin . [SEP]
[CLS] moreover , atomistic molecular dynamics simulations suggested that there was some correlation between the solvent - accessible surface area ( sasa ) and turnover rates between the dhla - peg - r ligands , indicating an important role for steric hindrance in determining rates of substrate turnover . [SEP]
[CLS] interestingly , the observation of steric hindrance with qd - [ substrate ] n conjugates has , to date , been limited to effects from ligands . [SEP]
[CLS] with compact ligands on qds , large numbers of peptide B-material per qd have not yet been reported to hinder protease activity versus smaller numbers . [SEP]
[CLS] the likely explanation is that maximum peptide B-material valences have been ≤60 per qd , whereas ligand numbers per qd have been estimated to be > 200 per qd . [SEP]
[CLS] higher densities of ligands are thus a likely factor ; however , differences in the overall np B-nanoparticle solvation for peg ligands versus compact ligands and / or a higher affinity of proteases for peptides B-material versus peg cannot be ruled out . [SEP]
[CLS] co - assembly of peptide B-material substrates with globular proteins B-material also did not introduce detectable steric hindrance , although there is not yet sufficient data to generalize this result . [SEP]
[CLS] in contrast to the above with qds , there is some indication that peptides B-material can be conjugated to au nps B-nanoparticle with sufficient density to cause steric hindrance , although these results are also not yet generalizable . [SEP]
[CLS] a study by yeh et al . found that chymotrypsin - catalyzed hydrolysis rates improved by 5 - fold when the length of a peptide B-material substrate conjugated to au nps B-nanoparticle increased from 9 residues to 13 residues , moving the cleavage sites further from the surface of the np B-nanoparticle . [SEP]
[CLS] an additional 100fold improvement was observed when the five inserted residues were converted from neutral to anionic . [SEP]
[CLS] one explanation for this marked improvement , proposed by yeh et al . , was that the five anionic residues interacted favorably with the halo of cationic B-material residues around the active site of chymotrypsin . [SEP]
[CLS] other possible explanations are that repulsion between neighboring polyanionic peptides B-material ( and potentially residual citrate ligands on the au np B-nanoparticle ) caused the peptides B-material to be conjugated at a lower density and / or adopt a conformation that was more upright relative to the au np B-nanoparticle surface , alleviating steric hindrance . [SEP]
[CLS] moreover , the au nps B-nanoparticle modified with 9 - mer and neutral 13 - mer peptides B-material had poor colloidal stability and required 0 . 1 % bovine serum albumin ( bsa ) in solution as a stabilizer , whereas the au np B-nanoparticle modified with the anionic 13 - mer probe did not . [SEP]
[CLS] bsa - stabilized aggregates of the former two np B-nanoparticle materials or bsa adsorbed onto individual au nps B-nanoparticle are potential steric hindrances for chymotrypsin , and bsa is a potential competitive inhibitor as a second substrate . [SEP]
[CLS] the initial 5 - fold improvement with the neutral 13 - mer peptide B-material therefore seems attributable to a steric effect , but the subsequent 100 - fold enhancement with the anionic 13 - mer peptide B-material is perhaps ambiguous or more complex in its origin . [SEP]
[CLS] nevertheless , the nature of peptide B-material binding to au nps B-nanoparticle versus qds , which is monothiol - gold B-material binding displacing citrate functionalization versus hexahistidine - zns shell B-material binding with a concomitant dithiol ligand , is such that a higher density of conjugated peptides B-material should be achievable with au nps B-nanoparticle . [SEP]
[CLS] steric effects from substrate density are therefore more likely to occur with au nps B-nanoparticle . [SEP]
[CLS] another example of steric hindrance with au nps B-nanoparticle was observed in assays for the activity of botulinum a light chain ( bolca ) protease activity . [SEP]
[CLS] the au nps B-nanoparticle were conjugated with peptide B-material substrates , and turnover was compared between au nps B-nanoparticle with average diameters of 1 . 4 , 6 , and 18 nm . [SEP]
[CLS] the turnover with the 6 and 18 nm au nps B-nanoparticle was 80 - fold less than with 1 . 4 nm au nps B-nanoparticle . [SEP]
[CLS] in analogous experiments with trypsin , the turnover was also less with the larger au nps B-nanoparticle but only 18 - fold less versus the 1 . 4 nm au np B-nanoparticle . [SEP]
[CLS] these different sensitivities to the steric effect of the np B-nanoparticle was attributed to the larger size of bolca , which was double the molecular weight of trypsin , as well as its 2 . 4 - nmdeep active site cleft . [SEP]
[CLS] presumptive steric effects have also been observed with bolca and qd - peptide B-material conjugates . [SEP]
[CLS] despite the above examples , steric hindrance is not a foregone conclusion with a high density of substrate on au nps B-nanoparticle . [SEP]
[CLS] prigodich et al . found that the high density of dna oligonucleotides around a np B-nanoparticle actually enhanced nuclease binding by a factor of about 2 , suggesting that there was either no steric hindrance or that it was overwhelmed by the effect of enhanced affinity ( e . g . , high local concentration ) . [SEP]
[CLS] interfacial environment [SEP]
[CLS] many enzymes have a ph , temperature , and other parameters that optimize their activity . [SEP]
[CLS] as noted earlier , the environment at the np B-nanoparticle interface is different than the environment of bulk solution , so this local environment may influence enzymatic activity toward a np B-nanoparticle - substrate conjugate . [SEP]
[CLS] one such example , shown in figure 11 , arises with spherical nucleic B-material acids I-material , which are densely multivalent au np B-nanoparticle - dna oligonucleotide conjugates . [SEP]
[CLS] compared to dna alone , these materials are ca . 4 - fold more resistant to hydrolysis by dnase i because the high local concentration of monovalent cations B-material at the np B-nanoparticle interface inhibits nuclease activity by displacing divalent cation B-material cofactors from the enzyme . [SEP]
[CLS] this high concentration of cations B-material arises from countering the polyanionic character of the oligonucleotides and from a local influx of anions B-material from osmotic pressure that must be balanced by additional cations B-material . [SEP]
[CLS] the inhibitory effect decreases with lower negative charge density on the au np B-nanoparticle and for nucleases that are engineered to be more tolerant of salt B-material . [SEP]
[CLS] although relatively few studies have ( so far ) uncovered an effect of the local np B-nanoparticle environment on enzyme B-property activity I-property toward substrate - on - np B-nanoparticle configurations , possibilities can be extrapolated from the converse enzyme - on - np B-nanoparticle configurations because the substrate−enzyme interaction occurs at the interface between the np B-nanoparticle and bulk solution in both cases . [SEP]
[CLS] for example , breger et al . found that phosphotriesterase activity was enhanced versus bulk solution when conjugated to a qd , concluding that different solvation at the np B-nanoparticle interface increased the rate of product dissociation from the enzyme , which was known to be slower than the rate of hydrolysis and thus limiting . [SEP]
[CLS] another study postulated that an enhancement in the activity of lipase adsorbed to au nps B-nanoparticle was from the interface lowering the activation energy barrier B-property for the formation of the enzyme−substrate complex . [SEP]
[CLS] a caution is that alterations of enzyme B-property activity I-property from the local environment at the np B-nanoparticle interface are not necessarily straightforward to distinguish from an allosteric or similar effect induced by contact with the np B-nanoparticle or other conjugated biomacromolecules . [SEP]
[CLS] this concept is more intuitive for enzyme - on - np B-nanoparticle configurations but may also be applicable to some np B-nanoparticle - substrate conjugates . [SEP]
[CLS] experiments that correlate enzyme B-property activity I-property with changes induced in the np B-nanoparticle interfacial environment by upstream changes in bulk solution are thus important supporting evidence . [SEP]
[CLS] acceleration of substrate turnover [SEP]
[CLS] the studies cited above have shown that many factors can affect enzyme B-property activity I-property toward np B-nanoparticle - substrate conjugates . [SEP]
[CLS] a feature common to several of these and other studies is an acceleration of substrate turnover with np B-nanoparticle - substrate conjugates versus equivalent quantities of enzyme and substrate in bulk solution without np B-nanoparticle . [SEP]
[CLS] examples include trypsin , thrombin , and plasmin activity toward qd - [ peptide ] n substrates , rnase h activity toward au np B-nanoparticle - [ oligonucleotide ] n conjugates , and tyrosinase activity toward qd - [ tyrosine ] n conjugates [SEP]
[CLS] the accelerations have been an approximate doubling of turnover rates at the low end and between 1 to 2 orders of magnitude at the high end , with each example listed in table 1 . [SEP]
[CLS] however , the acceleration of activity is not universal . [SEP]
[CLS] it has already been mentioned that the local environment of a au np B-nanoparticle - oligonucleotide conjugate can inhibit some nucleases , and one study found that dhla - cl4 - qd - [ peptide substrate ] n conjugates inhibited the activity of collagenase and elastase , although the same nominal materials accelerated trypsin activity 35 - fold in another study . [SEP]
[CLS] we have also seen indications that batch - to - batch variation can yield acceleration or inhibition for nominally similar materials . [SEP]
[CLS] these examples do not necessarily challenge the concept of enzymatic acceleration with np B-nanoparticle - [ substrate ] n conjugates , but once again highlight the complexity of the systems . [SEP]
[CLS] collision theory for reaction rates is a useful framework for analyzing the acceleration of enzymatic activity toward np B-nanoparticle - [ substrate ] n conjugates because , irrespective of a hopping or colliding model , a change in the kinetics rather than the thermodynamics of substrate to product conversion is expected . [SEP]
[CLS] to begin , consider an x μm concentration of the np B-nanoparticle - [ substrate ] n conjugate versus an nx μm concentration of only substrate , both mixed with e nm of enzyme . [SEP]
[CLS] the latter scenario has a greater frequency of collisions if the concepts of collision cross - section and productive orientation are momentarily neglected . [SEP]
[CLS] the np B-nanoparticle must therefore increase both of the latter parameters by a combined factor of greater than n if there is to be an acceleration of substrate turnover . [SEP]
[CLS] starting with the collision cross - section , the physical size of a np B-nanoparticle is typically much greater than that of a substrate alone and is made larger by multivalent conjugation of a substrate . [SEP]
[CLS] for example , a typical qd - [ peptide ] n conjugate has a qd in the range of 1 . 5−4 nm in radius , and fret data suggests that peptides B-material extend the radius by an additional 2 to 3 nm . [SEP]
[CLS] the collision cross - section is clearly much larger for the np B-nanoparticle - [ substrate ] n conjugate than for an individual substrate molecule , as depicted in figure 12a . [SEP]
[CLS] a rough estimate is between 3 - fold and 25 - fold larger for the example of a qd - [ peptide ] n conjugate . [SEP]
[CLS] the stokes−einstein equation also indicates that the cross - section for a np B-nanoparticle should increase at a faster rate than its diffusion coefficient decreases as the np B-nanoparticle diameter increases . [SEP]
[CLS] it is also expected that the likelihood of a productive collision is higher with a np B-nanoparticle - [ substrate ] n conjugate . [SEP]
[CLS] first , at the moment of an initial collision , the np B-nanoparticle conjugate will display multiple substrates in close proximity with different orientations relative to the enzyme . [SEP]
[CLS] this situation is expected to increase the probability of a productive mutual orientation between enzyme and substrate , as depicted in figure 12b . [SEP]
[CLS] second , transient interactions or migration along a np B-nanoparticle surface , or temporary capture within the hydration shell B-material of the np B-nanoparticle - substrate conjugate ( an effect reported with other nanoscale enzymatic systems ) , illustrated in figure 12c , have the potential to increase the residence time of the enzyme at the np B-nanoparticle , giving it more opportunity to rotate into a productive orientation and bind to a substrate molecule . [SEP]
[CLS] these factors also increase the likelihood that the enzyme will reassociate with a new substrate molecule after dissociation from another , as depicted in figure 12d , as does the high local concentration of substrate with larger n , depicted in figure 12e . [SEP]
[CLS] these overall concepts inspire the hypothesis that sufficiently weak but nonzero adsorption or another interaction with the np B-nanoparticle - [ substrate ] n conjugate may lead to an enhancement of turnover , even though moderate or strong adsorption is inhibitory . [SEP]
[CLS] a question that arises is if any of the above modifications to the parameters of collision theory represent a real change in the intrinsic k m and k cat for an enzyme−substrate combination . [SEP]
[CLS] the answer depends somewhat on perspective , and we will use the case of a qd - [ peptide ] n conjugate to illustrate . [SEP]
[CLS] if each peptide B-material molecule is to be a unique substrate , then the presumption is that there is no enhancement in either k m or k cat and that a good model must account for the features of the multivalent np B-nanoparticle configuration that lead to acceleration ( and perhaps even tolerate unfavorable changes in k m and k cat from loss of degrees of freedom or steric effects ) . [SEP]
[CLS] however , if the entire conjugate is to be treated as the substrate , then the acceleration of turnover is reconciled with new values of k m and k cat , preferably labeled as apparent values because of the different molecular - scale processes versus the conventional michaelis−menten model . [SEP]
[CLS] for example , many of the processes depicted in figure 12 suggest that the apparent value of the michaelis constant , k m , app , would , at minimum , be a convolution of the conventional enzyme−substrate k m and the adsorption constant , k ads , for the enzyme on the np B-nanoparticle . [SEP]
[CLS] we reserve judgment on which perspective is most practical until a hopping , colliding , or corresponding pseudomodel of turnover is confirmed . [SEP]
[CLS] in sum , multivalent np B-nanoparticle - substrate conjugates have the potential to yield large accelerations of enzyme B-property activity I-property because of the high local concentration of substrate at their interface , but strong adsorption and steric hindrance reduce or negate the acceleration , and further nuances remain to be determined . [SEP]
[CLS] evidence for a hopping model . [SEP]
[CLS] much of how to conceptualize and analyze the enzymatic turnover of npsubstrate conjugates seems to hinge on the confirmation of a model for how it occurs , whether hopping or otherwise . [SEP]
[CLS] the ideas of a high local concentration of substrate at the np B-nanoparticle interface and weak affinity interactions between the enzyme and np B-nanoparticle - substrate conjugate , discussed in the previous subsection , are the crux of the rationale for a hopping model of activity . [SEP]
[CLS] for many readers , a hopping or pseudohopping model of enzymatic turnover will seem intuitive for np B-nanoparticle - [ substrate ] n conjugates ; however , to our knowledge , there has yet to be a direct observation of two discrete populations of np B-nanoparticle - [ substrate ] n and np B-nanoparticle - [ product ] n that shrink and grow concurrently as the enzymatic reaction progresses . [SEP]
[CLS] rather , evidence has been indirect , as discussed below . [SEP]
[CLS] one of the most compelling bits of evidence is the observation that the complete turnover of qd - [ peptide ] n conjugates by proteases requires an equal or shorter amount of time as n increases , with n reaching values as high as 60 . [SEP]
[CLS] analogous results have been observed for increasing n with au np B-nanoparticle - [ oligonucleotide ] n conjugates and turnover by nucleases . [SEP]
[CLS] if only one or a small number of substrates were turned over in each encounter , as in colliding and pseudocolliding models , then larger n should require more encounters between enzyme and np B-nanoparticle and therefore a longer time for the reaction to reach completion . [SEP]
[CLS] in contrast , a hopping or pseudohopping model predicts these experimental results . [SEP]
[CLS] a potential counterargument posits that larger n progressively increases the probability of productive encounters and thus offsets the need for more encounters in a colliding or pseudocolliding model ; however , some observations are not consistent with this hypothesis . [SEP]
[CLS] the mobility B-property shifts for qd - [ peptide ] n conjugates observed by agarose B-technique gel I-technique electrophoresis I-technique diminish and become negligible as n increases , such that the effective size of the conjugates approximately saturates well before the maximum loading of peptide B-material per qd is reached , 133 so [SEP]
[CLS] invited feature article a putative larger size cannot account for the steady or increasing rate of turnover as n continues to increase . [SEP]
[CLS] moreover , when n was kept constant while the density of substrates in au np B-nanoparticle - [ oligonucleotide ] n conjugates was decreased by increasing the effective linker length between the substrate and au np B-nanoparticle , the rate of turnover by nuclease decreased despite the larger size of the conjugate . [SEP]
[CLS] the overall results of the above studies suggest that more substrate per np B-nanoparticle increases the frequency with which intersubstrate hopping occurs , as per figure 12e . [SEP]
[CLS] other evidence comes from the previously noted real and predicted adsorption or association of enzyme with np B-nanoparticle - [ substrate ] n conjugates . [SEP]
[CLS] excess components of a np B-nanoparticle - [ substrate ] n conjugate in bulk solution can also act as competitive inhibitors , as observed for certain surface chemistries of qd mixed with qd - [ peptide ] n conjugates and protease , and with ssdna mixed with au np B-nanoparticle - [ dna / rna ] n - [ ssdna ] m conjugates and rnase h . [SEP]
[CLS] these results suggest that the processes in figure 12c−e can occur , as can an analog of the process in figure 12e , between inequivalent biomacromolecules . [SEP]
[CLS] presumably , the energetics of these processes can be tuned to be either a contribution to the overall acceleration of enzyme B-property activity I-property or a deceleration that competes with other accelerating processes in figure 12 . [SEP]
[CLS] although preliminary , this idea is supported by the aforementioned observation by dı az et al . that there was secondary correlation between observed turnover rates and the calculated interaction energies between qds and trypsin . [SEP]
[CLS] certainly , a hopping mechanism is not unequivocal at this point and should not be taken as fact ; however , the results to date point in this direction , and the odds for a hopping or pseudohopping model appear to be better than for a colliding or pseudocolliding model for some types of multivalent npsubstrate conjugates . [SEP]
[CLS] the importance of enzymes as current and future biomarkers B-property and drug targets is undeniable , and nps B-nanoparticle of many kinds have both well - established and still - developing benefits for biological detection and targeting . [SEP]
[CLS] knowing more about how enzymes interact with nps B-nanoparticle will enable further advancement and innovation with respect to these application areas . [SEP]
[CLS] more fundamental research is needed and must address the complexity of the np B-nanoparticle interface and the limitations of current tools for elucidating its structure and properties . [SEP]
[CLS] so what is required to better understand the enzymatic turnover of np B-nanoparticle - substrate conjugates ? [SEP]
[CLS] one answer is more holistic studies . [SEP]
[CLS] taking our work as an example , we have utilized ensemble fret assays and gel B-technique electrophoresis I-technique supplemented by comparisons to kinetic models of enzyme B-property activity I-property and simple structural models of enzymes [SEP]
[CLS] these methods have been insightful but homogenized the np B-nanoparticle - enzyme systems and only scratched the surface of their overall complexity . [SEP]
[CLS] we have also made efforts to confirm that there is a qualitative persistence of trends across multiple batches of nps B-nanoparticle but have only been able to attribute quantitative differences in these trends to undetermined heterogeneity and batch - to - batch variation . [SEP]
[CLS] determining the root causes of batch - to - batch variation and more general differences in behavior between various np B-nanoparticle materials and methods of preparation will require an analysis that is both multifaceted and detailed : well - executed holistic studies are undoubtedly a substantial but worthwhile undertaking for developing a detailed understanding of enzymatic activity toward np B-nanoparticle - substrate conjugates . [SEP]
[CLS] aside from establishing the generality of results , a broader scope of experiments will help address domino effects and a hierarchy of trends . [SEP]
[CLS] the term " domino effect " refers to a nominal change in one property that leads to changes in other properties . [SEP]
[CLS] for example , a change in the number of macromolecular substrates per np B-nanoparticle has the potential to change not only the local concentration of substrate but also the average conformation of those substrates and the steric hindrance for enzyme−np and enzyme−substrate interactions . [SEP]
[CLS] other potential examples arise from the aforementioned propagation of changes from one layer of chemistry to another in the np B-nanoparticle - substrate conjugate : a change in the np B-nanoparticle material B-material alters the density of surface ligands , or a change in the identity of the surface ligand alters the average conformation of a substrate . [SEP]
[CLS] a hierarchy of trends will be useful for parsing domino effects by reliably predicting which of several possible effects is most important . [SEP]
[CLS] for example , steric hindrance may predominate differences in enzyme B-property activity I-property between two np B-nanoparticle - substrate conjugates with surface ligands of very different molecular weight , whereas the relative affinity of the enzyme toward the np B-nanoparticle may predominate if the surface ligands have similar molecular weight . [SEP]
[CLS] the ambition is that it will be possible to use a small number of discrete factors to predict behavior even if the real systems are influenced by a complex continuum of factors . [SEP]
[CLS] another answer to the question of how to better understand the enzymatic turnover of np B-nanoparticle - substrate conjugates is to shift away from ensemble methods and toward single - particle measurements . [SEP]
[CLS] these measurements avoid the ensemble averaging that homogenizes a heterogeneous system and are anticipated to reveal mechanistic details essential to the modeling of kinetics , including the determination of a hopping , colliding , or pseudomodel of turnover . [SEP]
[CLS] any distinct subpopulations of nps B-nanoparticle within an ensemble will also be revealed . [SEP]
[CLS] a second useful approach will be separation methods that divide a heterogeneous ensemble of nps B-nanoparticle into semihomogeneous fractions for enzymatic assays . [SEP]
[CLS] a comparison of progress curves or other data for these fractions versus the ensemble , in combination with a knowledge of the property by which the nps B-nanoparticle were fractionated , will provide insight into correlations between np B-nanoparticle properties and enzymatic activity . [SEP]
[CLS] in the same vein , highthroughput and automated methods for the preparation of langmuir libraries of np B-nanoparticle - substrate conjugates and for assays of enzymatic activity toward these conjugates will enable machine learning methods for elucidating how various properties affect enzymatic activity . [SEP]
[CLS] machine learning methods have , for example , already been used for applications in organic synthesis , 152−154 materials development , prediction of protein−ligand binding , and determination of the substrate specificity of enzymes . [SEP]
[CLS] an expanded role for computational modeling and simulation is also warranted . [SEP]
[CLS] there is the potential for this role to go beyond energy minimization and molecular dynamics modeling of enzyme−np interactions at a homogenized interface and instead address heterogeneity at individual facets , between subpopulations of nps B-nanoparticle in an ensemble , and between np B-nanoparticle identities . [SEP]
[CLS] these interactions can then be further extrapolated to simulate substrate turnover and ultimately produce a theoretical progress curve for an ensemble that can be matched to experimental data . [SEP]
[CLS] inference of mechanistic details then becomes possible through iterative tweaks of the parameters of the models until matched to experiments . [SEP]
[CLS] moreover , simulation is perhaps the best available method of extracting facet - dependent behaviors with nps B-nanoparticle . [SEP]
[CLS] these behaviors are potentially important , as has recently come to light with bulk interfacial systems with tethered biomolecules . [SEP]
[CLS] a detailed understanding of enzymatic activity toward npsubstrate conjugates leads to another question : what benefits will come from this fundamental knowledge ? [SEP]
[CLS] in the context of in vitro molecular diagnostics , more sensitive and more rapid assays of enzyme biomarkers B-property are possible with a np B-nanoparticle - substrate conjugate that optimizes and accelerates enzyme B-property activity I-property and concurrently resists or slows fouling with a protein B-material corona I-material in biological fluid . [SEP]
[CLS] such a material B-material has substantial value for point - of - care applications in particular . [SEP]
[CLS] another useful lens through which to view this question is the control that in vivo biological systems exert over enzyme B-property activity I-property toward substrates , mirrored against the comparatively poor substrate specificity when the same enzymes are used in vitro . [SEP]
[CLS] with a detailed understanding of np−enzyme B-nanoparticle and substrate− enzyme interactions , there is the potential for the rational design of a synthetic conjugate that achieves some of the in vivo substrate specificity of an enzyme : for example , a substrate optimized for the active site of an enzyme ( e . g . , amino B-material acid I-material sequence for a protease ) in combination with surface chemistry on the np B-nanoparticle that interacts either favorably with the target enzyme or unfavorably with nontarget enzymes of similar substrate specificity . [SEP]
[CLS] our observation that the functionalization of a qd surface with mpa ligands can virtually shut down thrombin activity but retain trypsin activity is an illustration of this general concept , albeit not suitable for biomedical application . [SEP]
[CLS] we also have recent results that show that it is possible to rationally functionalize a np B-nanoparticle to accelerate target enzyme B-property activity I-property rather than decelerate nontarget enzyme B-property activity I-property . [SEP]
[CLS] this behavior arose from cofunctionalizing a qd with a substrate for thrombin and peptide B-material fragments of a receptor B-material that allosterically interacted with thrombin to enhance its activity , although it was still strongly dependent on qd surface chemistry . [SEP]
[CLS] facet - dependent functionalization and interactions with enzymes , coupled with the synthesis of anisotropic nps B-nanoparticle such as plates and rods , may prove to be yet another route to generating selectivity . [SEP]
[CLS] overall , enhanced specificity will benefit in vitro assays and cellular and in vivo imaging probes for fundamental biological research and clinical application . [SEP]
[CLS] a better understanding of enzymatic activity toward nps B-nanoparticle also benefits enzyme - mediated drug delivery and theranostics . [SEP]
[CLS] here , the concept is that a substrate tethers a drug or prodrug payload to a np B-nanoparticle for release via enzymatic activity at the site of disease . [SEP]
[CLS] although organic nps B-nanoparticle such as liposomes B-nanoparticle and polymer B-material materials predominate np B-nanoparticle - mediated drug delivery , inorganic nps B-nanoparticle are also of interest and stand to enable chemotherapy in combination with other therapies ; for example , au nps B-nanoparticle and iron B-material oxide I-material nps B-nanoparticle have prospective application in photothermal and magnetic B-property hyperthermia therapies , respectively . [SEP]
[CLS] in addition , magnetic B-property nps B-nanoparticle and lanthanide B-material nps B-nanoparticle are also of interest as contrast B-technique agents I-technique for imaging . [SEP]
[CLS] current qd materials are of little interest for clinical drug delivery but are nonetheless promising as model np B-nanoparticle vectors for fundamental research on drug delivery . [SEP]
[CLS] with aberrant enzyme B-property activity I-property as a hallmark of many diseases ( e . g . , kinases and proteases in cancers and neurodegenerative disease ) [SEP]
[CLS] , the control of enzymatic activity toward np B-nanoparticle - substrate conjugates represents a possible means of controlling the location and rate of drug delivery . [SEP]
[CLS] moreover , there is also the possibility of using nps B-nanoparticle as a scaffold for enzyme inhibitors ( as many drugs are ) , analogous to what has been discussed at length for the np B-nanoparticle as a scaffold for enzyme substrates . [SEP]
[CLS] the high local concentration of inhibitor ( or drug ) at the np B-nanoparticle interface may increase potency , particularly when combined with np B-nanoparticle surface chemistry that is tailored to be very favorable and , ideally , somewhat selective for adsorption of the target enzyme . [SEP]
[CLS] to conclude , the activity B-property of I-property enzymes I-property toward np B-nanoparticle - substrate conjugates is an interesting and important avenue of research . [SEP]
[CLS] a full understanding of such systems represents a complex and nuanced problem to solve but will enable important advances in biomedical research and development and will be another step toward clinical applications of nps B-nanoparticle . [SEP]
[CLS] success in solving this problem will require a concerted effort that leverages a large variety of tools for colloid and interface science , with several potential rewards to reap . [SEP]
[CLS] the supporting information is available free of charge on the acs publications website at doi : 10 . 1021 / acs . langmuir . 8b02733 . [SEP]
[CLS] graphical summary of this article . [SEP]
[CLS] a detailed understanding of the turnover of multivalent np B-nanoparticle - substrate conjugates by enzymes will require a convergence of biochemistry , materials , and interface science . [SEP]
[CLS] the development of predictive models will significantly advance the applications of these conjugates , including assays , sensors , and imaging probes , as well as enzyme - mediated drug delivery . [SEP]
[CLS] ribbon and surface diagrams for the comparison of selected structural features between four members of the serine B-material protease family : chymotrypsin , trypsin , thrombin , and plasmin . [SEP]
[CLS] the ser - his - asp catalytic triads are colored red , the s1 substrate binding pockets are colored green , and select loops are colored yellow and orange . [SEP]
[CLS] the loops for chymotrypsin and trypsin are the l1 and l2 loops , which play a role in determining substrate specificity . [SEP]
[CLS] for thrombin , the loops are the 60 - loop and the γ - loop , which contribute to substrate binding . [SEP]
[CLS] loop - 5 is highlighted for plasmin and is most analogous to the 60 - loop for thrombin . [SEP]
[CLS] in addition , important residues for exosites i and ii are highlighted in blue and cyan for thrombin , and the five kringle domains are highlighted in various shades of blue for plasmin . [SEP]
[CLS] surfaces that are colored according to hydrophobicity B-property and electrostatic potential are also shown . [SEP]
[CLS] 3 . aspects of the interfacial chemistry of au nps B-nanoparticle and qds . [SEP]
[CLS] ( a ) general structure of a ligand for functionalizing a np B-nanoparticle and its organization at the interface of a np B-nanoparticle . [SEP]
[CLS] the examples of functional groups are not meant to be comprehensive in scope . [SEP]
[CLS] ( b ) cartoon of an idealized spherical 13 - nmdiameter au np B-nanoparticle ( left ) and two examples of how a real crystal may be faceted ( cuboctahedron , middle ; icosahedron , right ) . [SEP]
[CLS] chemical structures of citrate ligand 1 , and selected examples of ligands with thiol B-material anchors for tuning the surface chemistry , largely via the distal functionality . [SEP]
[CLS] 62−64 ( c ) cartoons of a wurtzite 3 . 5 - nm - diameter cdse qd ( far left ) and a 7 - nm - diameter cdse / zns ( middle right ) , both idealized to be spherical , and two examples of how real qds may be faceted ( middle left and far right ) . [SEP]
[CLS] facets that are charged ( terminated with either cd 2 + / zn 2 + or se 2− / s 2− ) or neutral ( terminated with both metal B-material and chalcogen ions B-material ) are highlighted . [SEP]
[CLS] chemical structures of selected examples of ligands with thiol B-material and dithiol anchors for tuning the surface chemistry of qds . [SEP]
[CLS] 74−81 for 12 , the average value of n typically varies from n = 12 to 15 . [SEP]
[CLS] the common names of the ligands shown for both au nps B-nanoparticle and qds can be found in the si . [SEP]
[CLS] invited feature article doi : 10 . 1021 / acs . langmuir . 8b02733 langmuir 2019 , 35 , 7067−7091 [SEP]
[CLS] 4 . high local concentration of a substrate around a np B-nanoparticle . [SEP]
[CLS] ( a ) simple model of a np B-nanoparticle - [ substrate ] n conjugate : the np B-nanoparticle has a radius r , and there are n substrates per np B-nanoparticle that occupy a local volume that extends radially 10 nm from the np B-nanoparticle surface . [SEP]
[CLS] the saturation of the np B-nanoparticle surface is based on a footprint of f ≈ 4 nm 2 for each substrate . [SEP]
[CLS] ( b ) plot of the local concentration as a function of np B-nanoparticle diameter and the value of n . details of the calculation can be found in the si . [SEP]
[CLS] examples of energy transfer ( et ) - based ( e . g . , fret , electron transfer ) assay formats for tracking enzymatic activity toward npsubstrate conjugates . [SEP]
[CLS] ( a ) release of an energy transfer partner from a np B-nanoparticle through cleavage of a labeled substrate by a hydrolase . [SEP]
[CLS] ( b ) conversion of a label from an energy transfer - active form to an inactive form ( or vice versa , not shown ) by an oxidoreductase . [SEP]
[CLS] ( c ) labeling of a substrate with an energy transfer partner by a transferase . [SEP]
[CLS] in principle , assay formats similar to those in panels a and c can be used for tracking lyase and ligase activity . [SEP]
[CLS] ( a ) hypothetical progress curves for the enzymatic turnover of np B-nanoparticle - [ substrate ] n conjugates with two different np B-nanoparticle materials and three enzyme concentrations . [SEP]
[CLS] measurement of only ( i ) the initial rates does not reveal significant differences between the two np B-nanoparticle materials , whereas the measurement of ( ii ) full progress curves clearly reveals differences . [SEP]
[CLS] the dashed box in panel ii indicates the region in panel i . [SEP]
[CLS] ( b ) kinetic data for the turnover of qd - [ peptide B-material substrate ] 13 conjugates by trypsin : ( left ) standard progress curves for different concentrations of trypsin ( 0 , 1 . 3−687 nm , scaling by a factor of 2 from purple to red ) and ( right ) progress curves plotted in enzyme time . [SEP]
[CLS] the blue line is the best fit to the mm model . [SEP]
[CLS] the red line is the mm prediction from assays of substrate only . [SEP]
[CLS] the inset shows a close - up of data early in the progress curves . [SEP]
[CLS] reproduced from ref 115 . [SEP]
[CLS] copyright 2012 american chemical society [SEP]
[CLS] illustrations of the progressive enzymatic turnover of np B-nanoparticle - [ substrate ] n conjugates according to the ( a ) colliding model and ( b ) hopping model . [SEP]
[CLS] for each model , cartoon snapshots are shown for the ( sub ) populations of np B-nanoparticle - substrate / product conjugates at multiple time points in the reaction as well as the poisson distribution ( s ) of the number of substrates per individual np B-nanoparticle , n , for the average values , n . [SEP]
[CLS] the reaction time listed for each value of n corresponds to the hypothetical progress curves in figure 8 . [SEP]
[CLS] 8 . thought experiment illustrating two different quantitative results for the same raw fret assay data : ( a ) raw data measured as qd and dye pl intensity and their ratio versus time ; ( b ) calibration data for the assumption of colliding and hopping models ; and ( c ) resulting progress curves . [SEP]
[CLS] details of the derivation of the data can be found in the si . [SEP]
[CLS] protease adsorption affects the turnover of multivalent qd - peptide substrate conjugates . [SEP]
[CLS] ( a ) simplified cartoon of the conjugates with dyelabeled peptide B-material substrates for a fret assay and four different surface ligands . [SEP]
[CLS] representative progress curves for the turnover of the gsh - qd - [ peptide B-material substrate ] 10 conjugates by trypsin and plasmin . [SEP]
[CLS] ( e1−e5 are different concentrations of enzyme , and c is a control without enzyme . [SEP]
[CLS] ) trypsin shows one - phase / convergent progress curves ; plasmin shows two - phase / nonconvergent progress curves . [SEP]
[CLS] the asterisk ( * ) highlights convergence . [SEP]
[CLS] ( b ) simplified cartoon of the conjugates ( ligands represented as a mesh shell B-material ) with added biomacromolecules and a dye - labeled peptide B-material substrate , sub ( a647 ) . [SEP]
[CLS] representative progress curves for x - qd - [ sub ( a647 ) ] 10 - [ sub ] n . [SEP]
[CLS] the progress curves show an enhancement in the rate or extent of substrate turnover . [SEP]
[CLS] ( c ) comparison of progress curves with plasmin for x - qd - [ peptide B-material substrate ] 10 conjugates with x = gsh versus dhla - sb and dhla - peg . [SEP]
[CLS] dhla - sb and dhla - peg convert the progress curves from two - phase / nonconvergent with gsh to one - phase / convergent . [SEP]
[CLS] ( d ) agarose gels illustrating the correlation between protease adsorption and kinetics : ( i ) comparison of trypsin ( weaker ) and plasmin ( stronger ) adsorption on gsh - qd ; ( ii ) dhla - qd - [ peptide ] 40 conjugates have lower adsorption than qd only ( cf . panel i ) ; and ( iii ) low adsorption of trypsin on dhla - sb - qd and dhla - sb - qd - [ peptide ] 20 conjugates . [SEP]
[CLS] adapted from ref 133 . [SEP]
[CLS] copyright 2017 american chemical society [SEP]
[CLS] 10 presents simple models of the sterically unhindered dhla - cl4 - qd - [ peptide substrate ] n conjugate and the hindered dhla - peg - ome - qd - [ peptide B-material substrate ] n conjugate as well as outputs of langmuir invited feature article doi : 10 . 1021 / acs . langmuir . 8b02733 langmuir 2019 , 35 , 7067−7091 the molecular dynamics simulations for sasa and adsorption energies ( vide supra ) . [SEP]
[CLS] 10 . [SEP]
[CLS] illustration of steric versus adsorption effects for the turnover of x - qd - [ peptide B-material substrate ] 7−12 conjugates for x = dhla - cl4 and x = dhla - peg - ome ( abbreviated as cl4 and ome ) . [SEP]
[CLS] ( a ) simple models of the conjugates . [SEP]
[CLS] ( b ) simulation results for the determination of solvent - accessible surface area ( sasa ) for the cl4 and ome ligands . [SEP]
[CLS] ( c ) simulation results for the adsorption of trypsin on the same surface chemistries . [SEP]
[CLS] ( d ) plots of sasa and interaction energy ( corresponding to panels b and c , respectively ) for four different ligands : dhla - cl4 , dhla - peg - cl4 , dhla - peg - ome , and dhla - peg - nh 2 . [SEP]
[CLS] sterics appeared to be the primary determinant of kinetics , with adsorption playing a secondary role . [SEP]
[CLS] adapted from ref 79 . [SEP]
[CLS] copyright 2017 american chemical society [SEP]
[CLS] 11 . [SEP]
[CLS] impact of monovalent cation B-material concentration in the interfacial environment of a multivalent np B-nanoparticle - oligonucleotide conjugate on nuclease activity . [SEP]
[CLS] ( a ) although dnase i has a higher affinity for a au np B-nanoparticle - dna conjugate versus an isolated oligonucleotide , the rate of hydrolysis is slower . [SEP]
[CLS] ( b ) progress curves for the assays illustrated in panel a . panels a and b are reproduced from ref 121 . [SEP]
[CLS] copyright 2009 american chemical society . [SEP]
[CLS] ( c ) schematic of a protein - spherical nucleic B-material acid I-material conjugate ( left ) and radial profile of monovalent cation B-material concentration from the center of the protein B-material . [SEP]
[CLS] a similar profile is expected for au np B-nanoparticle - [ oligonucleotide ] n conjugates . [SEP]
[CLS] reproduced from ref 147 . [SEP]
[CLS] copyright 2018 american chemical society [SEP]
[CLS] 12 . [SEP]
[CLS] illustrations of the potential mechanisms through which the turnover of a substrate is made more efficient by multivalent conjugation to a np B-nanoparticle . [SEP]
[CLS] ( a ) the np B-nanoparticle - [ substrate ] n conjugate has a larger collisional cross - section versus discrete substrate molecules . [SEP]
[CLS] ( b ) the close proximity between multiple substrates increases the likelihood of a collision with a productive orientation of enzyme . [SEP]
[CLS] ( c ) the enzyme collides with the np B-nanoparticle and diffuses along its surface to find the substrate . [SEP]
[CLS] ( d ) reversible adsorption to the np B-nanoparticle surface facilitates enzyme reassociation with substrate after dissociation . [SEP]
[CLS] ( e ) the close proximity between multiple substrates facilitates enzyme reassociation with substrate after dissociation . [SEP]
[CLS] the importance of each of these mechanisms is likely to depend on the details of the conjugate . [SEP]
[CLS] overview of cited studies on the turnover of np B-nanoparticle - substrate conjugates by enzyme [SEP]
[CLS] of ligands from figure 3 , calculation of local concentration , additional discussion of substrate design and properties , and calculations for the colliding versus hopping thought experiment ( pdf ) acknowledges the izaak walton killam memorial fund for a postdoctoral research fellowship and the university of glasgow for a lord kelvin adam smith fellowship . [SEP]
[CLS] advances in nanoparticle B-nanoparticle design have led to the development of nanoparticulate systems that can sense intracellular molecules , alter cellular B-event processes I-event , and release drugs to specific targets in vitro . [SEP]
[CLS] in this work , we demonstrate that oligonucleotide - coated gold B-nanoparticle nanoparticles I-nanoparticle are suitable for the detection of mrna in live hydra vulgaris , a model organism , without affecting the animal ' s integrity . [SEP]
[CLS] we specifically focus on the detection of hymyc1 mrna , which is responsible for the regulation of the balance between stem cell B-material self - renewal and differentiation . [SEP]
[CLS] myc deregulation is found in more than half of human cancers , thus the ability to detect in vivo related mrnas through innovative fluorescent B-property systems is of outmost interest . [SEP]
[CLS] in recent years , gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) have been at the forefront of scientific research because of their attractive properties , which stem from their tunable shape , size , and ligand functionalization . [SEP]
[CLS] in particular , spherical aunps B-nanoparticle have been extensively utilized because of their ease of synthesis as well as the ability to easily modify their surface with a variety of functional ligands using au−s chemistry . [SEP]
[CLS] 2−6 among various types of functional ligands , aunps B-nanoparticle coated with synthetic oligonucleotides are very attractive for biomedical applications because they combine the unique properties of oligonucleotides , such as selectivity and specificity , with the optoelectronic properties of the gold B-material core B-material . [SEP]
[CLS] oligonucleotide−aunp hybrids present enhanced colloidal stability , high cellular uptake without the requirement of cocarriers , and resistance to enzymatic degradation , advantages that have been explored for oligonucleotide detection as well as drug delivery in cells B-material . [SEP]
[CLS] specific detection is achieved by careful design considerations , which include the appropriate choice of oligonucleotides in terms of length and base content as well as the proper oligonucleotide density on the aunp B-nanoparticle surface , with the aim on the one hand to prevent nanoprobe B-nanoparticle degradation by enzymes and on the other hand to retain oligonucleotide functionality and nanoparticle B-nanoparticle stability . [SEP]
[CLS] well - designed oligonucleotidecoated nanoparticles B-nanoparticle have been shown to be effective in the regulation of gene expression . [SEP]
[CLS] for example , rosi et al . demonstrated the use of oligonucleotide - coated nanoprobes B-nanoparticle for the downregulation of enhanced green fluorescent B-property protein B-material ( egfp ) . [SEP]
[CLS] upon uptake , a significant knockdown of the gene was observed in a mouse endothelial cell B-material line ( c166 ) . [SEP]
[CLS] on the other hand , giljohann et al . demonstrated the significant downregulation of luciferase in hela cells B-material and cutler et al . showed how such systems could silence the epidermal growth B-material factor I-material receptor I-material ( egfr ) in scc12 cells B-material . [SEP]
[CLS] furthermore , gene silencing has also been successfully demonstrated in vivo where jensen et al . designed oligonucleotide - coated nanoprobes B-nanoparticle as a rnai therapy of glioblastoma multiform ( gbm ) by targeting and knocking down bcl2l12 mrna and the associated protein B-material levels , which tend to be overexpressed in gbm . [SEP]
[CLS] following this study , sita et al . demonstrated how the commonly administered drug for the treatment of gbm , temozolimide ( tmz ) , could be rendered more efficient by knocking down o6 - methylguanine - dna - methyltransferase ( mgmt ) , a protein B-material that hinders the drugs ' mechanism of action . [SEP]
[CLS] this is an open access article published under a creative commons attribution ( cc - by ) license , which permits unrestricted use , distribution and reproduction in any medium , provided the author and source are cited . [SEP]
[CLS] fuente and co - workers also reported the use of sirna nanoprobes B-nanoparticle to target tumor B-material cells B-material in lung cancer models via the downregulation of c - myc . [SEP]
[CLS] they succeeded to induce rnai both in vitro and in vivo , developing multiple strategies to bind sirna to the gold B-nanoparticle nanoparticle I-nanoparticle core B-material and achieving up to 80 % of gene downregulation . [SEP]
[CLS] oligonucleotide−gold nanoparticle B-nanoparticle hybrids have also been used for the detection of intracellular targets including microrna and mrna . mirkin and co - workers showed the utilization of such nanoprobes B-nanoparticle for the detection of survivin mrna as well as the detection of genetic markers of circulating tumor B-material cells B-material ( ctc ) in human blood including mesenchymal markers such as twist , vimentin , and fibronectin and the epithelial marker e - cadherin . [SEP]
[CLS] similarly , yang et al . used fret oligonucleotide - coated gold B-nanoparticle nanoparticles I-nanoparticle to target tk1 mrna , a target associated with cell B-event division I-event and tumor B-material growth , in hepg2 , mcf - 7 and l02 cells B-material , whereas wright and co - workers made use of hairpin dna - coated aunps B-nanoparticle for the detection of specific mrna sequences for glyceraldehyde 3phosphate dehydrogenase ( gapdh ) in hep - 2 cells B-material and the respiratory syncytial virus ( rsv ) in live rsv infected hep - 2 cells B-material . [SEP]
[CLS] on the other hand , zhou et al . used a hairpin system focusing on the detection of mdr1 mrna , which has been associated with the prediction of multidrug resistance of tumor B-material cells B-material . [SEP]
[CLS] moreover , research by our group has showed that oligonucleotide - coated aunps B-nanoparticle can be specifically designed to detect vimentin , desmocollin , and keratin8 mrnas , targets associated with the process of epithelial to mesenchymal transition ( emt ) in live cells B-material , whereas the detection of vimentin mrna was also successfully demonstrated in models of wounded skin . [SEP]
[CLS] this nanoparticle B-nanoparticle design has been extended beyond the confines of single target detection to achieve the simultaneous imaging of multiple mrna targets . [SEP]
[CLS] prigodich et al . showed the simultaneous detection of two targets , survivin and actin , by monitoring two separate fluorescence B-property outputs . [SEP]
[CLS] tang and co - workers have presented several studies focusing on multiplexed detection including the imaging of c - myc , tk1 , and galnac - t mrna in vitro in a number of different cell B-material lines as well as the simultaneous fluorescence B-property visualization of survivin and cyclin d1 mrna in sk - br - 3 and mcf - 10a cells B-material . [SEP]
[CLS] furthermore , work by our group has also demonstrated how vimentin and keratin8 mrna can be simultaneously detected via the use of aunp B-nanoparticle dimers . [SEP]
[CLS] apart from mrna , microrna has also been detected using oligonucleotide - coated nanoprobes B-nanoparticle . [SEP]
[CLS] tu et al . demonstrated the detection of mir - 122 in huh7 cells B-material using hairpin dna − coated aunps B-nanoparticle . [SEP]
[CLS] this target constitutes 70 % of the microrna in the liver and its potential reduction has been associated with hepatocellular carcinoma . [SEP]
[CLS] furthermore , huang et al . showed how two microrna targets , mir - 21 and mir - 141 microrna , could be simultaneously detected in live cancer cells B-material including hela cells and love - 1 cells . [SEP]
[CLS] although there is a significant amount of research focusing on the use of oligonucleotide - coated gold B-nanoparticle nanoparticles I-nanoparticle in in vitro systems there is less work associated with in vivo systems , which is highly important but experimentally more challenging . [SEP]
[CLS] in this work we demonstrate the targeted detection of mrna using oligonucleotide - coated gold B-nanoparticle nanoparticles I-nanoparticle in the freshwater polyp hydra vulgaris . [SEP]
[CLS] hydra is a small invertebrate classically used as model in developmental biology , which has also recently emerged as an amenable system to test the toxicity B-property and bioactivity of novel functional biomaterials and nanodevices B-nanoparticle , 32−39 reducing ethical concerns and economic costs related to vertebrate experimentation . [SEP]
[CLS] hydra structural anatomy , which presents a tissue grade organization with no organs or biological fluids , allows the study of the interaction between any medium suspended compound and the entire body cell B-material repertoire in a fast , simple , and reliable way . [SEP]
[CLS] the polyp is structured as a hollow and transparent tube with a basal foot and a mouth ( hypostome ) surrounded by a ring of tentacles in the apical zone ( see figure s1 ) . [SEP]
[CLS] the body wall is composed by two epithelial layers , an ectoderm facing outward , and an endoderm facing the inner cavity , plus a limited number of differentiated cell B-material types derived from interstitial stem cells B-material , a fast cycling pool of cells located in the central part of the body column . [SEP]
[CLS] these multipotent stem cells B-material can occur singly ( 1s ) or in clusters of 2 , 4 , 8 , and 16 cells B-material ( 2s , 4s , 8s , 16s ) and undergo self - renewal or differentiation pathways into either nematocytes , the stinging cells B-material characterizing the phylum , or neurons ( sensory and ganglionic ) , secretory cells B-material ( gland cells and mucous cells ) , and gametes . [SEP]
[CLS] interstitial cells B-material entering a nematocyte pathway undergo different cell B-material divisions ( 4s , 8s , 16s ) , which results in nests of cells B-material connected to each other by cytoplasmic bridges ( nematoblasts ) ( see figure s1 ) . [SEP]
[CLS] among the numerous genes controlling the stem cell B-material differentiation pathway , hymyc 1 is specifically expressed in nests of dividing nematoblasts and gland cells B-material , and it is involved in regulating the balance between stem cell B-material self - renewal and differentiation the well recognizable and defined expression pattern of hymyc 1 in hydra , its importance and the availability of its mrna sequence prompted us to select this gene for the real time study of mrna expression , in vivo . [SEP]
[CLS] the gene belongs to the myc proto - oncogene family ( c - myc , n - myc , and l - myc ) of transcription B-event factors controlling fundamental cellular B-event processes I-event including proliferation , growth , differentiation , metabolism , or apoptosis B-event , conferring high translational value to our study . [SEP]
[CLS] as myc deregulated expression occurs in the majority of human cancers , the availability of optimized detection methods may be of interest for the wide scientific community targeting c - myc for therapeutic purposes . [SEP]
[CLS] ■ experimental section synthesis of 13 . 9 ± 1 . 4 nm aunps B-nanoparticle . [SEP]
[CLS] a solution of sodium B-material tetrachloroaurate ( 1 mm , 100 ml ) was brought to the boil while stirring ( 700 rpm ) . [SEP]
[CLS] a solution of sodium B-material citrate ( 2 % wt , 5 ml ) was then injected into the gold B-material solution . [SEP]
[CLS] following a solution color change , stirring ( 700 rpm ) was continued for further 15 min . [SEP]
[CLS] once the reaction mixture reached room temperature , a solution of bissulfonatophenylphosphine ( bspp , 42 mg in 2 ml of milli - q water B-material ) was added and the solution was left to stir overnight to ensure successful ligand replacement . [SEP]
[CLS] the resulting bspp - coated spherical aunps B-nanoparticle were passed through a 0 . 45 ( μm ) millipore filter to remove large aggregates and further purified by two rounds of centrifugation ( 10 000 rpm , 20 min ) . [SEP]
[CLS] purification was assisted via the gradual addition of a concentrated nacl solution until a color change from red to blue was observed indicating particle precipitation . [SEP]
[CLS] synthesized aunps B-nanoparticle were finally redispersed in 3 ml of milli - q water B-material and stored at 4 °c . [SEP]
[CLS] oligonucleotide design . [SEP]
[CLS] sequences for hymyc1 - nanoprobes B-nanoparticle were designed based on hymyc 1 gene sequence ( genbank accession no . gq856263 ) . [SEP]
[CLS] " sense " strands were designed to have a length of 29 bases ( target sequence including a polya tail , see table s1 ) with a gc content < 50 % . [SEP]
[CLS] the " flare " strands were designed to have a melting temperature of > 40 °c and a length of 10 bases ( see table s1 for sequences ) . [SEP]
[CLS] the basic local assignment search tool ( blast ) tool was used to assess specificity and absence of off target sequences synthesis of dna - aunps B-nanoparticle for mrna detection . [SEP]
[CLS] synthesized aunps B-nanoparticle were modified with a shell B-material of oligonucleotide " sense " strands designed to detect specific mrna targets by adopting a salt - aging approach . [SEP]
[CLS] briefly , bspp - coated aunps B-nanoparticle in water B-material ( 10 nm , 1 ml ) were incubated B-technique with a solution of thiol - terminated oligonucleotide " sense " strands ( 3 μm , 1 ml ) and were left to shake for 24 h . bspp ( 1 mg / 20 μl , 10 μl ) was then added to the reaction mixture along with phosphate buffer ( 0 . 1 m , ph 7 . 4 ) and sds ( 10 % ) in order to achieve a final concentration of 0 . 01 m and 1 % of phosphate buffer and sds , respectively . [SEP]
[CLS] successful oligonucleotide attachment was then achieved by gradually increasing the salt B-material concentration . [SEP]
[CLS] six additions of nacl ( 2 m ) were performed over an 8 h period resulting in a final salt B-material concentration of 0 . 3 m . resulting oligonucleotide - coated aunps B-nanoparticle were purified by three rounds of centrifugation ( 16 400 rpm , 20 min ) and stored at 4 °c in hybridization buffer ( 5 mm phosphate buffer , 80 mm nacl ) . [SEP]
[CLS] oligonucleotide " sense " strands were hybridized to their complementary " flare " strands by incubating B-technique a solution of oligonucleotide - coated " sense " strands ( 40 nm , 500 μl ) , with an excess of the complementary " flare " strand ( 2 . 4 μm , 500 μl ) . [SEP]
[CLS] the solution was then heated to 65 °c for 5 min followed by slow cooling to room temperature . [SEP]
[CLS] the resulting probes were purified by two rounds of centrifugation ( 16 400 rpm , 15 min ) and finally redispersed in phosphate buffer saline ( pbs ) . [SEP]
[CLS] culture of animals . [SEP]
[CLS] hydra vulgaris was asexually cultured in hydra medium ( 1 mm cacl 2 and 0 . 1 mm nahco 3 ) at ph 7 . [SEP]
[CLS] the animals were kept at 18 ± 1 °c and fed thrice a week with freshly hatched artemia salina nauplii . [SEP]
[CLS] toxicity B-property assay . [SEP]
[CLS] groups of 10 polyps were placed in a plastic multiwell and incubated B-technique with the aunp B-nanoparticle probes ( 10 nm , 300 μl ) for 24 h . [SEP]
[CLS] after washing the polyps with hydra medium , the animals morphology was monitored using a stereomicroscope ( olympus szx - rfl2 ) and potential adverse effects were ranked assigning a numerical score as previously described . [SEP]
[CLS] in vivo imaging . [SEP]
[CLS] groups of 10 polyps from homogeneous populations were selected for the experiments and incubated B-technique with aunp B-nanoparticle probes ( 10 nm , 300 μl ) for 3 h in hydra medium . [SEP]
[CLS] animals were kept at 18 °c and protected from light . [SEP]
[CLS] following extensive washing , in vivo imaging was accomplished using an inverted fluorescence B-property microscope ( dmi 6000 , leica equipped with a leica dfc360fx camera ) or a nikon eclipse tie . [SEP]
[CLS] images were acquired with a cy3 / tritc filtercube ( λ exc = 552 nm , λ em = 578 nm ) and a fitc filtercube ( λ exc = 489 nm , λ em = 508 nm ) . [SEP]
[CLS] images were taken under the same conditions of acquisition ( light and exposure time ) and analysis was performed using the las as , nikon tsi and imagej software systems . [SEP]
[CLS] at least 4 biological replicas were carried out . [SEP]
[CLS] design and synthesis of dna - aunps B-nanoparticle . [SEP]
[CLS] 1 demonstrates the main principle of mrna detection using the dna - coated nanoprobes B-nanoparticle . [SEP]
[CLS] aunps B-nanoparticle were functionalized with thiol modified oligonucleotides , which have a terminal fam dye ( for simplicity these oligonucleotides are termed " sense " strands ) . [SEP]
[CLS] a shorter oligonucleotide strand modified with a cy3 dye ( termed " flare " strand ) was hybridized to the " sense " strand . [SEP]
[CLS] because of the close proximity of both dyes to the aunp B-nanoparticle surface their fluorescence B-property was suppressed by the gold B-material core B-material ( off state ) . [SEP]
[CLS] in the presence of the target mrna complementary to the sense strand , the flare strand was released from the nanoparticle B-nanoparticle , leading to an increase in its fluorescence B-property signature ( cy3 , on state ) that could be detected in live hydra via fluorescence B-technique microscopy I-technique . [SEP]
[CLS] in this study , dna - coated aunps B-nanoparticle were designed and synthesized for the targeted detection of hymyc1 mrna in hydra vulgaris . [SEP]
[CLS] by adopting a gradual salt - aging approach , we functionalized 14 ± 1 nm aunps B-nanoparticle with a layer of " sense " oligonucleotide strands ( see figures s3 and s4 and table s2 for qualitative characterization ) . [SEP]
[CLS] all dna - aunps B-nanoparticle were thoroughly characterized to determine successful " sense " strand attachment . [SEP]
[CLS] through degradation of the gold B-material core B-material and quantitative analysis of the oligonucleotide in solution it was determined that each aunp B-nanoparticle was coated with approximately 110 oligonucleotide strands with no significant variation for nanoparticles B-nanoparticle incubated B-technique with other sequences ( see table s3 for quantitative characterization ) . [SEP]
[CLS] for mrna detection , each nanoprobe B-nanoparticle consisted of approximately 60× " flare " strands as shown in table s4 , where hybridization was also assessed via fluorescence B-property melting analysis as seen in figure s5 . [SEP]
[CLS] hydra polyps were incubated B-technique with three types of nanoprobes B-nanoparticle . [SEP]
[CLS] the first batch was designed to detect the hymyc1 mrna ( hymyc1 nanoprobe B-nanoparticle ) , the second batch was designed with a scramble sequence ( scramble nanoprobe B-nanoparticle ) that does not detect any mrna in hydra ( negative control ) , and the third batch was designed to detect all intracellular mrna ( positive control ) . [SEP]
[CLS] the positive control ( gmrna nanoprobes B-nanoparticle ) was designed to display a polyt " sense " and polya " flare " strand capable of detecting all mature mrna via their characteristic polya tail ( see table s1 for detailed sequences ) . [SEP]
[CLS] 2 shows the strategy of our experiment . [SEP]
[CLS] upon incubation B-technique and uptake of the hymyc1 nanoprobes B-nanoparticle in live animals , the presence of the specific mrna would result in the targeted displacement of the " flare " strand . [SEP]
[CLS] on the other hand , in the case of the scramble nanoprobe B-nanoparticle , the absence of the target mrna would result in the lack of displacement of the " flare " strand , which would remain bound to its complementary " sense " strand . [SEP]
[CLS] prior to the experiment , an assessment of toxicity B-property was performed by incubating B-technique living polyps with the nanoprobes B-nanoparticle for 24 h . [SEP]
[CLS] the induction of putative morphological damages was assessed by assigning numerical scores to progressive morphological alteration . [SEP]
[CLS] the score ranges from 0 , where the polyp is disintegrated to 10 , where the polyps demonstrate an extended body and tentacles . [SEP]
[CLS] our research showed that none of the nanoprobes B-nanoparticle used throughout this study caused evident morphological changes after a 24 h incubation B-technique period , where all the polyp morphological scores were equal to 10 . [SEP]
[CLS] the high biocompatibility B-property of the gold B-material nanoprobes B-nanoparticle found in this study is in line with previous reports , which demonstrated that scheme 1 . schematic illustration of nanoprobe B-nanoparticle function a a when in close proximity to the aunp B-nanoparticle surface , the fluorescence B-property signal of the dyes on both the " sense " and " flare " strands is quenched . [SEP]
[CLS] upon mrna detection , competitive hybridization leads to the displacement of the " flare " strand and the concomitant restoration of its fluorescence B-property signature , which can be detected via fluorescence B-technique microscopy I-technique . [SEP]
[CLS] aunps B-nanoparticle are not toxic B-property to hydra even at higher concentrations and longer incubation B-technique times . [SEP]
[CLS] detection of hymyc1 mrna in hydra . [SEP]
[CLS] 1 shows that after 3 h of incubation B-technique , a red fluorescence B-property signal was observed in animals treated with the nanoprobes B-nanoparticle that detect hymyc1 mrna , but not in those treated with scramble− nanoprobes B-nanoparticle . [SEP]
[CLS] the signal was located mainly in the animal ' s body column , as expected since hymyc1 mrna is expressed in the cells B-material of the gastric region ( see figure s1a for a full image of the animal taken with an optical microscope ) and it is absent in head and tentacles ( see figure s2b ) . [SEP]
[CLS] on the other hand , when incubated B-technique with general mrna nanoprobes B-nanoparticle ( gmrna nanoprobes ) capable of detecting all mrna , a fluorescence B-property signal was located throughout the animals ' body including the head and tentacles ( see figure s2c ) . [SEP]
[CLS] as seen in figure 1 a , when incubated B-technique with the nanoprobes B-nanoparticle that detect hymyc1 mrna , hydra showed a strong fluorescence B-property signal in the body column due to the release of the cy3 - labeled " flare " strand . [SEP]
[CLS] on the other hand , figure 1d shows that upon incubation B-technique of live animals with scramble nanoprobes B-nanoparticle , no signal corresponding to " flare " release was detectable . [SEP]
[CLS] the faint fluorescence B-property detected in figure 1d was comparable to the autofluorescence signal detected from live animals prior to treatment with nanoprobes B-nanoparticle ( figure 1g ) . [SEP]
[CLS] furthermore , bright field images were also acquired and presented in figure 1c , f , and i showing the part of the hydra body being analyzed . [SEP]
[CLS] this data confirmed the specificity of the system toward the accurate detection of an mrna target . [SEP]
[CLS] in each case , the absence of a fluorescence B-property signal from the fammodified " sense " strand ( figure 1 b , e , and h ) confirmed that the " sense " strand remained on the nanoparticle B-nanoparticle surface without any signs of degradation in live tissue . [SEP]
[CLS] s 2 and 3 show images at higher magnification after incubation B-technique with hymyc1 nanoprobes B-nanoparticle . [SEP]
[CLS] as seen , the hymyc1 nanoprobe B-nanoparticle produced a precise and recognizable fluorescent B-property pattern due to single and nests of interstitial stem cells B-material , mirroring endogenous hymyc1 mrna . [SEP]
[CLS] similar expression pattern has been described by whole - mount in situ hybridization using digoxigenin - labeled hymyc1 rna probes . [SEP]
[CLS] overall , these results demonstrate the possibility to specifically detect hymyc1 mrna in real time in vivo . [SEP]
[CLS] the fact that within the animal body column no green fluorescence B-property could be observed means that the " sense " strand was not displaced from the aunp B-nanoparticle surface . [SEP]
[CLS] on the other hand , the detection of mrna in the cells B-material means cytoplasmic delivery of aunps B-nanoparticle , where the mrna is present . [SEP]
[CLS] although the mechanism of uptake of these nanoprobes B-nanoparticle has not been investigated yet , it has been already described that 14 nm spherical aunps B-nanoparticle bearing sirna can directly penetrate the plasma membrane of ectodermal cells B-material just after 30 min of incubation B-technique with hydra . [SEP]
[CLS] avoiding the classical endocytic pathways , aunps B-nanoparticle were able to deliver the sirna and induce a gene downregulation . [SEP]
[CLS] the aunps B-nanoparticle were also found on the membrane of interstitial cells B-material , where hymyc1 should be expressed . [SEP]
[CLS] in summary , we have shown that small amounts of dnacoated aunps B-nanoparticle as used within this study are not toxic B-property to hydra and they can be employed to detect specific mrnas in these animals . [SEP]
[CLS] here , we successfully detected the presence of the hymyc1 mrna using the corresponding oligonucleotide nanoprobes B-nanoparticle without any signs of nanoprobe B-nanoparticle degradation . [SEP]
[CLS] on the other hand , no fluorescence B-property was detected when scramble sequence nanoprobes B-nanoparticle were used . [SEP]
[CLS] our results demonstrate the possibility of using dna - coated aunps B-nanoparticle as a fast and reliable tool to qualitatively monitor the presence or not of specific mrna targets in hydra animals . [SEP]
[CLS] because of the key role played by the myc transcription B-event factor family in cell B-material and animal biology , the choice of myc as target gene for our methodology confers high translational impact to our results . [SEP]
[CLS] in vertebrates the myc protein B-material controls a variety of processes spanning from cell B-material cycle , to apoptosis B-event and the balance between stem cell B-material self - renewal / differentiation , thus the availability of safe and efficient tools to monitor in real time its expression levels may open the path to a wide use of these dna - coated aunps B-nanoparticle as novel investigation tools in stem cell B-material and cancer biology and in any physiological and pathological contexts demanding mrna detection tools . [SEP]
[CLS] the strength of our proposed approach relies on the fast kinetics of mrna detection . [SEP]
[CLS] previous studies have relied on in situ hybridization to assess mrna biodistribution , a technique that is costly and time - consuming as it relies on the in vitro cloning of doublestranded dna encoding for the gene of interest , synthesis of digoxigenin - labeled riboprobe ( antisense strand ) and finally on the hybridization of this riboprobe with the endogenous sense mrna in fixed tissue . [SEP]
[CLS] furthermore , that technique is prone to signal saturation and the generation of high signal backgrounds . [SEP]
[CLS] our work represents an important advance for the fast and accurate detection of mrna targets within an in vivo environment and it can pave the way for the broader exploitation of oligonucleotide coated gold B-nanoparticle nanoparticles I-nanoparticle in clinical applications , as a rapid method for the reliable assessment of mrna expression in living tissue . [SEP]
[CLS] 2 . schematic illustration of the process of mrna detection in hydra a [SEP]
[CLS] 1 . fluorescence B-technique microscopy I-technique images of the ( a , d , g ) cy3 and ( b , e , h ) fam channel of the gastric region of live hydra incubated B-technique with ( a−c ) nanoprobes B-nanoparticle specific for the detection of hymyc1 mrna , ( d−f ) nanoprobes B-nanoparticle designed with a scramble sequence that does not detect any mrna , and ( g−i ) hydra not treated with nanoprobes B-nanoparticle . [SEP]
[CLS] color guide for the different channels : red ( cy3 ) , fluorescence B-property signal corresponding to " flare " strand release . [SEP]
[CLS] green ( fam ) , fluorescence B-property signal corresponding to " sense " strand release . [SEP]
[CLS] bright - field images of the animal are also presented in c , f , and i . [SEP]
[CLS] scale bars are 100 μm . [SEP]
[CLS] associated content * s supporting information [SEP]
[CLS] the supporting information is available free of charge on the acs publications website at doi : 10 . 1021 / acsami . 8b17846 . [SEP]
[CLS] detailed dna - coated aunp B-nanoparticle characterization such as uv−vis spectra , gel B-technique electrophoresis I-technique images , and zeta B-property potential I-property measurements ; complementary fluorescence B-property images and experimental results of live hydra incubated B-technique with dna - coated nanoprobes B-nanoparticle ( pdf ) ■ author information corresponding authors * email : a . kanaras @ soton . ac . uk . [SEP]
[CLS] * email : claudia . tortiglione @ cnr . it . orcidafaf h . el - sagheer : 0000 - 0001 - 8706 - 1292 tom brown : 0000 - 0002 - 6538 - 3036 antonios g . kanaras : 0000 - 0002 - 9847 - 6706 [SEP]
[CLS] higher - magnification fluorescence B-technique microscopy I-technique images of the gastric region of live hydra incubated B-technique with nanoprobes B-nanoparticle specific for the detection of hymyc1 mrna . [SEP]
[CLS] the ( a , b ) cy3 , ( c , d ) bright - field and ( e , f ) merged channels are presented for images taken using a ( a , c , e ) 40× and ( b , d , f ) 63× oil immersion objective . [SEP]
[CLS] scale bars are 25 μm . [SEP]
[CLS] 3 . fluorescence B-technique microscopy I-technique images of the gastric region of live hydra incubated B-technique with nanoprobes B-nanoparticle specific for the detection of hymyc1 mrna where nests of nematoblasts are seen and enlarged in the right corner of the pictures . [SEP]
[CLS] ( a ) images obtained using a 10× objective . [SEP]
[CLS] ( b ) images obtained using a 20× objective . [SEP]
[CLS] color guide : red ( cy3 ) , fluorescence B-property signal corresponding to " flare " strand release . [SEP]
[CLS] scale bars are 100 μm . [SEP]
[CLS] the translation of proteins B-material as effective intracellular drug candidates is limited by the challenge of cellular entry and their vulnerability to degradation . [SEP]
[CLS] to advance their therapeutic potential , cell - impermeable proteins B-material can be readily transformed into protein B-material spherical nucleic B-material acids I-material ( prosnas ) by densely functionalizing their surfaces with dna , yielding structures that are efficiently taken up by cells B-material . [SEP]
[CLS] because small structural changes in the chemical makeup of a conjugated ligand can affect the bioactivity of the associated protein B-material , structure−activity relationships of the linker bridging the dna and the protein B-material surface and the dna sequence itself are investigated on the prosna system . [SEP]
[CLS] in terms of attachment chemistry , dna - based linkers promote a sevenfold increase in cellular uptake while maintaining enzymatic activity in vitro as opposed to hexaethylene glycol ( heg , spacer18 B-material ) linkers . [SEP]
[CLS] additionally , the employment of g - quadruplex - forming sequences increases cellular uptake in vitro up to fourfold . [SEP]
[CLS] when translating to murine models , the prosna with a dna - only shell B-material exhibits increased blood circulation times and higher accumulation in major organs , including lung , kidney , and spleen , regardless of sequence . [SEP]
[CLS] importantly , prosnas with an all - oligonucleotide shell B-material retain their enzymatic activity in tissue , whereas the native protein B-material loses all function . [SEP]
[CLS] taken together , these results highlight the value of structural design in guiding prosna biological fate and activity and represent a significant step forward in the development of intracellular protein - based therapeutics . [SEP]
[CLS] when materials are structured on the nanoscale , they exhibit unique properties that can be exploited for various applications . [SEP]
[CLS] dna is a notable illustration of this concept ; when densely radially functionalized onto a nanoparticle B-nanoparticle core B-material in the form of a spherical nucleic B-material acid I-material ( sna ) , dna achieves high cellular uptake through the engagement of scavenger receptors and is more resistant to nuclease degradation than its component nucleic B-material acids I-material . [SEP]
[CLS] these properties have enabled the development of snas as diagnostic tools and in therapeutic applications , ranging from gene regulation to immunotherapy . [SEP]
[CLS] because the three - dimensional architecture of the dna shell B-material is the primary driver for biological function , the sna platform has been expanded to include biocompatible B-property cores B-material such as liposomes B-nanoparticle , polymers B-material , and proteins B-material . [SEP]
[CLS] protein B-material snas ( prosnas ) are especially attractive because they address an unmet need in biologics development : facilitating the cellular delivery of large , hydro - philic , and charged macromolecules while maintaining their biological function . [SEP]
[CLS] the notion that dna shell B-material design parameters dictate the biological fate of snas is well - established . [SEP]
[CLS] one such example is that high oligonucleotide loadings correlate with enhanced cellular uptake . [SEP]
[CLS] in previous work with gold B-material nanoparticlebased snas , hexaethylene glycol ( heg ) , also known as spacer B-material 18 ( sp18 ) , were introduced at the interface between the nanoparticle B-nanoparticle core B-material and its oligonucleotide shell B-material to mitigate the electrostatic repulsion between the negatively charged gold B-material surface and the dna , enabling denser packing . [SEP]
[CLS] however , a pivot in dna shell B-material design is required when synthesizing an sna architecture from a protein B-material core B-material . [SEP]
[CLS] proteins B-material typically offer fewer attachment sites and thus may not be affected by electrostatic repulsion to the same degree . [SEP]
[CLS] moreover , with many proteins B-material , chemical modification of their surfaces can either positively or negatively influence different aspects of their function . [SEP]
[CLS] for example , in the development of proteinbased therapies , biocompatible B-property polymer B-material modification , or pegylation , has been utilized to extend the lifetime of susceptible proteins B-material . [SEP]
[CLS] however , such modifications generally lead to a loss in biological activity . [SEP]
[CLS] in addition to the introduction of a linker region to the dna shell B-material of snas , another design rule identified with gold B-material - based snas was their sequence - specific uptake behavior . [SEP]
[CLS] specifically that g - quadruplexes ( gqs ) , which are guanine - rich sequences that can fold into unique secondary structures , contributed to higher cellular uptake and a difference in biodistribution . [SEP]
[CLS] this phenomenon is attributed to a favorable interaction between the scavenger receptor B-material and a denser presentation of negative charge ascribed from the folding of the gq . [SEP]
[CLS] with insight into the potential benefits and detrimental effects of sequence design and polymer B-material modifications , we sought to study the effects of incorporating ( 1 ) spacer18 B-material ( a polymer B-material mimic ) in the dna shell B-material and ( 2 ) a gq sequence on the cellular uptake , enzymatic activity , and biodistribution of prosnas , utilizing the previously established β - galactosidase ( β - gal ) model system . [SEP]
[CLS] we hypothesized that the nature and length of the linker would modulate the properties of the oligonucleotide shell B-material of prosnas . [SEP]
[CLS] furthermore , the gqs will increase the uptake of the prosna akin to previous studies with the gold B-material core B-material . [SEP]
[CLS] for this purpose , we used in vitro and in vivo models to investigate tunable parameters in the prosna dna shell B-material ' s linker design space in the interest of developing a set of general dna shell B-material design rules for biologic drug development . [SEP]
[CLS] impact of linker design on cell B-material uptake and activity . [SEP]
[CLS] β - gal prosnas were synthesized using protocols previously established by our group . [SEP]
[CLS] the protein B-material was tagged with alexa fluor 647 ( af647 ) to facilitate tracking in vitro and in vivo . [SEP]
[CLS] to evaluate the impact of dna linker identity and length on the cellular uptake and enzyme B-property activity I-property of a β - gal prosna , dna was conjugated to the protein B-material through surface - accessible lysine B-material residues , with either t 4n or ( sp18 ) 2n ( n = 1 , 2 , 3 ) near the conjugation site ( figure 1a ) . [SEP]
[CLS] each linker , t 4 or ( sp18 ) 2 , is approximately equivalent in length but varies significantly in chemical identity and flexibility , which may impact the radial orientation of the oligonucleotide shell B-material and ultimately affect the biological properties of the resulting prosnas . [SEP]
[CLS] uv−vis spectroscopy B-technique showed that the dna loadings were identical ( 30 dna / β - gal , 3 . 7 pmol / cm 2 ) , regardless of the composition of the shell B-material ( figure s5 ) . [SEP]
[CLS] moreover , covalent conjugation of the dna to the protein B-material core B-material was confirmed via a denaturing sds - page gel , which shows mobility B-property shifts commensurate with the increased mass upon dna addition ( figure s6 ) . [SEP]
[CLS] importantly , in contrast to classical gold B-material - based snas , the absence of spacer18 B-material did not significantly affect loading density onto prosnas , suggesting that each surface - accessible lysine B-material on β - gal was readily available for conjugation , independent of linker type . [SEP]
[CLS] one of the defining features of the sna platform is its robust and unaided cellular uptake , which has been demonstrated in more than 60 cell B-material lines . [SEP]
[CLS] specifically , akin to other snas , the prosna was found to be taken up by cells B-material through scavenger receptor - a ( sr - a ) mediated endocytosis B-event through a receptor blocking study . [SEP]
[CLS] to establish the role of the linker on the cellular uptake of prosnas , hela cells B-material were incubated B-technique with the six β - gal prosna variants described above , and their uptake was monitored using flow B-technique cytometry I-technique by tracking the fluorescence B-property of the protein B-material core B-material ( figure 1b ) . [SEP]
[CLS] surprisingly , the prosna with no spacer18 B-material was taken up to the greatest extent . [SEP]
[CLS] furthermore , internalization dramatically decreased with increasing spacer18 B-material length , even though these modifications were made adjacent to the protein B-material surface and thus not expected to be accessible to the cell B-material surface . [SEP]
[CLS] in contrast , increasing the length of the dna linker imparted only minor changes in cellular uptake . [SEP]
[CLS] we speculate that these results originate from differences between the linkers in terms of persistence length and ability of the shell B-material to adopt a radial arrangement away from the protein B-material surface , resulting in a decreased affinity for sr - a . [SEP]
[CLS] the linker ' s deleterious uptake properties were further supported by switching the location of spacer18 B-material on the protein B-material : cellular uptake recovers , confirming that placing spacer18 B-material near the protein B-material surface is detrimental to internalization ( figures s11 and s12 ) . [SEP]
[CLS] these findings were further reproduced in another cell B-material line ( figure s15 ) . [SEP]
[CLS] based on these results , we find that while gold B-material - based snas benefited from the addition of spacer18 B-material linkers , the functionalization of protein B-material cores B-material should be carried out using linkers that are composed entirely of dna to maximize cellular uptake . [SEP]
[CLS] a critical aspect , unique to prosnas in the context of protein B-material delivery , is the retention of protein B-material function after conjugation . [SEP]
[CLS] to assess the effect of conjugating a large number of dna strands on the protein B-material surface on function , the enzymatic activity of the β - gal prosnas was measured utilizing an ortho - nitrophenyl - β - galactoside ( onpg ) assay , and michaelis−menten constants were calculated . [SEP]
[CLS] both linkers caused a loss of enzymatic activity as their length increased ; however , this effect is more pronounced for the spacer18 B-material linker ( figure 1c ) . [SEP]
[CLS] we speculate that spacer18 B-material is more detrimental to enzymatic activity because of an increased affinity of the spacer18 B-material linker for the protein B-material surface when compared to dna . [SEP]
[CLS] to test this hypothesis , we placed the spacer18 B-material linker away from the protein B-material surface by appending it at the end of a t 30 strand and found that activity was improved ( figure s18 ) . [SEP]
[CLS] considering that the reported isoelectric point for β - gal is approximately 5 . 5 , the protein B-material is overall negatively charged at physiological conditions . [SEP]
[CLS] similarly , the dna linkers provide a high density of negative charges through their phosphate backbone . [SEP]
[CLS] these two elements combined are thus expected to promote electrostatic repulsion and favor a radial orientation of the oligonucleotide shell B-material . [SEP]
[CLS] on the other hand , spacer18 linkers are less densely charged than dna and may have other interactions with the protein B-material surface . [SEP]
[CLS] based on these results , we conclude that with this protein B-material , short dnaonly linkers maximize the enzymatic activity of prosnas . [SEP]
[CLS] g - quadruplex shell B-material enhances cell B-material uptake in vitro . [SEP]
[CLS] we next assessed sequence - dependent cellular uptake and enzyme B-property activity I-property trends of prosnas under physiological conditions . [SEP]
[CLS] templated by their dna sequence , particular strands will fold into secondary structures that can bind preferentially to receptors on the cell B-material membrane . [SEP]
[CLS] in previous work , our group demonstrated that snas constructed using guanine - rich sequences resulted in significantly higher uptake compared to thymine - , adenine - , or cytosine - rich control snas . [SEP]
[CLS] g - rich sequences , which can fold into gqs , are a natural ligand for sr - a due to the proposed favorable electrostatic interaction between the gq and the lysine - rich collagenase region of the sr - a . [SEP]
[CLS] because gqs are expected to interact preferentially with sr - a , we hypothesized that prosnas with a dna shell B-material that can form gqs would be able to enter cells B-material in more significant amounts than snas composed of other nucleotides ( figure 2a ) . [SEP]
[CLS] gqs exist in various topologies based on their sequences ; therefore , we investigated the impact of gq folding on cellular uptake of the prosna in vitro . [SEP]
[CLS] to facilitate attachment to β - gal , all gq sequences were synthesized with a t 4 dna - only linker , which was found to maximize both cellular uptake and enzymatic activity ( vide supra ) . [SEP]
[CLS] to begin , we characterized all gq strands by circular dichroism to confirm the chosen sequences fold into the appropriate gq structure after the inclusion of a t 4 linker and dbco - dt conjugation group ( figure s9 ) . [SEP]
[CLS] then we synthesized prosnas using the previously reported method but under different buffer conditions . [SEP]
[CLS] the g - quartet structure in a gq ' s tetrad is selectively stabilized by a k + ion B-material , which coordinates to each of the guanine bases . [SEP]
[CLS] to linearize the strand , thus enabling more efficient loading , we replaced k + with li + as the counterion in the reaction buffer . [SEP]
[CLS] lithium B-material is the least stabilizing among type ia and iia cations B-material ; therefore , the gq does not form under these conditions . [SEP]
[CLS] after conjugation and prosna purification , the buffer was exchanged back to pbs ( a potassium B-material phosphate buffer ) to facilitate gq formation ( figure s10 ) . [SEP]
[CLS] to determine which gq topology results in the most significant enhancement in cellular internalization , hela cells B-material were treated with each of the prosna gq variants in serum containing media . [SEP]
[CLS] according to flow B-technique cytometry I-technique , all gqs demonstrated an approximately twofold increase in uptake compared to the t - rich prosna ( figure 2b ) . [SEP]
[CLS] more notably , the t 4 ( ggt ) 10 prosna , which folds into a parallel gq , increased uptake by fourfold . [SEP]
[CLS] this enhancement in uptake with the t 4 ( ggt ) 10 prosna was demonstrated over a range of loading densities with the highest cellular uptake seen at a loading of 20 dna / β - gal ( figure 2c ) . [SEP]
[CLS] the enhancement in cellular uptake with prosnas composed of a gq shell B-material stems from the dna ' s unique secondary structure . [SEP]
[CLS] however , this special folding imparts a small inhibitory effect on the enzyme B-property ' I-property s I-property activity I-property . [SEP]
[CLS] an onpg assay was used to calculate the k cat of the enzyme for prosnas with either t 4 t 30 or t 4 ( ggt ) 10 dna shells B-material . [SEP]
[CLS] after loading both prosnas equally ( 20 dna / β - gal ) , we calculated the k cat of the enzyme and found that there is an [UNK] % loss in activity with a gq shell B-material ( figure 2d ) . [SEP]
[CLS] this small loss in activity may stem from steric hindrance by the dense folding of the dna around the enzyme . [SEP]
[CLS] nevertheless , it is subject to the intentions of the application whether this marginal loss in activity depreciates the gain in cellular uptake for a g - quadruplex dna shell B-material . [SEP]
[CLS] prosnas demonstrate enhanced pharmacokinetics . [SEP]
[CLS] we assessed whether these shell B-material design considerations would translate from in vitro cell B-material studies to a more relevant in vivo mouse model . [SEP]
[CLS] previous work using gold B-material core snas highlighted the benefits of the oligonucleotide shell B-material in enabling the biodistribution of snas to all major organs , which have been applied for the treatment of many diseases , including glioblastoma and many other forms of cancer . [SEP]
[CLS] prosnas are a special case because there is an approximate order of magnitude difference in loading density ( 3 . 7 pmol / cm 2 ) when comparing to the classical gold B-material snas ( 30−60 pmol / cm 2 ) . [SEP]
[CLS] moreover , naked proteins B-material are susceptible to degradation and typically show low cellular uptake . [SEP]
[CLS] therefore , we evaluated if this loading density would be sufficient to enable applications of prosnas in vivo and if these newly devised design rules would be able to amplify these effects further . [SEP]
[CLS] based on cellular uptake and enzymatic activity findings in vitro , we hypothesized that spacer18 B-material would impede the internalization of prosnas within the tissue . [SEP]
[CLS] for this purpose , we started by comparing constructs bearing either a t 30 or ( sp18 ) 6 t 30 dna shell B-material , such that these two constructs would vary only in the presence of a spacer18 B-material linker . [SEP]
[CLS] we assessed both the blood circulation time and temporal biodistribution following a single tail vein intravenous injection of either β - gal prosnas or native protein B-material in cd - 1 mice , utilizing the alexa fluor 647 fluorescent B-property - tag on the protein B-material to track its distribution . [SEP]
[CLS] first , we measured the distribution half - life of these prosnas in plasma to determine the clearance rate of these protein B-material constructs . [SEP]
[CLS] notably , both prosnas exhibit an increase in circulation time ( 60 min ) , which is twice that of the native protein B-material ( 30 min ) , regardless of the linker identity ( figure 3a ) . [SEP]
[CLS] these similarities in the pharmacokinetic profiles of the snas highlight the influence of the dna shell B-material , regardless of linker , in transforming the biological properties of an sna ' s nanoparticle B-nanoparticle core B-material . [SEP]
[CLS] to evaluate the distribution of β - gal in tissue , we measured the fluorescence B-property of dissected and perfused organs utilizing an in vivo imaging system ( ivis ) . [SEP]
[CLS] quantification of radiant efficiency revealed that a majority of both prosna and native protein B-material were sequestered by the liver , akin to previous observations with other nanoparticle B-nanoparticle systems . [SEP]
[CLS] however , the prosnas , in particular the t 30 variant , exhibited distinct differences in biodistribution compared to the native protein B-material ( figure 3b ) . [SEP]
[CLS] consistent with our hypothesis , we observed that the native protein B-material is found primarily in the liver with limited distribution to other organs . [SEP]
[CLS] in contrast , the addition of the oligonucleotide shell B-material enabled a broader distribution of both prosnas . [SEP]
[CLS] importantly , an enhanced tissue accumulation was observed with the t 30 prosna , mirroring our previous in vitro cellular uptake results ( vide supra ) . [SEP]
[CLS] this finding is of significance because it highlights the importance of using an all - dna shell B-material when designing prosnas because it dictates their in vivo behavior . [SEP]
[CLS] in addition to linker identity being a determinant of uptake for the prosna , dna sequence was also established to enhance cellular uptake in vitro . [SEP]
[CLS] consistent with the previous experiment , we utilized ivis to study the distribution of the prosna with two different sequence identities : t 34 or t 4 ( ggt ) 10 . [SEP]
[CLS] both prosnas were loaded equally at 20 strands per protein B-material , which we demonstrated resulted in the highest internalization for the gq prosna in hela cells B-material . [SEP]
[CLS] cd - 1 mice were treated with each construct via the tail - vein , and after a 1 h treatment , mice were sacrificed and perfused , and organs were dissected for ivis analysis , which was used to track the af647 fluorophore covalently conjugated to the protein B-material . [SEP]
[CLS] based on fluorescence B-property counts , both prosnas accumulated in the dissected organs equally ( figure 3c ) . [SEP]
[CLS] from this experiment , it is apparent that the nature of the linker is a more important determinant of in vivo prosna behavior . [SEP]
[CLS] only prosnas exhibit enzymatic activity in tissue . [SEP]
[CLS] the ultimate test to determine the potential of the sna architecture as a delivery method for biologics was to assess whether the delivered enzyme retains its biological activity in tissue . [SEP]
[CLS] therefore , we performed a colorimetric activity assay specific to β - gal on cryo - sectioned organs , allowing us to gain information on the suborgan compartmentalization of the sna platform and its overall structural integrity . [SEP]
[CLS] for this purpose , mice were intravenously administered prosnas or native protein B-material . [SEP]
[CLS] after 1 h , mice were sacrificed and perfused , and organs were cryosectioned and subsequently stained with 5bromo - 4 - chloro - 3 - indolyl - β - d - galactopyranoside ( x - gal ) to visualize the enzyme B-property ' I-property s I-property activity I-property via the appearance of a bluecolored insoluble product . [SEP]
[CLS] strikingly , we observed blue coloring in both t 34 prosna and t 4 ( ggt ) 10 prosna treated mice ( n = 3 ) , which confirmed the successful delivery of active enzymes to tissues , while all organs treated with the native proteins B-material ( n = 3 ) exhibited no coloring ( figure 4 ) . [SEP]
[CLS] this is the first time that the prosna strategy has been tested in a living animal model , opening the door to various applications for relevant disease models . [SEP]
[CLS] in this context , we looked at the distribution of the blue stain within each organ , which revealed details about tissue localization of the prosna . [SEP]
[CLS] a first striking observation was the presence of blue in the prosna - treated liver indicative of enzyme B-property activity I-property , whereas it was completely absent in the case of the native protein B-material . [SEP]
[CLS] this was unexpected since ivis fluorescence B-property signals in the liver from all treatment groups were nearly identical , suggesting that the oligonucleotide shell B-material on the prosna was able to protect its structural integrity . [SEP]
[CLS] the liver and lung displayed diffuse staining throughout the organ , whereas in the kidney and spleen , it was localized to tissue substructures . [SEP]
[CLS] specifically , the prosna is trapped within the glomerulus in the kidney , which is a structural unit that acts as a filter with an average pore size of 3 nm . [SEP]
[CLS] we hypothesize that the prosnas are retained within this glomerular space and are thus prevented from entering the kidney tissue due to their inherent size . [SEP]
[CLS] however , when the filtration size thresholds of the organs are larger , as is the case with the spleen , the prosna readily enters . [SEP]
[CLS] indeed , the spleen exhibits two distinct regions based on nuclear fast red staining : red and white pulp . [SEP]
[CLS] the white pulp is predominantly a sheath of lymphocytes ( t and b ) surrounding the artery , while the red pulp envelops the white pulp and consists mostly of macrophages , plasma , and a few lymphocytes . [SEP]
[CLS] based on these images , the prosna is preferentially sequestered in the red pulp of the spleen . [SEP]
[CLS] in particular , there is a difference in spleen distribution between the t - rich and gq prosnas . [SEP]
[CLS] the t - rich prosna distributes evenly throughout the red pulp . [SEP]
[CLS] however , for the gq prosna , the enzyme predominantly accumulates in a marginal zone at the junction between the red and white pulp . [SEP]
[CLS] regardless , we hypothesize that both prosna variants are taken up by macrophages ( either marginal zone or red pulp ) , which take up the sna due to the abundance of scavenger receptors on their cell B-material membranes . [SEP]
[CLS] we established the importance of dna shell B-material design in tuning the activity , cellular uptake , and overall biodistribution of prosnas . [SEP]
[CLS] we find that the chemical composition of the linker is a crucial determinant of prosna properties : specifically , the replacement of the heg linker with dna leads to a recovery in both cellular uptake and enzymatic activity at no cost to loading density . [SEP]
[CLS] furthermore , we can harness the programmable topology of dna structures to modulate cell B-material interactions , as demonstrated with the gq prosnas acting as preferential ligands for the scavenger receptor B-material - a . in terms of biodistribution , the removal of the heg linker resulted in a greater distribution in organs other than the liver . [SEP]
[CLS] conversely , both types of sequences facilitated protein B-material delivery to a similar extent . [SEP]
[CLS] because the most critical parameter in protein B-material delivery consists in maintaining enzymatic activity , we demonstrate for the first time that the prosna strategy can be used to deliver functional enzymes to tissues , thus highlighting the relevance of this approach for biologics development . [SEP]
[CLS] considering the facile synthesis and conjugation of dna onto proteins B-material as well as its biocompatibility B-property , these findings emphasize the relevance of applying the sna architecture to therapeutically relevant proteins B-material . [SEP]
[CLS] we foresee that prosnas can be tailored to a breadth of applications from immunotherapy to enzyme replacement therapy in macrophage - relevant disease models . [SEP]
[CLS] the supporting information is available free of charge at https : / / pubs . acs . org / doi / 10 . 1021 / acscentsci . 0c00313 . [SEP]
[CLS] experimental procedures , oligonucleotide sequences , maldi - ms , uv−vis traces , sds page gel of purified prosnas , circular dichroism spectra of dna and prosna constructs , flow B-technique cytometry I-technique histograms , enzymatic activity analysis procedure , fluorescence B-property images from ivis , temporal biodistribution by ivis , and whole organ histogram images ( pdf ) [SEP]
[CLS] impact of linker design on cellular uptake and enzymatic activity . [SEP]
[CLS] ( a ) cartoon representation of the surface of β - gal at a lysine B-material residue , which is covalently modified with a dna strand . [SEP]
[CLS] the " linker region " , at the interface of β - gal and a t 30 oligonucleotide strand , varies in either increasing t 4 or 2 spacer18 phosphoramidite increments . [SEP]
[CLS] the representation was adapted from pdb id 1px3 . [SEP]
[CLS] ( b ) cellular uptake of prosnas with increasing t 4 ( purple ) or spacer18 B-material ( pink ) linkers , as determined by flow B-technique cytometry I-technique . [SEP]
[CLS] fluorescence B-property was measured in hela cells B-material ( n = 3 ) 4 h after treatment with 5 nm enzyme in serum containing media . [SEP]
[CLS] all prosnas have equal loading density of 30 dna / β - gal . [SEP]
[CLS] ( c ) relative k cat ( n = 3 ) determined by an onpg assay of prosnas with varying linker identity and the native protein B-material . [SEP]
[CLS] all prosnas have equal loading density of 30 dna / β - gal . [SEP]
[CLS] the bars in the graphs represent the mean , and error bars show the standard error of the mean ( sem ) . [SEP]
[CLS] an unpaired t test was performed with graphpad prism . [SEP]
[CLS] figure 1 . n . s . , not significant , * * p ≤ 0 . 01 . [SEP]
[CLS] impact of g - quadruplex sequences on cellular uptake and enzymatic activity . [SEP]
[CLS] ( a ) cartoon representation of the surface of β - gal at a lysine B-material residue which is covalently modified with a dna strand . [SEP]
[CLS] the inset represents either a t - rich and g - rich sequence structure . [SEP]
[CLS] in the gquadruplex topology , the guanine bases are selectively stabilized by a potassium B-material cation B-material . [SEP]
[CLS] the representation was adapted from pdb id 1px3 and 2n3m . [SEP]
[CLS] ( b ) cellular uptake of prosnas with different g - quadruplex topologies , as determined by flow B-technique cytometry I-technique . [SEP]
[CLS] 2kf7 ( antiparallel basket ) , 1kf1 ( parallel propeller ) , 148d ( antiparallel chair ) , and 2jpz ( mixed chair ) are named after their pdb id . [SEP]
[CLS] fluorescence B-property was measured in hela cells B-material ( n = 3 ) 4 h after treatment with 5 nm enzyme in serum containing media . [SEP]
[CLS] ( c ) cellular uptake of prosnas at different loading densities with either a t 4 t 30 or t 4 ( ggt ) 10 sequence , as determined by flow B-technique cytometry I-technique . [SEP]
[CLS] fluorescence B-property was measured in hela cells B-material ( n = 3 ) 2 h after treatment with 10 nm enzyme in serum containing media . [SEP]
[CLS] ( d ) relative k cat ( n = 3 ) determined by an onpg assay of prosnas ( both 20 dna / β - gal ) and the native protein B-material . [SEP]
[CLS] the bars in the graphs represent the mean , and error bars show sem . [SEP]
[CLS] an unpaired t test was performed with graphpad prism . [SEP]
[CLS] * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 . [SEP]
[CLS] pharmacokinetic profile of the prosna and native protein B-material . [SEP]
[CLS] ( a ) the clearance of two prosna variants with and without spacer18 B-material linker and native protein B-material were studied by measuring the fluorescence B-property in plasma . [SEP]
[CLS] raw data points as well as the geometric mean at each time point are depicted . [SEP]
[CLS] ( b , c ) [SEP]
[CLS] after a 1 h treatment with 4 mg of enzyme / kg mouse via a tail - vein injection ; mice ( n = 3 ) were sacrificed , perfused , and their organs dissected and fixed . [SEP]
[CLS] using ivis , a region of interest was drawn around each organ , and the fluorescence B-property counts were quantified . [SEP]
[CLS] the bars in the graphs represent the mean , and error bars show sem . [SEP]
[CLS] in vivo catalytic activity and tissue distribution of native β - gal and prosna . [SEP]
[CLS] representative light micrographs of histology slides after incubation B-technique with the β - gal substrate , x - gal . [SEP]
[CLS] the blue color apparent in tissue dissected from mice ( n = 3 ) 1 h after intravenous injection of 6 . 5 mg enzyme / kg mouse results from the hydrolysis of x - gal and the formation of an insoluble blue product . [SEP]
[CLS] scale = 250 μm . [SEP]
[CLS] liposomal B-nanoparticle spherical nucleic B-material acids I-material ( l - snas ) show significant promise as cancer immunotherapeutics . [SEP]
[CLS] l - snas are highly modular nanoscale assemblies defined by a dense , upright radial arrangement of oligonucleotides around a liposomal B-nanoparticle core B-material . [SEP]
[CLS] herein , we establish a set of l - sna design rules by studying the biological and immunological properties of l - snas as a function of liposome B-nanoparticle composition . [SEP]
[CLS] to achieve this , we synthesized liposomes B-nanoparticle where the lipid B-material phosphatidylcholine headgroup was held constant , while the diacyl lipid B-material tail chain length and degree of saturation were varied , using either 1 , 2 - dioleylphosphatidylcholine ( dopc ) , 1 , 2 - dimyristoyl - phosphatidylcholine ( dmpc ) , 1 , 2 - dipalmitoylphosphatidylcholine ( dppc ) , or 1 , 2 - distearoyl - phosphatidylcholine ( dspc ) . [SEP]
[CLS] these studies show that the identity of the constituent lipid B-material dictates the dna loading , cellular uptake , serum stability , in vitro immunostimulatory activity , and in vivo lymph node accumulation of the l - sna . [SEP]
[CLS] furthermore , in the 4t1 mouse model of triple - negative breast cancer ( tnbc ) , the subcutaneous administration of immunostimulatory l - snas synthesized with dppc significantly decreases the production of lung metastases B-event and delays tumor growth as compared to l - snas synthesized using dopc , due to the enhanced stability of l - snas synthesized with dppc over those synthesized with dopc . [SEP]
[CLS] moreover , the inclusion of cell B-material lysates derived from py8119 tnbc cells B-material as antigen sources in l - snas leads to a significant increase in antitumor efficacy in the py8119 model when lysates are encapsulated in the cores B-material of l - snas synthesized with dppc rather than dopc , presumably due to increased codelivery of adjuvant and antigen to dendritic cells B-material in vivo . [SEP]
[CLS] this difference is further amplified when using lysates from oxidized py8119 cells B-material as a more potent antigen source , revealing synergy between the lysate preparation method and liposome B-nanoparticle composition in synthesizing immunotherapeutic l - snas . [SEP]
[CLS] together , this work shows that the biological properties and immunomodulatory activity of l - snas can be modulated by exchanging liposome B-nanoparticle components , providing another handle for the rational design of nanoscale immunotherapeutics . [SEP]
[CLS] r egarded as one of the more successful biomolecule packing systems to date , [SEP]
[CLS] liposomes B-nanoparticle are useful in a wide variety of biomedical applications , including drug delivery , 2 , 3 biosensing , 4 , 5 diagnostics , gene delivery , and immunomodulation B-property . [SEP]
[CLS] the chemical composition of liposomes B-nanoparticle determines their performance in biological systems . [SEP]
[CLS] indeed , it has been well - established that the phase transition temperature ( t c ) of the constituent lipids B-material dictates the phospholipid bilayer membrane fluidity , which heavily influences the liposome B-nanoparticle ' s biological properties , including permeability and lipid B-material exchange . [SEP]
[CLS] the t c of a phospholipid is determined by the chemical identity and charge of the hydrophilic B-property headgroup as well as the chain length and degree of saturation of the diacyl lipid B-material tail . [SEP]
[CLS] at temperatures at or below the t c , liposomes B-nanoparticle exist in a gel phase , where lipid B-material exchange is limited and membrane fluidity is low . [SEP]
[CLS] at temperatures exceeding the t c , liposomes B-nanoparticle exist in a liquidcrystalline phase , where the membrane is more fluid and the dynamics of lipid B-material exchange are increased . [SEP]
[CLS] in general , the t c increases as the diacyl lipid B-material chain length increases , due to increased van der waals forces between the acyl chains . [SEP]
[CLS] however , introducing double bonds into the diacyl lipid B-material tail decreases the t c , as the packing of the hydrophobic B-property chains is disrupted by structural kinks introduced by the unsaturated bonds . [SEP]
[CLS] together , these subtle changes to the chemical structure of the individual lipid B-material components dictate the supramolecular properties of the liposome B-nanoparticle and are important to consider when designing liposome - scaffolded materials . [SEP]
[CLS] because of their biocompatibility B-property , modularity , and favorable physical properties , liposomes B-nanoparticle are attractive scaffolds for synthesizing spherical nucleic B-material acids I-material ( snas ) . snas are a unique class of nucleic B-material acid I-material defined by the dense , highly oriented arrangement of oligonucleotides around a nanoparticle B-nanoparticle core B-material . [SEP]
[CLS] snas exhibit markedly different biological properties from their linear nucleic B-material acid I-material analogues , including rapid cellular uptake without the need for transfection reagents [SEP]
[CLS] liposomal B-nanoparticle snas ( l - snas , figure 1a ) are synthesized by embedding oligonucleotides that have been functionalized with lipophilic B-property moieties ( e . g . , cholesterol ) into the outer membrane of a liposome B-nanoparticle ' s phospholipid bilayer . [SEP]
[CLS] diverse l - sna constructs with tunable properties can be rapidly generated by changing the lipophilic B-property oligonucleotide anchor or altering the sequence , backbone , and surface density of the oligonucleotide . [SEP]
[CLS] when comprised of immunostimulatory oligonucleotides ( cpg - 1826 ) , l - snas function as potent cancer immunotherapeutics . [SEP]
[CLS] prior research has revealed that the identity of the lipophilic B-property moiety used to embed dna into the liposomal B-nanoparticle membrane changes the stability of the l - sna , which in turn alters the biological and immunological properties of the material B-material . [SEP]
[CLS] moreover , when incorporating tumor - associated antigens ( taas ) into the l - sna , the nanoscale arrangement and attachment chemistry of immunomodulatory components on the scaffold can be used to modulate the kinetics of antigen presentation and costimulatory marker expression , which dictate their antitumor efficacy . [SEP]
[CLS] for cancers without identified taas , l - snas have been used to encapsulate tumor B-material cell B-material lysates as potent antigen sources . [SEP]
[CLS] in mouse models of triple - negative breast cancer ( tnbc ) , it has been found that the process by which lysates are generated prior to l - sna incorporation changes the available antigen pool , which impacts the immunotherapeutic potency of the construct . [SEP]
[CLS] herein , we sought to determine whether the molecular identity of the lipids B-material comprising l - snas could be used as an additional handle for controlling their biological properties and immunotherapeutic function . [SEP]
[CLS] toward this end , we synthesized a series of l - snas using liposomes B-nanoparticle comprised of lipids B-material with varying t c . [SEP]
[CLS] in this approach , the hydrophilic B-property headgroup of the phospholipids remained unchanged , while the chemical identity of the hydrophobic B-property acyl chains was systematically varied ( figure 1b ) . [SEP]
[CLS] in this way , the surface chemistry remained the same , but the bilayer composition differed . [SEP]
[CLS] we evaluated the resulting l - snas for their in vitro serum stability , cellular uptake , immune cell B-material activation , and immunotherapeutic function in orthotopic syngeneic mouse models of tnbc . [SEP]
[CLS] through these analyses , we determined that the liposome B-nanoparticle scaffold composition regulates the biological and immunological properties of l - snas ; namely , the more stable the l - sna , the better the in vivo performance . [SEP]
[CLS] significant antitumor efficacy and inhibition of lung metastasis B-event formation was observed in the 4t1 model of tnbc when animals were administered l - snas synthesized using lipids B-material with higher t c values , indicating that liposome B-nanoparticle stability governs l - sna efficacy in this model . [SEP]
[CLS] moreover , the inclusion of cell B-material lysates from py8119 tnbc cells B-material into the core B-material of l - snas synthesized from lipids B-material that support higher t c s significantly increased the resultant antitumor efficacy in the py8119 model , likely due to higher codelivery of immunotherapeutic components to dendritic cells B-material in vivo by the more stable l - sna scaffold . [SEP]
[CLS] this trend became more pronounced when lysates from oxidized py8119 cells B-material were utilized as the antigen source , indicating that the lysate preparation method and l - sna stability are additive contributors . [SEP]
[CLS] together , these results indicate that l - sna stability can be modulated by exchanging the lipid B-material components and that the most potent constructs are those synthesized using the most stable liposome B-nanoparticle scaffolds . [SEP]
[CLS] to assess the effect of liposome B-nanoparticle composition on the properties of l - snas , 80 nm liposomes B-nanoparticle comprised of lipids B-material with varying t c ( figure 1b ) were synthesized using 1 , 2 - dioleylphosphatidylcholine ( dopc , t c = −17 °c ) , 1 , 2 - dimyristoyl - phosphatidylcholine ( dmpc , t c = 24 °c ) , 1 , 2 - dipalmitoylphosphatidylcholine ( dppc , t c = 41 °c ) , or 1 , 2 - distearoylphosphatidylcholine ( dspc , t c = 55 °c ) [SEP]
[CLS] following overnight incubation B-technique with dna that is doubly functionalized with cholesterol and cy5 ( table s1 , entry 1 ) at t > t c for all lipids B-material , colloidally stable l - snas formed from all liposome B-nanoparticle scaffolds . the maximum dna loading per particle was observed for l - snas synthesized with dopc , as evidenced by the absence of free unincorporated dna by gel B-technique electrophoresis I-technique at the highest liposome to dna ratio ( figure 2 ) . [SEP]
[CLS] we hypothesize that this is because cholesterol is more readily intercalated into the membrane bilayer of dopc - based liposomes B-nanoparticle than those comprised of fully saturated phospholipids , due to conformational rearrangement of lipids B-material in the liquid - crystalline phase . [SEP]
[CLS] indeed , the maximum dna loading per particle was reduced in l - snas comprised of lipids B-material with higher t c values ( i . e . , dmpc , dppc , and dspc ) , as evidenced by the presence of free dna at loadings exceeding 150 strands per particle ( 0 . 31 pmol / cm 2 , figure 2 ) . [SEP]
[CLS] this is presumably due to decreased membrane fluidity of liposomes B-nanoparticle comprised of lipids B-material with higher t c . [SEP]
[CLS] dynamic B-technique light I-technique scattering I-technique ( dls ) analysis confirmed that the l - sna hydrodynamic diameter was identical regardless of core B-material composition ( table s2 ) . [SEP]
[CLS] to evaluate the dna dissociation from the l - sna scaffold in biological media , l - snas were synthesized using liposomes B-nanoparticle containing 1 % rhodamine - labeled lipids B-material ( figure s1 ) and dna doubly functionalized with cholesterol and cy5 ( table s1 , entry 1 ) . [SEP]
[CLS] on the l - sna scaffold , cy5 and rhodamine are within the radius required for forster resonance energy transfer ( fret ) , with the fret signal indicative of the presence of intact l - snas . [SEP]
[CLS] fret - capable l - snas were incubated B-technique in 10 % fetal bovine serum ( fbs ) at 37 °c , and their stability was evaluated as a function of the decrease in fret signal over time ( figure s2 ) . [SEP]
[CLS] to account for any potential variations in dye incorporation per particle and to normalize the data for comparison , the apparent rate constant , k , was calculated for each l - sna using a one - phase exponential decay equation ( figure s2 ) to determine the initial rate of fret decrease . [SEP]
[CLS] the calculated k values of the l - snas decrease as a function of increasing t c ( figure 3a ) , indicating that the dna dissociation rate is slower in higher - t c l - snas , and that these l - snas are more stable . [SEP]
[CLS] indeed , the initial rate of fret decay for l - snas synthesized with dspc was determined to be approximately 60 % lower than those synthesized with dopc at 37 °c . [SEP]
[CLS] importantly , the trends in the rate of dna dissociation from the sna scaffold as a function of t c become more pronounced at increased temperatures ( figure s3 ) . [SEP]
[CLS] the rate of dna dissociation from l - snas synthesized with dspc is [UNK] % lower than l - snas synthesized from dopc when serum stability is assessed at 65 °c . [SEP]
[CLS] moreover , the differences in the dna dissociation rate become negligible when l - snas are incubated B-technique at 20 °c ( figure s4 ) , due to decreased membrane fluidity of all l - sna constructs . [SEP]
[CLS] to correlate the in vitro serum stability with biological outcomes , the cellular uptake of all l - snas by primary immune cells B-material was evaluated as a function of liposome B-nanoparticle scaffold . [SEP]
[CLS] increased cellular uptake as a function of t c was observed in bone - marrow - derived dendritic cells B-material ( dcs ) , with an overall upward trend between t c and cellular uptake , showing an approximately 80 % increase in uptake of l - snas comprised of dspc as compared to dopc ( figure 3b ) . [SEP]
[CLS] intriguingly , the cellular uptake by dcs was consistently highest for l - snas synthesized from dppc in biological replicates ( n = 3 ) . [SEP]
[CLS] we hypothesize that this is because the t c of dppc ( 41 °c ) is close to the incubation B-technique temperature used for these assays ( 37 °c ) , and it is known that liposomes B-nanoparticle become increasingly permeable at temperatures close to the t c , thus allowing for increased l - sna membrane flexibility and greater association with cell B-material surfaces . [SEP]
[CLS] the effect of l - sna composition on downstream biological processes was assessed via in vitro dc activation , as these cells B-material are functional antigen - presenting cells B-material and have been shown to be efficiently activated by immunostimulatory l - snas . [SEP]
[CLS] l - snas were synthesized using immunostimulatory oligonucleotides ( cpg - 1826 ) functionalized with cholesterol ( table s1 , entry 2 ) and liposomes B-nanoparticle comprised of dopc , dmpc , dppc , or dspc . [SEP]
[CLS] following incubation B-technique with l - snas , dc activation was evaluated as a function of cd86 expression , which is a surface protein B-material present on mature immune cells B-material that promotes t cell differentiation and survival . [SEP]
[CLS] a dramatic increase in immune activation was observed when dcs were incubated B-technique with the more stable l - snas ( figure 3c ) , revealing a 6 . 5 - fold increase in cd86 expression by l - snas synthesized with dspc , as compared to those synthesized with dopc . [SEP]
[CLS] consistent with the cellular uptake trends observed in dcs , l - snas comprised of dppc induced the highest expression of cd86 , resulting in a nearly 10 - fold increase in cd86 expression as compared to l - snas comprised of dopc ( table s3 ) . [SEP]
[CLS] because immunostimulatory l - snas synthesized with dppc ( henceforth referred to as " dppc - snas " ) showed the highest in vitro stability , uptake , and cd86 expression , the immune uptake and responses generated by this construct were more comprehensively analyzed and compared to the standard l - sna construct , which is synthesized with dopc ( henceforth referred to as " dopc - sna " ) . [SEP]
[CLS] dppc - snas were consistently taken up to a greater extent by dcs than dopc - snas at all time points analyzed ( figure s5 ) . [SEP]
[CLS] in addition , dppc - snas showed increased immune activation over dopc - snas , as measured by the expression of surface proteins B-material cd80 and mhc - ii ( figure s6 ) and the secretion of the cytokine tnf - α ( figure s7 ) following 24 h of incubation B-technique . [SEP]
[CLS] to determine whether superior in vitro dc uptake correlates with higher in vivo lymph node targeting , the accumulation of dppc - snas in the lymph nodes was compared to that of dopc - snas as a function of time . [SEP]
[CLS] to achieve this , healthy mice were administered fluorophore - labeled snas via subcutaneous injection at a dose of 2 . 5 nmol of dna per construct ( n = 3 ) . [SEP]
[CLS] the lymph node accumulation of dppc - snas was 4 - fold higher than dopc - snas at 2 h postinjection ( figure s8 ) , while the concentration was equivalent at 24 h postinjection ( figure s9 ) . [SEP]
[CLS] together , this indicates that dppc - snas are shuttled more rapidly to the lymph nodes in vivo , without a loss in long - term retention . [SEP]
[CLS] the administration of cpg - 1826 in the 4t1 model of tnbc can suppress the spontaneous formation of lung metastases B-event , as a form of " adjuvant - only " immunotherapy . [SEP]
[CLS] thus , to evaluate whether l - sna stability and in vitro immunostimulatory activity correlated with in vivo outcomes , the activity of dopc - snas was compared to dppc - snas in the 4t1 mouse model ( figure 4 ) . [SEP]
[CLS] mice bearing 4t1 tumors B-material were peritumorally administered dopc - snas and dppc - snas via subcutaneous injection at a dose of 5 nmol of cpg - 1826 on days 6 , 10 , and 15 postinoculation ( n = 4 per group ) . [SEP]
[CLS] as a negative control , an additional set of animals ( n = 4 ) was administered saline . [SEP]
[CLS] at day 28 of the study , animals were sacrificed , lungs were perfused per literature protocol , and the number of lung nodules was counted . [SEP]
[CLS] dppc - snas significantly inhibited the formation of lung metastases B-event ( figure 4a , b ) , whereas dopc - snas were ineffective . [SEP]
[CLS] interestingly , this trend held when evaluating primary tumor B-material growth ( figure 4c , d ) , with dppc - snas significantly suppressing tumor B-material growth , while dopc - snas had no effect on primary tumor B-material growth throughout the duration of the study . [SEP]
[CLS] consistent with in vitro results ( vide supra ) , l - sna potency in the 4t1 model directly correlates with liposome B-nanoparticle stability . [SEP]
[CLS] to evaluate whether the observed differences in l - sna activity were tumor - model - dependent , the antitumor B-event activity I-event of l - snas in the py8119 model of tnbc was also evaluated . [SEP]
[CLS] unsurprisingly , administration of dppc - snas and dopc - snas as " adjuvant - only " immunotherapeutics was ineffective in this model ( figure 5a ) , as immunostimulation alone is often insufficient at raising an antitumor immune response , with a few notable exceptions that include the aforementioned 4t1 model . [SEP]
[CLS] since no common taas have been identified for tnbc , lysates from py8119 cells B-material were generated and then utilized as antigen sources . [SEP]
[CLS] these lysates were encapsulated into l - snas comprised of dopc ( dopc - lys - snas ) or dppc ( dppc - lys - snas ) . [SEP]
[CLS] the antitumor efficacy of both constructs was compared in vivo following l - sna administration at days 6 , 10 , and 15 at a dose of 5 nmol of cpg - 1826 and 10 μg of lysate . [SEP]
[CLS] excitingly , there was a [UNK] % reduction in tumor B-material growth when dppc - lys - snas were administered , as compared to when saline was administered to animals . [SEP]
[CLS] conversely , there was no difference in tumor B-material growth when dopc - lys - snas were administered to animals ( figure 5b ) , again showing the dependence of antitumor efficacy on l - sna stability . [SEP]
[CLS] oxidizing cancer cells B-material prior to lysate generation often increases their immunogenicity B-property , and incorporating oxidized lysates into the core B-material of l - snas leads to dramatic increases in the antitumor efficacy in mouse models of tnbc [SEP]
[CLS] therefore , to determine whether the effects of tumor B-material cell B-material oxidation and l - sna stability were additive , l - snas containing lysates from oxidized py8119 cells B-material were synthesized using dopc ( dopc - oxlys - snas ) and dppc ( dppc - oxlys - snas ) . [SEP]
[CLS] strikingly , dppc - oxlys - snas significantly suppressed tumor B-material growth over the duration of the study ( figure 5c ) , while dopc - oxlys - snas were ineffective at these doses of dna and lysate . [SEP]
[CLS] collectively , these data indicate that the effects of the lysate preparation method and l - sna stability are synergistic , a trend which becomes clearer when the study end point for all dppc - based l - snas is compared ( figure 5d ) . [SEP]
[CLS] while dppc - snas are ineffective at reducing tumor B-material growth in the py8119 model , the inclusion of lysates into the l - sna ( dppc - lys - snas ) renders these materials effective , and maximum antitumor efficacy is observed when lysates from oxidized cells B-material are used as the antigen sources ( dppc - oxlys - snas ) , revealing the importance of both the antigen processing method and liposome B-nanoparticle stability in these constructs . [SEP]
[CLS] synthesizing l - snas from liposomes B-nanoparticle comprised of lipids B-material with identical phosphatidylcholine headgroups , but a varied diacyl lipid B-material tail , allows for single - variable analysis of biological properties . [SEP]
[CLS] through these studies , we have determined that the serum stability , cellular uptake , immune activation , and antitumor B-event activity I-event of l - snas can be augmented by using liposome B-nanoparticle scaffolds comprised of lipids B-material with higher t c values while keeping the nanoparticle B-nanoparticle size and surface chemistry identical . [SEP]
[CLS] the dynamics of lipid B-material exchange in liposomes B-nanoparticle is a function of membrane fluidity , which decreases as t c increases . [SEP]
[CLS] thus , synthesizing l - snas from lipids B-material with higher t c produces structures whose dynamics of lipid B-material exchange are slower . [SEP]
[CLS] in turn , the rate of dna dissociation from the l - sna scaffold is significantly decreased when snas are synthesized from lipids B-material with higher t c , leading to greater interactions between the oligonucleotides and liposome B-nanoparticle core B-material and an overall increased structural stability . [SEP]
[CLS] because the l - sna architecture drives its biological properties , prolonged preservation of this structure in biological media likely leads to the observed enhancements in cellular uptake , immune cell B-material activation , and in vivo lymph node accumulation . [SEP]
[CLS] these results indicate that the biological interactions of nanomaterials B-material are not solely determined by nanoscale size and surface chemistry but rather that the chemical identity of the components of noncovalent assemblies also plays a substantial role . [SEP]
[CLS] we have previously shown that seemingly small chemical and structural changes to the l - sna platform can have profound impacts on resulting sna biological properties and functions . [SEP]
[CLS] for example , changing the chemistry used to anchor dna into the l - sna affects the in vitro serum stability , immune activation , and in vivo tissue distribution due to differences in l - sna stability . [SEP]
[CLS] moreover , altering the chemical bond that releases peptide B-material antigens from immunostimulatory l - snas enhances immune activation , and modifying the chemical composition and structural arrangement of antigenic cargo within immunostimulatory l - snas dramatically affects downstream antitumor efficacy because of differences in signaling kinetics . [SEP]
[CLS] in line with these findings , we now show that the composition of the liposomal B-nanoparticle core B-material can be used to modulate the biological properties of l - snas , providing another tunable handle in their rational design as immunotherapeutics . [SEP]
[CLS] in the 4t1 tnbc mouse model , administration of immunostimulatory l - snas synthesized with dppc significantly decreased tumor B-material growth and lung metastasis B-event formation , as compared to l - snas synthesized with dopc , indicating that more stable l - sna constructs are required for maximal immunostimulation in vivo . [SEP]
[CLS] moreover , in tnbc models where " adjuvant - only " therapy is ineffective , the inclusion of tumor B-material cell B-material lysates in the core B-material of the l - snas reveals a significant dependence on l - sna composition with respect to antitumor efficacy , where structural stability determines potency . [SEP]
[CLS] in the py8119 mouse model , lysates encapsulated in l - snas synthesized with dppc were more effective than their dopc analogues at stalling tumor B-material growth ; a trend that became more pronounced when incorporating lysates from oxidized py8119 cells B-material into the l - sna scaffold , revealing synergy between the lysate incorporation method and l - sna stability . [SEP]
[CLS] taken together , this work convincingly shows that seemingly subtle changes to the chemical structure of nanoscale immunotherapeutics can profoundly impact their performance and that liposome B-nanoparticle stability can be used as a variable to control the biological properties of l - snas . [SEP]
[CLS] these results have important implications for the development of therapeutic l - snas that extend beyond cancer immunotherapy . [SEP]
[CLS] for example , in applications where snas are systemically administered ( e . g . , gene regulation ) , l - snas synthesized using high - t c lipids B-material may maximize the serum stability and blood circulation . [SEP]
[CLS] furthermore , in applications where high stability is not advantageous , such as those where encapsulated cargo needs to be released from the core B-material of the l - sna following cellular uptake , 9 , 46 l - snas with intermediate B-property stability may be the most useful . [SEP]
[CLS] therefore , it is critical to carefully consider the liposome B-nanoparticle scaffold when designing both l - snas , as well as other liposome - based nanoparticle B-nanoparticle systems , for biomedical applications . [SEP]
[CLS] * sı supporting information [SEP]
[CLS] the supporting information is available free of charge at https : / / pubs . acs . org / doi / 10 . 1021 / acscentsci . 1c00181 . [SEP]
[CLS] materials and methods ; scheme s1 . [SEP]
[CLS] synthetic scheme of 1 , 2 - distearoyl - sn - glycero - 3 - phosphorylethanolamine ( dspe ) - rho ; table s1 . dna sequences ; table s2 . [SEP]
[CLS] dls analysis of l - snas ; table s3 [SEP]
[CLS] calculated increase ( % ) in cd86 expression ; [SEP]
[CLS] 1 . liposomal B-nanoparticle sna ( l - sna ) design parameters . [SEP]
[CLS] ( a ) schematic representation of the l - sna , wherein dna ( green ) is embedded into the outer phospholipid membrane of a liposome B-nanoparticle core B-material using a hydrophobic B-property dna anchor ( depicted in red ) . [SEP]
[CLS] the modularity of the l - sna architecture allows for the rapid generation of diverse constructs by using different phospholipids . [SEP]
[CLS] ( b ) chemical structure and phase transition temperature ( t c ) of the phospholipids used in the synthesis of the l - snas employed in these studies . [SEP]
[CLS] dna loading capacity of various liposome B-nanoparticle scaffolds . [SEP]
[CLS] the dna loading onto liposomes B-nanoparticle was assessed using native gel B-technique electrophoresis I-technique and cy5 - labeled dna . [SEP]
[CLS] l - snas can be successfully formed using liposomes B-nanoparticle comprised of all phospholipids tested ( dopc , dmpc , dppc , and dspc ) at dna loadings of up to 100 strands per particle , as evidenced by the reduction in dna mobility B-property without the presence of unincorporated dna , following incubation B-technique of cholesterol - functionalized , cy5 - labeled dna with liposomes B-nanoparticle at t > t c for all constructs . [SEP]
[CLS] in vitro serum stability , cellular uptake , and immune activation by l - snas . [SEP]
[CLS] ( a ) plot of the initial rate of decay , k , as a function of decrease in fret signal over time . [SEP]
[CLS] changing the liposome B-nanoparticle scaffold from dopc to one comprising phospholipids of higher t c significantly decreases the rate of dna dissociation from l - snas , thus increasing the stability of the overall construct . [SEP]
[CLS] ( b ) cellular uptake of l - snas by dcs as a function of liposome B-nanoparticle scaffold . [SEP]
[CLS] uptake is significantly increased by synthesizing l - snas from all higher - t c lipids B-material . [SEP]
[CLS] ( c ) dc activation as a function of l - sna composition . [SEP]
[CLS] changing the liposome B-nanoparticle scaffold from dopc to one comprising phospholipids of higher t c significantly increases the observed expression of cd86 . [SEP]
[CLS] statistical analysis was performed using an unpaired t test , where " * * " represents a p value of < 0 . 01 , " * * * " represents a p value of < 0 . 001 , and " * * * * " represents a p value of < 0 . 0001 . [SEP]
[CLS] error bars represent standard deviations . [SEP]
[CLS] mfi represents median fluorescence B-property intensity . [SEP]
[CLS] 4 . in vivo antimetastic and antitumor B-event activity I-event of l - snas in the 4t1 tnbc model . [SEP]
[CLS] ( a ) lung metastasis B-event production , as measured by the number of lung nodules identified at day 28 in the study , following administration of saline ( gray bar ) , dopc - snas ( blue bar ) , or dppc - snas ( pink bar ) . [SEP]
[CLS] white dots represent individual animals in each group . [SEP]
[CLS] ( b ) representative photos of lungs excised from each group . [SEP]
[CLS] ( c ) primary tumor B-material growth in the 4t1 model following administration of saline ( gray diamonds ) , dopc - snas ( blue square ) , or dppc - snas ( pink circle ) . [SEP]
[CLS] error bars represent standard errors of the mean . [SEP]
[CLS] ( d ) comparison of tumor B-material volume between treatment groups at day 28 in the study . [SEP]
[CLS] white dots represent individual animals in each group . [SEP]
[CLS] statistical analysis was performed using an unpaired t test , where " * " represents a p value of < 0 . 05 , and " ns " represents a p value of > 0 . 05 . [SEP]
[CLS] 5 . in vivo antitumor B-event activity I-event of l - snas in the py8119 tnbc model . [SEP]
[CLS] ( a ) antitumor efficacy of " adjuvant - only " l - snas as a function of liposome B-nanoparticle stability , following administration of saline ( gray triangle ) , dopc - snas ( blue square ) , or dppc - snas ( pink circle ) . [SEP]
[CLS] ( b ) antitumor efficacy of l - snas encapsulating py8119 lysates as a function of liposome B-nanoparticle stability . [SEP]
[CLS] animals were administered saline ( gray triangle ) , dopc - lys - snas ( blue square ) , or dppc - lys - snas ( pink circle ) . [SEP]
[CLS] ( c ) antitumor efficacy of l - snas encapsulating oxidized py8119 lysates as a function of liposome B-nanoparticle stability . [SEP]
[CLS] animals were administered saline ( gray triangle ) , dopc - oxlys - snas ( blue square ) , or dppc - oxlys - snas ( pink circle ) . [SEP]
[CLS] ( d ) comparison of tumor B-material volume between dppc - containing treatment groups at day 28 in the study . [SEP]
[CLS] white dots represent individual animals in each group . [SEP]
[CLS] error bars represent standard errors of the mean . [SEP]
[CLS] statistical analysis was performed using an unpaired t test , where " * " represents a p value of < 0 . 05 , " * * " represents a p value of < 0 . 01 , and " * * * " represents a p value of < 0 . 001 . [SEP]
[CLS] s1 . [SEP]
[CLS] structure of rhodaminelabeled lipids B-material ; figure s2 . fret decay curves ; figure s3 . [SEP]
[CLS] in vitro serum stability at 65 °c ; figure s4 . in vitro serum stability at 20 °c ; figure s5 [SEP]
[CLS] time - course analysis of l - sna uptake by dcs in vitro ; figure [SEP]
[CLS] s6 . [SEP]
[CLS] expression of cd80 and mhc - ii in vitro ; figure s7 . [SEP]
[CLS] secretion of tnf - α in vitro ; figure s8 . accumulation in lymph nodes at 2 h ; figure s9 . [SEP]
[CLS] accumulation in lymph nodes at 24 h ( pdf ) which could potentially benefit from the outcomes of this research . [SEP]
[CLS] ■ acknowledgmentsresearch reported in this publication was supported by the national cancer institute of the national institutes of health award u54ca199091 . [SEP]
[CLS] the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health . [SEP]
[CLS] the project was also supported by the prostate cancer foundation and the movember foundation award 17chal08 , the polsky urologic cancer institute of the robert h . lurie comprehensive cancer center of northwestern university at northwestern memorial hospital , and the air force research laboratory agreement fa8650 - 15 - 2 - 5518 . [SEP]
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[CLS] the views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements , either expressed or implied , of air force research laboratory or the u . s . government . [SEP]
[CLS] c . e . c . was supported by a postdoctoral fellowship , pf - 20 - 046 - 01 - lib , from the american cancer society , as well as the eden and steven romick postdoctoral fellowship through the american committee for the weizmann institute of science . [SEP]
[CLS] j . w . d . acknowledges support by the chemistry of life processes predoctoral training program at northwestern university . [SEP]
[CLS] l . b . acknowledges support from northwestern urg award 923acadyr1916683 . [SEP]
[CLS] the content is solely the responsibility of the authors and does not necessarily represent the official views of northwestern university . [SEP]
[CLS] fluorescent B-property dye labeling of dna oligonucleotides and nanostructures is one of the most used techniques to track their fate and cellular localization inside cells B-material . [SEP]
[CLS] here , we report that intracellular fluorescence B-property , and even fret signals , cannot be correlated with the cellular uptake of intact dna structures . [SEP]
[CLS] live cell B-material imaging revealed high colocalization of cyanine - labeled dna oligos and nanostructures with phosphorylated small - molecule cyanine dyes , one of the degradation products from these dna compounds . [SEP]
[CLS] nuclease degradation of the strands outside and inside the cell B-material results in a misleading intracellular fluorescent B-property signal . [SEP]
[CLS] the signal is saturated by the fluorescence B-property of the degradation product ( phosphorylated dye ) . [SEP]
[CLS] to test our hypothesis , we synthesized a range of dna structures , including cy3 - and cy5 - labeled dna cubes and dna tetrahedra , and oligonucleotides with different stabilities toward nucleases . [SEP]
[CLS] all give fluorescence B-property signals within the mitochondria after cellular uptake and strongly colocalize with a free phosphorylated dye control . [SEP]
[CLS] kinetics experiments revealed that uptake of stable dna structures is delayed . [SEP]
[CLS] we also studied several parameters influencing fluorescent B-property data : stability of the dna strand , fixation methods that can wash away the signal , position of the dye on the dna strand , and design of fret experiments . [SEP]
[CLS] dna nanostructures hold tremendous potential for biomedical applications and biotechnology because of their biocompatibility B-property , programmability , and easy synthesis . [SEP]
[CLS] however , few examples of successful dna machines in vivo have been reported . [SEP]
[CLS] we believe this contribution can be used as a guide to design better cellular uptake experiments when using fluorescent B-property dyes , in order to further propel the biological development , and application of dna nanostructures . [SEP]
[CLS] dna - and rna - based therapeutics , such as antisense oligonucleotides , aptamers , small interfering rnas , micro - rnas , and the recently developed crispr - cas9 editing tool , have emerged as highly promising strategies for disease treatment . [SEP]
[CLS] compared to small molecules , oligonucleotides are highly charged , have a high molecular weight , and are easily degradable by enzymes . [SEP]
[CLS] therefore , one essential element to their clinical success is their efficient intracellular delivery . [SEP]
[CLS] poor stability and cellular permeability have hampered the development of these technologies . [SEP]
[CLS] the fate of these molecules has been extensively studied , yet they remain extremely complex to elucidate : many different possible cellular uptake pathways have been discovered . [SEP]
[CLS] differences observed arise from the sequence , the length of the oligonucleotide , or the use of chemical modifications , making almost every molecule unique in its uptake profile . [SEP]
[CLS] more recently , the assembly of dna and rna into nanostructures has been explored as a method to deliver oligonucleotides and therapeutics . [SEP]
[CLS] dna nanostructures are easily synthesized and highly programmable , such that arbitrary shapes and sizes can be efficiently designed . [SEP]
[CLS] many examples in the literature have looked at their use in bioimaging , biosensing , or drug delivery . [SEP]
[CLS] these biocompatible B-property constructs are more resistant to nuclease degradation than their singlestranded components and offer complete control over the position of ligands . [SEP]
[CLS] they can be used to position drugencapsulating polymers B-material or protein B-event - binding I-event ligands . [SEP]
[CLS] they are also promising delivery tools for silencing oligonucleotides . [SEP]
[CLS] for example , we showed that antisense strands positioned on dna cages can induce higher gene silencing than the strands themselves , when transfected with lipofectamine . [SEP]
[CLS] dna nanostructures can also be designed to be dynamic and signal responsive : they can release a therapeutic upon the recognition of a mrna sequence , upon a change of ph , or with light . [SEP]
[CLS] all these results have demonstrated the potential to use dna nanostructures as drug delivery systems . [SEP]
[CLS] however , these structures face challenges similar to those faced by simple oligonucleotides because they are highly charged and degradable . [SEP]
[CLS] one essential element that differs from linear oligonucleotides is their 3d shape , which has been proposed to trigger new uptake profiles . [SEP]
[CLS] here again , structures will act differently according to sequence , length , and presence of chemical modification , but also its 3d shape and dna density , making the analysis of each of these structures unique . [SEP]
[CLS] in 2011 , tuberfield et al . reported the cellular uptake of a double - stranded dna tetrahedron in mammalian cells B-material . [SEP]
[CLS] the study showed that cyanine 5 dye ( cy5 ) - labeled structures were taken up via endocytosis B-event . by labeling the structure with biotin , the authors measured that 2 % of the initial tetrahedra was found inside the cell B-material . [SEP]
[CLS] following this important publication , many groups have explored the uptake of dna structures in cells B-material . [SEP]
[CLS] however , we found that the literature on cellular uptake of dna cages was somewhat ambiguous , uptake was not always quantified , and results greatly vary from one laboratory to another , due to differences in experimental design . [SEP]
[CLS] for example , for one structure ( tetrahedron ) , studies report minimal cell B-material uptake without transfection while other studies revealed high cell B-material internalization . [SEP]
[CLS] in some cases , uptake was increased by the positioning of aptamers on the structure . [SEP]
[CLS] other groups described no uptake of cyanine 3 dye ( cy3 ) - labeled dna duplex but observed uptake of the tetrahedron inside cells B-material , which is not consistent with the above - described first study , where single - and double - stranded controls were found inside the cell B-material . [SEP]
[CLS] the sequence and length of the oligonucleotide used as a control is an important parameter that needs to be carefully studied . [SEP]
[CLS] the uptake of larger structures , such as dna origami , also remains elusive , and recent studies reported that it depends on both the nanoparticle B-nanoparticle shape and the cell B-material line used . [SEP]
[CLS] bastings et al . used oligolysine - based coating B-material ( to prevent degradation ) on dna structures to study their uptake , while other groups studied the uptake of naked dna origami . [SEP]
[CLS] on the other hand , it is important to highlight some of the successes made with dna architectures . [SEP]
[CLS] in particular , in 2012 , a tetrahedron was decorated with folate to successfully deliver sirnas in vivo . [SEP]
[CLS] a dna icosahedron was used to encapsulate a fluorescent B-property polymer B-material to track ph changes in caenorhabditis elegans . [SEP]
[CLS] dna origami structures were also used as successful therapeutic robots by church et al . [SEP]
[CLS] more recently , a dna origami was functionalized with aptamers to target cancerous endothelial cells B-material , and inhibition of tumor B-material growth was demonstrated in mice and miniature pigs . [SEP]
[CLS] these examples demonstrate how careful design of either wireframe dna architectures or dna origami with chosen ligands can lead to a successful therapeutic device . [SEP]
[CLS] overall , despite the explosion in the number of designs of dna nanostructures , and some successes in vivo , the uptake of naked dna architectures in cells B-material remains ambiguous . [SEP]
[CLS] we believe there is a need to understand more precisely what governs the successful uptake of a 3d dna structure and its intracellular fate in cells B-material and in biological fluids . this will give greater insight into how to design better devices to achieve the desired therapeutic outcomes . [SEP]
[CLS] in this paper , we highlight some important considerations that need to be taken into account when examining the uptake of dna structures . [SEP]
[CLS] under non - transfected conditions , uptake of naked dna nanostructures is , in general , too low to observe any gene silencing , similar to oligonucleotides . [SEP]
[CLS] therefore , many groups have looked at uptake pathways by positioning a fluorescent B-property dye on the structure and tracking the intracellular signal . [SEP]
[CLS] cyanine dyes , and fluorescein to a lesser extent , have been mostly used since they are available as phosphoramidites and therefore easy to attach to nucleic B-material acids I-material . [SEP]
[CLS] however , the fluorophores themselves can cross the cellular membrane and accumulate in cell B-material organelles . [SEP]
[CLS] we initially questioned whether the intracellular fluorescent B-property signal corresponds to ( i ) dye - guided uptake of the structure ( the dye interacts with the cellular membrane ) , ( ii ) degradation of the structure followed by uptake of the dye , or ( iii ) unaided cellular uptake of the intact dna structure ( the small - molecule dye does not play an important role ) . [SEP]
[CLS] our experiments revealed that the intracellular fluorescence B-property signal was caused by degradation of the dna by extracellular nucleases , leading to release and uptake of the cyanine dyes . [SEP]
[CLS] phosphorylated dyes as models of degradation products were synthesized , incubated B-technique with cells B-material , and colocalized perfectly with the signal from dna structures . [SEP]
[CLS] we showed that stabilizing the structure toward nuclease degradation simply delayed the intracellular fluorescent B-property signal . [SEP]
[CLS] serum - free conditions , where extracellular nucleases are removed , or chemical fixation caused the disappearance of the signal . [SEP]
[CLS] finally , we showed that forster resonance energy transfer ( fret ) , often used to assess integrity of a dna structure inside the cells B-material , needs more careful controls when performed . [SEP]
[CLS] notably , we could observe fret between two separate , free phosphorylated dyes ( cy3 and cy5 ) when they were co - incubated B-technique with cells B-material . [SEP]
[CLS] assembly of structure and design . [SEP]
[CLS] in this study , we will focus on wireframe dna architectures . [SEP]
[CLS] our group has developed dna minimal nanocages B-nanoparticle such as cubes , prisms , and nanotubes B-nanoparticle . [SEP]
[CLS] in particular , the dna nanocube is composed of four dna strands ( 96 - mers component strands called " clips " ) that can self - assemble in a one - pot reaction with quantitative yields . [SEP]
[CLS] the dna cube displays eight singlestranded regions ( four on the top , and four on the bottom ) . [SEP]
[CLS] a dna tetrahedron was also prepared , as it is one of the most used structures in the literature . [SEP]
[CLS] the tetrahedron is also composed of four dna strands but is fully double - stranded . [SEP]
[CLS] we labeled both structures at the 5 ′ - end with a cyanine dye using phosphoramidite chemistry : the tetrahedron was labeled with cy5 , and two clips were prepared for the cube , one with cy3 and one with cy5 ( figure 1 ) . [SEP]
[CLS] this labeling procedure has been commonly used in the literature . [SEP]
[CLS] thermal assemblies were performed following previous protocols and assessed using gel B-technique electrophoresis I-technique ( figure s1 ) . [SEP]
[CLS] cellular uptake of cyanine - labeled oligonucleotide and dna nanostructures ( 5 ′ - end ) . [SEP]
[CLS] first , we compared the cellular uptake of the assembled nanostructure with the corresponding component strand ( clip strand , 96 - mer for the cube , 63 - mer for the tetrahedron ) . [SEP]
[CLS] if dna nanostructures were taken up to a higher extent than their single - stranded components , via new internalization pathways due to their 3d shape , we would observe a difference in their uptake profile . [SEP]
[CLS] structures were added to fbs - containing media and tracked using confocal live microscopy B-technique in hela cells B-material at different time points ( 1 . 5 , 4 , 6 , and 24 h ) . [SEP]
[CLS] after 1 . 5 h , we did not detect any signal for the structures , and the signal for the clips was very low , suggesting that uptake requires a longer time ( figure s9 ) . [SEP]
[CLS] at later time points ( 4 , 6 , and 24 h ) , we observed a similar uptake profile of the clip strands and the respective dna nanostructures , consisting of very bright dots but also long filaments inside the cytoplasm of the cell B-material ( figure 2 and figures s2−s4 and s6−s8 ) . [SEP]
[CLS] the signals of the clip and the structure colocalize well ( figure s5 ) . [SEP]
[CLS] to ensure that the cellular uptake of the component clip is not caused by their folding into secondary structures , which may mimic a 3d scaffold , we looked at the uptake of shorter 5 ′ - end labeled dna strands ( 20 - mers ) and a dinucleotide ( cy3 - tg ) ( figures s10 and s11 ) . [SEP]
[CLS] these short strands revealed the same fluorescent B-property profile inside cells B-material with dots and filaments , suggesting that the pattern observed is not structure - dependent . [SEP]
[CLS] cy5 - labeled structures and oligonucleotides gave signal distribution similar to that of cy3 - labeled structures ( figure s4 ) . [SEP]
[CLS] to gain more insight into the fate of these structures inside cells B-material , we investigated in which organelles the structures are accumulating . [SEP]
[CLS] colocalization analysis revealed mitochondrial accumulation . [SEP]
[CLS] positively charged lipophilic B-property dyes are known to enter the mitochondria due to electrostatic interactions with the mitochondrial membrane . [SEP]
[CLS] 3 , 3 ′ - dihexyloxacarbocyanine iodide B-material ( dioc6 ) , cyanine dyes , and some alexa dyes can be used to stain the mitochondria . oligonucleotides labeled with cy5 or cy3 have been previously reported as mitochondrial markers in different cell B-material lines , resulting in long filamentous fluorescent B-property signal . [SEP]
[CLS] therefore , we tested whether our structures can accumulate in the mitochondria and used celllight mitochondria - gfp ( bacmam 2 . 0 ) and celllight lysosomes - gfp ( bacmam 2 . 0 ) to stain the two organelles ( figure s12 ) . [SEP]
[CLS] the bacmam constructs are plasmids encoding for proteins B-material of the membrane of the organelles fused to green fluorescent B-property protein B-material . [SEP]
[CLS] one cube clip , the dna nanocube , and the dna tetrahedron were tested ( figure 3 [SEP]
[CLS] pearson ' s coefficients ( pccs ) and mander ' s coefficients ( mccs ) , revealed partial colocalization with lysosomes and the mitochondria . [SEP]
[CLS] for the mitochondria , the structures gave similar pccs ranging from 0 . 4 to 0 . 5 after 4 h incubation B-technique and 0 . 6−0 . 8 after 24 h incubation B-technique . [SEP]
[CLS] the increase over time suggests slow accumulation in this organelle . [SEP]
[CLS] the partial colocalization with the lysosome ( pcc remains around 0 . 5−0 . 6 ) is consistent with previous reports in the literature , indicating uptake of cyanine dyes , oligos , and structures via endosome / lysosome pathways ( figures s16−s18 ) . [SEP]
[CLS] while cyanine - labeled oligonucleotides have been reported to result in mitochondrial fluorescence B-property signals , finding dna nanostructures within the mitochondria was surprising and had never been reported previously in the literature . [SEP]
[CLS] as pointed out by bao et al . when they studied cy3 - labeled short oligonucleotides , the positive charge of the dye should be largely compensated by the negative charge of the dna . [SEP]
[CLS] this makes an entire dna strand , and to a larger extent a dna nanostructure , unlikely to interact with the negatively charged mitochondrial membranes . [SEP]
[CLS] however , the results were the same for the component strands and dna structures . [SEP]
[CLS] this may imply that the overall charge of the molecules does not impact accumulation to the mitochondria . [SEP]
[CLS] another hypothesis is that the fluorescent B-property signal may simply arise from degradation of the dna structure with release of the cyanine dye , which is known to cross to the mitochondrial membranes . [SEP]
[CLS] we decided to investigate whether the fluorescent B-property degradation products , i . e . , the cyanine dye being cleaved from the oligo , can cross the cellular membrane and stain the mitochondria . [SEP]
[CLS] typically , previous studies have used commercially available versions of the cyanine dye as controls . [SEP]
[CLS] phosphorylated small - molecule cyanine dyes colocalize with nanostructures . [SEP]
[CLS] upon recognition by exonucleases and subsequent hydrolysis of the phosphate linkage B-property , two fluorescent B-property degradation products may be produced from cyanine - labeled oligonucleotides : cy3 - phosphate and cy3hydroxyl ( figure s19 ) . [SEP]
[CLS] the cy3 - hydroxyl is positively charged , has almost the same structure than the cy3 molecule , and is already known as a mitochondrial marker . [SEP]
[CLS] the cy3phosphate carries at least one negative charge ( phosphoester pk a1 [UNK] ) that will neutralize the positive charge of the dye . [SEP]
[CLS] to our knowledge , its interaction with and within cells B-material remains unknown . [SEP]
[CLS] therefore , we synthesized cy3 - phosphate ( cy3 - p ) and cy5 - phosphate ( cy5 - p ) ( figure 4a and supporting information ) . [SEP]
[CLS] fluorescence B-property spectra reveal emission peaks for these molecules similar to those for cy3 and cy5 dyes , as well as similar binding B-event to I-event serum I-event proteins I-event in the cellular media ( figures s22 and s23 ) . [SEP]
[CLS] the dyes have a lower fluorescence B-property intensity compared to the dna - labeled strands ( figures s82 and s83 ) . [SEP]
[CLS] indeed , attaching these fluorophores to dna likely increased the local viscosity B-property resulting in higher fluorescence B-property emission . [SEP]
[CLS] we then incubated B-technique the dyes with hela cells B-material in fbscontaining media ( 1 . 5 h , 6 h , 24 h ) . [SEP]
[CLS] dyes were taken up quickly ( less than 1 . 5 h ) and gave the exact same filamentous signal with some bright spots as dye - labeled dna strands ( figures s24 and s25 ) . [SEP]
[CLS] partial colocalization with mitochondria ( cy5 - p ) and lysosomes ( cy3 - p and cy5 - p ) was measured and showed results similar to those obtained with oligonucleotides and dna structures ( figures s26−s28 ) . [SEP]
[CLS] co - incubating B-technique the two dyes cy3 - p and cy5 - p with cells B-material resulted in complete colocalization of their respective signals ( pcc is ranging from 0 . 85 to 0 . 95 ) . [SEP]
[CLS] ( figure s29 ) . [SEP]
[CLS] interestingly , upon co - incubation B-technique , each of our previously tested oligonucleotides and nanostructures was found to colocalize with the dye ( figure 4b and figures s30−s35 ) . [SEP]
[CLS] finally , we tested the cellular uptake of the cy3 - labeled dinucleotide ( cy3 - tg ) , another model for dna degradation as it should lead to both potential degradation products quickly ( cy3 - phosphate and cy3 - hydroxyl ) . [SEP]
[CLS] cy3 - tg strongly colocalized with the cy5 - p signal . [SEP]
[CLS] ( figure s36 ) [SEP]
[CLS] in brief , our degradation model compounds for cyaninelabeled dna strands ( the phosphorylated dyes ) colocalize with dna structures . [SEP]
[CLS] this suggests that the intracellular fluorescent B-property signal may be caused by the degradation product entering the cell B-material , i . e . , the dye getting cleaved from the dna , and not by uptake of the dna structure . [SEP]
[CLS] therefore , it favors the degradation hypothesis over the hypothesis of the dye guiding the entire structure to the mitochondria . [SEP]
[CLS] to confirm this , we looked at uptake kinetics and synthesized structures that are more resistant to nuclease degradation . [SEP]
[CLS] these structures will lead to a slower release of the cyanine dye within the cell B-material media , potentially allowing the dna structure to enter cells B-material before its degradation by nucleases . [SEP]
[CLS] more - resistant dna cubes and clip components result in slower intracellular fluorescence B-property . [SEP]
[CLS] when we examined the kinetics of cellular uptake , we noted that the free dyes and the free dinucleotide cy3 - tg were taken up in less than 2 h . [SEP]
[CLS] intracellular fluorescence B-property from the 5 ′ - end labeled clip appears and increases after 2 h , while it takes more than 4 h for the 3d structures to give a detectable signal ( figure s9 ) . [SEP]
[CLS] at 24 h , the signal from the clip and the structures are the same . [SEP]
[CLS] similar results were observed by bao et al . as they studied different length oligonucleotides . [SEP]
[CLS] we hypothesize that the delayed signal is caused by a delayed degradation . [SEP]
[CLS] indeed , dna 3d constructs are more stable than their single - stranded components . [SEP]
[CLS] therefore , as it takes more time for nucleases to digest the strand and for the dye to get cleaved , the fluorescence B-property signal is delayed . [SEP]
[CLS] to test this hypothesis , we synthesized new structures with different serum stabilities , resulting in different release rates of the cyanine dye [SEP]
[CLS] in serum , 3 ′ - exonucleases are the main cause for dna degradation , while inside the cell B-material both 5 ′ - and 3 ′ - exonucleases will degrade oligonucleotides . [SEP]
[CLS] therefore , labeling the strand on the 5 ′ - end does not protect it from the main source of nuclease degradation in cell B-material media , leading to a fast release of the dye . [SEP]
[CLS] to overcome this stability issue , we synthesized cube clips with cy3 and cy5 positioned at the 3 ′ - end . [SEP]
[CLS] we also synthesized two dna clips with the dye " buried " in the sequence by changing the position to two different internal positions : one at the corner of the cube structure ( clip " cor " ) , and one in the single - stranded region ( clip " mid " ) ( figure 5a and table s - i ) . [SEP]
[CLS] to verify increased nuclease resistance , we performed serum stability experiments : strands were mixed in cell B-material media ( supplemented with serum , that contains nucleases ) , samples were collected at different time points and analyzed using gel B-technique electrophoresis I-technique experiments . [SEP]
[CLS] the halflife extracted from these experiments reflect the time at which 50 % of the full product is still intact , but it does not give us the exact time of release of the phosphorylated dye degradation product ( cy3 - p or cy5 - p ) . [SEP]
[CLS] it is still , however , a good indication of the relative stabilities of each of the strands . [SEP]
[CLS] the half - life of the clip with cy3 positioned at the 5 ′ - end is the shortest ( 52 min ) , and the 3 ′ - modification dramatically increased the resistance to extracellular enzymes ( 236 min ) . [SEP]
[CLS] the two internal positions also increased serum stability but gave different results ( 69 min ( mid ) and 120 min ( cor ) ) ( figures s37 and s38 and table s - ii ) , most likely due to sequence dependence of nuclease activity . [SEP]
[CLS] the assembled dna cubes have higher stabilities than the clip counterparts ( table s - iii ) . [SEP]
[CLS] the different modifications only slightly affected the overall stability of the cube structure ( figures s41 and s42 ) . [SEP]
[CLS] in denaturing conditions of the self - assembled structures , the 3 ′ - end and the corner modification still have the highest stabilities , meaning that the dye from these structures should be released last ( figures s43 and s44 ) . [SEP]
[CLS] we incubated B-technique the different clips and structures with hela cells B-material and looked at the fluorescent B-property signal using confocal live microscopy B-technique ( figures s53−s64 ) . [SEP]
[CLS] after 4−5 h incubation B-technique , the uptake of the more stable clips or structures is barely detectable while the 5 ′ - end clip gives a strong fluorescent B-property signal . [SEP]
[CLS] when comparing the intracellular fluorescence B-property , by fixing the laser settings , the signal of the 5 ′ - end labeled strands and cubes seems much higher than for the other clips ( figure 5a and figure s65 ) . [SEP]
[CLS] we believe that this difference in intracellular fluorescence B-property is caused by the slower degradation of the strands , causing delayed signal . [SEP]
[CLS] indeed , at 24 h , our serum stability experiments revealed that the 5 ′ - end labeled strand is fully degraded while a smearing on the gel can be observed for the other strands ( 3 ′ - , cor , and mid ) , indicating the presence of a variety of different length oligonucleotides ( figure s37 ) . [SEP]
[CLS] finally , we co - incubated B-technique the phosphorylated dyes with the different constructs and confirmed the colocalization of the signal for all the dna structures at 24 h , by measuring the pccs and mccs ( figure 5b and figures s53−s64 ) . [SEP]
[CLS] in short , as we slow down degradation and dye release , the signal appears slower for cube clips and cubes , strengthening the hypothesis of degradation of the structure followed by cellular uptake of the dye ( or short dye - labeled oligonucleotides ) . [SEP]
[CLS] results were confirmed in another cancer cell B-material line , hepg2 cells B-material ( liver hepatocellular cells B-material ) . [SEP]
[CLS] we observed cellular uptake of the dyes and delayed uptake of the 3 ′ - end labeled structure . [SEP]
[CLS] ( figures s94−s98 ) . [SEP]
[CLS] to confirm further that stable structures are taken up later , we looked at the dna tetrahedron , which is fully double - stranded and more resistant to nucleases . [SEP]
[CLS] hexaethylene glycol to prevent dna tetrahedron degradation . [SEP]
[CLS] the tetrahedron structure has been extensively investigated in biological studies . [SEP]
[CLS] however , in our hands , the assembly of the tetrahedron led to multiple higher mobility B-property products in the gel , indicating the formation of higher - order assemblies ( figure s1 ) . [SEP]
[CLS] we used this mix of products for our studies , as the protocol has been widely used by the community ( supporting information ) . [SEP]
[CLS] we also purified the tetrahedron structure using electrophoretic methods , to further confirm our results with the monodisperse structure ( figure s1 ) . [SEP]
[CLS] to prevent the release of the dye , we synthesized a fluorescently B-property labeled clip protected by hexaethylene glycol units ( heg ) at both the 3 ′ - and 5 ′ - ends . [SEP]
[CLS] the heg modification increased resistance to nuclease degradation ( figures s39 and s40 , and tables s2 and s3 ) . [SEP]
[CLS] the modification did not affect the overall stability of the tetrahedron , but the clip from the structure is more stable ( figures s45 , s46 , s48 , and s49 ) . [SEP]
[CLS] incubation B-technique with cells B-material led to similar conclusions : the heg protection , as it increased the nuclease resistance , significantly delayed the cellular uptake ( figure 6 and figures s67 and s68 ) . [SEP]
[CLS] co - incubation B-technique of cy3 - p with tetrahedron , purified tetrahedron , and heg - protected tetrahedron confirmed colocalization with the degradation product ( figures s66−s72 ) . [SEP]
[CLS] results were confirmed in hepg2 cell B-material line as well ( figures s99−s102 ) . [SEP]
[CLS] at early time points ( 4 h ) , we observed very faint dots ( laser power and gain were increased ) from the dna structure that do not colocalize with the dye ( figures s67 and s68 ) , possibly due to very low uptake of intact structures or oligos , consistent with the 1−2 % uptake observed by turberfield et al . [SEP]
[CLS] these strands eventually degrade after cellular uptake ( endosomal ph and cytoplasmic nucleases ) , releasing the dye as well . [SEP]
[CLS] all these experiments indicate that as we stabilize the dye - labeled dna strand , the fluorescent B-property signal is simply delayed . [SEP]
[CLS] we then investigated other methods to prevent dna degradation and dye uptake , such as changing serum conditions or using negatively charged fluorescent B-property dyes , to further assess the serum degradation hypothesis . [SEP]
[CLS] changing serum conditions to prevent dna degradation . [SEP]
[CLS] to prevent extracellular nuclease degradation , we tested uptake in low - serum conditions ( 0 . 1 % fbs instead of 10 % ) . [SEP]
[CLS] at 0 . 1 % fbs concentration , nuclease degradation should be dramatically reduced . [SEP]
[CLS] we did observe uptake of the free dyes ; however , uptake of 5 ′ - end - labeled clip and cube , as well as of the 3 ′ - end - labeled clips , was considerably reduced ( figure 7a and figures s73−s75 ) . [SEP]
[CLS] this seems to indicate that our dna constructs need to be degraded to produce a detectable signal . [SEP]
[CLS] we also performed " pulse - chase " experiments where structures are incubated B-technique in serum - free conditions ( 0 % fbs ) for 15−20 min , cells B-material are washed , and new cell B-material culture media ( 10 % fbs ) is then added . [SEP]
[CLS] much lower signal could be detected , and after 1 day incubation B-technique , we did observe the same pattern than for degraded products ( filament and dots ) ( figure s76 ) . more stable structures gave a delayed signal compared to less stable ones ( 3 ′ - vs 5 ′ - cy3 cubes ) , suggesting uptake of the degraded product ( figure s77 ) . [SEP]
[CLS] curiously , this result suggests binding or low uptake of some structures into the cell B-material , which are not washed away by the washing steps and are eventually getting degraded . [SEP]
[CLS] sulfonated cyanine dyes do not accumulate in the mitochondria . [SEP]
[CLS] negatively charged sulfonate groups are typically placed on cyanine dyes to increase their solubility B-property in I-property water I-property , avoid dye aggregation , and prevent their cellular uptake . [SEP]
[CLS] we used click chemistry and one of our previously developed phosphoramidites to label the cube clip with a sulfonated cy5 at its 5 ′ - end or in an internal position ( figure 7b and figures s91 and s92 ) . [SEP]
[CLS] the sulfonated clip and cube gave little fluorescent B-property signal inside cells B-material compared to the nonsulfonated cy5 ( figure 7c and figure s93 ) . [SEP]
[CLS] this means that the intracellular fluorescence B-property is dye - dependent and that low fluorescent B-property signal , sometimes observed from other dyes can not be correlated with uptake of the structure . [SEP]
[CLS] we believe that the observation of a fluorescent B-property signal should always be followed by a quantification of the uptake . [SEP]
[CLS] for example , quantification methods such as streptavidin−biotin labeling or qpcr methods revealed low uptake of nanostructures inside cells B-material . [SEP]
[CLS] in conclusion , all our experiments are consistent with prior degradation of the cyanine - labeled dye , followed by uptake of the fluorescent B-property dye inside the cell B-material , leading to most of the observed fluorescent B-property signal . [SEP]
[CLS] previous research from various laboratories has indicated uptake of dna nanostructures through endosomal pathways and used fret experiments to prove their integrity inside cells B-material . [SEP]
[CLS] these results are in apparent contradiction with our observations , and we therefore examined fret experimental design in more details . [SEP]
[CLS] free dyes cy3 - p and cy5 - p can give an intracellular fret signal . [SEP]
[CLS] dna structures can easily be labeled with a fret pair , for example cy3 / cy5 . [SEP]
[CLS] precisely two components strands are labeled : one with cy3 ( donor ) , one with cy5 ( acceptor ) , such that the two dyes are close enough ( 1−10 nm ) to allow energy transfer upon excitation of cy3 . [SEP]
[CLS] if the structure is disassembled , no energy transfer will happen . [SEP]
[CLS] therefore , the observation of fret signal intracellularly is in general explained by invoking the uptake of an intact structure . [SEP]
[CLS] as shown above , we observed that the two free dyes highly colocalize in cells B-material ( figure s29 ) . [SEP]
[CLS] we believe this is due to their high local concentration in the lysosome and their accumulation in the mitochondria . [SEP]
[CLS] we decided to test whether these two separate dye molecules can give a fret signal inside live cells B-material , giving rise to an " incorrect fret signal " ( sometimes called random fret ) . [SEP]
[CLS] we synthesized a positive control for fret : a dna strand labeled with cy3 and cy5 directly attached to one another via a phosphodiester bond at the 5 ′ end called strand cy3 - cy5 ( table s - i ) . [SEP]
[CLS] emission spectra confirmed that the strand cy3 - cy5 causes fret emission in dmem ( cell B-material media ) and fbs - supplemented dmem , while no signal was observed for the free dyes or for mixture of dyes with labeled dna strands ( figures s82−s84 ) . [SEP]
[CLS] the cy3 - cy5 phosphate bond in the strand cy3 - cy5 remains stable upon addition of serum ( figure s85 ) , confirming it can be used as a positive control . [SEP]
[CLS] we then examined fret signals in live cells B-material at 6 and 24 h , using confocal microscopy B-technique . [SEP]
[CLS] structures and dyes were incubated B-technique at 150 nm final concentration . [SEP]
[CLS] after background and cross - talk corrections , we calculated the fret / cy3 ratio to quantify energy transfer . [SEP]
[CLS] surprisingly , we observed high fret signal when the two f ree phosphorylated dyes cy3 - p and cy5 - p were coincubated , while the fret - positive strand gave a smaller signal ( figure 8 and figures s88−s90 ) . [SEP]
[CLS] at 6 h , the ratio is high for the two co - incubated B-technique dyes and for the 5 ′ - end - labeled clip coincubated with free dye . [SEP]
[CLS] the positive control , the strand cy3 - cy5 , gives almost no signal . [SEP]
[CLS] we believe full dna degradation was not reached at this time point and that the linked dyes from the degraded dna strand had not yet entered the cell B-material to a high extent ( figure s85 ) . [SEP]
[CLS] at 24 h , the strand cy3 - cy5 gives a small signal , less than that of the two free dyes . [SEP]
[CLS] interestingly , coincubating a cy3 - labeled dna strand ( 5 ′ - end , less stable ) with a separate cy5 - labeled dna strand ( 3 ′ - end , more stable ) resulted in a measurable fret / cy3 ratio , albeit lower than the two f ree dyes , consistent with the degradation hypothesis . [SEP]
[CLS] overall , the fact that we can observe random fret signal with two separate smallmolecule dyes supports the need of carefully designed controls for fret experiments with dna strands and structures . [SEP]
[CLS] the signals from the free dyes should be studied , or two chemically different dyes should be used . [SEP]
[CLS] fluorescent B-property signal is washed away by chemical fixation . [SEP]
[CLS] another important parameter to compare our work to other studies in the literature is the use of live confocal microscopy B-technique to prevent effects from the fixative agents on the fluorescent B-property signal . [SEP]
[CLS] cyanine dye accumulation inside the mitochondria does not resist chemical fixative agents like formaldehyde . [SEP]
[CLS] we tested whether the signal is disrupted by chemical fixation . when we fixed cells B-material with formaldehyde , the filamentous signal of the phosphorylated dyes disappeared , and we saw only bright dots ( endosome / lysosomes ) and diffuse cytoplasmic signal ( figure s78 ) . [SEP]
[CLS] the result was similar for oligonucleotides ( figure s79 ) , but cy3 - p and cy5 - p still colocalized ( figure s80 ) . [SEP]
[CLS] methanol fixation removed all the fluorescent B-property signal or caused dna precipitation ( figure s81 ) . [SEP]
[CLS] this emphasizes that the effect of chemical fixation needs to be carefully studied since it can cause misleading information . [SEP]
[CLS] safety statement . no unexpected or unusually high safety hazards were encountered in the course of this work . [SEP]
[CLS] the observation of colocalized signals when dna structures and their degradation products are administered to cells B-material indicates that intracellular fluorescence B-property does not necessarily correlate with cellular uptake . [SEP]
[CLS] instead , experiments revealed that most of the signal arises from degradation of dna structures by nucleases , releasing the fluorescent B-property dye that is then taken up inside cells B-material . [SEP]
[CLS] in particular , we observed that , as we stabilized the dye within the structure , uptake was delayed . [SEP]
[CLS] changing the dye to its cell - impermeable version ( sulfonated ) or preventing serum degradation considerably reduced the intracellular fluorescence B-property . [SEP]
[CLS] interestingly , fret signals were observed between the separate free cyanine dyes inside cells B-material , revealing the need for more carefully designed controls when using fret experiments to assess uptake of intact dna structures . [SEP]
[CLS] protocol design , such as the use of chemical fixation instead of live conditions , can also dramatically change the pattern of the fluorescence B-property signal . [SEP]
[CLS] in this study , we focused on wireframe dna - minimal architectures . [SEP]
[CLS] however , we believe that the findings can be extended to any nucleic acid - based material B-material , such as dna origami or rna therapeutics , as nucleases can still access and degrade the labeled strands . [SEP]
[CLS] dna - dense structures such as dna origami may have different degradation profiles than wireframe structures , but they are still prone to nuclease degradation within a few hours and disassembly as the dication concentration is lower in biological media . [SEP]
[CLS] the fluorescent B-property dye to track dna origami cellular uptake is often placed at the 5 ′ - or 3 ′ - end of the short staple strands and can protrude from the surface of the origami . [SEP]
[CLS] embedding the dye within the structure was shown to result in a longer stability of the fret signal , coherent with longer structural integrity . [SEP]
[CLS] beside the signal from the degradation product , the fluorescence B-property signal and intensity will depend on many parameters linked to the nature of the dye : binding B-event to I-event serum I-event proteins I-event can increase fluorescence B-property , as can sequence - specific attachment to a dna strand . [SEP]
[CLS] cellular localization is known to influence the brightness of the dye , for example because of changes of ph in the different organelles . [SEP]
[CLS] therefore , altered fluorescence B-property intensity could simply arise from longer retention in the endosomal compartment , where the dye may be brighter or dimmer ( lowed ph ) . [SEP]
[CLS] we believe that measuring the total fluorescence B-property intensity in a cell B-material , for example by using flow B-technique cytometry I-technique , cannot be simply correlated with higher uptake of a structure . [SEP]
[CLS] precise colocalization and fluorescence B-property analysis need to be performed . [SEP]
[CLS] furthermore , our results may indicate that cyanine dyes may not be an ideal choice for the investigation of cellular uptake of dna structures . [SEP]
[CLS] cyanine dyes fluorescence B-property is strongly dependent on its local environment . [SEP]
[CLS] on the other hand , the cell B-material permeability of the cyanine dyes allowed us to detect the uptake of the degradation products inside the cell B-material . [SEP]
[CLS] it is important to note , however , that using other types of dyes would not prevent the fluorescent B-property molecule from getting cleaved off the dna strand ; they would simply change the intensity or location of the intracellular fluorescent B-property signal arising from degradation . [SEP]
[CLS] the absence of a signal can be difficult to study , as it could just indicate issues with the experimental setup . [SEP]
[CLS] overall , we believe that , in addition to choosing the dye carefully , control experiments involving a phosphorylated f ree dye as a model of degradation , rather than only unsubstituted dye , should be systematically performed when using fluorescence B-property methods such as microscopy B-technique , flow B-technique cytometry I-technique , or fret . [SEP]
[CLS] two chemically different dyes can be used to confirm that the results observed are not dyedependent . [SEP]
[CLS] thorough quantification of the uptake of the dna structure is also needed if the structure is thought to enter the cell B-material . [SEP]
[CLS] nonfluorescent methods such as gene silencing experiments , qpcr , or biotin - labeling can be used for quantification . [SEP]
[CLS] overall , our results can be used as a cautionary tale on how to design and analyze fluorescence B-property experiments when examining the uptake profile of dna - based materials . [SEP]
[CLS] for fluorescence - based assays , we recommend ( a ) comparison of uptake with model of degradation products ( such as the phosphorylated dye , even for fret experiments ) , ( b ) correlation of degradation kinetics with cellular uptake kinetics , ( c ) thorough controls and optimization of the cellular work conditions ( serum , fixation , temperature ) , and ( d ) changing the nature and position of the dye to track whether the signal is dye - dependent or not ( and trying to place the dye in less accessible positions on the labeled oligonucleotide ) . [SEP]
[CLS] finally , our findings bring new important considerations for the use of dna nanostructures in biological systems . [SEP]
[CLS] first , we believe that our results may not be contradictory with previously reported successful examples of dna nanostructures inside cells B-material , but instead they provide clues on the design of more reliable fluorescence - based assays . [SEP]
[CLS] we hope they will help the community deciphering the rules that govern the successful uptake of certain dna structures compared to others . [SEP]
[CLS] we also believe that revealing that wireframe dna nanostructures do not enter cells B-material to a high extent can be turned into a real advantage in using them as drug delivery devices . [SEP]
[CLS] nonspecific cellular uptake of dna cages is not desirable , as it would mean that cages can penetrate different cells B-material in vivo . [SEP]
[CLS] instead , it gives us the opportunity to attach targeting B-material ligands I-material on these cages to promote their entry into specific cellsfor B-material example , folate decoration can lead to internalization into cancer cells B-material . [SEP]
[CLS] to increase cellular uptake , inspiration can also be taken from the extensive work done by the oligonucleotide therapeutics field . [SEP]
[CLS] specifically , we believe that inserting chemical modifications B-event in I-event dna I-event ( nucleobase , backbone , 3 ′ - and 5 ′ - end modifications ) could reduce nuclease degradation while substantially increasing uptake of dna cages . [SEP]
[CLS] on the other hand , because of their poor uptake , dna nanostructures could be used as biosensors or bioimaging systems outside of the cell B-material . [SEP]
[CLS] their programmability and higher nuclease resistance compared to single - stranded dna make them excellent biodegradable B-property materials to sense the cell B-material surface or extracellular proteins B-material . [SEP]
[CLS] they could also be used to promote cell−cell interaction , by targeting membrane receptors . [SEP]
[CLS] finally , as drug delivery systems , they could also be designed to carry small - molecule drugs . [SEP]
[CLS] the dna cage degradation could lead to a slow release of the drug that can then be internalized by the targeted cells B-material . [SEP]
[CLS] stabilizing the structure , to control the rate of drug release , can be achieved by the introduction of chemical modifications in the bases or the dna backbone , chemical coating B-material , or binding B-event to I-event serum I-event proteins I-event . [SEP]
[CLS] ■ associated content * s supporting information [SEP]
[CLS] the supporting information is available free of charge on the acs publications website at doi : 10 . 1021 / acscentsci . 9b00174 . [SEP]
[CLS] general protocols and instrumentation information , dna strands and structures synthesis and characterization , live confocal microscopy B-technique images ( cellular uptake of dyes , oligonucleotides , and structures ) and colocalization analysis , serum stability assays , serum - free experiments , analysis of effect of fixating agents , fret experiments , fluorescence B-property spectra , sulfonated dyes synthesis and uptake , and results in hepg2 cell B-material line , including [SEP]
[CLS] cyanine - labeled dna nanostructures . [SEP]
[CLS] ( a ) two wireframe dna - minimal nanostructures were used in the study : the dna nanocube , composed of four 96 - mers , and the dna tetrahedron , composed of four 63 - mers . [SEP]
[CLS] ( b ) cyanine dyes ( cy3 on the schema ) were attached at the 5 ′ - end of one of the dna component clips , using phosphoramidite chemistry and resulting in a phosphate linkage B-property between the dye and the base . [SEP]
[CLS] uptake of dna oligonucleotides and dna nanostructures . [SEP]
[CLS] ( a ) experimental setup : dna structures are incubated B-technique with mammalian cells B-material , directly in the cellular media ( fbs - supplemented ) . [SEP]
[CLS] fluorescent B-property signal is detected with confocal microscopy B-technique . [SEP]
[CLS] ( microscope image credit : zeiss microscopy B-technique ) [SEP]
[CLS] ( b ) after 6 h incubation B-technique with hela cells B-material , we observed the same fluorescent B-property signal for each dna nanostructures and their component clip , using live confocal microscopy B-technique . [SEP]
[CLS] representative images are shown in the figure . [SEP]
[CLS] scale bars are 20 μm . [SEP]
[CLS] mitochondrial and lysosomal localization of dna structures . [SEP]
[CLS] live confocal microscopy B-technique in hela cells B-material revealed colocalization of dna structures . [SEP]
[CLS] in the image , we represent the cy5 - labeled dna nanocube colocalizing with mitochondria and the cy3 - labeled dna nanocube with the lysosomes , both after 24 h incubation B-technique . [SEP]
[CLS] representative images are shown in the figure . [SEP]
[CLS] phosphorylated dyes colocalize with dna structures . [SEP]
[CLS] ( a ) structures of synthesized phosphorylated cyanine 3 ( cy3 - p ) and cyanine 5 ( cy5 - p ) dyes . [SEP]
[CLS] ( b ) co - incubation B-technique of structures and model for degradation products ( free dyes ) revealed high colocalization , using live confocal microscopy B-technique in hela cells B-material . [SEP]
[CLS] representative images are shown in the figure . [SEP]
[CLS] changing the dye position within the structure . [SEP]
[CLS] representative images are shown in the figure . [SEP]
[CLS] ( a ) live confocal microscopy B-technique of hela cells B-material ( 24 h incubation B-technique ) with the different clips and cubes at fixed laser settings . [SEP]
[CLS] the experiment revealed that release of the dye at later time points caused a delay in the appearance of the fluorescent B-property signal . [SEP]
[CLS] ( b ) co - incubation B-technique with the phosphorylated dye confirmed colocalization of the more stable structures with this model for degradation products . [SEP]
[CLS] heg - protection of the dna tetrahedron . [SEP]
[CLS] ( a ) chemical structure of heg modification . [SEP]
[CLS] ( b ) gel B-technique electrophoresis I-technique from serum stability experiments . [SEP]
[CLS] the component clip of the tetrahedron revealed higher stability of the clip component upon heg - labeling at 5 ′ - and 3 ′ - ends . [SEP]
[CLS] cy5 channel is displayed . [SEP]
[CLS] ( c ) live confocal microscopy B-technique ( hela cells B-material , fixed laser settings ) revealed that the more stable structures caused a delayed cellular uptake . [SEP]
[CLS] scale bars at 20 μm . [SEP]
[CLS] representative images are shown in the figure . [SEP]
[CLS] lowering serum conditions and changing the dye to its sulfonated version . [SEP]
[CLS] representative images are shown in the figure . [SEP]
[CLS] ( a ) live confocal microscopy B-technique in hela cells B-material in low - serum conditions ( 0 . 1 % fbs ) revealed that with reduced degradation , no fluorescent B-property signal is observed . [SEP]
[CLS] ( b ) chemical structure of sulfonated cy5 dye , negatively charged . [SEP]
[CLS] ( c ) structures labeled with sulfonated dye did not produce any intracellular fluorescent B-property signal compared to nonsulfonated labeled structures . [SEP]
[CLS] forster resonance energy transfer ( fret ) experiments . [SEP]
[CLS] ( a ) live confocal microscopy B-technique after 24 h incubation B-technique in hela cells B-material . [SEP]
[CLS] representative images are shown in the figure . [SEP]
[CLS] ( b ) quantification of the gray level intensity from the microscopy B-technique experiments ( detailed protocols in the supporting information ) . [SEP]
[CLS] these two experiments revealed that the two free separate small molecules can give a fret signal , even though they are not in close proximity before incubation B-technique . [SEP]
[CLS] s s1−s102 and tables s - i−s - iii ( pdf ) ■ author information corresponding author * e - mail : hanadi . sleiman @ mcgill . ca . orcid hanadi f . sleiman : 0000 - 0002 - 5100 - 0532 notes the authors declare no competing financial interest . [SEP]
[CLS] ■ acknowledgments the authors thank nserc , cihr , cfi , cancer research society , canada research chairs program and frqnt for funding . e . v . - c . would like to thank " fundacion alfonso martı n escudero " for a postdoctoral fellowship . [SEP]
[CLS] the authors would like to thank dr . erika wee and the advanced bioimaging facility ( abif ) at mcgill university for training and help with the confocal imaging and fret experiments . [SEP]
[CLS] we thank dr . katherine bujold and hassan fakih for providing the fret - positive dna strand , prof . karine auclair for use of equipment , and dr . johans fakhoury for useful and helpful discussions . [SEP]
[CLS] ■ note added after asap publication this paper was published asap on april 26 , 2019 , with the figure 2 graphic in the place of figure 1 . [SEP]
[CLS] the corrected version was reposted on april 30 , 2019 . [SEP]
[CLS] spherical nucleic B-material acids I-material ( snas ) are nanomaterials B-material typically consisting of a nanoparticle B-nanoparticle core B-material and a functional , dense , and highly oriented oligonucleotide shell B-material with unusual biological properties that make them appealing for many applications , including sequence - specific gene silencing , mrna quantification , and immunostimulation . [SEP]
[CLS] when placed in biological fluids , snas readily interact with serum proteins B-material , leading to the formation of ill - defined protein B-material coronae I-material on the surface , which can influence the targeting capabilities of the conjugate . [SEP]
[CLS] in this work , snas were designed and synthesized with functional proteins B-material , such as antibodies B-material and serum albumin , deliberately adsorbed onto their surfaces . [SEP]
[CLS] these particles exhibit increased resistance to protease degradation compared with native snas but still remain functional , as they can engage in hybridization with complementary oligonucleotides . [SEP]
[CLS] snas with adsorbed targeting antibodies B-material exhibit improved cellular selectivity within mixed cell B-material populations . [SEP]
[CLS] similarly , snas coated with the dysopsonizing protein B-material serum albumin show reduced macrophage uptake , providing a strategy for tailoring selective sna delivery . [SEP]
[CLS] importantly , the protein B-material coronae I-material remain stable on the snas in human serum , exhibiting a less than 45 % loss of protein B-material through exchange after 12 h at 37 °c . [SEP]
[CLS] taken together , these results show that protein−sna complexes and the method used to prepare them provide a new avenue for enhancing sna stability , targeting , and biodistribution . [SEP]
[CLS] certain nanomaterials B-material can carry and present peptides B-material , proteins B-material , oligonucleotides , and small molecules within highly engineered structures to target tissues , making them appealing for biomedical and life science applications . [SEP]
[CLS] however , many nanomaterials B-material , when introduced to biological fluids , nonspecifically adsorb biomolecules , resulting in the formation of a protein B-material corona I-material around the structure . [SEP]
[CLS] the protein B-material corona I-material alters the biological stability , 3−7 biodistribution , and targeting efficiency of a nanomaterial B-material , sometimes diminishing its therapeutic potential . [SEP]
[CLS] though the surface charge , size , and shape of a nanomaterial B-material can modulate the composition of the protein B-material corona I-material , its formation is largely unavoidable in biological environments . [SEP]
[CLS] careful modification of the nanoparticle B-nanoparticle surface , however , can help dictate protein B-material corona I-material formation and mediate its effects on pharmacokinetics , yielding constructs with improved targeting capabilities that sometimes exceed covalent attachment methods or reduced nonspecific cellular uptake [SEP]
[CLS] spherical nucleic B-material acids I-material ( snas ) , a unique class of nanomaterials B-material consisting of a spherical B-nanoparticle nanoparticle I-nanoparticle core B-material densely functionalized with a highly oriented nucleic B-material acid I-material shell B-material , have enhanced biological properties , including increased resistance to nuclease degradation compared with linear oligonucleotides of the same sequence , the ability to rapidly enter cells B-material in high quantities without transfection agents , and tailorable immunogenicity B-property . [SEP]
[CLS] these properties have positioned snas for use in applications , such as gene silencing , immunomodulation B-property , drug delivery , and nucleic B-material acid I-material detection in vitro and in live cells B-material [SEP]
[CLS] however , snas , like many other nanomaterials B-material , interact with serum proteins B-material , resulting in the formation of a protein B-material corona I-material that can alter their uptake properties . [SEP]
[CLS] antibody−dna conjugates have been hybridized onto the surface of snas to improve their targeting capabilities and direct them to cancer cells B-material . [SEP]
[CLS] however , in this approach , the antibody B-material densities utilized were so low ( 1−2 antibodies B-material / particle ) that they would be unlikely to alter protein B-material corona I-material formation . [SEP]
[CLS] alternatively , pegylation of the nanoparticle B-nanoparticle core B-material has shown the ability to reduce nonspecific adsorption of serum proteins B-material , thus extending blood circulation times , but such modifications compromise sna uptake efficiency by target cells B-material . [SEP]
[CLS] herein we report a universal method for targeting snas to specific cell B-material types . [SEP]
[CLS] to accomplish this goal , we designed and synthesized snas with predefined protein B-material coronae I-material consisting of functional proteins B-material immobilized on the oligonucleotide shell B-material . [SEP]
[CLS] we then explored the stability of these structures in buffer and human serum . [SEP]
[CLS] in addition , we studied their ability to hybridize with complementary oligonucleotides as well as selectively target cell B-material populations based on molecular signatures present on the cell B-material surface . [SEP]
[CLS] for example , snas with adsorbed human epithelial growth B-material factor I-material receptor I-material 2 ( her2 ) monoclonal antibodies B-material ( mabs ) exhibited selectivity for her2 - positive breast cancer cells B-material in mixed cell B-material cultures with her2 - negative breast cancer cells B-material . [SEP]
[CLS] taken together , our results show that this approach provides an easy , efficient , and flexible method for controlling sna interactions within cells B-material that has the potential to improve sna stability , cell B-material targeting , and biodistribution . [SEP]
[CLS] particle synthesis and characterization [SEP]
[CLS] in order to generate particles with defined protein B-material coronae I-material , we first synthesized snas by functionalizing 13 nm gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) with thiolated oligodeoxynucleotides ( odns ) using established synthetic methods . [SEP]
[CLS] we then incubated B-technique these snas with different functional proteins B-material , such as human epithelial growth B-material factor I-material receptor I-material 2 monoclonal antibodies B-material ( anti - her2 ) , immunoglobulin B-material g I-material ( igg ) , and human serum albumin ( hsa ) . [SEP]
[CLS] each protein B-material was selected on the basis of its potential ability to confer specific targeting properties to snas . [SEP]
[CLS] specifically , anti - her2 was used as a model protein B-material for cell B-material targeting because it is a clinically relevant target for her2positive breast cancer . [SEP]
[CLS] igg was used because it is an immunogenic B-property protein B-material that attracts macrophages , while hsa was explored because it can shield nanoparticles B-nanoparticle from liver clearance and macrophage uptake . [SEP]
[CLS] after incubating B-technique the individual proteins B-material with the snas , we washed away the loosely bound proteins B-material by pelleting the particles via centrifugation , leaving primarily a hard corona ( defined as one with a high affinity for a particle ) adsorbed on the sna surface . [SEP]
[CLS] to verify the formation of a hard corona , we measured the diameter and ζ potential of the snas before and after incubation B-technique with the different proteins B-material ( figure 1 ) . [SEP]
[CLS] dynamic B-technique light I-technique scattering I-technique ( dls ) revealed that the average diameters of igg - adsorbed snas ( 28 . 3 ± 4 . 1 nm ) , anti - her2 - adsorbed snas ( 23 . 5 ± 2 . 1 nm ) , and hsa - adsorbed snas ( 19 . 6 ± 1 . 4 nm ) were larger than those of the bare snas ( 15 . 1 ± 2 . 2 nm ) ( figure 1b ) . [SEP]
[CLS] furthermore , the snas with adsorbed igg ( −28 . 0 ± 0 . 6 mv ) and hsa ( −24 . 0 ± 3 . 6 mv ) displayed a more positive ζ potential compared with bare snas ( −35 . 4 ± 2 . 4 mv ) ( figure 1c ) . [SEP]
[CLS] since igg has an isoelectric point between 6 . 6 and 7 . 2 and that of hsa is 4 . 7 , both proteins B-material are negatively charged at physiological ph . [SEP]
[CLS] the shielding of the highly negatively charged odn by the protein B-material coronae can contribute to the observed elevated ζ potential . [SEP]
[CLS] noticeably , neither igg nor hsa undergoes significant conformational changes in the ph ranges used in this study . [SEP]
[CLS] a lack of significant change in the ζ potential of the anti - her2 - adsorbed snas ( −35 . 2 ± 0 . 7 mv ) may be due to the charge variants of the mab obtained from the manufacturer , which could result in a less positive surface charge of the mab ( the isoelectric point of the main isoform is 8 . 7 ) . [SEP]
[CLS] notably , charge variants of mabs do not seem to significantly affect their binding affinity for target receptors . [SEP]
[CLS] as further verification of protein B-material corona I-material formation , we examined the electrophoretic B-property mobility I-property of snas after incubation B-technique with proteins B-material . [SEP]
[CLS] all of the snas with protein B-material coronae had reduced mobility B-property in an agarose gel compared with bare snas ( figure s1a ) , further confirming that protein B-material adsorption occurred . [SEP]
[CLS] notably , the number of dna strands per particle did not change appreciably upon protein B-material adsorption , indicating that all shifts in electrophoretic B-property mobility I-property were due to the protein B-material coronae I-material and not changes in the total number of dna strands ( table s2 ) . [SEP]
[CLS] furthermore , the number of proteins B-material adsorbed onto each sna was quantified via a fluorescence - based assay using fluorophore ( i . e . , texas red - x ) - labeled proteins B-material . [SEP]
[CLS] for a 13 nm aunp B-nanoparticle functionalized with [UNK] odns , approximately 40 ± 2 , 26 ± 2 , and 23 ± 2 igg , anti - her2 , and hsa proteins B-material / np B-nanoparticle were found to be adsorbed , respectively . [SEP]
[CLS] the reproducibility of this coating B-material method was confirmed with hsa , as additional separate batches yielded similar numbers of proteins B-material per np B-nanoparticle ( table s3 ) . [SEP]
[CLS] the higher - density igg coating B-material is likely due to the higher affinity of igg for the odn shell B-material . [SEP]
[CLS] the interactions driving binding B-event of I-event the I-event proteins I-event and snas likely consist of both electrostatic and hydrogen B-material - bonding interactions , which have been reported for interactions between proteins B-material and dna ; however , the exact mechanisms are still unclear . [SEP]
[CLS] in our studies , increased salt B-material concentrations ( 1 m nacl , mgcl 2 ) , nonionic detergents ( 0 . 1 % triton x - 100 ) , and ethanol ( 1−20 % ) had negligible effects on the coronae . [SEP]
[CLS] only harsh denaturing conditions consisting of 0 . 1 % sodium B-material dodecyl sulfate ( sds ) removed the coronae . [SEP]
[CLS] these results suggest that there is a strong multifaceted interaction between the proteins B-material and snas . [SEP]
[CLS] stability of protein B-material coronae I-material in biological fluids . [SEP]
[CLS] after establishing that we could adsorb an initial protein B-material corona I-material on the snas , we examined the stability of the different protein B-material coronae by studying the exchange dynamics of proteins B-material on the surface of snas under serum - rich conditions ( i . e . , 10 % human serum ( hs ) ) . [SEP]
[CLS] to accomplish this objective , we synthesized snas with cy5 - labeled odns , to which we adsorbed proteins B-material labeled with texas red - x , a fluorophore that can transfer emitted energy to the cy5 fluorophore on the odn shell B-material when attached to the snas . [SEP]
[CLS] thus , as proteins B-material were displaced from the particle surface , an increase in texas red - x fluorescence B-property was observed . [SEP]
[CLS] we incubated B-technique the snas in 10 % hs at 37 °c and tracked the change in fluorescence B-property for 12 h , at which point the change in fluorescence B-property had plateaued ( figure 1e ) . [SEP]
[CLS] after 12 h , we found that more than 55 % of the initial corona remained ( full protein B-material dissociation was established in 0 . 1 % sds ) , indicating that a stable hard protein B-material corona I-material had formed on the snas . [SEP]
[CLS] compared with igg and anti - her2 , adsorbed hsa tends to dissociate from the sna surface even without serum , indicating its weaker affinity for the odn shell B-material ; the addition of serum increases the hsa desorption by about 20 % . [SEP]
[CLS] significantly , the stable adhesion of the functional proteins B-material ensures that active protein B-material coronae are retained even in physiologically relevant media . [SEP]
[CLS] to assess the composition of the protein B-material coronae I-material following incubation B-technique in serum , the protein - coated snas were incubated B-technique in 10 % hs for 4 h at 37 °c , the unbound protein B-material was then removed through centrifugation , and the bound protein B-material was dissociated using sds . [SEP]
[CLS] the extracted protein B-material was then analyzed using an sds - page gel running with tris−glycine−sds buffer ( figure s1b ) . [SEP]
[CLS] darker protein B-material bands at [UNK] kda indicate antibody B-material enrichment for the igg @ snas and anti - her2 @ snas . [SEP]
[CLS] enrichment of hsa was not observed for hsa @ snas , most likely because of the abundance of albumin in hs . [SEP]
[CLS] albumin composes 50−60 % of blood plasma proteins B-material , and igg is about one - fifth of the albumin content . [SEP]
[CLS] we explored the characteristics of snas precoated with corona B-material proteins I-material to ensure that they retained some of the same key biological properties as the original snas that make them valuable in biology and medicine . [SEP]
[CLS] first , we examined their resistance to nucleases by quantifying the amount of odns degraded . [SEP]
[CLS] to measure degradation , we synthesized snas with cy5 - labeled odns ; the cy5 dye is quenched when the labeled strand is attached to the gold B-material core B-material . [SEP]
[CLS] upon incubation B-technique with deoxyribonuclease i ( dnase i ) , an endonuclease , odns are cleaved from the aunp B-nanoparticle core B-material , resulting in increased cy5 emission intensity . [SEP]
[CLS] in this experiment , preadsorption of igg or hsa significantly reduced both the rate and efficiency of odn degradation , and the anti - her2 coating B-material lessened the odn degradation efficiency compared with the bare snas ( figure 2b ) ; this result indicates that preadsorption of proteins B-material enhances sna resistance to nucleases . [SEP]
[CLS] this presumably occurs because of the increased steric hindrance of the protein B-material corona I-material that prevents nucleases from accessing the odns . [SEP]
[CLS] given that the odns are potentially sterically hindered when a protein B-material corona I-material is adsorbed to the structures , we examined whether a protein B-material corona I-material reduced their ability to recognize complementary binding partners , a necessary step for antisense and rna interference pathways as well as mrna sensing . [SEP]
[CLS] to assess this property , we designed a aunp B-nanoparticle - based fluorescence B-property quenching assay in which the hybridization of fluorophore - labeled ( i . e . , cy3 . 5 ) strands complementary to those making up the sna shell B-material results in quenching due to the proximity of the cy3 . 5 fluorophore to the aunp B-nanoparticle core B-material . [SEP]
[CLS] the quenching of the cy3 . 5 fluorescence B-property by the aunp B-nanoparticle core B-material is an indicator B-property of the amount of hybridization and therefore a measure of the surface dna accessibility ( figure 2c ) . [SEP]
[CLS] the percentage of dna hybridized to protein - immobilized snas was calculated in comparison with hybridization measured for protein - free snas and was normalized to the hybridization of protein - free snas to noncomplementary ( i . e . , t20 ) strands . [SEP]
[CLS] surprisingly , we found that the preadsorbed protein B-material coronae I-material decreased dna accessibility by only [UNK] % ( figure 2d ) compared with bare snas . [SEP]
[CLS] furthermore , when we assessed whether the most - dense protein B-material corona I-material ( i . e . , igg @ sna ) altered the specificity , we found no apparent increase in binding of noncomplementary strands compared to a proteinfree sna ( figure s2 ) . [SEP]
[CLS] together , these experiments imply that the protein B-material corona I-material does not significantly alter recognition of complementary strands . [SEP]
[CLS] cellular selectivity of the snas with immobilized antibodies B-material . [SEP]
[CLS] on a cellular level , we investigated whether we could use preadsorbed protein B-material coronae I-material on snas to modulate their uptake by targeted cell B-material types . [SEP]
[CLS] a key attribute of snas is that they enter nearly any cell B-material type ( over 50 to date ) , an especially powerful property for many therapeutic and diagnostic applications ; however , selective targeting could impart an enhanced therapeutic effect . [SEP]
[CLS] as a first test , we examined the targeting capabilities of snas with immobilized antibodies B-material . [SEP]
[CLS] for these experiments , snas were synthesized with an odn shell B-material containing 10 % cy5 - labeled strands for flow B-technique cytometry I-technique detection , thereby minimizing potential perturbation to the uptake pathway caused by the fluorophore . [SEP]
[CLS] we then incubated B-technique these constructs with either anti - her2 mabs or a nontargeting antibody B-material , igg . we assessed the selectivity of the snas by treating the cocultured breast cancer cell B-material lines sk - br - 3 ( her2 overexpressing ) and mda - mb - 231 ( her2 negative ) with the particles ( figure 3a ) . [SEP]
[CLS] furthermore , the mda - mb - 231 cells B-material were engineered to express green fluorescent B-property protein B-material ( gfp ) , such that the two cell B-material lines could be separated by flow B-technique cytometry I-technique on the basis of gfp fluorescence B-property intensity . [SEP]
[CLS] significantly , the anti - her2 - adsorbed snas preferentially entered the her2 - positive cells B-material compared with the her2 - negative ones over an 8 h treatment time ( figure 3b ) , even under conditions with greater numbers of her2 - negative cells B-material ( figure s3 ) . [SEP]
[CLS] in contrast , nontargeting igg - adsorbed snas and bare snas showed no cellular selectivity . [SEP]
[CLS] importantly , precoating snas with nontargeting proteins B-material did not seem to reduce their cancer cell B-material uptake efficiency compared to that for bare snas ( figure 3c ) . [SEP]
[CLS] this finding is consistent with a previous report that mab functionalization improves cellular selectivity , but the physical adsorption of mabs demonstrated here is easier and more adaptable than the reported conjugation approach . [SEP]
[CLS] significantly , cells B-material were treated with mab - adsorbed snas in complete growth medium supplemented with 10 % serum , and the targeting capabilities still persisted . [SEP]
[CLS] these conditions show that mab - adsorbed snas are able to retain cellular selectivity even in the presence of other serum proteins B-material . [SEP]
[CLS] evasion of macrophage clearance of the dysopsonin - adsorbed snas . [SEP]
[CLS] lastly , we investigated the potential utility of our approach for creating snas that can target or avoid macrophages , which play central roles in immunomodulation B-property and clearance . [SEP]
[CLS] for this purpose , we preadsorbed either a recognized opsonin , igg , or a dysopsonin , hsa , on the odn shell B-material of snas and incubated B-technique them with human macrophages ( figure 4a ) . [SEP]
[CLS] typically , an opsonin marks a construct as foreign and induces macrophage clearance , while dysopsonins do the opposite . [SEP]
[CLS] to perform this experiment , human thp - 1 monocytes were first differentiated into macrophages using phorbol 12 - myristate 13 - acetate ( pma ) ( figure s4 ) . [SEP]
[CLS] we then treated the macrophages with bare and active protein - coated snas for 1−8 h at 37 °c . [SEP]
[CLS] we hypothesized that igg preadsorption would improve the sna uptake efficiency by macrophages while hsa preadsorption would reduce it . [SEP]
[CLS] interestingly , both igg and hsa adsorption lowered the uptake of the snas into the macrophages , with hsa adsorption having a more significant impact on reducing sna clearance by macrophages ( figure 4b ) . [SEP]
[CLS] igg is an opsonin that can be cleared by macrophages through fc receptor B-material recognition , while non - protein - coated snas and hsa alone are reported to enter cells B-material through scavenger receptor B-material a ( sr - a ) recognition . [SEP]
[CLS] previous findings showed that changes in protein B-material corona I-material composition change the cellular uptake pathway for nanoparticles B-nanoparticle . [SEP]
[CLS] therefore , we speculated that coating B-material snas with igg could alter their preferred uptake pathway . [SEP]
[CLS] indeed , significantly diminished uptake efficiency was observed for these snas when the fc receptors were blocked by fcx ( figure 4c ) , meaning that the igg - coated snas entered cells B-material through a different route than observed with typical snas . [SEP]
[CLS] as a comparison , when sr - a was inhibited by fucoidan , the reduction in the uptake efficiency of bare snas and hsaimmobilized snas is more significant than for igg - adsorbed snas . [SEP]
[CLS] since macrophages are phagocytic , when phagocytosis B-event is inhibited by cytochalasin d , the uptake of all sna types is suppressed . [SEP]
[CLS] taken together , these results indicate that igg immobilization alters the major cellular uptake pathway of snas , which could be the reason for the overall reduction of uptake efficiency . [SEP]
[CLS] significantly , hsa coating B-material of snas reduces nonspecific macrophage uptake compared with bare snas , opening new avenues to explore for increasing blood circulation half - life . [SEP]
[CLS] this work has introduced a straightforward and flexible method for incorporating active protein B-material coronae I-material on sna surfaces with relatively high stability even in the presence of serum . [SEP]
[CLS] importantly , this method increases the cellular selectivity of snas and reduces nonspecific macrophage clearance without significantly affecting the accessibility of the oligonucleotide shell B-material . [SEP]
[CLS] therefore , this work points toward a potential strategy for improving sna targeting and distribution in vivo , which could impact ongoing clinical efforts aimed at sna therapeutic development . [SEP]
[CLS] finally , looking forward , this methodology , depending upon particle surface characteristics , could be generally applied to other nanomaterials B-material to improve cellular selectivity . [SEP]
[CLS] 1 . synthesis and characterization of protein - adsorbed snas . [SEP]
[CLS] ( a ) schematic representation of monoclonal B-material antibody I-material ( top ) or igg ( bottom ) immobilization on the odn shell B-material of snas . [SEP]
[CLS] ( b ) distributions of the hydrodynamic diameters of bare , igg - ( igg @ sna ) , anti - her2 - ( her2 @ sna ) , and hsa - immobilized snas ( hsa @ sna ) . [SEP]
[CLS] ( c ) ζ potentials of bare snas , igg @ snas , her2 @ snas , and hsa @ snas . [SEP]
[CLS] ( d ) schematic depicting the displacement of texas red - x - labeled proteins B-material from the surface of cy5 - labeled snas by serum proteins B-material . [SEP]
[CLS] the fluorescence B-property of texas red - x increases as protein B-material displacement occurs . [SEP]
[CLS] ( e ) kinetic fluorescence B-property profiles of texas red - x - labeled igg , anti - her2 , and hsa during incubation B-technique with 10 % serum proteins B-material . [SEP]
[CLS] 2 . in vitro properties of snas preadsorbed with functional proteins B-material . [SEP]
[CLS] ( a ) schematic representation of the degradation of the odn shell B-material in the presence of dnase i , in which the cy5 fluorophore attached to the outer shell B-material is no longer quenched by aunps B-nanoparticle following protease degradation . [SEP]
[CLS] ( b ) fluorescence B-property kinetic profiles of the bare , igg - ( igg @ sna ) , anti - her2 - ( her2 @ sna ) , and hsa - immobilized snas ( hsa @ sna ) with and without dnase i treatment . [SEP]
[CLS] ( c ) schematic representation of the hybridization of cy3 . 5 - labeled complementary strands to the odns immobilized on aunps B-nanoparticle . [SEP]
[CLS] fluorescence B-property is quenched as hybridization occurs . [SEP]
[CLS] ( d ) degrees of hybridization of snas with complementary strands for igg @ snas , her2 @ snas , and hsa @ snas compared with that for bare snas . [SEP]
[CLS] selective cellular uptake of the monoclonal her2 antibody - adsorbed snas . [SEP]
[CLS] ( a ) schematic describing the sna treatment of the cocultured her2 - expressing breast cancer cells B-material , sk - br - 3 , and non - her2 - expressing breast cancer cells B-material , mda - mb - 231 . [SEP]
[CLS] ( b ) uptake profiles of anti - her2 - adsorbed ( her2 @ sna ) , igg - adsorbed ( igg @ sna ) , and bare snas following incubation B-technique for 1−8 h with the cocultured cells B-material ( solid circles , her2 - positive cells ; open circles , her2 - negative cells B-material ) . [SEP]
[CLS] mfi = median fluorescence B-property intensity [SEP]
[CLS] ( c ) representative overlaid cellular uptake histograms ( as measured by cy5 intensity ) for cocultured breast cancer cells B-material after treatment with her2 @ snas , igg @ snas , and bare snas for 6 h . [SEP]
[CLS] the distributions of cy5 fluorescence B-property of her2 + cells B-material are denoted by the darker - shaded histograms , while those for the her2− cells B-material are shown in a lighter shade . [SEP]
[CLS] 4 . cellular uptake of the igg ( opsonin ) - and hsa ( dysopsonin ) - adsorbed snas . [SEP]
[CLS] ( a ) schematic describing the sna treatment of thp - 1 - derived macrophages . [SEP]
[CLS] ( b ) uptake of hsaadsorbed ( hsa @ sna ) , igg - adsorbed ( igg @ sna ) , and bare snas following 1−8 h incubation B-technique with thp - 1 - derived macrophages . [SEP]
[CLS] ( c ) receptor inhibited thp - 1 macrophage uptake of hsa @ sna , igg @ sna , and bare snas following pretreatment with cytochalasin d , fucoidan , or fc receptor B-material blocker ( fcx ) . [SEP]
[CLS] the broad deployment of nanotechnology and nanomaterials B-material in modern society is increasing day by day to the point that some have seen in this process the transition from the silicon B-material age to a new nano age . [SEP]
[CLS] nanocrystalsa distinct class of nanomaterialsare forecast to play a pivotal role in the next generation of devices such as liquid crystal displays , lightemitting diodes , lasers , and luminescent B-property solar concentrators . [SEP]
[CLS] however , it is not to be forgotten that this cutting - edge technology is rooted in empirical knowledge and craftsmanship developed over the millennia . [SEP]
[CLS] this review aims to span the major applications in which nanocrystals were consistently employed by our forebears . [SEP]
[CLS] through an analysis of these examples , we show that the modern - age discoveries stem from multimillennial experience passed on from our proto - chemist ancestors to us . [SEP]
[CLS] t he birth of nanotechnologythat is , the use of materials with nanoscale dimensions and / or whose properties rely on their structural organization at the nanoscaleis usually associated with two events : the speech by richard feynman in 1959 at caltech ( " there is plenty of room at the bottom " , december 29 , american physical society meeting ) , and the speech by norio taniguchi in 1974 ( " on the basic concept of nanotechnology " ) at the international conference on production engineering in tokyo . [SEP]
[CLS] since these two events , the use of the word nano ( denoting a factor of 10 −9 ) emerged in both scientific and nonscientific literature . [SEP]
[CLS] in the 1990s , thanks to actions encouraging and coordinating research in the nanodomain , such as the national nanotechnology initiative in the united states , an association between the terms " nanotechnology " and " future " was promoted in the collective image . [SEP]
[CLS] currently , the use of nanotechnologies and nanomaterials B-material is seen in a growing number of commercial applications . [SEP]
[CLS] for instance , nanosized metal−oxide−semiconductor field - effect transistors have been implemented in the last generations of computers and smartphones . [SEP]
[CLS] semiconductor and metal B-material nanocrystals ( ncs ) have evolved into much - appraised material B-material ' s building blocks in nanoscience and nanotechnology . [SEP]
[CLS] in recent decades , giant leaps have been made in the development , optimization , and commercialization of nanocrystals for diverse purposes ; for example , sunscreens contain zno and tio 2 nanoparticles B-nanoparticle as absorbers of the ultraviolet fraction of sunlight , while semiconductor nanocrystals can be found in some of the most modern liquid - crystal displays ( lcds ) to achieve a better color gamut . [SEP]
[CLS] this drive is motivated by the awareness that their macroscopic properties depend on the nanocrystal dimensions and size . [SEP]
[CLS] for instance , the much higher surfaceto - volume ratio compared to their bulk counterparts produces unmatched catalytic properties in nanoparticles B-nanoparticle , as was shown , for example , by paul sabatier when he used ultrafine nickel B-material particles to perform catalytic hydrogenation B-event ( which gained him the nobel prize in 1912 ) . [SEP]
[CLS] furthermore , nanotechnology and , in particular , nanocrystals are forecast to play a pivotal role in the next generation of devices in electronics , medicine , 30−34 photonics , and catalysis as well as in less conventional fields 39−41 such as textiles . [SEP]
[CLS] the use of nanomaterials B-material is shaping our society to the point that some have recognized in it the birth of a new age ( i . e . , the nano age ) , distinguishing it from the current " silicon B-material age " . [SEP]
[CLS] however , as is often the case in science , the use of nanotechnology can be traced back far earlier in time . [SEP]
[CLS] in fact , history presents a plethora of situations in which nanotechnology was used in purely empirical and unwitting , though effective , approaches . [SEP]
[CLS] during the fifth millennium bc the inhabitants of cyprus would bleach wool and fleece with nanoporous clay , while corsica has a long - standing tradition of ( nano ) asbestos - enriched pottery as a way to enhance the mechanical properties of clay . [SEP]
[CLS] the mesoamerican civilization of the maya developed two weather - resistant pigments based on the incorporation of natural dyes into nanostructured clay . [SEP]
[CLS] at the same time , the christian crusade warriors could probably not conceive that the superior properties of the moorish damascus steel were due to embedded carbon B-nanoparticle nanotubes I-nanoparticle , although this type of nanotechnology was already known and used almost a millennium earlier in india . [SEP]
[CLS] concerning nanocrystals , despite the common notion that they are a relatively recent technology , they have also been used , though ( again ) purely empirically , for at least three millennia . [SEP]
[CLS] we will also show that their modern ( re ) discovery stems from a long - standing traditional use . [SEP]
[CLS] as we will show in this review , the modern field of nanocrystals relies on an ancient , deep , and factual know - how that can be traced back historically as far as the ancient world . [SEP]
[CLS] we will first discuss how nanocrystals were widely used in the manufacturing of glass . [SEP]
[CLS] notably , the case of gold B-material will be discussed more extensively as a prototypical example of the development of nanocrystals through the centuries . [SEP]
[CLS] related to glass and gold B-material , we will then examine the use of nanocrystals in the coloration of ceramics . [SEP]
[CLS] an analysis of the use of nanocrystals for cosmetics will follow . [SEP]
[CLS] we will then proceed to the brief history of semiconductor nanocrystals and , in particular , how it evolved from the glass - making industry . [SEP]
[CLS] the development of nanocrystals is intimately related to the field of glass manufacturing : already in the ancient world , glass was , in fact , colored through the addition of metals B-material to the melt . [SEP]
[CLS] some of the earliest archeological evidence suggests that , already during the bronze age , in italy , our ancestors enriched silica pastes with chromophores ( e . g . , cu , co , fe , and mo ) in order to color glass . [SEP]
[CLS] in particular , red glass was obtained through the formation of metallic copper B-material nanocrystals from the reduction of copper B-material oxides I-material under a reducing atmosphere . [SEP]
[CLS] these metallic copper B-material nanocrystals , typically located at the surface of the glass ( figure 1a ) , rendered blue colored glasses with a thin red colored layer originating either from the excitation of localized surface plasmon modes in the nanoparticles B-nanoparticle or by scattering from the nanoparticles B-nanoparticle themselves . [SEP]
[CLS] this evidence shows that , even though our ancestors were not able to produce glass in a standardized , scalable manner , they mastered this art well enough to manufacture colored glass with controlled macroscopic properties , using an empirical technique very similar to that used to produce luster ceramics and modern colored glass more than two millennia later . [SEP]
[CLS] the same technique of coloration developed in the bronze age and relying on metallic cu nanocrystals ( figure 1b ) was maintained throughout the centuries , when it was , for example , used to produce red enamels and mosaic tesserae in both celtic and roman gaul ( current - day france ) . [SEP]
[CLS] however , the technique of coloring with cu nanocrystals was finally exploited in all its potentiality with the production of the huge stained glass windows of the romanic and , especially , gothic cathedrals ( e . g . , sainte - chapelle in paris ) during the middle age . [SEP]
[CLS] in the whole christendom , glassmakers refined and improved the manufacturing of colored glass through the centuries , extending it to incorporate the use of silver B-material to obtain a yellow / amber coloration . [SEP]
[CLS] the use of silver B-material was most probably adopted from the middle east , where it was already known from the eighth century ad in the production of luster ceramics . [SEP]
[CLS] the first report in the christian world seems to appear in the 13th century in the " el lapidario " of king alfonso x , where a stone called ecce ( most probably pyrargyrite , ag 3 sbs 3 ) is suggested to be ground up , mixed with honey , and spread over glass in order to dye it in yellow / gold B-material . [SEP]
[CLS] recent studies unearthed the mechanism of production of these types of glass : a silver - based slurry paste was applied to soda - lime glass ( rich in na and k compounds ) , which was then annealed ( 300−600 °c ) . [SEP]
[CLS] in a process that is very similar to that used for luster pottery , the ag + ions B-material would diffuse into the glass , while na + and k + ions B-material would migrate toward the surface ; the thermoreducing agents in the glass ( e . g . , as 3 + , sb 3 + , sn 2 + ) would then reduce the silver B-material ions B-material to ag°nanocrystals with a diameter of 5−10 nm . [SEP]
[CLS] the glassmakers were able to control the resulting color of the glass through the presence of additives in the glass : the presence of cu ions B-material , for example , would slow down ag + diffusion , thus producing larger nanocrystals and hence obtaining more vivid colors . [SEP]
[CLS] the same technique was used to obtain dichroic glasses ( figure 1c ) : a longer growth of ag nanocrystals would typically result in nanocrystal populations with different sizes in the same glass and therefore with optical properties dominated , in different measures , by absorption and scattering ( i . e . , resulting in different colors in transmission and reflection ) . [SEP]
[CLS] similarly to the silver B-material - stained glass , also the copper - based red ruby glass was produced through a similar mechanism involving the diffusion of copper B-material ions B-material in the glass and their successive reduction to metallic copper B-material ( figure 1d , e ) to form thin red - colored multilayers rich in cu nanocrystals ( figure 1f ) , as shown by experimental and archeological evidence . [SEP]
[CLS] all in all , the arcane and highly empirical procedure to form red and yellow colored glass , along with the difficulty to create a highly reducing working environment , ensured that glass fabrication remained exclusive to a limited number of artisans who , though unaware of it , can be rightfully seen as proto - nanochemists . [SEP]
[CLS] metallic gold B-material has accompanied the development of human civilization throughout the millennia under different forms . [SEP]
[CLS] thanks to its physical properties and its rarity , it has often been imbued with strong symbolism associated with royalty and religion , which partly explains its fate throughout history . [SEP]
[CLS] for these reasons , this paragraph will be dedicated to exploring the uses of gold B-material , in the form of nanocrystals , in diverse materials and applications . [SEP]
[CLS] recent studies have investigated the role of gold B-material , in the form of nanocrystals , in the coloration of glass . [SEP]
[CLS] the earliest report on red - colored glass via the presence of gold B-material nanocrystals is attributed to the assyrians : thompson translated a cuneiform clay tablet from 700 bc describing a recipe to make red glass through the addition of gold B-material . however , it was the romans who truly mastered the technique of coloring glass in red with gold B-material : the lycurgus cup , in this sense , represents an exquisite , well - known example ( figure 2a ) . the cup depicts a wellknown episode from greek mythology where king lycurgus , threatening the life of one of the priestesses of the god dionysus , is restrained by the god himself through the use of vines and is eventually killed . [SEP]
[CLS] the cup , besides the mythological symbolism , is also interesting because it is a fine example of a dichroic glass : when observed in reflection , it looks green , while it is red when observed in transmission ( figure 2a ) . [SEP]
[CLS] the optical properties of the cup have attracted scholarly attention since the second half of the 20th century , when these optical properties had already been correctly attributed to the presence of silver B-material and gold B-material in the glass , possibly in a colloidal form . [SEP]
[CLS] however , it was only in 1990 that the dichroic properties of the cup were unambiguously attributed to the presence of alloyed ag / au metallic B-nanoparticle nanoparticles I-nanoparticle in the glass by means of electron B-technique microscopy I-technique ( figure 2b ) . [SEP]
[CLS] the color is produced by the interaction of light with metallic B-nanoparticle nanoparticles I-nanoparticle dispersed in the glass by means of absorption ( of the localized surface plasmon modes ) and scattering . [SEP]
[CLS] despite the outstanding craftsmanship of this cup , the scarcity of archeological findings of comparable quality let us tend toward the idea that the control of the colorant process was probably very difficult , and similar objects could only be produced in extremely limited numbers . [SEP]
[CLS] nevertheless , the ability to color the glass red via the formation of gold B-nanoparticle nanoparticles I-nanoparticle in situ ( from which we have the name gold B-material ruby glass or gold red ruby ) was preserved during the political and cultural fragmentation of the roman empire and was employed to color roman mosaic tesserae hundreds of years later . [SEP]
[CLS] a few examples of glass mosaics and panels ranging from the fourth to the 12th century ad have been reported to rely on gold B-material for coloration . [SEP]
[CLS] in particular , the most typical technique consisted of embedding extremely thin gold B-material foils in glass matrices , thus obtaining a golden appearance . [SEP]
[CLS] this appearance was enriched and complemented by tiny red droplets produced by the precipitation of colloidal gold B-material in the glass matrix through a thermal process ( figure 2c ) . [SEP]
[CLS] however , a more frequent use of gold B-material for coloration is found when analyzing pale , reddish - white colored mosaic tesserae throughout the mediterranean basin ( figure 2d , e ) : several studies in fact attribute this color to the presence of colloidal metallic B-nanoparticle nanoparticles I-nanoparticle of gold B-material ( and mixed alloys of gold−silver ) in the glass matrix . [SEP]
[CLS] the methodology used by the ancient glassmakers to produce these tesserae is still under debate , as is the state of preservation and transmission of this knowledge throughout the centuries , as it is possible that earlier mosaics have been disassembled and their pieces reused for more recent ones [SEP]
[CLS] however , it is clear that roman glassmakers did possess and master the knowledge to produce gold B-material red ruby glass several centuries ago . [SEP]
[CLS] more systemic use of gold B-material as a chromophore in glass and ceramics directly emerged from the development of alchemy in the middle east . [SEP]
[CLS] this discipline revolved around the search for the philosopher ' s stone : according to the tradition , this stone was supposed to transform any common metal B-material into gold B-material , the purest of all metals B-material , and it was often imagined looking like garnets and rubies . [SEP]
[CLS] in this sense , ganzenmuller has suggested a link between the search for the philosopher ' s stone and the creation of gold B-material ruby glass , where the latter is a product of the former . [SEP]
[CLS] according to some sparse studies , the ninth century persian manuscript " secret of secrets " , attributed to the alchemist al - razi , contains both one of the earliest modern descriptions of the preparation of gold B-material ruby glass and one of the oldest recipes for the preparation of pure sulfuric B-material , nitric , and hydrochloric acids , from which aqua regia was later prepared . [SEP]
[CLS] during the 14th and 15th centuries , with the translation and the diffusion of arabic texts in europe , the first mentions of gold B-material ruby glass appear in literature , especially in italy and germany , even though the truthfulness of these publications is debated . [SEP]
[CLS] at the same time , descriptions of how to prepare powerful acids also appear in europe : in 1530 the alchemist georg bauer , better known as georgius agricola , described in his de re metallica how to prepare aqua valens ( i . e . , poweful water B-material , figure 2f ) , a generic formulation to indicate powerful acids such as aqua fortis ( nitric acid ) and aqua regia ( hydrochloric acid / nitric acid , 3 : 1 ) . [SEP]
[CLS] the preparation of the latter , capable of dissolving gold B-material , among other things , opened the doors for the preparation of the first dispersions of colloidal gold B-material during the 17th century . [SEP]
[CLS] in 1685 , andreas cassius from leiden published in the de auro a recipe to produce colloidal dispersions of gold B-nanoparticle nanoparticles I-nanoparticle from the dissolution of gold B-material in aqua regia followed by reprecipitation via the addition of a mixture of stannic and stannous chloride B-material , which took the name " purple of cassius " . [SEP]
[CLS] this preparation has been believed to be the first reported preparation of colloidal gold B-material for a long time ; however , hunt recently showed that johann glauber ( figure 2 ) discovered how to make these preparations at least 25 years before cassius ( in 1659 ) . [SEP]
[CLS] the ability to dissolve gold B-material and to produce dispersions of colloidal gold B-material had two major consequences . [SEP]
[CLS] on one side , strongly fuelled by exoteric and alchemical beliefs , colloidal dispersions of gold B-material have been deployed for medical purposes : diluted solutions of gold B-material dissolved in aqua regia ( in this context called aurum potabile , potable gold B-material ) were believed to be a universal panacea , capable of treating diseases in men and animals . [SEP]
[CLS] on the more technological side , such solutionprocessing of gold B-material paved the way for the relatively reproducible fabrication of high - quality gold B-material ruby glass . [SEP]
[CLS] although some earlier reports exist on producing gold B-material ruby glass from colloidal dispersions of gold B-material ( e . g . , antonio neri ) , johann kunckel ( figure 2 ) was the first glassmaker able to use this technique to produce gold B-material ruby glass on a large scale ( figure 2g ) . [SEP]
[CLS] the son of an alchemist and alchemist himself , familiar with the work of cassius , kunckel employed gold B-material chloride B-material , obtained from the dissolution of gold B-material into aqua regia , as a precursor . [SEP]
[CLS] the red ruby glass would then be obtained by reducing the gold B-material chloride B-material in the molten glass via the addition of metallic tin B-material . [SEP]
[CLS] by varying the thermal annealing steps , he could obtain , very reproducibly , different tints of red . [SEP]
[CLS] at the end of the 17th century , gold B-material ruby glass became fashionable among the rich elites of europe , and in the 18th century the production of gold B-material ruby glass expanded in france , with the glass of bernard perrot 99 ( a ligurian glassmaker who used arsenic B-material instead of tin B-material to precipitate the gold B-material ) , in italy , with the murano glassmakers , and in england , where it was produced in a less saturated tint called cranberry . [SEP]
[CLS] the use of gold B-material ruby glass was so widespread that , during the french revolution , the convention ( the french parliament between 1792 and 1795 ) considered melting the stained glass from french churches with the purpose of extracting gold B-material , before they discovered that copper B-material was the chromophore , and not gold B-material . [SEP]
[CLS] in the meantime , the same solution - based technique for coloring the glass was used to produce gold B-material ruby enamels on ceramics . [SEP]
[CLS] this craft developed mainly in france , where la manufacture royale de sevres gave origin to the rose pompadour series 101 in 1757 using purple of cassius , and in china , where gold B-material tincture ( probably imported by the jesuits ) was used to decorate porcelain , under the name of famille rose ( figure 2h ) , from 1735 . [SEP]
[CLS] during the 18th century the empirical scientific approach introduced by the enlightenment led to the first investigations of the origin of the color of the purple of cassius . [SEP]
[CLS] in 1857 michael faraday ( figure 2 ) was the first to suggest both that " finely - divided gold B-material " could be at the origin of the red color and that " a mere variation in the size of its particles gave rise to a variety of resultant colours " , thus consciously linking , to the best of our knowledge for the first time , color and size in nanoparticles B-nanoparticle . [SEP]
[CLS] despite correctly suggesting the size of the nanoparticles B-nanoparticle to be smaller than the wavelength of light , faraday incorrectly attributed the color of the nanocrystals to their relation with vibrations of ether B-material particles : " besides , the waves of light are so large compared to the dimensions of the particles of gold B-material which in various conditions can be subjected to a ray , that it seemed probable the particles might come into ef fective relations to the much smaller vibrations of the ether B-material particles " . [SEP]
[CLS] however , it was only in 1898 , with the discovery of the slit ultramicroscope by richard zsigmondy , that the colloidal nature of gold B-material in the purple of cassius was finally unravelled ; this discovery earned him the nobel prize in chemistry in 1925 . to complete the picture , in 1908 gustav mie ( figure 2 ) developed the first rigorous mathematical description of the optical behavior of colloids that scatter light . [SEP]
[CLS] according to his theory , when a spherical particle is much smaller than the wavelength of light , an incident electromagnetic radiation can generate a coherent oscillation of the free electron cloud on the surface of the particle , which takes the name of localized surface plasmon resonance . [SEP]
[CLS] the wavelength of this oscillation , being size - , shape - , and materialdependent , strongly influences the absorption and the scattering of the particles themselves , thus resulting in remarkable optical properties . [SEP]
[CLS] after the abundant use of gold B-material nanocrystals for the coloration of materials over the centuries , the past century has faced the rise of colloidal gold B-material for biological and medical applications . [SEP]
[CLS] the intrinsic chemical inertia of this metal B-material ( i . e . , low toxicity B-property ) , quickly promoted it as an ideal candidate for applications spanning from drug delivery to tumor B-material detection and biosensing . [SEP]
[CLS] in this case , for example , colloidal gold B-material nanocrystals are widely used to label antigens for biological electron B-technique microscopy I-technique and for viral antibody B-material and antigen tests ( e . g . , some covid - 19 rapid tests ) . [SEP]
[CLS] the prolificity of this application field is reflected in the rich literature concerning the functionalization of the surface of gold B-material nanocrystals in order to interact with biological probes and receptors . [SEP]
[CLS] the popularity of gold - like decorations on ceramic and glass has been suggested to have fostered the development of luster ceramic . [SEP]
[CLS] this technique produces a peculiar metallic gold - like glaze on ceramic materials using ag and cu ; for this reason , this technique might have been developed to circumvent the prohibition , by islamic rule , of making profane utensils out of gold B-material . in fact , the first examples of such a luster ceramic or lustreware , characterized by a peculiar metallic glaze , date back to the ninth century ad to the city of samarra ( as well as baghdad , basra , kufa , and susa ) , under the domination of the abbasid caliphate , in modern iraq ( figure 3 ) . after the fracture of the abbasid caliphate , luster ceramics disappeared in the iraqi area to appear in fustat , egypt , under the domination of the fatimids . it has been suggested that , this technique being too difficult to imitate , a direct transmission of knowledge likely occurred . from this area , luster ceramics spread to hispano - moorish spain , in particular , valencia , as did also the manufacturing of paper . [SEP]
[CLS] in valencia luster ceramics appeared in the 13th century and survived the centuries of the reconquista until the 18th century , when it was replaced by porcelain from the orient . [SEP]
[CLS] it is probably during this period that the know - how was transmitted to christian artisans and , from spain , diffused to italy in the 15th century , where it became particularly popular in the productions of gubbio and deruta . [SEP]
[CLS] even though the technique to color ceramics with nanocrystals is in many ways similar to that used to produce colored glass , the resulting structure and optical properties are quite different due to the nature of the material B-material . [SEP]
[CLS] a very scarce number of recipes have survived the passage of time . [SEP]
[CLS] nevertheless , via the investigation of surviving samples and via experimental archeology , [SEP]
[CLS] nanocrystals in ceramicswe now have an understanding of how luster ceramic was produced . [SEP]
[CLS] typically , a paste of clay and ochre , cu and ag salts B-material , water B-material , vinegar , and lye ( likely with an excess of vinegar ) was applied to the outer vitrified surface of the ceramic pot ( figure 3b ) . [SEP]
[CLS] during this step , the acetic acid of the mixture would partially dissolve , at 80−100 °c , the pb - rich surface of the pot , effectively increasing its porosity and therefore maximizing the diffusion of the ag / cu - rich melt . [SEP]
[CLS] this step would be followed by an increase in the temperature to allow the silver B-material and copper B-material salts B-material to diffuse into the glass matrix , where they would undergo an ionic exchange with the alkaline ions B-material ( k + and na + ) composing the ceramic . [SEP]
[CLS] this process would produce layers of metallic ag and cu nanocrystals of different shapes and sizes . [SEP]
[CLS] to promote the reduction to metallic silver B-material and copper B-material , iron B-material and tin B-material oxides B-material would be added to the ceramic paste . [SEP]
[CLS] several " firing " steps at different temperatures ( 600−1000 °c ) would then follow in order to burn the acetic acid residuals and melt the ag and cu , allowing them to reprecipitate as nanocrystals so to obtain the targeted coloration ; then the remaining paste would be washed away from the surface , thus leaving a beautiful iridescent color ( figure 3c ) . [SEP]
[CLS] this technique , compared to that used to color glasses , would result in several nanometersthick multilayers ( spacing of few hundreds of nm ) [SEP]
[CLS] highly concentrated nanocrystals [SEP]
[CLS] ( figure 3d , e ) . [SEP]
[CLS] for this reason luster ceramic has been viewed by many as the first example of a high - density nanocluster thin film ever developed by humans . [SEP]
[CLS] furthermore , several reports state that the multilayered structure , combined with the presence of silver B-material and copper B-material nanocrystals , is the origin of the angledependent coloration of luster ceramics . [SEP]
[CLS] in particular , while the absorption of the localized surface plasmon resonances of the nanocrystals ( figure 3f ) produces a diffused incoherent coloration , the interference of the multilayered structures generates a strong angle - dependent coloration that is tuned by the interlayer distance ( figure 3g ) . [SEP]
[CLS] historical samples of luster ceramics are very heterogeneous both geographically and chronologically : while the early productions of egypt and mesopotamia usually have a more complex structure along with smaller nanocrystals ( 10−15 nm ) , the later samples from spain and italy have simpler structures with larger nanocrystals ( 50−100 nm ) [SEP]
[CLS] this difference is a result of the fact that , although in all the productions , ag and cu ( and ag / cu alloys ) are the chromophores ( typically ag for yellow color and cu for red ) , different manufacturing techniques were used . this is mainly demonstrated by the presence of different elements and impurities B-property in the glaze itself . typical of the italian production is , for example , the presence of bi to reduce silver B-material and copper B-material , while the presence of feo and hgs impurities B-property has been connected to the balancing of the chemical environment between reducing and oxidizing . [SEP]
[CLS] tin B-material and lead would be further added to the glass to tune the diffusion of the ions B-material and the reducing power of the glass , thus resulting , when combined with different firing steps , in nanocrystals of different sizes . [SEP]
[CLS] as a result , different glass compositions and firing techniques would result in different colors for the same amount of chromophores added . [SEP]
[CLS] overall , the production of luster ceramics required a high level of technical skill as well as strong empirical knowledge from craftsmen of the middle age . [SEP]
[CLS] these proto - solid - state chemists had to subtly balance all these experimental parameters in order to produce high - quality ceramics in a process that , in the words of a 16th century potter , " is so uncertain that often , out of one hundred pieces , hardly six are good " . [SEP]
[CLS] beauty and health care of the body has been one of the main focuses of mankind throughout the whole of human history . [SEP]
[CLS] it is , therefore , natural to find examples of the use of nanocrystals for healthcare purposes . [SEP]
[CLS] in the previous paragraphs , we have already discussed the use of colloidal dispersions of gold B-material during the modern age as a panacea ; however , supposed healing properties were not a prerogative of gold B-material , and other metals B-material and compounds have been used for similar purposes . [SEP]
[CLS] it is well - known that galena , the natural mineral form of pbs , was widely used in the ancient world as a pigment . [SEP]
[CLS] it therefore does not come as a surprise that pbs is also one of the earliest pieces of historical evidence for the use of semiconductor nanocrystals . [SEP]
[CLS] in particular , it has been shown that , during greco - roman times , a mixture of pbo , ca ( oh ) 2 , and water B-material was used as a hair dyeing mixture . [SEP]
[CLS] researchers showed that the alkaline environment created by ca ( oh ) 2 in water B-material would promote the reaction of pbo with the sulfurbased amino B-material acids I-material of keratine , thus producing in situ nanocrystals of pbs . the nanocrystals , with an average size of [UNK] nm , would blacken the hair while retaining its mechanical properties . [SEP]
[CLS] another popular use of galena was as a component to make kohl , a powder cosmetic comparable to modern mascara widely used by the egyptians . [SEP]
[CLS] although there is no scientific evidence to support the statement that pbs was used for this purpose in its nanocrystalline form , we do not think this theory is to be rejected completely . [SEP]
[CLS] we note that , until the modern age , there was no awareness about lead toxicity B-property ; this explains the widespread use of lead for various applications , ranging from plumbing to artificial sweeteners . [SEP]
[CLS] a cosmetic for the eyes similar to kohl , kajal , is widespread in india for its supposed antifungal B-property and antibacterial properties . [SEP]
[CLS] kajal is prepared from the incineration of the monosha plant leaves covered in oil , and it has been shown to contain carbon B-material nanocrystals with sizes below 100 nm . [SEP]
[CLS] more generally , a category of preparations of the traditional indian ayurvedic medicine , the bhasmas , make much use of nanocrystals for therapeutic purposes . [SEP]
[CLS] bhasmas are described as herbomineral preparations ( i . e . , consisting of grinded metals B-material and herbs ) produced through incineration , and they are employed to treat several diseases . [SEP]
[CLS] the metals B-material used for the bhasmas are really diverse ( e . g . , zn , pb , hg , and , obviously , au ) , but they have been shown to enhance the absorption by the human body ( i . e . , assimilation ) of the active ingredients contained in the herbs thanks to their nanoscale dimensions . [SEP]
[CLS] interestingly , the use of metals B-material for therapeutic and diseasepreventative reasons is not limited to the indian subcontinent . [SEP]
[CLS] several literary sources show how the antimicrobial B-property properties of silver B-material were already well - known from the sixth century bc , when water B-material was often stored in silver B-material containers ; for the same reason , silver B-material nitrate was prescribed by medieval doctors for the treatment of wounds . [SEP]
[CLS] despite this widespread knowledge it is striking that there is no evidence for the use of silver B-material in its colloidal form in history , unlike colloidal gold B-material . [SEP]
[CLS] the few mentions that exist are not supported by factual evidence . [SEP]
[CLS] the well - known antibacterial properties of silver B-nanoparticle nanoparticles I-nanoparticle has recently promoted them as a common ingredient in modern deodorants and toothpastes , along with gold B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] however , the current use of nanocrystals in cosmetics is not only limited to silver B-material and gold B-material : at present a wide variety of nanocrystals are widely used in this industry . [SEP]
[CLS] we have already mentioned the use of various nanocrystals ( e . g . , tio 2 , zno , ceo 2 , and zro 2 ) in sunscreens due to their strong light absorption in the uv region , but for the same reasons they are also extensively used in lip balms and moisturizers . [SEP]
[CLS] furthermore , several modern cosmetic products rely on nanocrystals ( e . g . , in the form of nanocapsules B-nanoparticle or nanosuspensions ) as agents to carry and manipulate the release and action of the cosmetic active substances . [SEP]
[CLS] in particular , nanocrystals offer superior properties compared to bulk materials , for instance , improved penetration in the skin and in the mucosal surfaces , as observed for organic nanocrystals such as rutin ( i . e . , quercetin - 3 - o - rutinoside ) nanocrystals . [SEP]
[CLS] the early 1980s mark the inception of research in the field of semiconductor nanocrystals ( or quantum B-nanoparticle dots I-nanoparticle ) . [SEP]
[CLS] indeed , four decades ago , the size - dependent optical properties of solvent - and glass - dispersed colloidal semiconductors have been unambiguously attributed to the quantum - size effect . [SEP]
[CLS] nevertheless , it is also instructive to highlight the earlier history and the continuity of research that led to the modern field of quantum B-nanoparticle dots I-nanoparticle . [SEP]
[CLS] during the 19th and 20th centuries , interest in glass manufacturing received a substantial boost ; the development of optical B-technique microscopy I-technique and spectroscopy B-technique , in fact , promoted research oriented to the production of high - quality glass , both colored and transparent . [SEP]
[CLS] for example , at the end of the 19th century , otto schott established the " glastechnisches laboratorium schott & gen jena " , which soon became the leader in optical glass manufacturing , to the point that " schott glasses " soon , by the transmission of meaning , became the term to refer more generally to optical filters . [SEP]
[CLS] in this context , as the heirs of a multimillennial tradition from artists and craftsmen , researchers were investigating the composition dependence ( via the incorporation of additives such as chalcogens and halogens B-material ) on the optical properties of glasses . [SEP]
[CLS] to the best of our knowledge , it is in this framework that researchers in the field of glass coloration started to observe size - dependent optical properties in glasses containing cds additives ( figure 4a ) [SEP]
[CLS] in particular , they noticed that the absorption edge and emission of the colored glasses could be tuned by temporally varying the tempering step . [SEP]
[CLS] as the works of zsigmondy and mie were already known , the authors hypothesized that the origin of the color shift toward longer wavelengths was to be found in the formation and growth in size of cds colloids in the glass , producing , to the best of our knowledge , the first observation of quantum confinement effects in semiconductor nanocrystals . [SEP]
[CLS] a similar observation was made a few years later for samples of ruby - colored se - containing glass . [SEP]
[CLS] in this case , researchers correctly attributed the coloration of the glass to the precipitation of cdse and cds crystallites in the glass itself and recorded one of the first micrographs of sub - micrometer semiconductor crystals ( figure 4b ) . [SEP]
[CLS] moreover , they hypothesized a link between the color variation ( from pale yellow to red ) observed during tempering of the glasses and a variation in the size of the crystals over time . [SEP]
[CLS] in 1950 , victor lamer developed a theory explaining the formation of monodisperse hydrosols through a separation between nucleation and growth ( figure 4c ) . [SEP]
[CLS] this theory , as we will see , has been pivotal in the development of monodisperse colloidal nanocrystals . [SEP]
[CLS] in the second half of the 20th century researchers started focusing on the investigation of the sizedependent optical properties of ( nano ) crystals embedded in solid matrixes . [SEP]
[CLS] in a few years crystals of very different compositions , and in matrices or as colloids , were characterized : cubr ( figure 4d ) , cucl , agi , and agbr ( figure 4e ) . [SEP]
[CLS] for cdse nanocrystals ( with radius between 1 and 5 nm ) dispersed in glass , the inverse size dependence of optical absorption with the squared radius of the nanocrystals was classified as an " optical anomaly " by katzschmann . [SEP]
[CLS] similar observations on size - dependent optical properties were also collected in the case of thin films ; for example , stasenko unambiguously identified the dependence of the energy band gap of cds on the thickness of the thin films ( 1 . 7 nm ) . in 1984 , itoh and co - workers observed the effects of exciton confinement in cucl microcrystals embedded in nacl matrixes . all of these observations throughout the decades , together , paved the way for the work of alexey ekimov and louis brus , who successfully and intentionally synthesized semiconductor nanocrystals in glass and in water B-material and correctly attributed their size - dependent properties to the quantum confinement effect ( figure 4f ) . [SEP]
[CLS] furthermore , collaborations between ekimov and alexander efros resulted in the very first theory describing the " quantum size effect " in semiconductor nanocrystals . [SEP]
[CLS] the size - dependent optical properties were linked to the concept of the confinement of the exciton wave function into a potential well smaller than the exciton bohr radius . [SEP]
[CLS] interestingly , despite independently investigating and discovering quantum B-nanoparticle dots I-nanoparticle , these two groups of scientists ( ekimov ' s / efros and brus ' ) approached the quantum B-nanoparticle dot I-nanoparticle field as successors of two different research currents . [SEP]
[CLS] ekimov was investigating the physicochemical properties of halogen - doped colored glasses , which , as we previously showed , derives from a long - standing tradition of colored ( optical ) glass manufacturing . [SEP]
[CLS] brus ' research instead was rather motivated by the anticipated utility of colloidal semiconductors in photocatalysis . [SEP]
[CLS] in both cases , it is essential to highlight the role of the cross - fertilization of ideas across scientific disciplines ( i . e . , the influence of concepts present in a specific scientific discipline in the development of a new idea in a different scientific discipline ) . [SEP]
[CLS] in particular , brus was also influenced by the contemporary observations of the optical effects in molecular - beam - epitaxy - grown quantum wells ( onedimensional ( 1d ) quantum confinement ) . [SEP]
[CLS] the independent research of these two groups of scientists ( brus ' and ekimov ' s / efros ) represents a milestone for the quantum B-nanoparticle dot I-nanoparticle field : their work correctly linked , for the first time , empirical evidence with scientific understanding of the underlying process . [SEP]
[CLS] furthermore , their work was crucial in sparking the interest of the scientific community and successfully igniting research in this field . [SEP]
[CLS] this milestone has been recently described in a highly detailed review by the leading actors themselves ; we refer to this work for a more specific and more personal account of their discoveries . [SEP]
[CLS] in the years immediately after ekimov ' s and brus ' publications , the field saw a rapid acceleration : the colloidal synthesis was extended to a plethora of other semiconductors , while the nanocrystals began to be extensively characterized both optically and structurally . [SEP]
[CLS] however , the main bottleneck to the complete establishment of the field was the high polydispersity of the nanocrystals produced , which hampered efforts to consistently link the optical properties to a well - defined size of the nanocrystals . [SEP]
[CLS] this problem was effectively solved in 1993 for ncs both in glass and in solution . [SEP]
[CLS] ekimov solved this impasse for ncs in glass by using different stages , characterized by different temperatures , in the production of the nanocrystal - doped glass , thus separating the nucleation and growth phases of the nanocrystals ; this technique decreased the size dispersion from 15 % to 5 % . [SEP]
[CLS] a similar approach was independently developed for colloidal quantum B-nanoparticle dots I-nanoparticle . [SEP]
[CLS] inspired by the theory of lamer , the research group of bawendi developed the highly reliable synthetic method known as " hot injection " for the production of monodisperse colloidal nanocrystals in high concentrations ( figure 4g ) . [SEP]
[CLS] this work represents another key benchmark in the development of this field , since it allowed the production of consistently monodisperse ( semiconductor , but also metal B-material and oxide B-material ) nanocrystals with different chemical compositions . [SEP]
[CLS] the year after , in 1994 , the first synthetic procedure to produce inp colloidal nanocrystals was published ( figure 4h ) , thus introducing what would become one of the main competitors to the ii−vi semiconductor ncs family . [SEP]
[CLS] on the path toward better technical developments , in 1996 the very first core / shell nanocrystals were synthesized . [SEP]
[CLS] this enabled the possibility to tune the quantum confinement effect by varying the shell B-material thickness and material B-material , opening the doors to core / shell heterostructures with spatial delocalization of the charges . [SEP]
[CLS] furthermore , the possibility to confine the exciton in the core B-material of these heterostructures ( i . e . , away from surface defects ) permitted one to achieve much higher quantum yields and , overall , to make nanocrystals more chemically stable ( figure 4i ) . [SEP]
[CLS] the next important milestone was the transition , in the years 2000−2010 , toward safer and more benign precursors . [SEP]
[CLS] organometallic reagents were thus substituted with carboxylates B-material , while , for example , elemental selenium B-material was introduced as a potent selenium B-material source . [SEP]
[CLS] the possibility to produce high - quality quantum B-nanoparticle dots I-nanoparticle with commonplace chemical equipment ( e . g . , with schlenk lines , fume hoods ) and safe reagents made them readily accessible to researchers of different disciplines , promoting the interdisciplinarity of the quantum B-nanoparticle dot I-nanoparticle field . [SEP]
[CLS] from this point onward , the number of publications grew exponentially , mainly thanks to interdisciplinary collaborations between synthetic chemists , physicists , and engineers . [SEP]
[CLS] among significant subsequent advances , we mention the development of anisotropic particles : colloidal nanorods B-nanoparticle in 2000 by peng and colloidal semiconductor nanoplatelets by ithurria in 2008 ( figure 4j ) . [SEP]
[CLS] these types of nanocrystals , characterized by atomically defined thickness ( for nanoplatelets ) and strong exciton confinement in only one or two directions , are particularly important because they introduced the shape of the nanocrystals as an additional parameter toward the manipulation of the optical properties of semiconductor nanocrystals ( in addition to their size ) . [SEP]
[CLS] furthermore , from the mid - 2000s researchers also started to work on the development of organic and inorganic ligands to deploy semiconductor nanocrystals in commercial applications . [SEP]
[CLS] thanks to these new ligands , charge transport in quantum B-nanoparticle dot I-nanoparticle solids was strongly improved , and quantum B-nanoparticle dots I-nanoparticle could finally be efficiently used for optoelectronic applications involving the production and harvesting of light . [SEP]
[CLS] it has to be noted , though , that the long - term stability of quantum B-nanoparticle dots I-nanoparticle is still a major issue , hampering their effective widespread use for commercial applications . [SEP]
[CLS] notable progress in this field is seen as the very first quantum B-nanoparticle dot I-nanoparticle displays in the past decade . [SEP]
[CLS] the continued emergence of size - and shape - uniform nanocrystals led to their use as " artificial atoms B-material " for constructing mesoscale crystalline structures by the spontaneous self - assembly of nanocrystals . [SEP]
[CLS] these superlattices are of mono , binary , or ternary compositions and exhibit periodic and quasi - crystalline order . [SEP]
[CLS] such efforts pave the path to tailoring collective physical properties emerging from the close packing of nanocrystals and their periodic arrangements , such as miniband transport and super - radiance . [SEP]
[CLS] lead - halide perovskite ( lhp ) semiconductor nanocrystals are the most recent generation of colloidal semiconductors . [SEP]
[CLS] in particular , their near - unity photoluminescence B-property quantum yield without any shell B-material passivation , their intriguing optical properties on the single - particle level as well as on a collective scale , and a facile synthesis quickly promoted them to become one of the most interesting materials for scientists in the field of semiconductor nanocrystals . [SEP]
[CLS] as we have tried to show with this review , the evolution of the scientific knowledge in the field of nanocrystals can be traced back to much earlier times , as the product of a continuity of observations and discoveries ; this is also true for perovskite nanocrystals . [SEP]
[CLS] the first mention of cesium - based lead halide compounds is attributed to h . l . wells , who identified them and prepared them from aqueous solutions in 1893 ( figure 5a ) . [SEP]
[CLS] however , it was only at the end of the 1950s that the crystal structure of bulk cspbx 3 ( x = cl , br , i ) was clearly identified as perovskite . [SEP]
[CLS] at the same time , the first observations of a crystalline phase transition and photoconductivity properties were recorded ( figure 5b ) . [SEP]
[CLS] relevant for the development of colloidal cesium - based perovskite nanocrystals was the research investigating the optical properties of bulk cesium halide doped with lead . [SEP]
[CLS] at the latest in 1976 , investigators observed that any excitation in the absorption band of cesium bromide B-material , doped with lead , at room temperature resulted in one single emission band at 2 . 45 ev ( green region ) . this photoluminescence B-property is now attributed to the inclusion of cspbbr 3 nanocrystals in the csbr matrix . in the 1990s , in fact , the concept of the confinement of the excitons was invoked to explain the optical properties of lead halide perovskites , and spectroscopical measurements were performed on few - nanometer large cspbx 3 inclusions in cscl , csbr , and csi hosts . [SEP]
[CLS] for the first time , the concept of " quantum size effect " was introduced to explain the discrepancy between the optical properties of these inclusions and the corresponding cspbx 3 bulk ( figure 5c ) . [SEP]
[CLS] in general , starting from 1997 , the bright emission of cspbbr 3 nanocrystals , formed in situ in csbr / pb matrixes , was investigated systematically both in thin films and single crystals . [SEP]
[CLS] at the same time , also thin films of stoichiometric cspbx 3 were studied . [SEP]
[CLS] the interest for thin films was also boosted from the fact that this geometry , combined with the high density of emission centers , was ideal for stimulated emission experiments ( lasing ) . [SEP]
[CLS] analogously to conventional quantum B-nanoparticle dots I-nanoparticle , also in the case of cesium - based perovskite nanocrystals the conceptualization of quantum B-nanoparticle dots I-nanoparticle in solid matrixes was the prelude to the development of colloidal nanocrystals . [SEP]
[CLS] inspired by the aforementioned works as well as by the surge in attention for methylammonium lead halide compounds for photovoltaic applications , in 2015 , size - tunable , monodisperse , and shape - uniform cspbx 3 nanocrystals were produced for the first time ( figure 5d ) . the extraordinary control on their photoluminescence B-property , their near - unity quantum yield , and their facile synthesis quickly captured the interest of researchers , thus promoting them as the herald of a new generation of colloidal quantum B-nanoparticle dots I-nanoparticle . [SEP]
[CLS] soon after , the colloidal synthesis was extended to other lead - based perovskite materials , such as formamidinium - and methylammonium - [SEP]
[CLS] based lhp , whose bulk forms were already known from much earlier [SEP]
[CLS] the first commercial application of semiconductor nanocrystals appeared in 2013 , when sony developed the first display employing quantum B-nanoparticle dots I-nanoparticle ( from qd vision−cdsebased ) to improve the light - emitting diode ( led ) backlighting in lcd televisions so to achieve a better color gamut . [SEP]
[CLS] since then , quantum dot - based displays have slowly penetrated the market , and they can now be found in upmarket displays distributed mainly by samsung ( who acquired qd vision in 2016 ) under the " qled " brand ( using inp - based quantum B-nanoparticle dots I-nanoparticle instead of the cdse - based ones ) . [SEP]
[CLS] in the foreseeable future novel quantum B-nanoparticle dot I-nanoparticle displays are expected to penetrate the market , where quantum B-nanoparticle dots I-nanoparticle are employed as color filters / converters ; in this light should be seen the integration of qled and organic light - emitting diode ( oled ) technologies , as announced by samsung , and the use of lhp nanocrystals , as developed by several companies ( e . g . , avantama , nanolumi , helio display materials , peroled , brightcomsol , quantum solutions ) . furthermore , if the main issues related to stability over time can be overcome , quantum B-nanoparticle dots I-nanoparticle can also be expected to play a pivotal role in the development of micro - leds , flexible displays , and displays based on an electroluminescent B-property quantum B-nanoparticle dot I-nanoparticle film . [SEP]
[CLS] notably also infraredemitting quantum B-nanoparticle dots I-nanoparticle have recently found a commercial deployment ; in particular , quantum solutions , and other companies , currently sell them ( i . e . , pbs nanocrystals ) as colloidal materials , and emberion has introduced them in infrared cameras and other solid - state devices . [SEP]
[CLS] currently , nanocrystal research proceeds at a vertiginous speed , as witnessed by the increasing number of scientific publications published each year , delivering new findings and opening up new questions at a pace never before experienced in the history of mankind . [SEP]
[CLS] we are now able to produce nanocrystals , both in solid matrices and in solution , with exquisite size and morphological control and diverse properties , allowing us to manufacture objects that would have been attributed to magic by our ancestors . [SEP]
[CLS] however , in our progress , we must not lose the retrospective view of the path that led us here . [SEP]
[CLS] the history of nanocrystals is not just defined by a few scarce breakthroughs but is also the product of a continuous process of improvement and refinement that , from the technical crafts of our ancestors , leads to modern - age laboratories . [SEP]
[CLS] we view this interplay between past and present as crucial for informing and inspiring novel research that is also grounded on insights from the past . [SEP]
[CLS] in particular , we envisage three main directions along which future research in this vein should develop : ( 1 ) investigating the link between the nature of nanocrystals ( i . e . , size and composition ) and their optical properties in ancient artifacts , ( 2 ) retrieving and reproducing ancient recipes and methods for the production of such artifacts , and ( 3 ) tracing the evolution of knowledge through the centuries by reviewing historical scientific accounts through the lens of our modern theoretical understanding . [SEP]
[CLS] we believe that this can only be achieved through a combined interdisciplinary effort from chemists , physicists , materials scientists , and historians , in order to unveil the legacy that , through the millennia , connects us to our ancestors . [SEP]
[CLS] nanocrystal , a piece of material B-material whose dimensions ( at least one ) are in the nanometer ( 10 −9 m ) range and whose atoms B-material are arranged in an ordered structure ; gold B-material ruby glass , a red glass whose coloration originates from the presence of gold B-material nanocrystals embedded in the material B-material ; localized surface plasmon resonance , a coherent oscillation of the free electron cloud on the surface of a nanoparticle B-nanoparticle , induced by light excitation , and confined by the size of the nanoparticle B-nanoparticle being smaller than the wavelength of the light used to excite the plasmon ; luster ceramic , a particular type of ceramic and pottery characterized by a gold - like glaze and produced via the incorporation of silver B-material and copper B-material in the material B-material ; exciton , a bound state of an electron and a hole , also called electron− hole pair , attracting each other via electrostatic coulomb interaction ; quantum confinement , a variation of the properties of a material B-material due to the confinement of the exciton into a piece of material B-material whose dimensions are smaller than the exciton bohr radius [SEP]
[CLS] 1 . examples of the use of nanocrystals in glass throughout history . [SEP]
[CLS] ( a ) scanning electron B-technique microscopy I-technique ( sem ) image of cu nanocrystals dispersed in a bronze age colored glass from frattesina di rovigo ( italy ) ; reprinted with permission from ref 61 . [SEP]
[CLS] copyright ( 2004 ) , elsevier . [SEP]
[CLS] ( b ) transmission B-technique electron I-technique microscopy I-technique ( tem ) image of a metallic cu crystallite in a gallo - roman glass ( 4th century ad ) ; reprinted with permission from ref 66 . [SEP]
[CLS] copyright ( 1991 ) chapman & hall , with permission of springer nature . [SEP]
[CLS] ( c ) stained glass piece from the cathedral of avila ( 16th century ) , spain , observed through optical B-technique microscopy I-technique in transmitted light ( left ) and reflected light ( right ) ; reprinted with permission from ref 67 . [SEP]
[CLS] copyright ( 2013 ) springer science business media dordrecht . [SEP]
[CLS] ( d ) scheme representing the distribution , form , and concentration of cu in red glass . [SEP]
[CLS] the darker shade zone represents the development of red color ; reprinted with permission from ref 68 . [SEP]
[CLS] copyright ( 2013 ) elsevier . [SEP]
[CLS] ( e ) composite tem image of a cu red glass from burgos , spain , 13th century . [SEP]
[CLS] the black dots , whose size increases from bottom left to top right , are cu nanocrystals ; reprinted with permission from ref 68 . [SEP]
[CLS] copyright ( 2013 ) elsevier . [SEP]
[CLS] ( f ) cobalt B-material blue glass from york minister ( england ) , 15th century , presenting red striae from precipitation of cu nanocrystals ; reprinted with permission from ref 68 . [SEP]
[CLS] copyright ( 2013 ) elsevier [SEP]
[CLS] 2 . use of gold B-material nanocrystals throughout the centuries . [SEP]
[CLS] ( a ) photograph of the lycurgus cup in reflected light ( left ) and transmitted light ( right ) ; reprinted with permission of the british museum . [SEP]
[CLS] copyright trustees of the british museum . [SEP]
[CLS] ( b ) tem image of a ag / au alloy nanocrystal from the lycurgus cup ; reprinted with permission . [SEP]
[CLS] 81 copyright ( 2007 ) , john wiley and sons . [SEP]
[CLS] ( c ) digital image of a saint sabina church mosaic tessera in rome , italy . [SEP]
[CLS] the gold B-material - foil tessera presents few gold B-material ruby droplets from gold B-material nanocrystals precipitation ; reprinted with permission . [SEP]
[CLS] 84 copyright istituto centrale per il restauro , photographer marcello leotta . [SEP]
[CLS] ( d , e ) face from the saint pudentiana church mosaic in rome , italy . [SEP]
[CLS] the flesh tone tesserae are colored by gold B-material nanocrystals dispersed in the glass matrix ; reprinted with permission . [SEP]
[CLS] 84 copyright ( 2010 ) susanna sarmati . [SEP]
[CLS] inset portraits ( from left to right ) : rhazes ( al - razi ) ( wellcome collection . attribution 4 . 0 international , cc by 4 . 0 ) ; johann rudolph glauber ( public domain ) ; johannes kunckel ( public domain ) ; michael faraday ( public domain ) ; gustav mie ( reprinted ; 87 attribution 4 . 0 international , cc by 4 . 0 ) . [SEP]
[CLS] ( f ) representation of the distillation of aqua valens from the 1912 hoover translation of agricola ' s de re metallica ( 1556 ) ; reprinted with permission . 88 copyright ( 2006 ) john wiley and sons . [SEP]
[CLS] ( g ) photograph of ruby glass goblet ( 1690−1700 ) from potsdam , germany , probably gottfried spiller . [SEP]
[CLS] cmog 79 . 3 . 258 . gift of the ruth bryan strauss memorial foundation . [SEP]
[CLS] image licensed by the corning museum of glass , corning , ny ( www . cmog . org ) under cc by - nc - sa 4 . 0 . ( h ) digital image of famille rose porcelain set , qing dynasty , 18th century ; reprinted with permission from the british museum . [SEP]
[CLS] copyright trustees of the british museum . [SEP]
[CLS] 3 . nanocrystals in luster ceramics . [SEP]
[CLS] ( a ) geographical map showing the location of the main centers of luster ceramic over the centuries . [SEP]
[CLS] adapted with permission from ref 128 . [SEP]
[CLS] copyright ( 2012 ) philippe sciau . creative commons license cc 3 . 0 . [SEP]
[CLS] ( b ) schematic of luster ceramic production : ( 1 ) outermost glassy fired glaze of lead - based ceramic ; ( 2 ) the precursor paste , containing ag and cu salts B-material , lye , vinegar , water B-material is applied onto the glassy surface ; ( 3 ) the acetic acid , in combination with the lye , attacks the surface , increasing its porosity ; ( 4 ) the temperature is increased to melt the ag / cu - rich liquid ; ( 5 ) the surface of the pot is locally flashed to burn the residuals of acetic acid and to promote the recrystallization of ag and cu as nanocrystals ; ( 6 ) luster is revealed after washing away the residual paste . [SEP]
[CLS] reprinted with permission . 124 copyright ( 2008 ) with permission from taylor & francis . [SEP]
[CLS] ( c ) example of 12th century luster ceramic from fustat ( cairo , egypt ) at ( left ) nondiffracting and ( right ) diffracting observation angles ; reprinted with permission . 124 copyright ( 2008 ) with permission from taylor & francis . [SEP]
[CLS] ( d ) transmission B-technique electron I-technique microscopy I-technique image of the multilayered structure of a luster ceramic from the 12th century . [SEP]
[CLS] reprinted with permission . 133 copyright ( 2008 ) trans tech publications , ltd . ( e ) transmission B-technique electron I-technique microscopy I-technique image of the inner part of a 15th century gold - like luster from deruta , italy . [SEP]
[CLS] reprinted with permission . 134 copyright ( 2004 ) john wiley & sons . [SEP]
[CLS] ( f ) absorption spectrum of a gold - like luster decoration from gubbio , italy ( 16th century ) . [SEP]
[CLS] the absorption features associated with localized surface plasmon resonance of ag and cu nanocrystals are indicated . [SEP]
[CLS] reprinted with permission . [SEP]
[CLS] 129 copyright ( 2002 ) elsevier science b . v . [SEP]
[CLS] ( g ) schematic representation of the optical effects observed in luster ceramic . [SEP]
[CLS] reprinted with permission . 128 copyright ( 2012 ) philippe sciau . [SEP]
[CLS] creative commons license cc 3 . 0 . [SEP]
[CLS] 4 . semiconductor nanocrystals in the modern age . [SEP]
[CLS] ( a ) absorption spectra of different glasses containing cds . reprinted with permission . [SEP]
[CLS] 167 ( b ) micrographs of selenium B-material - containing ruby - colored glasses ( magnification × 5000 ) . [SEP]
[CLS] the black dots are cdse microcrystals in the glass matrix ( smallest size [UNK] nm ) . [SEP]
[CLS] reprinted with permission . [SEP]
[CLS] 168 copyright ( 1932 ) society of glass technology . [SEP]
[CLS] ( c ) schematic representation of the concentration of monomers B-material as a function of time during the synthesis of hydrosols ; adapted with permission . [SEP]
[CLS] 169 copyright ( 1950 ) american chemical society . [SEP]
[CLS] ( d ) emission spectra of cubr / nabr mixtures showing narrow line width emission , associated with the presence of quantum B-nanoparticle dots I-nanoparticle . [SEP]
[CLS] reproduced with permission . [SEP]
[CLS] 170 copyright ( 1957 ) aip publishing . [SEP]
[CLS] ( e ) absorption spectra of agi ( nano ) crystals of different sizes . [SEP]
[CLS] the sharp absorption features are associated with excitonic features . [SEP]
[CLS] reprinted with permission . [SEP]
[CLS] 171 copyright ( 1967 ) american physical society . [SEP]
[CLS] ( f ) ( left ) absorption spectra of cucl nanocrystals , with a radius between 2 . 5 and 31 nm , embedded in a glass matrix . [SEP]
[CLS] the sharp excitonic features become more prominent with smaller sizes . [SEP]
[CLS] reprinted with permission . [SEP]
[CLS] 172 copyright ( 1983 ) aip publishing . [SEP]
[CLS] ( right ) absorption spectra of cds nanocrystals dispersed in water B-material . [SEP]
[CLS] the size dependence of the absorption edge is clearly visible . [SEP]
[CLS] reprinted with permission . [SEP]
[CLS] 173 copyright ( 1982 ) jetp letters . [SEP]
[CLS] ( g ) ( top ) absorption spectra of colloidal cdse nanocrystals ranging from 1 . 2 to 11 . 5 nm . [SEP]
[CLS] ( bottom ) transmission electron micrograph of colloidal nanocrystals . [SEP]
[CLS] reprinted with permission . [SEP]
[CLS] copyright ( 1993 ) american chemical society . [SEP]
[CLS] ( h ) absorption spectra of inp nanocrystals . [SEP]
[CLS] reprinted with permission . [SEP]
[CLS] copyright ( 1994 ) american chemical society . [SEP]
[CLS] ( i ) photoluminescence B-property of cdse ( dotted line ) and cdse / cds ( solid line ) nanocrystals . [SEP]
[CLS] reprinted with permission . [SEP]
[CLS] copyright ( 1996 ) american chemical society . [SEP]
[CLS] ( j ) absorption ( solid lines ) and photoluminescent B-property excitation ( dotted lines ) spectra of nanoplatelets of different thicknesses . [SEP]
[CLS] reprinted with permission . [SEP]
[CLS] copyright ( 2008 ) american chemical society . [SEP]
[CLS] 5 . perovskite nanocrystals . [SEP]
[CLS] ( a ) header of the scientific article by h . l . wells , where cesium - based lhps are mentioned for the first time . reproduced with permission . [SEP]
[CLS] 236 copyright ( 1893 ) verlag gmbh & co . kgaa , weinheim ; with permission from john wiley and sons . [SEP]
[CLS] ( b ) header of the scientific article by møller in 1958 , when the crystal structure of cspbx 3 was identified for the first time as perovskite . [SEP]
[CLS] reproduce with permission . [SEP]
[CLS] 237 copyright ( 1958 ) nature publishing group . [SEP]
[CLS] ( c ) absorption ( left ) and photoluminescence B-property ( right ) of csbr / pb 0 . 05 mol % samples measured at various temperatures . [SEP]
[CLS] reprinted with permission . [SEP]
[CLS] 238 copyright ( 2001 ) elsevier science b . v . [SEP]
[CLS] ( d ) photoluminescence B-property of cspbx 3 colloidal nanocrystals . [SEP]
[CLS] adapted with permission . [SEP]
[CLS] 232 copyright ( 2015 ) american chemical society . [SEP]
[CLS] scanometric detection of tomato leaf curl new delhi viral dna using aunp B-nanoparticle - conjugated mono - and bifunctional oligo probes through direct dna hybridization assay ( ddh assay ) and sandwich dna hybridization assay ( sdh assay ) with silver B-material enhancement was developed . [SEP]
[CLS] tomato leaf curl new delhi virus ( tolcndv ) coat B-material protein gene - specific thiol - modified ssoligo probes were used for the preparation of mono - and bifunctional aunp B-nanoparticle - ssoligo probe conjugates ( signal probes ) . [SEP]
[CLS] ssdna arrays were prepared using polymerase chain reaction ( pcr ) , rolling circle amplification ( rca ) , genomic dnas fragments , and phosphate - modified positive control / capture probes through 1 - ethyl - 3 - ( 3 - dimethylaminopropyl ) - carbodiimide / 1 - methylimidazole conjugation on the amine - modified glass slide ( gs ) surface . [SEP]
[CLS] in the ddh assay , signal probes were directly hybridized with ssdna array of positive control and tolcndv dna samples and the detection signals were amplified by silver B-material enhancement . [SEP]
[CLS] dark black / gray colors were developed on the gs by the result of ag enhancement , which can be visualized and discriminated by the naked eye . [SEP]
[CLS] the images were captured using a simple flatbed scanner , and the determined amounts of signal probes were hybridized with their target dna . [SEP]
[CLS] similarly , the sdh assay also performed through two rounds of hybridization between capture probes and target dna ; target dna and signal probes followed by silver B-material enhancement . [SEP]
[CLS] the detection signals were found higher in the pcr sample than the rca and genomic dna samples because of the presence of increased copy numbers of complementary dnas in pcr samples . [SEP]
[CLS] further , bifunctional aunp B-nanoparticle - ssoligo probe shows higher intensity of detection signal than monofunctional probes because it can be hybridized with both strands of dsdna targets . [SEP]
[CLS] moreover , the ddh - based scanometric method showed higher detection sensitivity than the sdh assay - based scanometric method . [SEP]
[CLS] overall , bifunctional signal probes showed more detection sensitivity than monofunctional probes in scanometric methods based on both ddh and sdh assays . [SEP]
[CLS] the limit of detection of this developed scanometric method was optimized ( 100 zm to 100 pm concentration ) . [SEP]
[CLS] further , ddh assay - based scanometric method shows significant advantages over the sdh assay method , such as costeffectiveness , because it requires only single probes ( signal probes ) , less time - consuming by the need of only single - step hybridization , and higher detection sensitivity ( up to zm ) . [SEP]
[CLS] to the best of our knowledge , this is the first attempt made to develop a scanometric - based nanoassay method for the detection of plant viral dna . [SEP]
[CLS] this approach will be a remarkable milestone for the application of nanotechnology in the development of nanobiosensor for plant pathogen detection . [SEP]
[CLS] the detection of dna sequences is very important in various fields , such as clinical diagnosis of microbial infections , metabolic disorders , genetic diseases , as well as in environmental and forensic applications . [SEP]
[CLS] methods such as polymerase chain reaction ( pcr ) and southern blotting with fluorescence tagged molecules are extensively used for the detection of specific dna . [SEP]
[CLS] however , these methods have their own disadvantages in the view of degree of accuracy , time , complexity , labor - intensiveness , and cost . [SEP]
[CLS] over the past several years , nanotechnology has been making a number of exciting advances in the field of molecular diagnostics , enhancing the specificity and sensitivity of the existing conventional detection methods . [SEP]
[CLS] particularly , nanometersized gold B-material particles exhibit unique size and shape , fascinating optical and surface physicochemical properties , strong spr band , and biocompatibility B-property . [SEP]
[CLS] nowadays , gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) have been widely used in biosensors for the detection of target dna / protein B-material and other molecules . [SEP]
[CLS] the detection of the target dna sequence was performed using label - free unmodified aunps B-nanoparticle with oligonucleotide probes , 5−8 but the detection limit was very low . [SEP]
[CLS] to overcome the limitations , mirkin and co - workers developed the scanometric method for the detection of target dna with aunp B-nanoparticle - conjugated oligonucleotide probes through silver B-material enhancement . [SEP]
[CLS] the detection limit was optimized up to 50 fm , and the results can be read out through the naked eye and flatbed scanner without the need of sophisticated instruments , which were 100 times more sensitive than the fluorescence B-property probe - based method , where aunps B-nanoparticle act as an optical sensing element and the oligonucleotide probe acts as a recognizing unit . [SEP]
[CLS] finally , the detection sensitivity was increased up to atomolar level through the array - based detection of dna by the scanometric method with electrical readout . [SEP]
[CLS] moreover , the scanometric method has been used to detect single nucleotide mismatch , specific rna targets , and prostate cancer markers ( micro - rna ) . [SEP]
[CLS] scanometric - based biobarcode assay is used for mrna expression analysis and alzheimer ' s disease diagnosis . [SEP]
[CLS] it is also used to carry out imaging of dna hybridization , and high - level mismatch discrimination of anthrax lethal factor dna , light scattering property - based homogenous detection of dna , microarray detection of double - and triple - helix dna binders B-property , visual detection of dna with molecular beacon , and analysis of dna microarray with dna intercalator - conjugated aunps B-nanoparticle were reported . [SEP]
[CLS] however , the application of scanometric methods for the detection of plant viruses is not yet reported so far . [SEP]
[CLS] particularly , whitefly - transmitted tomato leaf curl new delhi virus ( tolcndv ) belongs to the genus begomovirus and the family geminivirus infecting tomato causing severe leaf curl disease , where 100 % yield loss was reported . [SEP]
[CLS] tolcndv is considered as economically important because it can spread easily and lead to uncontrolled infection due to the absence of an effective detection method and disease management strategies . [SEP]
[CLS] also , a high degree of nucleotide diversity was found among the different isolates of tolcv in india and worldwide . [SEP]
[CLS] in this circumstance , degenerate probe has been used in the existing conventional detection methods to overcome the diversity among isolates of this virus . [SEP]
[CLS] recently , we have demonstrated label - free colorimetric detection of begomovirus with cholate - caped aunps B-nanoparticle using degenerate probe with a detection sensitivity of ≥600 pm . [SEP]
[CLS] however , it is difficult to control hybridization reaction and elimination of unhybridized dna , which may interfere with the results , and moreover requires a huge volume of sample dna . [SEP]
[CLS] hence , the development of a new detection method with ultrasensitivity is very important . [SEP]
[CLS] so far , scanometric method - based detection was demonstrated using monofunctional aunp B-nanoparticle - oligo probes conjugate with synthetic and amplified targets through silver B-material and gold B-material enhancement methods . [SEP]
[CLS] more recently , we have demonstrated colorimetric detection of tolcndv dna using bifunctional aunp B-nanoparticle - oligo probe conjugates , which detect both strands of dsdna targets at the same time with increased sensitivity . [SEP]
[CLS] however , bifunctional aunp B-nanoparticle - oligo probe conjugate using scanometric detection of target dna is not yet reported . [SEP]
[CLS] in continuation of our previous works , herein , we have demonstrated scanometric method - based detection of tolcndv dna from amplified pcr and rolling circle amplification ( rca ) dna and genomic dna using monoand bifunctional aunp B-nanoparticle - conjugated virus - specific oligo probes further detection signals were amplified using silver B-material enhancement . [SEP]
[CLS] ■ experimental section [SEP]
[CLS] materials . [SEP]
[CLS] trisodium citrate , hydrogen B-material tetrachloroaurate trihydrate ( haucl 4 • 3h 2 o ) , disodium hydrogen B-material phosphate ( na 2 hpo 4 ) , monosodium dihydrogen phosphate ( nah 2 po 4 ) , silver B-material nitrate , hydroquinone ( 0 . 3 % ) , citric acid ( 0 . 4 % ) , sucrose , acetone , ethanol , sodium B-material chloride I-material ( nacl ) , sodium B-material hydroxide B-material ( naoh ) , and sodium B-material dodecyl sulfate ( sds ) were obtained from loba chemie ( mumbai , india ) . [SEP]
[CLS] microscopic glass slide , ethylenediaminetetraacetic acid ( edta ) , sodium B-material acetate , bovine serum albumin ( bsa ) , and agarose were purchased from himedia laboratories pvt . ltd . ( mumbai , india ) . [SEP]
[CLS] nitric acid ( hno 3 ) , sulfuric B-material acid ( h 2 so 4 ) , and hydrogen B-material peroxide ( h 2 o 2 ) were purchased from thermo fisher scientific india pvt . ltd . ( mumbai , india ) . [SEP]
[CLS] ( 3 - aminopropyl ) triethoxylsilane ( aptes ) , 1 - methylimidazole ( 1 - meim ) , and carbodiimide [ 1ethyl - 3 - ( 3 - dimethylaminopropyl ) - carbodiimide ( edc ) ] were obtained from sigma - aldrich co . ( st . louis ) . [SEP]
[CLS] templiphi dna amplification kit , illustra , was obtained from ge healthcare limited ( u . k . ) . [SEP]
[CLS] the primers encoding the coat B-material protein B-material ( cp ) gene ( tolcvfp / tolcvrp ) were synthesized commercially ( eurofins mwg operon , india pvt . ltd . , bangalore , india ) . [SEP]
[CLS] red dye pcr master mix was purchased from ampliqon a / s ( denmark ) . [SEP]
[CLS] polymerase chain reaction ( pcr ) was performed in l1996ggd peltier model thermocycler purchased from lark innovative fine teknowledge pvt . ltd . ( india ) . [SEP]
[CLS] thiol B-material and phospahte modified oligonucleotide probes were synthesized from vbc - biotech service gmbh ( vienna , austria ) . [SEP]
[CLS] preparations and characterizations of aunp B-nanoparticle - conjugated mono - and bifunctional ssoligo probes . [SEP]
[CLS] citratecapped aunps B-nanoparticle were synthesized through heat reduction method and characterized by a uv−visible spectrophotometer , high - resolution transmission B-technique electron I-technique microscopy I-technique ( hr - tem ) , dynamic B-technique light I-technique scattering I-technique ( dls ) , and ζ potential analysis . [SEP]
[CLS] the synthesized aunps B-nanoparticle molar concentration was calculated . [SEP]
[CLS] signal probe 1 for forward strand and signal probe 2 for reverse strand were designed with thiol B-material modifications at 3 ′ ends , which were used for the preparation of aunp B-nanoparticle - conjugated oligo probes with mono - and bifunctionalities . [SEP]
[CLS] complementary strands specific for signal probes were also designed with phosphate functional B-material group I-material at 5 ′ ends . [SEP]
[CLS] similarly , positive control c2 - ssdna 1 and 2 complementary to capture probe 1 and signal probe 1 as well as capture probe 2 and signal probe 2 were designed in 72 and 74 mer , respectively , and all of the designed oligos were custom - synthesized ( table s1 ) . [SEP]
[CLS] the prepared aunp B-nanoparticle - conjugated virus - specific monofunctional signal probe 1 , signal probe 2 , and bifunctional signal probe were characterized by a uv−visible spectrophotometer , hr - tem , dls , ζ potential analysis , and agarose B-technique gel I-technique electrophoresis I-technique analysis . [SEP]
[CLS] the number of ssoligo probes attached on single aunp B-nanoparticle surface was calculated ( preparation and characterizations of aunps B-nanoparticle and aunp B-nanoparticle - ssoligo probes with bifunctionalities were already published ) . [SEP]
[CLS] pcr , rca dnas , and genomic dna of tolcndv prepared and fragmented through the heat fragmentation method are described in the supporting information ( figure s1agrose gel image for the analysis of isolated dna from infected sample ; figure s2 agarose gel image for the analysis of pcr amplified 0 . 5 kb size of cp gene of tolcndv for the confirmation of viral infection ; and figure s3agarose gel for the analysis of rca products amplified from tolcndv - infected samples ) . [SEP]
[CLS] modification of glass slide surface with amine B-material functional I-material groups I-material . [SEP]
[CLS] glass slide ( gs ) surface was modified with amine B-material functional I-material groups I-material using aptes followed in our earlier report . [SEP]
[CLS] in detail , glass slides were first treated with piranha solution ( h 2 o 2 / h 2 so 4 , 30 : 70 ratio ) for 12 h , washed thoroughly with ddh 2 o , and dried under a stream of n 2 gas . [SEP]
[CLS] for the silanization , the treated gss were immersed in a solution containing 2 . 0 % of aptes at room temperature ( rt ) for 30 min . [SEP]
[CLS] after incubation B-technique , the slides were rinsed with ddh 2 o , dried under a stream of n 2 gas , and the slides were kept at 120 °c for 30 min for strong binding . [SEP]
[CLS] preparation of sample dna array . [SEP]
[CLS] different concentrations of 5 ′ - phosphate - modified ssoligoes were immobilized on the nh 2 - gs surface . [SEP]
[CLS] amine - modified glass slide was divided into three rows and eight columns using acrylic cover slide and adhesive glow . [SEP]
[CLS] in detail , ice - cold 10 mm 1methylimidazole ( 1 - meim ) ph 7 . 0 was prepared as the final concentration from 0 . 1 m ice - cold 1 - meim . [SEP]
[CLS] the ssoligo solution was loaded on amine - functionalized gs ( 10 μl per spot ) on ice . [SEP]
[CLS] carbodiimide ( edc ) ( 0 . 2 m ) was dissolved in freshly prepared ice - cold 10 mm 1 - meim , and 10 μl of edc was added to each spot in the slides and incubated B-technique at 50 °c for 5 h . [SEP]
[CLS] after incubation B-technique , the slides were washed three times with washing solution 1 ( 0 . 4 n naoh + 0 . 25 % sodium B-material dodecyl sulfate ) for 5 min at 50 °c followed by three times washing with sterile water B-material . [SEP]
[CLS] finally , the unbounded surface was blocked by adding 10 μl of 5 % bsa and allowed to rest for 30 min at rt , and then the slide was washed with sterile water B-material . [SEP]
[CLS] hybridization of aunp B-nanoparticle - conjugated oligo probe was optimized through direct dna hybridization ( ddh ) assay associated with silver B-material enhancement . hybridization was performed with optimized parameters like temperature , ph , and time , as reported earlier with slight modification . [SEP]
[CLS] aunpconjugated signal probe 1 ( 5 μl ) was mixed with 5 μl of hybridization buffer ( 4× saline sodium B-material citrate ( ssc ) buffer , 0 . 05 % tween - 20 , and 35 % formamide : 0 . 6 m nacl and 0 . 06 m trisodium citrate were dissolved , ph 7 . 0 was adjusted with hcl , and the final volume was made up to 100 ml with ddh 2 o ) and added to each well of different concentrations of c - ssdna 1 ( 1 nm , 100 pm , 100 fm , 100 am , and 100 zm ) immobilized on gs . [SEP]
[CLS] it was incubated B-technique at 40 °c for 1 h for hybridization . [SEP]
[CLS] after incubation B-technique , the slide was subjected to highstringency wash with 20 μl of washing solution 2 ( 0 . 5 m nano 3 containing 0 . 05 % of tween - 20 ) twice at rt ( 2 min for each washing step ) and one - time low - stringency wash with 0 . 4× ssc buffer for 30 s . [SEP]
[CLS] scanometric detection . [SEP]
[CLS] the presence of hybridized signal probes was amplified through silver B-material enhancement . [SEP]
[CLS] in brief , silver B-material nitrate ( 0 . 04 % ) , hydroquinone ( 0 . 3 % ) , citric acid ( 0 . 4 % ) , and sucrose ( 0 . 1 % ) were prepared in pure acetone . [SEP]
[CLS] solutions were protected from light , mixed on a rocking platform for 4 h , and then cooled to −20 °c overnight . [SEP]
[CLS] cooled solutions were centrifuged at 600g for 10 min to remove insoluble material B-material and stored at −50 °c . [SEP]
[CLS] for silver B-material enhancement , these four solutions were mixed in the following ratio : two parts of silver B-material nitrate / two parts of hydroquinone / one part of citric acid / one part of sucrose ( 2 : 2 : 1 : 1 ) . [SEP]
[CLS] this mixed solution of 10 μl was loaded on each spot and the slide was incubated B-technique at 4 °c for 2−3 min . [SEP]
[CLS] after the incubation B-technique , the slide was washed thoroughly with sterile water B-material . [SEP]
[CLS] finally , the slide was dried at rt and the developed black spots on the slides were scanned using canon flatbed scanner ( lide 110 ) . [SEP]
[CLS] the scanned image was converted to grayscale , and the intensity of grayscale of silver B-material enhancement was measured by using imagej software . [SEP]
[CLS] specificities of aunp B-nanoparticle signal probes 1 and 2 and bifunctional aunp B-nanoparticle signal probe were studied using tolcndv viral dna and noncomplementary dna through ddh and sdh methods . [SEP]
[CLS] in detail , 10 ng / μl concentration fragmented tolcndv dna and noncomplementary dna were immobilized on gs surface for ddh , and 100 fm capture probes 1 and 2 and equal mixture of 1 and 2 were immobilized on gs surface for sdh assay , followed by hybridization of 5 μl of mono - and bifunctional aunp B-nanoparticle signal probes with 5 μl of hybridization buffer for ddh assay . [SEP]
[CLS] similarly , complementary dna 10 ng / μl mixed with 5 μl of hybridization buffer for the first round of hybridization was followed by the second round of hybridization with mono - and bifunctional aunp B-nanoparticle signal probes . [SEP]
[CLS] silver B-material enhancement was carried out following the above - mentioned procedure , and enhanced slides were scanned for analysis . [SEP]
[CLS] ddh assay - based scanometric detection of tolcndv dna . [SEP]
[CLS] different concentrations of c - ssdna 1 , c - ssdna 2 , and c - ssdna 1 and 2 ( 100 pm , 100 fm , 100 am , 100 zm , and 0 zm ) were immobilized on amine B-material - modified gs surfaces following the above - described methods . [SEP]
[CLS] similarly , fragmented pcr , rca , and genomic dna products were immobilized on the amine B-material - modified gs surface . [SEP]
[CLS] in detail , 7 . 5 ng / μl concentration of dna was prepared and denatured at 95 °c for 10 min using a thermocycler , followed by cooling in ice for 10 min . [SEP]
[CLS] the denatured 10 μl of dna from pcr , rca , and genomic samples were loaded on each spot separately and immobilized by following the above - described procedure . [SEP]
[CLS] the amount of sample dna immobilized on each spot was also measured spectrophotometrically . [SEP]
[CLS] after immobilization of dna samples , the slides were subjected to hybridization with aunp B-nanoparticle signal probe 1 , aunp B-nanoparticle signal probe 2 , and bifunctional aunp B-nanoparticle signal probe . [SEP]
[CLS] then , the slides were pretreated by incubating B-technique at 95 °c for 5 min in a thermal cycler ( pcr machine ) and hybridized at 40 °c for 1 h . [SEP]
[CLS] after the incubation B-technique , the unbounded signal probes were washed by high - and low - stringency washes . [SEP]
[CLS] then , the slides were subjected to silver B-material enhancement . [SEP]
[CLS] sdh assay - based scanometric detection of tolcndv dna . [SEP]
[CLS] sandwich ( double - step ) hybridizationbased scanometric detection of tolcndv dna was performed using two kinds of probes , viz . , ( 1 ) capture probes and ( 2 ) signal probes . [SEP]
[CLS] in the present study , 27 - mer capture probe 1 was selected 110 bp away from signal probe 1 in the cp gene region of tolcndv ( 309−420 nt ) and 28 - mer capture probe 2 was selected at 138 bp away from signal probe 2 in the cp region ( 683−821 nt ) . [SEP]
[CLS] these two capture probes were custom - synthesized with 3 ′ phosphate group functionalization . [SEP]
[CLS] the capture probes stock solutions were prepared with a final concentration of 100 pm / μl . [SEP]
[CLS] in this method , different concentrations of capture probe 1 and 2 and equal mixture of capture probes 1 and 2 ( 100 pm , 100 fm , 100 am , 100 zm , and 0 zm ) were immobilized on the amine - modified gs surface through edc / 1 - meim condensation reaction . [SEP]
[CLS] further , unbounded surface was passivated using 5 % bsa . [SEP]
[CLS] capture probe - immobilized slides were subjected to first - step hybridization with c2 - ssdna / samples dna . [SEP]
[CLS] similarly , capture probes 1 and 2 and equal mixture of 1 and 2 ( 100 pm / well ) were immobilized separately on gs surface for pcr , rca , and genomic dna hybridization throughout the study . [SEP]
[CLS] complementary dna ( c2 - ssdna - 1 , c2 - ssdna - 2 , and mixture of c2 - ssdna - 1 and 2 ) ( 5 μl ) were mixed with 5 μl of 4× ssc hybridization buffer and added to each well in the corresponding slides . [SEP]
[CLS] then , the slides were pretreated by incubating B-technique at 95 °c for 5 min in a thermal cycler and allowed for hybridization at 40 °c for 1 h . [SEP]
[CLS] after the incubation B-technique , the unbounded c2 - ssdnas underwent high - and low - stringency washes . [SEP]
[CLS] similarly , 5 μl of heat - fragmented pcr , rca , and genomic dna were also mixed with 5 μl of hybridization buffer for the hybridization , and unbounded strands of dsdna were removed by washing with high - and low - stringency wash buffers . [SEP]
[CLS] then , the slides were subjected to the second - step hybridization with aunp B-nanoparticle - conjugated monofunctional signal probe 1 , signal probe 2 , and bifunctional signal probe . [SEP]
[CLS] after the hybridization , the slides were subjected to high - and lowstringency washes . [SEP]
[CLS] silver B-material enhancement was carried out to enhance detection signals and characterized by atomic B-technique force I-technique microscopy I-technique ( afm ) and hr - tem . [SEP]
[CLS] preparation of mono - and bifunctional aunp B-nanoparticle - conjugated signal probes . [SEP]
[CLS] synthesis and characterization of citrate - capped aunps B-nanoparticle . [SEP]
[CLS] citrate - capped aunps B-nanoparticle were prepared through the heat reduction method and used for the preparation of mono - and bifunctional signal probes . [SEP]
[CLS] gradual change in the color of the reaction solution ( haucl 4 ) from yellow to purple and then to red after the addition of trisodium citrate at the boiling condition confirmed the formation of aunps B-nanoparticle . [SEP]
[CLS] the synthesized aunps B-nanoparticle solution was analyzed by a uv−visible spectrophotometer , and the absorbance peak of the red aunp B-nanoparticle solution was found at 520 nm . [SEP]
[CLS] the size and shape of the synthesized aunps B-nanoparticle were analyzed by hr - tem . [SEP]
[CLS] it shows that citrate - capped aunps B-nanoparticle are spherical in shape with an average size of [UNK] nm diameter in a highly monodispersed manner . [SEP]
[CLS] the hydrodynamic diameter of the synthesized citrate - capped aunps B-nanoparticle was studied using the dls method . [SEP]
[CLS] the average hydrodynamic diameter of the monodispersed citrate - capped aunps B-nanoparticle was found to be [UNK] nm with an electronegative B-property ζ potential −18 mv ( figure s4a−d ) . [SEP]
[CLS] the molar concentration of the synthesized aunps B-nanoparticle was calculated to be 3 . 75 nm , which has been already published . [SEP]
[CLS] ssoligo probes designing and synthesis . [SEP]
[CLS] tolcndvspecific oligonucleotide probes were designed by multiple - sequence alignment of retrieved nucleotide sequences of tolcndv from ncbi using bioedit 7 . 2 . 2 software . [SEP]
[CLS] oligo probe 1 ( 20 mer ) was selected at the beginning of coat B-material protein B-material ( cp ) gene , and oligo probe 2 ( 20 mer ) was selected at the end of the cp gene after sequence alignment . [SEP]
[CLS] however , six nucleotide bases were added at the 5 ′ end of probe sequences as a linker to enhance the recognition efficiency for hybridization . [SEP]
[CLS] in addition , self - dimer ( self - complementation ) formation between probe 1 , probes 1 and 2 , and probe 2 was analyzed and the designed oligo probes were customsynthesized with thiol B-material modification at the 5 ′ end . [SEP]
[CLS] similarly , complementary sequences for these two probes were also synthesized ( table s1 , supporting information ) . [SEP]
[CLS] finally , the stock solutions were prepared at a concentration of 100 pm / μl for all of the probes . [SEP]
[CLS] preparation of aunp B-nanoparticle - conjugated mono - and bifunctional ssoligo probes . [SEP]
[CLS] aunp B-nanoparticle - conjugated oligonucleotide probes that are monofunctional signal probe 1 , signal probe 2 , and bifunctional signal probe ( equal mixture of oligo probe 1 and 2 ) were prepared by following the standard protocol reported earlier based on the au−s chemistry . [SEP]
[CLS] thiolmodified oligonucleotide probes at the 5 ′ end was chemisorbed on aunp B-nanoparticle surface and formed a self - assembled oligo thiol monolayer . [SEP]
[CLS] aunps B-nanoparticle and thiol - modified oligo probes 1 and 2 , and equal mixture of probes 1 and 2 were mixed individually and incubated B-technique in the presence of phosphatebuffered saline . [SEP]
[CLS] salt B-material aging process was used to reduce the intermolecular repulsion forces between ssdna oligonucleotide probes and aunps B-nanoparticle and enhance the chemisorption . [SEP]
[CLS] further , unbounded aunps B-nanoparticle were removed through aggregation by salt aging ( figure 1a ) . [SEP]
[CLS] the optical absorbance properties of monofunctional signal probes 1 and 2 and bifunctional signal probe were analyzed with control and reference after the salt B-material aging process using a uv−visible spectrophotometer ( figure 1b ) . [SEP]
[CLS] aunps B-nanoparticle show different light absorbance properties with and without salt B-material treatment . [SEP]
[CLS] in the absence of salt B-material , aunps B-nanoparticle show a sharp absorbance band at 520 nm with high intensity , but in the presence of salt B-material , aunps B-nanoparticle show a broadening peak toward the longer - wavelength region ( above 600 nm ) with a small peak at 520 nm due to aggregation . [SEP]
[CLS] the absorbance spectra of signal probe 1 , signal probe 2 , and bifunctional signal probe ( aunp B-nanoparticle probe conjugates ) show an spr band at 524 nm even after the salt B-material treatment . [SEP]
[CLS] the red band shift of aunps B-nanoparticle from 520 to 524 nm found after the conjugation of aunps B-nanoparticle with thiol - modified oligonucleotide probes could be due to the change of refractive B-property index I-property surrounding the aunps B-nanoparticle and the formation of dielectric layers around the particles . [SEP]
[CLS] this can be further implied to the successful adsorption of ssdna probes onto the surface of aunps B-nanoparticle . [SEP]
[CLS] dispersibility and aggregation of monofunctional signal probes 1 and 2 and bifunctional signal probe in high - salt B-material environment were analyzed by dynamic B-technique light I-technique scattering I-technique ( dls ) technology along with control and a reference ( figure 1c ) . [SEP]
[CLS] the average hydrodynamic diameter of aunps B-nanoparticle without salt B-material treatment ( reference ) was found as [UNK] nm and that of aunps B-nanoparticle with salt B-material treatment ( 0 . 1 m final concentration ) was found as [UNK] nm ( data not shown ) . [SEP]
[CLS] this could be attributed to the salt B-material - induced irreversible aggregation of uniformly dispersed aunps B-nanoparticle in aqueous medium . [SEP]
[CLS] the average hydrodynamic diameters of monofunctional signal probes 1 and 2 and bifunctional signal probes after the salt B-material treatment were found to be [UNK] , [UNK] , and [UNK] nm , respectively . [SEP]
[CLS] this could be due to the formation of oligo thiol monolayer on aunps B-nanoparticle surface . [SEP]
[CLS] the significant difference in the particles size ( hydrodynamic diameter ) is mainly due to the variation in the number of oligo probes attached on the surface . [SEP]
[CLS] further , highdensity chemisorption of oligo probes on aunp B-nanoparticle surface depends on the size , shape , ph , and base compositions . [SEP]
[CLS] in these , oligo probe sequence is the most important factor that determines the adsorption potentials . [SEP]
[CLS] in detail , thymidine ( dt ) - rich oligo probes attached more numbers than da - , dc - , and dg - rich oligo probes . [SEP]
[CLS] simultaneously , ζ potentials of mono - and bifunctional signal probes were measured . [SEP]
[CLS] the ζ potential of citrate - capped aunps B-nanoparticle was found to be −18 mv ( figure s4d , inset ) . [SEP]
[CLS] this electronegativity B-property of aunps B-nanoparticle is attributed by the anionic nature of citrate molecules , which stabilize on the aunps B-nanoparticle surface . [SEP]
[CLS] the ζ potential of aunps B-nanoparticle significantly increased after the conjugation of ssdna oligo probes . [SEP]
[CLS] monofunctional signal probes 1 and 2 and bifunctional signal probe showed electronegative B-property ζ potentials of −32 , −45 , and −35 mv , respectively ( figure 1c , inset ) . [SEP]
[CLS] this could be due to the presence of more electronegative B-property ssdna oligo probes on the surface of aunps B-nanoparticle as a result of chemisorption . [SEP]
[CLS] mono - and bifunctional signal probes sizes were investigated by hr - tem analysis ( figure 1d ) . [SEP]
[CLS] the unconjugated aunps B-nanoparticle were found to be monodispersed with a diameter of [UNK] nm before the salt B-material treatment , which aggregated with increased size ( more than [UNK] nm ) after the salt B-material treatment ( data not shown ) . [SEP]
[CLS] however , oligo probe - functionalized aunp B-nanoparticle conjugates were not aggregated in the high - salt B-material environment and they were found to be in 2d array arrangements . [SEP]
[CLS] in addition , thin faint bands were clearly observed surrounding the aunpoligo probe conjugates ( inset images of figure 1d ) . [SEP]
[CLS] the thicknesses of the clearly visible layer were found to be 1 . 9 , 2 . 0 , and 1 . 7 nm , respectively . [SEP]
[CLS] this is due to the conjugation of aunps B-nanoparticle with thiol - modified ssdna oligonucleotide probes . [SEP]
[CLS] this functionalization prevents particle overlapping and aggregation by maintaining interparticle distance , and it proves that thiol - modified oligonucleotide - functionalized aunps B-nanoparticle were not aggregated and resist the salt - induced aggregation . [SEP]
[CLS] twodimensional array arrangements of aunp B-nanoparticle probe conjugates were mainly due to the weaker interactions between ssdna oligonucleotide probes immobilized on aunps B-nanoparticle . [SEP]
[CLS] aunp B-nanoparticle - conjugated mono - and bifunctional signal probes were analyzed using agarose B-technique gel I-technique electrophoresis I-technique . [SEP]
[CLS] s5a , b shows the electrophoretic separation of unmodified aunps B-nanoparticle ( lane 1 ) , monofunctional signal probe 1 ( lane 2 ) , monofunctional signal probe 2 ( lane 3 ) , and bifunctional signal probe ( lane 4 ) . [SEP]
[CLS] uniform red color bands were observed only in lanes 2 , 3 , and 4 of aunp B-nanoparticle - ssoligo probe conjugates . [SEP]
[CLS] unmodified aunps B-nanoparticle move faster than ssoligo probe - functionalized aunp B-nanoparticle and appeared as shearing without any identical bands ( lane 1 ) . [SEP]
[CLS] moreover , partially and completely functionalized aunps B-nanoparticle move according to their size and mass . [SEP]
[CLS] particularly , completely functionalized aunps B-nanoparticle - ssoligo probe possesses more mass and moves very slowly than the partially functionalized aunps B-nanoparticle . [SEP]
[CLS] a great number of probes on aunps B-nanoparticle ( spherical nucleic B-material acid I-material ) restrict the mobility B-property of aunps B-nanoparticle . [SEP]
[CLS] s5b shows measured band intensities in all of the four lanes . [SEP]
[CLS] very less intensity was found in lane 1 with unmodified aunps B-nanoparticle than the lanes 2 , 3 , and 4 , which show higher band intensities with similar pattern of mobility B-property . [SEP]
[CLS] these results prove that the ssoligo - functionalized aunps B-nanoparticle are more stable than the unmodified aunps B-nanoparticle under the electrophoretic condition . [SEP]
[CLS] the number of oligonucleotide probes chemisorbed on single aunp B-nanoparticle ( [UNK] nm ) was calculated using the uv−visible spectroscopy B-technique . [SEP]
[CLS] aunp B-nanoparticle - conjugated oligonucleotide probes ( mono - and bifunctional signal probes ) were treated with βmercaptoethanol , a strong reducing B-property agent I-property , to break the au−s bond and for the release of surface - bounded oligo probes and aggregation of aunps B-nanoparticle . [SEP]
[CLS] with assistance of molar concen - tration of aunps B-nanoparticle , ssoligoes , and the number of aunps B-nanoparticle in synthesized solution , the number of oligonucleotide probes immobilized on single aunp B-nanoparticle were calculated as 103 , 110 , and 105 / aunp B-nanoparticle for signal probe 1 , signal probe 2 , and bifunctional signal probe , respectively . [SEP]
[CLS] the adsorbance efficiency of ssoligo probes depends on the size and surface volume ratio of aunps B-nanoparticle , length of probes , and ionic strength of the medium . [SEP]
[CLS] more surface area of aunps B-nanoparticle facilitates the adsorbance of a greater number of ssoligo probes . [SEP]
[CLS] the amine B-material group I-material was functionalized on gs surfaces through the silane monolayer method using aptes for the immobilization of dna , which was already published . [SEP]
[CLS] briefly , the gs surface was treated with piranha solution to clean organic residues and other impurities B-property present on gs surface . [SEP]
[CLS] in addition , it is also used to increase the hydrophilic B-property property of glass by hydroxylating the surface , thus increasing the number of silanol group B-nanoparticle on I-nanoparticle the I-nanoparticle surface I-nanoparticle . [SEP]
[CLS] the treated gs surface was functionalized with amine B-material functional I-material group I-material through the silanization using aptes . [SEP]
[CLS] the goal of silanization ( or siliconization B-event ) of glassware is to increase its hydrophobicity B-property and is used for covalent immobilization of biomolecules . [SEP]
[CLS] it was reported that the 5 ′ end phosphate functional B-material group I-material of dna gets immobilized on the amine - functionalized gs surface through condensation reaction using carbodiimide and imidazole as coupling and catalytic agents . [SEP]
[CLS] the general mechanism for the immobilization of 5 ′ phosphate of dna on aminated gs surface is depicted schematically in figure 2 . [SEP]
[CLS] initially , 5 ′ phosphate group was attacked by edc to form active ester intermediate B-property . [SEP]
[CLS] then , imidazole reacts with active ester to release edc in the form of isourea and produce reactive phosphorylimidazole . [SEP]
[CLS] this phosphorylimidazole reacts with aminated gs surface to form a phosphoramidate covalent bond between dna and gs surface , and imidazole is released without any changes . [SEP]
[CLS] in this mechanism , edc acts as a coupling / condensation agent and it was hydrated to form isourea , where imidazole acts as a catalyst B-property and it activates dna to form a covalent bond with gs surface . [SEP]
[CLS] the amount of dna immobilized on the defined surface was optimized to prepare dna / probe array for scanometric detection of dna . [SEP]
[CLS] the 5 ′ end phosphate group - functionalized complementary ssdna specific for signal probes 1 and 2 and bifunctional signal probe were immobilized on aminefunctionalized gs surface . [SEP]
[CLS] in detail , different concentrations of c - ssdna ( 1 nm , 100 pm , 100 fm , 100 am , and 100 zm ) were immobilized onto 5 mm diameter surface of aminefunctionalized gs surface . [SEP]
[CLS] edc absorbs a molecule of water B-material to form isourea in this condensation reaction . [SEP]
[CLS] the formation of phosphoramidate B-material bond was catalyzed by imidazole to activate the phosphate group . [SEP]
[CLS] in this method , only 5 ′ phosphate end of c - ssdna alone is immobilized on the surface and another end ( 3 ′ ) is left free as such . [SEP]
[CLS] the amount of c - ssdna immobilized on the 5 mm diameter of reaction well on the aminefunctionalized gs surface was calculated using a standard ssdna concentration graph . [SEP]
[CLS] the amounts of c - ssdna present in washed buffer solutions collected from 1 nm ( or 1000 pm ) c - ssdna ( 1 , 2 , and equal mixture of 1 and 2 ) immobilized reaction wells were determined to be 56 . 05 , 54 . 56 , and 195 pm for c - ssdna 1 , 2 , and mixture of 1 and 2 , respectively . [SEP]
[CLS] after the subtraction of these values from the initial concentration of c - ssdna ( 1000 pm ) , the amounts of ssdna immobilized on surface were determined to be 943 . 95 , 945 . 44 , and 805 pm for c - ssdna for 1 , 2 , and the mixture of 1 and 2 , respectively . [SEP]
[CLS] the overall concentration of c - ssdna immobilized on 5 mm diameter aminated gs reaction well was found to be 80 . 5−94 . 54 % in this edc - mediated immobilization method ( figure s6a , b ) . [SEP]
[CLS] the amounts of c - ssdna for probes 1 and 2 were found to be similar and higher than that of the mixture of probes 1 and 2 . [SEP]
[CLS] this could be due to the formation of dimer to some extent between probes 1 and 2 , which might interfere in the immobilization process . [SEP]
[CLS] edcmediated selective immobilization of dna at the point of terminal phosphate group onto a solid surface is involved extensively in clinical diagnosis . [SEP]
[CLS] the perfect and selective immobilization of unmodified dna gives a new insight into the preparation of ssdna arrays . [SEP]
[CLS] likewise , pcr - and rca - amplified tolcndv dna products and isolated total genomic dna from infection suspected plant leaf samples were also immobilized on the amine - functionalized gs surface to perform the detection of specific nucleotide sequence through ddh assay - based scanometric method . [SEP]
[CLS] prior to immobilization of dna samples , it should be fragmented into small pieces to increase the number of 5 ′ terminal phosphate groups and increase the efficiency of immobilization , because the number of functional phosphate groups at the 5 ′ end of dna and length of the dna are the important factors in determining the efficiency of dna immobilization on the surface . [SEP]
[CLS] therefore , pcr , rca , and genomic dna were fragmented by heat treatment . [SEP]
[CLS] heat fragmentation profiles of pcr , rca , and genomic dna samples at 95 °c for different time durations ( 10 , 20 , 30 , and 40 min ) are shown in figure s6a−c , respectively . [SEP]
[CLS] the complete disappearance of band intensity in 40 min clearly indicates the complete fragmentation of dna in a wide size range . [SEP]
[CLS] heat fragmentation efficiency was found to be higher in genomic dna than rca and pcr products reflected by the band intensity . [SEP]
[CLS] the intensity of genomic dna was found to be lower than the rca and pcr products after 40 min heat treatment ( figure s6d ) . [SEP]
[CLS] the corresponding agarose gel images also confirm the disintegration ( figure s7 ) and indicate that the larger dna ( rca and genomic dna ) gets fragmented completely to 0 . 5−1 . 0 kb in size . [SEP]
[CLS] all of the previous reports deal with the heat fragmentation of genomic dna into different sizes from 100 bp to 1 . 0 kb . [SEP]
[CLS] however , fragmentation of pcr dna and rca dna has not been investigated so far . [SEP]
[CLS] in this study , we observed that the fragmentation of genomic and rca dna occurred more rapidly than the pcr dna , and the size of pcr product was 0 . 5 kb , which required more time ( 40 min ) than larger dna ( 30 min ) . [SEP]
[CLS] recently , the pretreatment ( fragmentation and denaturation ) of genomic dna has become necessary for the hybridization assay . [SEP]
[CLS] this immobilized dsdna fragment was converted to ssdna through alkaline treatment using naoh . [SEP]
[CLS] where dsdna fragments were denatured into ssdna , unimmobilized strand was removed by frequent washing . [SEP]
[CLS] in the case of pcr dna , the complete quantity of dna fragments ( [UNK] ng ) was immobilized on gs surface and optical absorbance of free dna present in collected washing solution was almost zero or the quantity of dna in the solution might be below detection sensitivity of instruments . [SEP]
[CLS] this is because the complete fragmentation of pcr dna with short length posses a greater number of phosphate ends , which enhance the immobilization efficiency . [SEP]
[CLS] the amounts of rca and genomic dna immobilized onto the surface was calculated to be 1 . 98 and 2 . 43 μg , respectively , from 2 . 01 μg of rca dna and 4 . 14 μg of genomic dna ( figure s9 ) . [SEP]
[CLS] however , the size of fragments was not uniform in genomic and rca samples that lead to differences in the immobilization efficiency . [SEP]
[CLS] the amine - modified and ssdna - immobilized gs surface properties and morphologies were investigated by afm analysis ( figure s10 ) . [SEP]
[CLS] the afm image of ssdna - immobilized gs had a surface roughness of rms 33 . 8 nm , which is 16 times greater than the surface roughness of unmodified gs and 5 times higher than the amine B-material - modified gs . [SEP]
[CLS] the increased roughness of gs surface is mainly due to the ssdna immobilization and ssdna appearing in a bulblike shape on the surface . [SEP]
[CLS] this is due to the highly flexible nature of ssdna , leading to the formation of self - folding or compressed bulblike structure through physical or weak interactions in nonaqueous environment . [SEP]
[CLS] moreover , dense immobilization of ssdna or dense ssdna array on the surface needs to be avoided and it is not desirable for the hybridization assay due to the steric hindrance interference . [SEP]
[CLS] scanometric detection of tolcndv dna . [SEP]
[CLS] scanometric detection of tolcndv dna was done through ddh and sdh assays using aunp B-nanoparticle - conjugated virus - specific signal probes and silver B-material enhancement . [SEP]
[CLS] 3a shows the diagrammatic representation of selected target regions for capture and sensing probes for both forward and reverse strands of cp gene in tolcndv dna - a component . [SEP]
[CLS] 3b , c shows schematic representation of ddh assay and sdh assay - based scanometric method . [SEP]
[CLS] ddh assay requires only signal probes ( aunp B-nanoparticle - conjugated probe ) and sdh assay requires two probes , which are capture probes as well as signal probes . [SEP]
[CLS] the limit of detection for this scanometric - based nanoassay method was optimized for an effective detection of dna . [SEP]
[CLS] different concentrations of c - ssdna - 1 ( 1 nm , 100 pm , 100 fm , 100 am , and 100 zm ) were immobilized on gs surface and hybridized with signal probe 1 . [SEP]
[CLS] further , the hybridized signal probe 1 was measured by silver B-material enhancement ( figure 4 ) . [SEP]
[CLS] the development of black - gray color by the result of silver B-material enhancement and its intensity of ag enhancement increased gradually while increasing the concentration of c - ssdna from 0 zm to 100 pm . further increasing the concentration of c - ssdna 1 to 1 nm , the intensity of black color decreased considerably ( figure 4a ) and the results could be easily discriminated by the naked eye . [SEP]
[CLS] the intensity of ag enhancement increased ( 102 , 193 , 201 , and 211 au ) with increasing concentration of c - ssdna 1 from 0 zm , 100 zm , 100 am , 100 fm , and 100 pm , respectively ( figure 4b ) . [SEP]
[CLS] this is because increase in the signal probe 1 hybridization with its respective concentration leads to increase in an autocatalytic reduction of silver B-material ions B-material to metals B-material as well as deposition on aunp B-nanoparticle . [SEP]
[CLS] this ag enhancement increases the detection limit of scanometry - based nanoassay method to [UNK] times greater sensitivity than fluorescence - based methods . [SEP]
[CLS] because silver B-material metal B-material strongly reflects the visible spectrum , it can be detected using a flatbed scanner . [SEP]
[CLS] further , when the concentration of c - ssdna 1 increased to 1 nm , the intensity of ag enhancement decreased to 198 au . [SEP]
[CLS] this could be attributed to the steric hindrance of c - ssdna1 array and reduces the hybridization efficiency of signal probe 1 . [SEP]
[CLS] the surface / volume ratio and steric hindrance are the important factors that determine the hybridization efficiency of dna strands with aunp B-nanoparticle probe conjugates . [SEP]
[CLS] in detail , 1 nm ssdna targets tightly packed on 5 mm glass slide surface generate steric hindrance , which restricts the access and hybridization efficiency of signal probes . [SEP]
[CLS] further , specificity analysis of mono - and bifunctional signal probes was performed through ddh - and sdh - based scanometric methods . [SEP]
[CLS] high intensity of ag enhancement was found only in the presence of target tolcndv dna than the noncomplementary dna and control . [SEP]
[CLS] this is mainly due to the hybridization of a great number of aunp B-nanoparticle - ssoligo probe conjugates with its target dna than the noncomplementary dna . [SEP]
[CLS] the results were easily discriminated by the naked eye ( figure s10 ) . [SEP]
[CLS] tolcndv dna using monofunctional signal probes 1 and 2 [SEP]
[CLS] scanometric detection of tolcndv pcr , rca , and genomic dna was done through ddh assay associated with silver B-material enhancement using three kinds of signal probes , which are monofunctional aunp B-nanoparticle probe 1 ( specific for forward strand at the position of 310 nt of cp gene ) , monofunctional aunp B-nanoparticle probe 2 ( specific for reverse strand at the position of 810 nt of cp gene ) , and bifunctional aunp B-nanoparticle probe ( mixture of oligo probes 1 and 2 ) . [SEP]
[CLS] initially , ssdna arrays were made by the immobilization of different concentrations of c - ssdna and heat - fragmented pcr , rca , and genomic dna on gs surface . [SEP]
[CLS] immobilized pcr , rca , and genomic dsdna samples were converted to ssdna by alkaline denaturation , followed by hybridization . [SEP]
[CLS] 5a shows the scanometric detection of tolcndv target sequence using signal probe 1 . [SEP]
[CLS] intensity of black - gray color construed as a result of ag enhancement , which was found to increase by increasing the concentration of c - ssdna 1 from 0 zm to 100 pm . the intensity of ag enhancement for pcr dna was higher than those for rca ( slightly above 100 zm ) and genomic dna ( below 100 zm ) , which were very close to 100 pm concentration of positive control ( c - ssdna 1 ) . [SEP]
[CLS] similar results were found in the scanometric detection of tolcndv pcr , rca , and genomic dna with different concentrations of c - ssdna 2 ( 0 zm , 100 zm , 100 am , 100 fm , and 100 pm ) using signal probe 2 . [SEP]
[CLS] a black - gray spot on the scanned image is construed as a result of ag enhancement ( figure 5b ) . [SEP]
[CLS] the intensity of color was found to gradually increase from 0 zm to 100 pm . tolcndv pcr , rca , and genomic dna also showed different intensities of black - gray color development , indicating that hybridization of signal probe 2 with its target sequence . [SEP]
[CLS] the measured color intensities at different concentrations of c - ssdna 2 ( 0 zm to 100 pm ) and tolcndv pcr , rca , and genomic dna are presented in figure 5b . [SEP]
[CLS] the intensity of ag enhancement for pcr and rca samples was very close to 100 fm and 100 zm concentration of c - ssdna 2 , respectively . [SEP]
[CLS] however , color intensity of genomic dna sample less than 100 zm ddh assay - based scanometric detection of tolcndv using bifunctional signal probe . [SEP]
[CLS] signal probe with bifunctional property can recognize and hybridize with both strands of dsdna of tolcndv pcr , rca , and genomic dna samples as well as increase detection sensitivity , because any one strand of dsdna was immobilized on gs surface at a time while preparing ssdna array . [SEP]
[CLS] scanometric detection of tolcndv pcr , rca , and genomic dna with different concentrations of mixture of c - ssdna 1 and c - ssdna 2 ( 0 zm to 100 pm ) using bifunctional signal probe and ag enhancement is presented in figure 5c . [SEP]
[CLS] the intensity of ag enhancement for pcr sample was found to be higher than rca and genomic dna samples . [SEP]
[CLS] further , the concentration of target in pcr product was estimated approximately between 100 fm and 100 pm . [SEP]
[CLS] however , the concentration of target sequence in rac was around 100 zm , and in genomic dna samples , it was very much lower than 100 zm . [SEP]
[CLS] interestingly , the bifunctional signal probe - based scanometric detection of tolcndv dna showed increased intensity , particularly for pcr , rca , and genomic dna than monofunctional signal probe 1 and signal probe 2 . [SEP]
[CLS] this indicates that the amount of bifunctional signal probe hybridization with its targets , particularly pcr and rca and genomic dna , was high . [SEP]
[CLS] hence , this can be hybridized with both strands of dsdna sample , leading to an increased ag enhancement . [SEP]
[CLS] further , bifunctional oligos probe - functionalized aunps B-nanoparticle show more sensitivity and similar hybridization efficiency with monofunctional signal probes ( figure 5d ) . [SEP]
[CLS] there are two main factors determining the hybridization efficiency of signal probe : ( 1 ) the concentration of target dna and ( 2 ) the length of dna fragments immobilized on gs [SEP]
[CLS] the amount of pcr product immobilized on gs ( 0 . 9 μg ) was less than rca dna ( 1 . 98 μg ) and genomic dna ( 2 . 01 μg ) . [SEP]
[CLS] however , the concentration of complementary strands specific for signal probes in pcr product was found to be higher than that for rca dna product . [SEP]
[CLS] therefore , the hybridization was more with intense detection signal [SEP]
[CLS] in rca dna products , copy numbers of targets might be less and have immobilized other regions of dna fragments , which causes the steric hindrance on gs surface that reduces the hybridization of signal probes . [SEP]
[CLS] usually , the genomic dna has less concentration of viral genome and is not subjected to any amplification . [SEP]
[CLS] therefore , ag - enhanced intensity of genomic dna was found to be very low . [SEP]
[CLS] moreover , the length of dna fragments immobilized on the surface also plays a vital role in the dna hybridization . [SEP]
[CLS] the size of fragments was not uniform in genomic and rca samples , leading to differences in the immobilization and hybridization efficiency . [SEP]
[CLS] hence , the length of rca and genomic dna fragments was found to be few hundred base pairs to a few kb , which were higher than pcr product , which is less than 100 bp . [SEP]
[CLS] larger dna fragments have a high tendency to form a self - coiled secondary structure than the smaller dna fragments , and they occupy more space than the smaller one . [SEP]
[CLS] secondary structure formation within the targets can reduce the binding constant of a specific probe by as much as 105−106 times . [SEP]
[CLS] hence , it was found that fragmented dna can minimize the possible inhibitory effects of secondary structure on hybridization efficiency than the unfragmented dna sample . [SEP]
[CLS] the overall gibbs free energy was changed during hybridization of probe with targets . [SEP]
[CLS] shortening amplified product / dna fragments shows a high potential of hybridization with more negative value of gibbs free energy because the lower gibbs free energy ( more in negative value ) favors the formation of probe−target duplex in higher potential than the longer dna fragments . [SEP]
[CLS] color intensity of ag enhancement of genomic dna was very low , that is , one - third of 100 zm concentration because genomic dna obtained from tolcndv - infected sample has only a few copy numbers targets . [SEP]
[CLS] after the amplification by pcr and rca , the number of complementary strand specific for signal probes was increased in logarithmic scale and as a result the intensity of ag enhancement was found to be increased . [SEP]
[CLS] further , the detection sensitivity of bifunctional signal probes significantly increased compared to the monofunctional signal probes 1 and 2 . [SEP]
[CLS] this is because single nanoparticle B-nanoparticle carries both signal probes , with the ability to recognize both forward and reverse strands of dsdna samples . [SEP]
[CLS] moreover , both stands of dsdna sample have equal ( 50 % ) possibility of getting immobilized on gs surface at the time of ssdna array preparation . [SEP]
[CLS] also , strand - specific immobilization of dsdna samples is not possible at aqueous state because both strands look similar at terminals . [SEP]
[CLS] sdh assay - based scanometric detection of tolcndv dna using monofunctional signal probes 1 and 2 . [SEP]
[CLS] this sdh assay - based scanometric detection of tolcndv dna was performed and compared with the ddh assay - based scanometric detection method to review the cost and sensitivity as well as time factors . [SEP]
[CLS] unlike the ddh assay , the sdh assay requires two probes : ( 1 ) capture probe , which is immobilized on gs surface , and ( 2 ) signal probe , which is labeled with aunps B-nanoparticle and hybridized with target dna . [SEP]
[CLS] both capture and signal probes are partially complementary to the target sequences of tolcndv dna cp gene . [SEP]
[CLS] the capture probes 1 and 2 were modified with the phosphate functional B-material group I-material at 3 ′ end and partially complementary to 5 ′ end of tolcndv dna . [SEP]
[CLS] further , the capture probes were immobilized on aminated gs surface and subjected to first - step dna hybridization with target dna , and they formed a capture probe−target ssdna complex . [SEP]
[CLS] followed by the second - step hybridization of mono - and bifunctional signal probes with 3 ′ end of target dna , it formed a capture probe−ssdna target− signal probe complex . [SEP]
[CLS] this complex was subjected to silver B-material enhancement to increase the detection signal ( figure 3c ) . [SEP]
[CLS] different concentrations of capture probe 1 ( 0 zm , 100 zm , 100 am , 100 fm , and 100 pm ) were immobilized on aminated surface through phosphoramidate B-material bond using edc and imidazole as coupling and catalytic agents , respectively . [SEP]
[CLS] similarly , 100 pm concentration of capture probe 1 was arrayed in a separate well for pcr , rca , and genomic dna of tolcndv . [SEP]
[CLS] arrayed gs were subjected to hybridization with positive complementary ssdna ( c2ssdna - 1 ) and fragmented tolcndv pcr , rca , and genomic dna . [SEP]
[CLS] the second - step hybridization was carried out with signal probe 1 , and the detection signal was amplified through ag enhancement . [SEP]
[CLS] finally , the intensity of ag enhancement was quantified through the scanometric method ( figure 6a ) . [SEP]
[CLS] the resulting black - gray color of ag enhancement was found to gradually increase with increasing concentrations of capture probe 1 at 0 zm to 100 pm . at initial concentration ( 0 zm ) , intensity of ag enhancement increased with increasing concentrations of capture probe 1 , viz . , 100 zm , 100 am , 100 fm , and 100 pm . [SEP]
[CLS] however , intensity of ag enhancement was increased by increasing the copy number of target dna sequence in tolcndv dna sample in the order from genomic dna ( 123 au ) , which was below 100 zm concentration of target . [SEP]
[CLS] moreover , the color intensity of pcr dna was very close to 100 pm of positive control and higher than rca dna product , which was between 100 zm and 100 am . [SEP]
[CLS] signal probe 2 was also used in the scanometric detection of tolcndv dna through the sdh assay method ( figure 6b ) . [SEP]
[CLS] the concentrations of hybridized signal probe 2 and c2 - ssdna 2 were increased by increasing the concentration of capture probe 2 on gs surface that led to an increase in the color intensity ( figure 6b ) . [SEP]
[CLS] the intensities of ag enhancement for tolcndv pcr , rca , and genomic dna samples were found to be 158 , 149 , and 104 au , respectively . [SEP]
[CLS] even though they show similar pattern of ag enhancement , which decreases from pcr to rca and genomic dna samples , their concentrations were estimated to be below 100 zm for all of the three dna samples . [SEP]
[CLS] sdh - based scanometric detection of tolcndv dna using bifunctional signal probe . [SEP]
[CLS] the bifunctional signal probe has the capacity to hybridize with both strands of dsdna simultaneously . [SEP]
[CLS] to increase the detection signal with less quantity of target dsdna samples , equal mixture of capture probe 1 and 2 were immobilized on gs surface . [SEP]
[CLS] the scanometric detection of tolcndv pcr , rca , and genomic dna samples with different concentrations of equal mixture of capture probes 1 and 2 ( 0 zm to 100 pm ) hybridized with a constant concentration of equal mixture of c2 - ssdna 1 and 2 and aunp B-nanoparticle bifunctional signal probe with ag enhancement is presented in figure 6c . [SEP]
[CLS] the measured intensity of ag enhancement for the different concentrations ( 0 zm to 100 pm ) of equal mixture of capture probes 1 and 2 also shows similar results , that is , the intensity increases with increasing concentration ( figure 6c ) . [SEP]
[CLS] however , the calculated target concentrations for all of these three samples were found to be below 100 zm . [SEP]
[CLS] this observation was similar to the abovediscussed concept , where the increase in the concentration of capture probes 1 and 2 also increased the hybridization of probes as well as detection signal . [SEP]
[CLS] however , the pcr dna has shown higher signal intensity than rca and genomic dna samples . [SEP]
[CLS] moreover , the high detection intensity shown by the bifunctional signal probe than the monofunctional signal probe - based scanometric detection of tolcndv through sdh assay method ( figure 6d ) could be due to the hybridization of capture and signal probes with both strands of dsdna samples even in the presence of low - quantity targets . [SEP]
[CLS] in these sdh assay - based scanometric methods , many factors restrict the detection efficiency and sensitivity . [SEP]
[CLS] the rca and genomic dna fragments are very large , which favors the formation of self - coiled secondary structure that decreases hybridization efficiency as well as ag enhancement . [SEP]
[CLS] in addition , there is a significant loss of target dna during more number of washing steps . [SEP]
[CLS] in addition , target sequence concentration and copy numbers are very less in rca products and genomic dna than the pcr dna . [SEP]
[CLS] these reduces the detection sensitivity of sdh assay - based scanometric methods . [SEP]
[CLS] the scanometry - based detection of dna through silver B-material enhancement showed sensitivity up to 50 fm , which is 100 times higher than the fluorescence - based detection , as already reported . [SEP]
[CLS] scanometric - based detection of bio - bar - code dna was reported with detection sensitivity up to 500 zm . 9 , 57 more recently , early detection of plant pathogen ( pseudomonas syringae ) dna using aunp B-nanoparticle - conjugated probe through recombinase polymerase amplification and differential pulse voltammetry has been reported with 10 000 times more sensitivity than the conventional pcr method . [SEP]
[CLS] this experimental evidence proves that bifunctional signal probe has great advantages over the monofunctional probes . [SEP]
[CLS] overall , the ddh assay - based scanometric method with bifunctional signal probe is more sensitive and cost - effective than the sdh assay - based scanometric method . [SEP]
[CLS] moreover , this is the first report for scanometric detection of plant virus ( tolcndv - dna ) using aunp B-nanoparticle - conjugated probe with higher sensitivity ( 100 zm ) than the previous reports . [SEP]
[CLS] afm characterization of ag - enhanced signal probe in scanometric detection of dna . [SEP]
[CLS] afm was used to characterize the signal probe - hybridized scanometric slide before and after ag enhancement . [SEP]
[CLS] an afm image ( histogram and three - dimensional ( 3d ) view ) of signal probe - hybridized slide before and after ag enhancement is presented in figure 7a , b . [SEP]
[CLS] the surface roughness of signal probe - hybridized slide was found to be [UNK] . 9 nm , with spherical - shaped particles ( size ca . 50−103 nm ) . [SEP]
[CLS] after the sliver enhancement , surface roughness was increased to [UNK] . 2 nm . [SEP]
[CLS] particles with size [UNK] nm were found in the form of huge cluster or precipitate ( ca . 183−255 nm in size ) . [SEP]
[CLS] this indicates the surface deposition of nanoscale ag metal B-material on aunp B-nanoparticle of signal probe as a result of particle appeared in cluster or in precipitated topology . [SEP]
[CLS] this proves that the au acted as template and autocatalyst for the reduction of ag ions B-material to metal B-material and deposition . [SEP]
[CLS] this process could have occurred only in the presence of signal probe on the scanometric slide . [SEP]
[CLS] hr - tem characterization of ag - enhanced signal probe in scanometric detection of tolcndv dna . [SEP]
[CLS] agenhanced signal probe in the scanometric detection of tolcndv dna was analyzed by hr - tem . [SEP]
[CLS] the light microscopic images show the presence of black microscopic particles formed as the result of ag enhancement on the scanometric slide in two different magnifications ( 10× and 40× ) ( figure 8a ) . [SEP]
[CLS] this is clearly interpreted due to the reduction of ag ions B-material to metal B-material that deposits on aunps B-nanoparticle surface . [SEP]
[CLS] the hr - tem images show ag enhancement of signal probe in the scanometric detection of tolcndv dna ( figure 8b ) . [SEP]
[CLS] in these images , a strong au core B-material was present in the center with size 18−25 nm and a ag shell B-material was present around the au core B-material with 6−35 nm width . [SEP]
[CLS] the total diameter of ag - enhanced signal probe was 40−71 nm . [SEP]
[CLS] these results clearly indicate that reduced metal B-material was deposited on the au surface and also prove that au acted as metal B-material catalyst B-property for the reduction of ag ions B-material as well as template for surface deposition . [SEP]
[CLS] this nanometal hybrid has high electron density , which could be a reason for the black - gray appearance of ag - enhanced spot on the scanometric slide . [SEP]
[CLS] energy - dispersive x - ray ( edx ) spectroscopy B-technique analysis was used for the qualitative and quantitative analyses of elemental composition of ag - enhanced signal probe ( figure 8c ) . [SEP]
[CLS] edx spectrum also shows the presence of both au and ag elements , confirming that the particles shown in the image were au core B-material / ag shell B-material . [SEP]
[CLS] the ratio of au to ag of particle was 1 . 44 : 98 . 55 , and their atomic percentages were 0 . 79 % and 99 . 20 % , respectively , which indicates that ag was present predominately than au in this particle . [SEP]
[CLS] these data have correlation with tem image , which shows larger diameter of ag shell B-material ( 35 nm than au core B-material 18 nm ) . [SEP]
[CLS] the au core B-material has strong electron density , and the ag shell B-material has less density and therefore it appears as layer . [SEP]
[CLS] ■ conclusions [SEP]
[CLS] development of scanometric method for the detection of plant pathogen , especially tolcndv through direct and sandwich dna hybridization using virus - specific aunp B-nanoparticle - oligo probe conjugates as signal probes was attempted in the present study . [SEP]
[CLS] scanometric detection of tolcndv dna was developed through ddh and sdh assays with silver B-material enhancement using mono - and bifunctional signal probes . [SEP]
[CLS] direct hybridization required only signal probe and sample ssdna arrays that were prepared through edc - based immobilization of fragmented pcr , rca , and genomic dna obtained from tolcndvinfected plant sample with positive control on amine - modified gs surface . [SEP]
[CLS] sandwich hybridization required two probes , viz . , capture and signal probes , and it undergoes two steps of hybridization : first , capture probes array on gs surface , followed by hybridization with sample ssdna fragments and signal probes . [SEP]
[CLS] the scanometric detection range was optimized using different concentrations of positive control from 0 zm to 1 nm . [SEP]
[CLS] detection signal of 1 nm was lower than 100 pm due to the steric hindrance at higher concentration of probe immobilized on the defined gs surface . [SEP]
[CLS] therefore , the detection limit was optimized between 100 zm and 100 pm . the hybridization signal was amplified through ag enhancement - based autocatalytic reduction of ag ions B-material by aunps B-nanoparticle and surface deposition . [SEP]
[CLS] the intensity of ag enhancement in the direct dna hybridization using monofunctional signal probes 1 and 2 were found similar to the detection sensitivity up to 100 zm concentration of positive control . [SEP]
[CLS] however , the bifunctional signal probe showed significantly increased detection signal intensity compared to the monofunctional signal probes . [SEP]
[CLS] in particular , pcr , rca , and genomic dna sample bifunctional signal probes showed high hybridization efficiency with higher detection signal intensity than monofunctional signal probes . [SEP]
[CLS] in this , pcr dna showed higher intensity than rca and genomic dna because of a number of complementary strands specific for signal probe amplified in logarithmic scale by pcr and short length of fragments . [SEP]
[CLS] genomic dna sample with less viral genome with larger fragments would lead to low detection intensity . [SEP]
[CLS] similar results were found in sdh - based scanometric detection of tolcndv dna . [SEP]
[CLS] bifunctional signal probe showed better results than the monofunctional signal probe in all samples . [SEP]
[CLS] however , sdh - based scanometric detection required two kinds of probes , which increases the detection cost . [SEP]
[CLS] direct dna hybridization - based scanometric detection of tolcndv required only one kind of probe and sample dna can be directly immobilized without any modification to prepare the dna array . [SEP]
[CLS] therefore , this method is advantageous in view of the detection cost and time , and it also has similar detection signal intensity of sandwich hybridization . [SEP]
[CLS] this is the first report on scanometric detection of tolcndv dna using aunp B-nanoparticle - conjugated oligo probes with a high sensitivity of up to 100 zm . the application of this developed method will be extended for detection of any other target dna . [SEP]
[CLS] the supporting information is available free of charge on the acs publications website at doi : 10 . 1021 / acsomega . 9b00340 . [SEP]
[CLS] experiments and results of primer and probe designing and synthesis , viral dna isolation , pcr and rca amplification , dna fragmentation , aunp B-nanoparticle synthesis , [SEP]
[CLS] preparation of aunp B-nanoparticle - conjugated oligonucleotide probes . [SEP]
[CLS] ( a ) stability of aunp B-nanoparticle probe conjugates after the salt B-material treatment . [SEP]
[CLS] 1aunps with salt B-material , 2signal probe 1 with salt B-material , 3signal probe 2 with salt B-material , 4bifunctional signal probe with salt B-material , and 5aunps without salt B-material . [SEP]
[CLS] ( b ) uv−visible spectra of aunps B-nanoparticle " a " , aunps B-nanoparticle with nacl " b " , signal probe 1 with nacl " c " , signal probe 2 with nacl " d " , and bifunctional probe with nacl . [SEP]
[CLS] ( c ) dls characterization of aunp B-nanoparticle - conjugated probes . [SEP]
[CLS] hydrodynamic diameter of aunp B-nanoparticle - sp1 ( [UNK] nm ) , aunp B-nanoparticle - sp 2 ( [UNK] nm ) , and bifunctional signal probe aunp B-nanoparticle - sp 1 and 2 ( [UNK] nm ) and its corresponding hr - tem images of two - dimensional ( 2d ) arrangement of aunp B-nanoparticle - conjugated signal probe 1 , signal probe 2 , and bifunctional signal probe ( d ) . ( the blue line indicates that part of figure is already published and permission obtained from rsc advance ) . 8 [SEP]
[CLS] preparation of aunp B-nanoparticle - conjugated oligonucleotide probes . [SEP]
[CLS] ( a ) stability of aunp B-nanoparticle probe conjugates after the salt B-material treatment . [SEP]
[CLS] 1aunps with salt B-material , 2signal probe 1 with salt B-material , 3signal probe 2 with salt B-material , 4bifunctional signal probe with salt B-material , and 5aunps without salt B-material . [SEP]
[CLS] ( b ) uv−visible spectra of aunps B-nanoparticle " a " , aunps B-nanoparticle with nacl " b " , signal probe 1 with nacl " c " , signal probe 2 with nacl " d " , and bifunctional probe with nacl . [SEP]
[CLS] ( c ) dls characterization of aunp B-nanoparticle - conjugated probes . [SEP]
[CLS] hydrodynamic diameter of aunp B-nanoparticle - sp1 ( [UNK] nm ) , aunp B-nanoparticle - sp 2 ( [UNK] nm ) , and bifunctional signal probe aunp B-nanoparticle - sp 1 and 2 ( [UNK] nm ) and its corresponding hr - tem images of two - dimensional ( 2d ) arrangement of aunp B-nanoparticle - conjugated signal probe 1 , signal probe 2 , and bifunctional signal probe ( d ) . ( the blue line indicates that part of figure is already published and permission obtained from rsc advance ) . 8 [SEP]
[CLS] 2 . schematic representation of covalent immobilization of dna on the amine B-material - modified gs surface . [SEP]
[CLS] ( a ) graphical representation of capture and signal probes ( 1 and 2 ) specific sites at cp gene of tolcndv . [SEP]
[CLS] ( b ) schematic representation of scanometric detection of tolcndv dna through direct ( single - step ) dna hybridization method and sandwich ( double - step ) dna hybridization methods with silver B-material enhancement ( c ) . [SEP]
[CLS] 4 . optimization of detection limits of scanometric detection of tolcndv dna using different concentrations of positive control ( c - ssdna 1 ) ( 0 zm , 100 zm , 100 am , 100 fm , 100 pm , and 1 nm ) . [SEP]
[CLS] ( a ) ag - enhanced scanometric slide with different concentrations of positive control ( 0−1 nm ) and ( b ) bar graph of measured intensity of ag enhancement of different concentrations of positive control ( p value ≥ 0 . 05 ) . [SEP]
[CLS] scanometric detection of tolcndv pcr , rca , and genomic dna samples by ddh assay using signal probe 1 ( a ) , signal probe 2 ( b ) , and bifunctional signal probe ( c ) with different concentrations of c - ssdna 1 , c - ssdna 2 , and c - ssdna 1 and 2 ( 0 zm , 100 zm , 100 am , 100 fm , and 100 pm ) . [SEP]
[CLS] ( d ) degree of potential of hybridization efficiency of monofunctional signal probes 1 and 2 and bifunctional signal probe with different concentrations of c - ssdna 1 , c - ssdna - 2 , equal mixture of c - ssdna 1 and 2 , tolcndv pcr , rca , and genomic dna samples . [SEP]
[CLS] the error bar indicates p value ≥0 . 05 . [SEP]
[CLS] scanometric detection of tolcndv pcr , rca , and genomic dna samples by sdh assay using signal probe 1 ( a ) , signal probe 2 ( b ) , and bifunctional signal probe ( c ) with different concentrations of capture probe 1 , capture probe 2 , and equal mixture of capture probe 1 and 2 ( 0 zm , 100 zm , 100 am , 100 fm , and 100 pm ) , as well as constant volume of positive control . [SEP]
[CLS] ( d ) degree of potential of hybridization efficiency of capture probes as well as monofunctional signal probes 1 and 2 and bifunctional signal probe with positive controls , tolcndv pcr , rca , and genomic dna samples . [SEP]
[CLS] the error bar indicates p value ≥0 . 05 . [SEP]
[CLS] ( a ) afm analysis signal probe - hybridized dna array before ag enhancement and after ag enhancement ( b ) with histogram and 3d view [SEP]
[CLS] increases of particle size ( nm ) and height were clearly found after the ag enhancement . [SEP]
[CLS] 8 . microscopy B-technique analysis of ag - enhanced signal probe in scanometric detection of tolcndv dna . [SEP]
[CLS] ( a ) light microscopy B-technique images of ag - enhanced spot on the scanometric slide in different magnifications 10× and 40× showing microscopic particles . [SEP]
[CLS] ( b ) hrtem analysis of ag enhancement of scanometric slide at different magnifications with size of au core B-material ( 18 nm ) / ag shell B-material ( 35 nm ) in the images indicating the surface deposition of ag metal B-material by autocatalytic reduction of ag ions B-material by aunps B-nanoparticle . [SEP]
[CLS] ( c ) edx analysis of ag - enhanced particles in scanometric detection of tolcndv dna , and the spectrum shows predominant presence of ag over au in au core / ag shell B-material nanoparticles B-nanoparticle . [SEP]
[CLS] we describe a novel two - step method , starting from bulk silicon B-material wafers , to construct dna conjugated silicon B-nanoparticle nanoparticles I-nanoparticle ( sinps ) . [SEP]
[CLS] this method first utilizes reactive high - energy ball milling ( rhebm ) to obtain alkene grafted sinps . [SEP]
[CLS] the alkene moieties are subsequently reacted with commercially available thiol - functionalized dna via thiol−ene click chemistry to produce sinp dna conjugates wherein the dna is attached through a covalent thioether B-material bond . [SEP]
[CLS] further , to show the utility of this synthetic strategy , we illustrate how these sinp odn conjugates can detect cancer - associated mir - 21 via a fluorescence on strategy . [SEP]
[CLS] given that an array of biological molecules can be prepared with thiol B-material termini and that sinps are biocompatible B-property and biodegradable B-property , we envision that this synthetic protocol will find utility in salient sinp systems for potential therapeutic and diagnostic applications . [SEP]
[CLS] spherical nucleic B-material acids I-material composed of a nanoparticle B-nanoparticle scaffold conjugated with a dna shellare B-material currently being investigated as functional nanomaterials B-material in applications ranging from in vitro biosensors to in vivo transfection , diagnostic , and theranostic agents . [SEP]
[CLS] the reason these hybrid materials are considered for use in such technologies is that they not only possess the unique biomolecular recognition properties of oligonucleotides ( odns ) , but often have emergent properties that are not present in free odns , such as increased binding affinity to target sequences , enhanced nuclease resistance , and entrance into cells B-material without the need for ancilliary transfectants . [SEP]
[CLS] in terms of the core B-material nanomaterial B-material scaffold , a variety of heavy metal B-material inorganic B-nanoparticle nanoparticles I-nanoparticle ( e . g . , au , ag , cdse , fe 3 o 4 ) have been explored with the goal of imparting additional physiochemical properties to the system ( such as plasmonics , photoluminescence B-property , scattering , and catalysis ) . [SEP]
[CLS] although these cores B-material have shown demonstrated use in spherical nucleic B-material acid I-material systems , the potential toxicity B-property and biodegradability B-property issues of heavy metal inorganic particles remain a concern and judicious passivation techniques are required . [SEP]
[CLS] in this regard , the construction of water - soluble , heavy - metal free , silicon B-nanoparticle nanoparticles I-nanoparticle ( sinps ) conjugated with dna is highly attractive since silicon B-material is well - established to be biocompatible B-property , 22−24 biodegradable B-property , and earth - abundant , and can exhibit photoluminescence B-property . [SEP]
[CLS] a number of synthetic methods ( including electrostatic interactions , postsynthesis linking , and automated solid - phase synthesis ) have been explored to functionalize odns onto bulk silicon B-material substrates . [SEP]
[CLS] in addition , methods have been established to obtain sinps . [SEP]
[CLS] however , the effective and site - selective conjugation of sinps with odns remains a formidable challenge since typical hydrogen - or halogenterminated sinps are readily oxidized and are also prone toward nonselective nucleophilic attack . [SEP]
[CLS] in fact , literature on sinp odn conjugates is rare and the reported syntheses have involved either multiple synthetic steps and / or harsh conditions ( such as the use of high concentrations of hf , 36 bromine B-material , or laser ablation ) . [SEP]
[CLS] in addition to the paucity of synthetic methods to obtain sinp based spherical nucleic B-material acids I-material , to the best of our knowledge , there has been no report on utilizing dna conjugated sinps as functional systems . [SEP]
[CLS] with this communication , we first disclose a mild , two - step method , featuring reactive high - energy ball milling ( rhebm ) followed by thiol−ene click chemistry , to prepare sinp dna conjugates from readily available silicon B-material wafers . [SEP]
[CLS] these silicon - based spherical nucleic B-material acids I-material have been characterized via a combination of microscopy B-technique ( tem and afm ) , spectroscopy B-technique ( uv−vis and fluorescence B-property ) , and gel B-technique electrophoresis I-technique . [SEP]
[CLS] furthermore , we demonstrate the utility of these sinp odn conjugates by illustrating how these particles can be utilized to detect oncogenic microrna - 21 ( mir - 21 ) via a fluorescence on strategy . [SEP]
[CLS] the preparation of the sinp odn conjugates is illustrated in scheme 1 . [SEP]
[CLS] first , rhebm of silicon B-material wafers in the presence of 1hexene and 1 , 7 - octadiene ( [UNK] : 2 v / v ) generated alkene terminated sinps . [SEP]
[CLS] after removal of insoluble sediments via centrifugation , the resultant sinps were covalently functionalized with dna by reacting an excess ( 110 equiv ) of 3 ′ - thiol modified 27mer odn ( 5 ′ - tcaacatcagtctga - taagct −fl aaaaaa - sh - 3 ′ ) that also contains a fluorescein ( fl ) unit as a spectroscopic handleto the surface alkene moieties through the thiol−ene click reaction ( initiated by 365 nm light in the presence of dmpa ) . [SEP]
[CLS] the resultant sinp odn conjugates were purified via a 30k amicon centrifugal filter to remove unreacted odns . [SEP]
[CLS] the successful coupling of the odns to the sinp was first inferred from uv−vis spectroscopy B-technique . [SEP]
[CLS] as shown in figure 1a , the purified sinp odn conjugate clearly shows absorption bands for both the odn unit ( λ max = 260 nm ) as well as the fluorescein reporter group ( λ max = 490 nm ) . [SEP]
[CLS] although the core B-material sinp does absorb in the 200−400 nm region ( figure 1a , inset ) , the extinction coefficient of the odn is significantly higher ( e . g . , at 260 nm the free odn has an ε of 3 . 33 × 10 5 l • mole −1 • cm −1 which is ca . 5 . 5 - fold higher than that of the sinp ) . [SEP]
[CLS] thus , using the absorption of the dna at 260 nm in conjunction with the calculated concentration of the core B-material sinp , we estimated that 4−5 odn strands are loaded onto each sinp core B-material . [SEP]
[CLS] a polyacrylamide B-technique gel I-technique electrophoresis I-technique ( page ) study was performed to further confirm the production of sinp odn conjugates . [SEP]
[CLS] as can be observed from figure 1b , the major band in lane 2 is a distinct green band ( due to the emission from the fluorescein unit of the odn ) that runs slower than the unconjugated odn ( lane 1 ) , as would be expected for a nanoparticle B-nanoparticle containing multiple odn conjugates . [SEP]
[CLS] transmission B-technique electron I-technique microscopy I-technique ( tem ) was first applied to characterize the morphology and size of the sinp odn conjugates . [SEP]
[CLS] shown in figure 2 are images of sinps that are unconjugated ( a : after step 1 of synthesis ) and odn conjugated ( b : after step 2 ) . [SEP]
[CLS] the unconjugated sinps display spheroid particles with an average diameter of 3 nm . [SEP]
[CLS] in contrast , the odn conjugated sinps exhibit a significantly larger diameter ( 10 nm ) . [SEP]
[CLS] the increase in nanoparticle B-nanoparticle size provides further evidence for the successful conjugation reaction . [SEP]
[CLS] afm measurements ( figure 2c , d ) , performed under tapping mode , gave additional information about the size and distribution of the sinps . [SEP]
[CLS] these measurements are consistent with the tem data and display an average height of 3 nm for the unconjugated sinps and 10 nm for the spherical nucleic B-material acids I-material . [SEP]
[CLS] with evidence in hand for the formation of sinp odn conjugates , we were keen on exploring the capacity of these silicon based spherical nucleic B-material acids I-material as sensing agents for biologically relevant rna . [SEP]
[CLS] as a proof - of concept , we focused on detecting mir - 21 since this noncoding rna is overexpressed in a of cancers , as it downregulates the production of tumor B-material suppressor proteins B-material . [SEP]
[CLS] in fact , due to its integral nature in cancer , sensing agents for mir - 21 are an important are of research interest . [SEP]
[CLS] our mir - 21 detection scheme is shown in figure 3 and relies on a fluorescence on strategy . [SEP]
[CLS] while the core B-material sinp does fluoresce B-property , the quantum yield of fluorescence B-property is not substantial ( 2 % ) and thus we chose to use the fluorescein moiety on the conjugated odns as the reporter group . [SEP]
[CLS] in stage 1 , a 15 - mer quencher strand ( 5 ′ - dabcyl - tagcttatcagactg - 3 ′ ) hybridizes with the odns conjugated to the sinp . [SEP]
[CLS] since the fluorophore and dark quencher are in proximity , a significant decrease in the fluorescence B-property intensity is observed with a plateau at 1 equiv of the quencher strand ( figure 3b ) . [SEP]
[CLS] this off state , which contains a 7 base toe - hold on the 5 ′ terminus of the sinp odn conjugate , transitions to a fluorescence B-property on state upon introduction of mir - 21 which displaces the quencher strand ( figure 3c ) since the conjugated odn forms a more stable dna : rna duplex with mir - 21 . [SEP]
[CLS] in contrast to the clear binding of the sinp odn conjugate to mir - 21 , which displays saturation behavior , when a negative control ( mir - 155 ) is added , the silicon based spherical nucleic B-material acid I-material system does not turn on as the conjugated odn on the sinp is not complementary to mir - 155 . [SEP]
[CLS] in summary , we have disclosed a facile two - step synthesis from bulk silicon B-material waferto prepare sinp odn conjugates , by performing tandem rhebm and thiol−ene click chemistry . [SEP]
[CLS] in addition to characterizing the sinp odn conjugates by a series of spectroscopic and microscopic studies , we have for the first time demonstrated that sinp dna conjugates can serve as fluorescence B-property on sensors that detect oncogenic mir - 21 . [SEP]
[CLS] given that ( a ) sinp cores B-material have been found to have minimal toxicity B-property and favorable biodegradable B-property characteristics , these sinps are attached to odns via nonlabile thioether B-material bonds , and ( c ) spherical nucleic B-material acids I-material with a variety of cores B-material are known to transfect into cells B-material , we envision that these silicon based spherical nucleic B-material acids I-material may serve as potential diagnostic and / or therapeutic agents that can be used in cellular environments . [SEP]
[CLS] we are currently exploring these possibilities . [SEP]
[CLS] it is also important to note that , in general , many biological molecules can be functionalized with thiols B-material ( e . g . , cysteine linked peptides B-material and thiol terminated glycosides ) and thus this simple two - step strategy may pave the way for the rapid investigation of a variety of sinp bioconjugates for biomedical applications . [SEP]
[CLS] 1 . straightforward two - step synthesis for the production of sinp odn conjugates [SEP]
[CLS] 2 . tem and afm images of the unconjugated ( a and c , respectively ) and the odn conjugated ( b and d , respectively ) sinps [SEP]
[CLS] the inset within panels c and d display the height histogram from the afm images . [SEP]
[CLS] ( a ) schematic illustrating the use of sinp odn conjugates to detect mir - 21 . [SEP]
[CLS] ( i ) the quencher strand hybridizes with the sinp odn conjugate leading to a fluorescence B-property off state . [SEP]
[CLS] ( ii ) mir - 21 binds to the sinp odn conjugate leading to displacement of the quencher strand , resulting in a fluorescence B-property on state . [SEP]
[CLS] ( b ) quenching of sinp odn conjugate upon addition of increasing equivalents of the quencher odn . [SEP]
[CLS] inset : fluorescence B-property quenching profile with increasing equiv of quencher odn , followed at 520 nm . [SEP]
[CLS] ( c ) fluorescence B-property enhancement profile in the presence of increasing amounts of mir - 21 ( black ) and control mir - 155 ( red ) . [SEP]
[CLS] note : for these fluorescence B-property experiments the fluorescein unit on the sinp odn conjugates were excited at 490 nm and the concentration of conjugated odns was 500 nm . [SEP]
[CLS] ovarian cancer is the leading cause of death among women with gynecological malignancies . [SEP]
[CLS] acquired resistance to chemotherapy is a major limitation for ovarian cancer treatment . [SEP]
[CLS] we report here the first use of nanoscale metal−organic frameworks ( nmofs ) for the co - delivery of cisplatin B-material and pooled small interfering rnas ( sirnas ) to enhance therapeutic efficacy by silencing multiple drug resistance ( mdr ) genes and resensitizing resistant ovarian cancer cells B-material to cisplatin B-material treatment . [SEP]
[CLS] uio nmofs with hexagonal - plate morphologies were loaded with a cisplatin B-material prodrug and mdr gene - silencing sirnas ( bcl - 2 , p - glycoprotein [ p - gp ] , and survivin ) via encapsulation and surface coordination , respectively . [SEP]
[CLS] nmofs protect sirnas from nuclease degradation , enhance sirna cellular uptake , and promote sirna escape from endosomes to silence mdr genes in cisplatin - resistant ovarian cancer cells B-material . [SEP]
[CLS] co - delivery of cisplatin B-material and sirnas with nmofs led to an order of magnitude enhancement in chemotherapeutic efficacy in vitro , as indicated by cell B-technique viability I-technique assay I-technique , dna laddering , and annexin v staining . [SEP]
[CLS] this work shows that nmofs hold great promise in the co - delivery of multiple therapeutics for effective treatment of drug - resistant cancers . [SEP]
[CLS] o varian cancer is the deadliest gynecologic cancer with a high - mortality rate that has remained unchanged in the past four decades . [SEP]
[CLS] the dismal prognosis of ovarian cancer is in large part due to the acquired resistance to chemotherapy . [SEP]
[CLS] epithelial ovarian cancer , the most common type of ovarian cancer , is initially responsive to cisplatin B-material therapy . [SEP]
[CLS] the recurrent disease , however , is often refractory to treatment and leads to mortality . [SEP]
[CLS] new strategies to overcome drug resistance are urgently needed in order to reduce the mortality rate of ovarian cancer . [SEP]
[CLS] the discovery of small interfering rna ( sirna ) in 1998 has provided new avenues of combating resistant cancers . [SEP]
[CLS] silencing genes that are involved in drug resistance using rna interference ( rnai ) can reverse cisplatin B-material resistance in ovarian cancer . [SEP]
[CLS] successful treatment of ovarian cancer cells B-material with multidrug resistance ( mdr ) gene - silencing sirnas and cisplatin B-material requires the development of novel vehicles B-material that can specifically and effectively deliver cisplatin B-material to cell B-material nuclei and sirnas to cell cytoplasms , respectively . [SEP]
[CLS] we report here the first use of nanoscale metal−organic frameworks ( nmofs ) for the co - delivery of cisplatin B-material and pooled sirnas to overcome drug resistance in ovarian cancer cells B-material . [SEP]
[CLS] mofs are an emerging class of self - assembled , porous materials whose properties can be readily tuned by varying the molecular building blocks . [SEP]
[CLS] when scaled down to the nanoregime , nmofs serve as efficient nanocarriers for the delivery of imaging contrast B-technique agents I-technique and chemotherapeutics . [SEP]
[CLS] we surmised that nmofs represent a unique nanocarrier platform by virtue of their high porosity and controllable surface functionalities : the large pores of nmofs can be used to load chemotherapeutics , while the metal B-material ions B-material on the nmof surfaces can be used to bind sirnas . the simultaneous and efficient delivery of cisplatin B-material and pooled sirnas to ovarian cancer cells B-material can allow for enhanced anticancer B-property efficacy by blocking multiple drug resistance pathways . [SEP]
[CLS] in this work , a cisplatin B-material prodrug and sirna were sequentially loaded into uio nmofs by encapsulation inside the nmofs and coordinating to metal B-material sites on the nmof surfaces , respectively . [SEP]
[CLS] uio protects sirnas from nuclease degradation , enhances sirna cellular uptake , and promotes sirna escape from endosomes to silence mdr genes in cisplatin - resistant ovarian cancer cells B-material . [SEP]
[CLS] as a result , co - delivery of cisplatin B-material and sirnas with uio led to an order of magnitude enhancement in chemotherapeutic efficacy in vitro . [SEP]
[CLS] the uio series of mofs based on zr 6 ( μ 3 - o ) 4 ( μ 3 - oh ) 4 secondary building units ( sbus ) and dicarboxylate bridging ligands first reported by lillerud et al . are highly porous and stable in aqueous environment due to the high connectivity of the sbus and the strong interaction between zirconium and oxygen B-material . [SEP]
[CLS] uio nmofs thus represent potential nanocarriers for anticancer B-property drugs . [SEP]
[CLS] in this work , the uio nmof with aminotriphenyldicarboxylic acid ( amino - tpdc ) bridging ligand was synthesized by heating a n , n - dimethylformamide ( dmf ) solution of zrcl 4 and amino - tpdc at 80 °c for 5 days ( figure 1a and scheme s1 ) . [SEP]
[CLS] the as - synthesized nmof adopted a distorted uio structure as manifested by powder xray diffraction ( pxrd ) , which was likely responsible for its hexagonal plate - like morphology as shown in transmission B-technique electron I-technique microscopy I-technique ( tem ) images ( figure 1b−g ) . [SEP]
[CLS] tem micrographs gave the diameter of the plate at [UNK] nm and the thickness at [UNK] nm ( figure 1b−d ) . [SEP]
[CLS] high - resolution tem images showed that the distances between the lattice fringes are 1 . 83 nm ( figures 1f and s6 and 10 ) , corresponding to the predicted d ( 111 ) value of 1 . 85 nm . [SEP]
[CLS] the fast fourier transform pattern ( fft ) proved a 3 - fold symmetry along the observation direction ( figure 1e ) . [SEP]
[CLS] the cisplatin B-material prodrug , cis , cis , trans - [ pt ( nh 3 ) 2 cl 2 ( oet ) ( ococh 2 ch 2 cooh ) ] , was loaded into the pores of uio via encapsulation to form uio - cis ( figure 1a and scheme s2 ) . [SEP]
[CLS] nmr spectroscopy B-technique supported the noncovalent encapsulation of cisplatin B-material prodrug ( figures s11− 13 ) , whereas pxrd indicated that uio - cis is isostructural to uio - 68 ( figure 1g ) . [SEP]
[CLS] the cisplatin B-material prodrug loading in uio - cis was determined to be 12 . 3 ± 1 . 2 wt % by inductively coupled plasma mass spectrometry . [SEP]
[CLS] drug resistance often involves multiple and dynamically acquired mdr mechanisms . [SEP]
[CLS] among them , p - gp is overexpressed in the malignant tissues and is responsible for decreased drug accumulation in cells B-material . [SEP]
[CLS] bcl - 2 is responsible for the activation of cellular antiapoptotic defense . [SEP]
[CLS] survivin has a functional role in both cell B-event division I-event and apoptosis B-event control and is often found to be upregulated in cancers . [SEP]
[CLS] simultaneously blocking these mdr - relevant molecular signaling pathways can provide an effective approach for overcoming drug resistance in cancer cells B-material . [SEP]
[CLS] sirna was loaded onto uio - cis by simply mixing uio - cis and sirna in water B-material at a cisplatin B-material : sirna mass ratio of 4 . 5 : 1 to form sirna / uio - cis ( figure 1a ) . [SEP]
[CLS] sirna is believed to bind to nmof surface via multiple coordination bonds between phosphate residues on the sirna backbone and vacant zr sites on the nmof surface . [SEP]
[CLS] the sirna loading did not change the morphology of nmofs as shown by tem ( figure 1d ) . [SEP]
[CLS] dynamic B-technique light I-technique scattering I-technique ( dls ) measurements gave average diameters of 98 ± 11 nm ( pdi = 0 . 070 ) , 103 ± 17 nm ( pdi = 0 . 124 ) , and 128 ± 3 nm ( pdi = 0 . 116 ) for uio , uio - cis , and sirna / uio - cis , respectively ( figures s8 , s10 , and s15 ) . [SEP]
[CLS] the increase in the dls diameter for sirna / uio - cis is consistent with the presence of sirna on the uio surface . [SEP]
[CLS] the sirna binding capabilities of nmofs were confirmed by gel B-technique electrophoresis I-technique , which showed that nmofs could efficiently " capture " sirna on the surface as evidenced by the complete retardation of sirna band migration for sirna / uio - cis ( figure s16 ) . [SEP]
[CLS] the sirna loading B-property efficiency I-property ( le ) was also quantitatively examined by fluorimetry . [SEP]
[CLS] fluorescently B-property labeled sirna ( tamra - sirna ) was used to form sirna / uio - cis , and the le was determined to be as high as 81 . 6 ± 0 . 6 % . [SEP]
[CLS] as a result of steric hindrance on surfaces , nmofs protected sirna from rnase degradation : a sirna band was clearly visible upon incubating B-technique sirna / uio - cis in serum for up to 4 h , while the naked sirna was completely degraded under the same condition ( figure 1h ) . [SEP]
[CLS] interestingly , sirna " coating B-material " on the nmof surface significantly retarded protein B-material adsorption , suggesting possible stabilization of nmofs via sirna binding ( figure 1i ) . [SEP]
[CLS] high sirna uptake levels and successful endosomal escape are two prerequisites for efficient sirna - mediated gene silencing . [SEP]
[CLS] compared to the naked sirna solution , cellular uptake of sirna / uio - cis was significantly enhanced ( figure 2a ) , indicating that the nmof facilitates the sirna internalization via endocytosis B-event pathways . [SEP]
[CLS] the sirna uptake was also directly observed by confocal B-technique laser I-technique scanning I-technique microscopy I-technique ( clsm ) . [SEP]
[CLS] as illustrated in figures 2b and s17 , large amounts of sirna ( red fluorescence B-property ) were located in the cytoplasms of skov - 3 cells B-material . [SEP]
[CLS] in addition , zirconium phosphate has extremely low solubility B-property ( k sp = 10 −134 ) , which demonstrates a high affinity of zr ( iv ) to phosphate ions B-material . [SEP]
[CLS] as illustrated in figure 2c , phosphate buffer saline ( pbs ) containing relatively high phosphate group concentration ( 2 mm ) significantly promoted sirna release compared to water B-material . [SEP]
[CLS] it is reasonable to expect that sirna could dissociate from uio - cis and that uio - cis could decompose after internalization and entrapment in endosomes due to the presence of much higher concentrations of endogenous phosphate ions B-material in endosomes than in extracellular environments ( figure s12 ) . [SEP]
[CLS] the dissociated zr ions B-material can bind to the negatively charged and phosphate - groupenriched endosome membrane to disrupt the endosome structure and facilitate the release of entrapped sirnas . [SEP]
[CLS] this hypothesis was supported by clsm studies and timedependent endosome / sirna colocalization studies ( figure s19 and 20 ) . [SEP]
[CLS] after a 2 h incubation B-technique , sirna in the sirna / uio - cis was able to escape from the endo / lysosome entrapment , as demonstrated by the separation of red ( sirna ) and green ( lysotracker green stained lysosome ) fluorescence B-property in the cytoplasm ( figures 2d and we further evaluated the transfection B-property efficiency I-property mediated by sirna / uio - cis in skov - 3 cells B-material . [SEP]
[CLS] as shown in figure 3a , sirna / uio - cis evoked potent gene silencing in skov - 3 cells B-material at 0 . 4 μg / ml ( 30 nm ) of sirna as determined by elisa . [SEP]
[CLS] interestingly , by using one - third of the sirna dose for the pooled sirnas / uio - cis compared to single sirna / uio - cis , equivalent gene silencing efficiencies were achieved , suggesting the synergistic silencing effects of pooled sirnas . [SEP]
[CLS] in comparison , none of the free sirna solution , uio - cis , and uio was able to down regulate the gene expression . [SEP]
[CLS] to examine whether the efficient and simultaneous knockdown of three mdr - relevant genes including survivin , bcl - 2 , and p - gp could effectively reverse the cisplatin B-material resistance in ovarian cancer cells B-material , the cytotoxicity B-property of free cisplatin B-material , uio - cis , and sirna / uio - cis was assessed by 3 - ( 4 , 5 - dimethylthiazol - 2yl ) - 5 - ( 3 - carboxymethoxyphenyl ) - 2 - ( 4 - sulfophenyl ) - 2h - tetrazolium ( mts ) assay ( figure 3b ) and by flow B-technique cytometry I-technique ( figure s26 ) . [SEP]
[CLS] the cisplatin B-material ic 50 values of free cisplatin B-material , uio - cis , pooled sirnas / uio - cis , free cisplatin B-material plus free pooled sirnas , and free cisplatin B-material plus pooled sirnas / uio were calculated to be 53 . 9 sirna / uio at 12 times higher sirna dose . [SEP]
[CLS] by co - delivering pooled sirnas and cisplatin B-material utilizing nmofs , the ic 50 value dramatically decreased ( by more than 11 - fold ) compared to free cisplatin B-material and uio - cis . [SEP]
[CLS] in control experiments , pooled sirnas / uio - cis and uio - cis exhibited a similar level of cytotoxicity B-property in cisplatin - sensitive cancer cell B-material lines including a2780 , pc - 3 , mcf - 7 , and h460 cells B-material but significantly lower ic 50 values in cisplatin - resistant a2780 / cddp cells B-material ( 4 . 2 ± 0 . 6 vs 21 . 4 ± 1 . 4 μm for pooled sirnas / uio - cis and uio - cis , respectively ; figure s21−24 , table s1 ) . [SEP]
[CLS] this result suggested that the cisplatin - resistant ovarian cancer cells B-material could be resensitized after being transfected with sirna / uio - cis , and the synergistic effects of sirna and cisplatin B-material significantly enhanced the in vitro chemotherapeutic efficacy . [SEP]
[CLS] at 50 times higher uio dose , cell B-property viability I-property was determined to be 98 . 1 ± 5 . 4 % , indicating a lack of toxicity B-property for uio . [SEP]
[CLS] we carried out dna ladder and annexin v conjugate staining assays in order to demonstrate that the enhanced cytotoxicity B-property of sirna / uio - cis was caused by cell B-material apoptosis B-event rather than necrosis . [SEP]
[CLS] as indicated in figure 3c , no dna fragmentation was detectable in the control , uio - cis , and free cisplatin B-material groups . [SEP]
[CLS] cells B-material treated with sirna / uio - cis displayed characteristic dna fragmentation or laddering , demonstrating that the cytotoxicity B-property induced by sirna / uio - cis was associated with apoptosis B-event . [SEP]
[CLS] annexin v conjugate staining provided further evidence to the apoptosis B-event induced by sirna / uio - cis ( figures 3d and s25 ) . [SEP]
[CLS] journal of the american chemical societysirna ( red fluorescence B-property ) loaded in the nmofs was efficiently internalized into the cytoplasm after a 24 h incubation B-technique to trigger mdrrelevant gene silencing . [SEP]
[CLS] annexin v conjugate ( green fluorescence B-property ) was clearly visible in cells B-material treated with sirna / uio - cis but not in cells B-material treated with sirna / uio ( pooled sirnas alone ) or uio - cis ( cisplatin B-material alone ) . [SEP]
[CLS] this result indicates that co - delivery of cisplatin B-material and pooled sirnas would induce cell B-material apoptosis B-event in cisplatin B-material - resistant cells B-material by combining the synergistic effects of down - regulating the expressions of mdr - relevant genes and chemotherapeutics . [SEP]
[CLS] importantly , uio and pooled sirnas / uio - cis evoked no immunogenic B-property response in raw 264 . 7 macrophage and skov - 3 cells B-material ( figure s27 ) . [SEP]
[CLS] in summary , we have shown that uio nmofs with high porosity and surface binding sites represent a unique nanocarrier platform for the co - delivery of chemotherapeutic agents and pooled mdr gene silencing sirnas to drugresistant ovarian cancer cells B-material . [SEP]
[CLS] cisplatin B-material prodrug was efficiently loaded into uio by encapsulation , whereas sirna was loaded by coordinating to metal B-material sites on the nmof surfaces . [SEP]
[CLS] the uio protects sirnas from nuclease degradation , enhances sirna cellular uptake , and promotes sirna escape from endosomes to silence mdr genes , leading to an order - of - magnitude enhancement in chemotherapeutic efficacy of cispltain . [SEP]
[CLS] the simplicity of the present approach makes it amenable to codelivery of chemotherapeutics and other nucleic B-material acid I-material drugs such as sirna , microrna , and plasmid dna by nmofs . [SEP]
[CLS] we believe that this unique nmof platform holds great promise in the treatment of difficult to cure cancers by co - delivering therapeutic cargoes of disparate characteristics and functions . [SEP]
[CLS] 1 . preparation and characterization of sirna / uio - cis . [SEP]
[CLS] ( a ) schematic presentation of sirna / uio - cis synthesis and drug loading . [SEP]
[CLS] tem images of uio ( b ) , uio - cis ( c ) , and sirna / uio - cis ( d ) . [SEP]
[CLS] bars : 200 nm . [SEP]
[CLS] ( e ) electron diffraction pattern of uio by fft . [SEP]
[CLS] ( f ) high - resolution tem image of uio . [SEP]
[CLS] bar : 20 nm . [SEP]
[CLS] ( g ) pxrd patterns of uio - 68 ( black ) , uio ( red ) , and uio - cis ( blue ) . [SEP]
[CLS] ( h ) serum stability of pooled sirnas in sirna / uio - cis as evaluated by electrophoresis B-technique . [SEP]
[CLS] ( i ) bsa binding to uio - cis with and without sirna loading . [SEP]
[CLS] the results were expressed as the amount of bsa ( μg ) adsorbed to per μg of uio - cis . [SEP]
[CLS] communication dx . doi . org / 10 . 1021 / ja4098862 | j . am . chem . soc . 2014 , 136 , 5181−5184 [SEP]
[CLS] ± 4 . 7 , 53 . 2 ± 4 . 4 , 4 . 7 ± 1 . 8 , 45 . 1 ± 7 . 0 , and 6 . 6 ± 0 . 3 μm , [SEP]
[CLS] respectively [SEP]
[CLS] no cytotoxicity B-property ( cell B-property viability I-property of 96 . 2 ± 3 . 4 % ) was observed in skov - 3 cells B-material when treated with [SEP]
[CLS] cellular uptake and endosomal escape of sirna / uio - cis in skov - 3 cells B-material . [SEP]
[CLS] ( a ) sirna / uio - cis significantly increase ( by > 11 - fold ) the sirna uptake amount compared to naked sirna ( n = 3 ) . [SEP]
[CLS] ( b ) clsm image showing the internalization of sirna ( tamra - labeled ) into the cytoplasm . [SEP]
[CLS] nuclei were stained with dapi . [SEP]
[CLS] bar represents 20 μm . [SEP]
[CLS] ( c ) sirna release from nmofs was dramatically promoted in 2 mm pbs compared to water B-material . [SEP]
[CLS] ( d ) sirna ( tamra - labeled , red ) successfully escaped from endosomes as evidenced by the separation of green and red fluorescence B-property ( white arrows ) . [SEP]
[CLS] endosome / lysosome and nuclei were stained with lysotracker green and dapi , respectively . [SEP]
[CLS] bar represents 5 μm . [SEP]
[CLS] 3 . in vitro gene silencing efficiency and anticancer B-property efficacy . [SEP]
[CLS] ( a ) sirna / uio - cis - mediated efficient gene silencing in skov - 3 cells B-material at a 30 nm sirna dose . [SEP]
[CLS] silencing efficiency was expressed as percentage values of control group treated with pbs . [SEP]
[CLS] ( b ) skov - 3 cells B-material were incubated B-technique with free cisplatin B-material , uio - cis , pooled sirnas / uio - cis , free cisplatin B-material plus free pooled sirnas , and free cisplatin B-material plus pooled sirnas / uio at different concentrations for 72 h , and then the cytotoxicity B-property was determined by mts assay . [SEP]
[CLS] ( c ) analysis of dna ladder on 2 % ( w / v ) agarose gel at 35 v for 5 h after dna extraction from skov - 3 cells B-material treated with sirna / uio - cis at an equivalent cisplatin B-material concentration of 10 μm . lanes 1−5 : dna marker , control , uio - cis , sirna / uio - cis , and free cisplatin B-material . [SEP]
[CLS] ( d−f ) clsm images showing cell B-material apoptosis B-event and sirna ( tamra - labeled , red ) internalization in skov - 3 cells B-material after incubation B-technique with uio - cis ( d ) , sirna / uio ( e ) , and sirna / uio - cis ( f ) for 24 h . the apoptotic cells B-material were stained with alexa fluor 488 annexin v conjugate , and the nuclei were stained with dapi . [SEP]
[CLS] bar represented 10 μm . [SEP]
[CLS] spherical nucleic B-material acid I-material ( sna ) nanoparticle B-nanoparticle conjugates are a class of bionanomaterials that are extremely potent in many biomedical applications . [SEP]
[CLS] their unique ability to enter multiple mammalian cell B-material types as single - entity agents arises from their novel three - dimensional architecture , which consists of a dense shell B-material of highly oriented oligonucleotides chemically attached typically to a gold B-nanoparticle nanoparticle I-nanoparticle core B-material . [SEP]
[CLS] this architecture allows snas to engage certain cell B-material surface I-material receptors I-material to facilitate entry . [SEP]
[CLS] here , we report studies aimed at determining the intracellular fate of snas and the trafficking events that occur inside c166 mouse endothelial cells B-material after cellular entry . [SEP]
[CLS] we show that snas traffic through the endocytic pathway into late endosomes and reside there for up to 24 h after incubation B-technique . [SEP]
[CLS] disassembly of oligonucleotides from the nanoparticle B-nanoparticle core B-material is observed 16 h after cellular entry , most likely due to degradation by enzymes such as dnase ii localized in late endosomes . [SEP]
[CLS] our observations point to these events being likely independent of core B-material composition and treatment conditions , and they do not seem to be particularly dependent upon oligonucleotide sequence . [SEP]
[CLS] significantly and surprisingly , the snas do not enter the lysosomes under the conditions studied . [SEP]
[CLS] to independently track the fate of the particle core B-material and the fluorophore - labeled oligonucleotides that comprise its shell B-material , we synthesized a novel class of quantum B-nanoparticle dot I-nanoparticle snas to determine that as the sna structures are broken down over the 24 h time course of the experiment , the oligonucleotide fragments are recycled out of the cell B-material while the nanoparticle B-nanoparticle core B-material is not . [SEP]
[CLS] this mechanistic insight points to the importance of designing and synthesizing next - generation snas that can bypass the degradation bottleneck imposed by their residency in late endosomes , and it also suggests that such structures might be extremely useful for endosomal signaling pathways by engaging receptors that are localized within the endosome . [SEP]
[CLS] spherical nucleic B-material acid I-material ( sna ) conjugates represent an emerging class of bionanomaterials that typically consist of two types of fundamental building blocks , oligonucleotides and inorganic B-material nanoparticle I-material cores I-material . [SEP]
[CLS] due to their ability to naturally enter multiple mammalian cell B-material types without the aid of lipid B-material or cationic B-material transfection agents , snas are extremely potent and useful in a variety of biomedical applications . [SEP]
[CLS] snas have been shown to be effective for intracellular diagnostic assays and as novel gene regulation agents . [SEP]
[CLS] we previously postulated a mechanism for endocytosis B-event of snas , which involves the specific recognition of their dense oligonucleotide shell B-material by class a scavenger receptors on the cell B-material membrane as well as their subsequent uptake via the lipid - raft / caveolae pathway . [SEP]
[CLS] unfortunately , very little is known about the intracellular events of snas following cell B-material entry . [SEP]
[CLS] previous work by others on different classes of nanoparticles B-nanoparticle has suggested that particle size , shape , [SEP]
[CLS] density , and surface chemistry can impact cellular entry [SEP]
[CLS] however , investigations of the intracellular events that happen to nanoparticles B-nanoparticle subsequent to their cellular entry are scarce and do not seem to reach a universal consensus . [SEP]
[CLS] indeed , it is likely that there is no universal description of biotrafficking for nanoparticles B-nanoparticle and that all variables must be considered . [SEP]
[CLS] for example , polymeric B-nanoparticle nanoparticles I-nanoparticle can access multiple intracellular compartments , such as the golgi apparatus , cytosol , and endoplasmic reticulum . [SEP]
[CLS] chitosan B-nanoparticle nanoparticles I-nanoparticle modified with hydrophobic B-property moieties end up in lysosomes . [SEP]
[CLS] protein - coated gold B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) often get trapped inside endosomes but can be directed elsewhere in the cell B-material through the addition of cellpenetrating peptides B-material or a liposome B-nanoparticle exterior , or even recycled out of the cell B-material . [SEP]
[CLS] similarly , lipid B-nanoparticle nanoparticles I-nanoparticle containing sirna have been shown to be largely limited to the endocytic pathway , resulting in recycling . [SEP]
[CLS] due to their three - dimensional ( 3d ) oligonucleotide shells B-material , snas represent a fundamentally different class of nanomaterial B-material with the ability to engage receptors that other classes of particles often do not . [SEP]
[CLS] therefore , snas may exhibit a unique profile of intracellular fate , a topic worthy of exploration . [SEP]
[CLS] in this work , we synthesized a new class of sna consisting of a quantum B-nanoparticle dot I-nanoparticle ( qd ) core B-material and a fluorescent B-property oligonucleotide shell B-material ( fl - qd - sna ) to support our investigation of the intracellular events that occur following the cellular uptake of our classic gold B-material sna ( au - sna ) construct . [SEP]
[CLS] specifically , we studied the intracellular trafficking of snas in c166 mouse endothelial cells B-material , the signals which dictate trafficking routes , the rate and origins of sna degradation , and the recycling potential of the material B-material . [SEP]
[CLS] based upon the results of these studies , we propose a mechanism by which snas are recognized , processed , and distributed within the cell B-material . [SEP]
[CLS] snas primarily traverse to and accumulate in late endosomes upon cellular entry . [SEP]
[CLS] we first studied the intracellular locations of snas as a function of incubation B-technique time by using c166 cells B-material ( mouse endothelial ) as a model cell B-material line , under conditions where cells B-material were continuously incubated B-technique ( no change in medium ) with snas ( 10 nm ) . [SEP]
[CLS] for this purpose , we prepared cyanine 5 ( cy5 ) - labeled snas ( cy5 - snas ) made from 5 ′ cy5 - labeled , single - stranded dna oligonucleotides ( cy5 - ssdnas ) covalently attached to the surface of 13 nm aunps B-nanoparticle ( 80 ± 5 oligonucleotides per particle ) . [SEP]
[CLS] in addition to the cy5 moiety , these oligonucleotides bear a nontargeting sequence that is antisense to the mrna transcript B-event of the green fluorescent B-property protein B-material ( gfp ) ( sequence information listed in table s1 ) . [SEP]
[CLS] we chose the gfp sequence for our studies because it is irrelevant and non - targeting for the c166 cell B-material - line , which does not contain the gene . [SEP]
[CLS] subsequent discussion of snas will refer to this sequence unless otherwise specified . [SEP]
[CLS] by examining whether the fluorescent B-property signals of cy5 - snas colocalized with the immunofluorescence of specific protein B-material markers for intracellular compartments with strong literature precedent for nanoparticle B-nanoparticle accumulation , we were able to delineate the route of trafficking of snas inside cells B-material . [SEP]
[CLS] these protein B-material markers include early endosome antigen 1 ( eea1 ) , ras - related protein B-material 9 ( rab9 ) , lysosomal - associated membrane protein B-material 1 ( lamp1 ) , and giantin . [SEP]
[CLS] their corresponding intracellular compartments are early endosome , late endosome , lysosome , and the trans - golgi network , respectively . [SEP]
[CLS] our previous work has shown that snas are primarily localized in early endosomes after 1−2 h of incubation B-technique with cells B-material . [SEP]
[CLS] in these experiments , the snas carry a cy5 dye ( red ) , and the cells B-material are stained with a complementary dye - labeled ( green ) marker of interest ( in figure 1a , the marker of interest is eea1 , a protein B-material that localizes exclusively in early endosomes ) . [SEP]
[CLS] here , we demonstrate that snas show moderate colocalization with early endosomes from the fourth to 24th hour of incubation B-technique ( figure 1a ) . [SEP]
[CLS] this continual association with early endosomes suggests that uptake and trafficking are continuous processes . [SEP]
[CLS] importantly , strong colocalization of snas with rab9 ( protein B-material localizes in late endosomes ) was observed after the fourth hour of incubation B-technique , and colocalization persisted 24 h post - incubation B-technique . [SEP]
[CLS] experimentally , we observe a typical cell B-material doubling time of 18 h for c166 cells B-material , and we believe that snas largely reside within late endosomes after their departure from early endosomes ( figure 1b , note the orangeyellow color , indicating colocalization of snas and rab - 9 marker ) . [SEP]
[CLS] significantly , we did not observe appreciable colocalization between the fluorescent B-property signals of snas and markers for lysosomes ( lamp - 1 ) or trans - golgi network ( giantin ) over the entire incubation B-technique period of 24 h ( figure 1c , d ) . [SEP]
[CLS] from these data , we conclude that the probability of snas trafficking from early endosomes to the lysosome or trans - golgi network is low . [SEP]
[CLS] it is also interesting to note that our data suggest that snas do not fully progress through the typical route of the endolysosomal pathway for the degradation of biological entities . [SEP]
[CLS] instead , over this time period , snas stall inside of late endosomes and do not traffic extensively beyond this point . [SEP]
[CLS] transmission B-technique electron I-technique microscopy I-technique ( tem ) images for cells B-material collected after continuous treatments of au - snas further reinforce the confocal imaging data . [SEP]
[CLS] at most time points , the overwhelming majority of snas is observed inside perinuclear vesicles measuring from 400 nm to 1 μm in diameter ( figure 2a , c ) , a size range consistent with the reported average dimension of late endosomes ( 700 nm ) . [SEP]
[CLS] only a tiny portion of these particles escape the endosomes and are found in the cytosol ( figure s1 ) . [SEP]
[CLS] moreover , high - magnification tem images reveal a key defining ultrastructural feature of late endosomes , the presence of numerous luminal vesicles that resemble multivesicular bodies ( mvbs ) encapsulated by the late endosomes ( figure 2b ) . [SEP]
[CLS] these luminal vesicles mostly measure 50−100 nm across ( figure 2c ) , fitting literature reports for luminal vesicles characteristic of late endosomes . [SEP]
[CLS] structures such as cisternae and electron dense lumens were not seen in association with snas , indicating no intracellular trafficking to the trans - golgi network and mature lysosome , respectively . [SEP]
[CLS] moreover , tem micrographs reveal nanoparticle B-nanoparticle clusters of increasing sizes as well as decreasing interparticle distances inside late endosomes as a function of incubation B-technique time . [SEP]
[CLS] after 4 h of incubation B-technique , snas in clusters of 20−30 particles are localized in late endosomes without apparent contact with each other . [SEP]
[CLS] by contrast , snas typically manifest as clusters of [UNK] particles accumulated inside late endosomes after 16 h of incubation B-technique . [SEP]
[CLS] in short , upon cellular entry , au - snas that exist in early endosomes as individual particles gradually traverse to late endosomes as clusters of particles in close proximity to one another . ( lysosomes ) , and giantin ( trans - golgi network . [SEP]
[CLS] ) most snas colocalize with late endosomes during continuous incubation B-technique in c166 cells B-material . [SEP]
[CLS] some sna colocalization with early endosomes is observed . [SEP]
[CLS] snas do not colocalize at any time with lysosomes or the trans - golgi network . [SEP]
[CLS] mander ' s colocalization coefficients are displayed in yellow ( > 0 . 6 indicates substantial colocalization . [SEP]
[CLS] intracellular location likely does not depend significantly on the chemical composition of the nanoparticle B-nanoparticle core B-material or oligonucleotide sequence . [SEP]
[CLS] following identification of intracellular compartments which house our conventional au - snas after their cellular entry , we set out to preliminarily determine whether trafficking to as well as accumulation in late endosomes without further departure was a phenomenon generalizable to other snas with different nanoparticle B-nanoparticle cores B-material . [SEP]
[CLS] to do so , we treated cells B-material with two structural variants of the au - sna , namely a hollow sna and an sna with a cdse / zns qd core B-material ( scheme 1 ) . [SEP]
[CLS] hollow snas are 3d oligonucleotide - based nanoconstructs obtained by crosslinking multiple alkyne - terminated cy5 - ssdnas on the surface of an aunp B-nanoparticle core B-material that is subsequently dissolved by kcn . [SEP]
[CLS] quantum B-nanoparticle dot I-nanoparticle snas ( qd - snas ) were first synthesized by our group in 1999 by covalently attaching oligonucleotides directly to the qd core B-material . [SEP]
[CLS] subsequent to that work , we reported a method for non - covalently immobilizing ssdna on the surface of aliphatic - ligand protected cdse / zns quantum B-nanoparticle dots I-nanoparticle by reacting them with amphiphillic B-property polymers B-material , functionalized with dna . [SEP]
[CLS] significantly , we have used qd - snas and the hollow snas to show that the composition and even absence of the nanoparticle B-nanoparticle core B-material have no appreciable effect on the intracellular fate of snas ; both sna structure variants were found to be within late endosomes after 4 h of incubation B-technique ( figure 3a , compare figure 1b ) . [SEP]
[CLS] next , we studied two snas composed of different oligonucleotide sequences to determine if there was sequence - specific sorting within the cell B-material . [SEP]
[CLS] for these experiments , we functionalized aunp B-nanoparticle cores B-material with two distinct ssdna sequences , namely a repeated thymidine sequence ( t 30 ) and a sequence antisense to the transcript B-event of the survivin oncogene . [SEP]
[CLS] t 30 is not gene targeting , and the survivin sequence exists but is not overexpressed in this cell B-material line . [SEP]
[CLS] therefore , these two types of snas allow one to probe if sequence makes a significant difference with regard to intracellular sna fate . [SEP]
[CLS] confocal immunofluorescence studies show that these snas behave analogously to our gfp sequence snas , also trafficking into late endosomes after 4 h of continuous incubation B-technique ( figure 3b ) . [SEP]
[CLS] tem micrographs also show the presence of t 30 snas inside of large membrane - bound vesicles ( figure s2 ) . [SEP]
[CLS] therefore , the sheer 3d architecture of the oligonucleotides , rather than the sequence information encoded by them , is likely the primary attribute which governs the intracellular trafficking behavior of snas . [SEP]
[CLS] further in - depth studies are needed to confirm these preliminary findings . [SEP]
[CLS] uptake and trafficking are independent processes . [SEP]
[CLS] next , we considered other mechanisms which could drive endocytic sorting . [SEP]
[CLS] previous work has shown that sna cellular entry is a continuous process . [SEP]
[CLS] we hypothesized that sna trafficking into the late endosome could potentially be driven by subsequent waves of sna uptake . [SEP]
[CLS] to investigate the effect of continuous uptake on the trafficking of snas , we utilized a pulse - chase setup to follow a small window of uptake events . [SEP]
[CLS] cells B-material were treated with au - snas ( 10 nm ) for 1 h only and then placed into clean , fresh sna - free media for different durations of time . [SEP]
[CLS] immunofluorescence analysis shows colocalization of snas with the early endosomes at the end of the 1 h treatment , but strong colocalization with late endosomes 24 h after the initial treatment with snas has ended ( figure 3c ) . [SEP]
[CLS] tem images support this conclusion as au - snas are in small , featureless vesicles as isolated entities at the end of 1 h treatment but collect inside of larger , multivesicular endosomes after the 24 h treatment - free incubation B-technique period ( figure 3d ) . [SEP]
[CLS] treating cells B-material with au - snas for only 4 h also gives similar results ( figure s3 ) . [SEP]
[CLS] these data show that cells B-material sort snas independent of uptake duration or quantity . [SEP]
[CLS] in other words , c166 cells B-material will naturally sort an overwhelming majority of snas from early endosomes to late endosomes unbiased by the stimulus induced by the uptake of subsequent waves of snas . [SEP]
[CLS] intracellular disassembly of snas is likely due to degradation by dnaseii . [SEP]
[CLS] the late endosome represents an integral part of the cellular degradation pathway and has been shown to eventually fuse with lysosomes . [SEP]
[CLS] the lumen of the late endosome is known to be an environment that facilitates degradation of biomacromolecules . [SEP]
[CLS] acidic ph , presence of catabolic enzymes , and redox active species are just a few characteristic features of the late endosome . [SEP]
[CLS] proteins B-material and oligonucleotides have been shown to be extensively degraded due to this environment . [SEP]
[CLS] we were interested in probing the susceptibility of snas to degradation due to their prolonged accumulation inside the late endosomes . [SEP]
[CLS] from our tem imaging data in figure 2 , the intracellular aggregation of the aunp B-nanoparticle core B-material becomes increasingly prevalent as a function of incubation B-technique time , suggesting the possibility of intracellular degradation of the oligonucleotide shell B-material that originally provided steric stabilization to and electrostatic repulsion between sna nanostructures . [SEP]
[CLS] to address the issue of degradation , we incubated B-technique cy5 - labeled au - snas ( also used for the confocal imaging studies in figure 1 ) under different chemical conditions and measured how many cy5 - ssdna strands remain on the surface of the aunp B-nanoparticle core B-material after the treatment . [SEP]
[CLS] briefly , we centrifuged the chemically treated sna solution to recover the sna pellet , dissolved the aunp B-nanoparticle core B-material , and quantified the cy5 fluorescence B-property of the solution against a standard calibration curve . [SEP]
[CLS] we first subjected cy5 - labeled au - snas to various buffers with ph values ranging from 7 . 5 to 4 . 5 , a window that covers the essential intracellular compartments expected to be traversed by a sna along the endolysosomal pathway , including extracellular fluid ( ph = 7 . 4 ) , early endosomes ( ph = 5 . 5−6 . 0 ) , and late endosomes / lysosomes ( ph = 4 . 5−5 . 0 ) . [SEP]
[CLS] after 3 d of incubation B-technique , cy5 - snas did not show reduction in oligonucleotide loading . [SEP]
[CLS] we next added cy5 - snas to a degassed pbs solution that contains intracellular concentrations of glutathione ( 1−10 mm ) to analyze whether the surplus of thiol B-material groups from glutathione ( gsh ) in the cell B-material would displace thiolated ssdna strands off the aunp B-nanoparticle surface . [SEP]
[CLS] for this experiment , the pbs was thoroughly degassed by repeated freeze−thaw cycles to prevent the oxidation of gsh to form dimers ( gssg ) in the cell - free environment . [SEP]
[CLS] again , after incubation B-technique for 1 d , the fluorescence B-property associated with the solution of cy5 - snas did not significantly increase , indicating no appreciable oligonucleotide displacement from the particle surface ( figure s4 ) . [SEP]
[CLS] thus , change in ph and thiol B-material displacement by glutathione cannot account for the aggregation of au - snas inside the cell B-material . [SEP]
[CLS] finally , we investigated if dna nucleases natively found in late endosomes or lysosomes may contribute to the degradation of snas . [SEP]
[CLS] two common nucleases pertinent to dna degradation are deoxyribonuclease i ( dnase i ) and deoxyribonuclease ii ( dnase ii ) . [SEP]
[CLS] dnase i has been implicated in dna degradation in the serum , extracellular space , and also in the cytosol of cells B-material . [SEP]
[CLS] an acidic endonuclease , dnase ii is usually found within intracellular compartments , most notably lysosomes . [SEP]
[CLS] since late endosomes are able to fuse with other late endosomes or lysosomes , we hypothesize that dnase ii is responsible for dna degradation when snas are shuttled to and stalled in the late endosomes . to test this hypothesis , we introduced cy5 - labeled au - snas into a cell - free solution that contains the same concentration of either dnase i or dnase ii , each buffered at the appropriate ph required for its proper functioning . after 4 h of incubation B-technique , cy5 - labeled au - snas treated with dnase ii lost [UNK] % of their original oligonucleotide loading , whereas those treated with dnase i lost only [UNK] % ( figure 4a ) . [SEP]
[CLS] we further showed that , by contrast to the sna architecture , more than 80 % of cy5 - ssdna of the same nucleotide sequence was degraded after incubation B-technique with both enzymes for 4 h . [SEP]
[CLS] for this assay , free dna was synthesized with a 3 ′ molecular quencher of the 5 ′ dye ( see supporting information for sequence information ) in order to allow for similar percentage degradation calculations as those done with snas . [SEP]
[CLS] thus , the arrangement of dna oligonucleotides in the form a dense 3d shell B-material can endow snas with additional stability against enzymatic degradation , but snas are less resistant to enzymatic attack by dnase ii than dnase i , a conclusion supported by early , less comprehensive work . [SEP]
[CLS] note that the time points in these studies may or may not be relevant to the cellular studies since the intracellular concentration of these enzymes has not been reported to date . [SEP]
[CLS] we wish to further visualize how intracellular nucleases like dnase ii may disassemble the sna architecture . [SEP]
[CLS] to achieve this goal , we need to independently track the movement of both the np B-nanoparticle core B-material and dna strands in the cell B-material . [SEP]
[CLS] while dna strands can be labeled fluorescently B-property , the innate lack of fluorescence B-property of aunps B-nanoparticle precluded us from visualizing how the dna strands are falling off the np B-nanoparticle core B-material for our classic au - snas by confocal imaging . [SEP]
[CLS] to circumvent this bottleneck , we synthesized fl - qd - snas , a new class of sna nanostructure that consists of a cdse / zns core B-material ( with an emission wavelength of 630 nm ) covalently functionalized with fluorescein - labeled ssdnas . [SEP]
[CLS] we believe that the fl - qd - sna serves as a reasonable proxy for the classic au - sna due to its similar hydrodynamic diameter ( [UNK] nm ) and oligonucleotide loading ( 70 ssdna ±4 / particle ) as previously described . [SEP]
[CLS] indeed , dnases exhibit similar activity profiles for both constructs , whereby incubation B-technique in dnase ii led to more significant reduction in oligonucleotide loading than dnase i ( figure 4a , b ) . [SEP]
[CLS] we then treated c166 cells B-material continuously with fl - qd - snas and imaged them at various time points . [SEP]
[CLS] confocal microscopy B-technique shows the fl - qd - snas remain largely intact for up to 16 h , as evidenced by overlapping fluorescence B-property signals of the qd core B-material and fl - ssdnas ( figure 5 ) . [SEP]
[CLS] after 16 h , the fluorescein signal from the oligonucleotides separates from that of the qd core B-material , with an even larger effect after 24 h . [SEP]
[CLS] this separation is likely due to dna cleavage from the sna by enzymes in the late endosomes , particularly dnase ii , which has high activity at low ph . it is important to note that the treatment in this study is continuous , and any newly uptaken qd - snas likely influence the true time scale of degradation . [SEP]
[CLS] in this case , degradation products must build up for a time before they are observable by confocal microscopy B-technique . [SEP]
[CLS] differential recycling of sna components . [SEP]
[CLS] once we utilized qd - snas to establish that the sna architecture disassemble on the time scale of roughly 16−24 h after entering c166 cells B-material , we returned to au - snas and explored whether their individual components are expelled from the cell B-material . [SEP]
[CLS] using the same pulse - chase setup previously described in figure 3 , we tracked the gold B-material content in cells B-material using inductively coupled plasma mass spectrometry ( icp - ms ) and quantified the cy5 fluorescence B-property in cells B-material by spectroscopic analysis . [SEP]
[CLS] after a 4 h pulse treatment of cells B-material with cy5 - snas , we washed out the nanoparticles B-nanoparticle , grew the treated cells B-material in clean medium , and then divided cell B-material samples for icp - ms and fluorescence B-property analysis . [SEP]
[CLS] to exclude the possibility that any observed reduction in the intracellular gold B-material or dna amount is due to cell B-event division I-event but not actual exocytosis , we treated the cell B-material sample as a population rather than on a single cell basis by ensuring a near - constant density of c166 cells B-material plated for this experiment . [SEP]
[CLS] after the 4 h pulse incubation B-technique , intracellular gold B-material content remains relatively constant in the cell B-material population over the course of 24 h of growth , indicating no net exocytosis of the aunp B-nanoparticle core B-material ( figure 6a ) . [SEP]
[CLS] given our tem data that reveal increased clustering of aunp B-nanoparticle cores B-material , this near - perfect mass balance of intracellular gold B-material content across the entire period of incubation B-technique time suggests that the aunp B-nanoparticle core B-material of snas is continuously sorted from multiple early endosomes of smaller sizes to significantly fewer late endosomes of much larger sizes . [SEP]
[CLS] the lack of recycling of the aunp B-nanoparticle core B-material may stem from the degradation of a large portion of the oligonucleotide shell B-material by intracellular nucleases ( most likely , dnase ii ) . [SEP]
[CLS] such degradation likely leads to the loss of biological recognition of the sna architecture and renders the aunp B-nanoparticle core B-material susceptible to colloidal aggregation in the presence of intracellular amounts of salt B-material . [SEP]
[CLS] ultimately , these aunp B-nanoparticle clusters may not traffic efficiently due to there being no known cellular receptors for this material B-material . [SEP]
[CLS] by contrast , cy5 fluorescence B-property is observed to rapidly decrease in the cell B-material lysates if the sna treatment is discontinued after 4 h ( figure 6b ) . [SEP]
[CLS] this decline in fluorescence B-property likely results from degradation products of dnase cleavage that are expelled from the cell B-material either by active transport or diffusion , but not via any recycling pathway based on our confocal imaging data presented in figure 1 . [SEP]
[CLS] diffusion seems unlikely as the confocal images presented in figure 5 reveal punctate spots rather than homogeneous patches of fluorescence B-property , which would be indicative of release into the cytoplasm . [SEP]
[CLS] moreover , we measured the fluorescence B-property of the culture medium collected from the same pulse - chase experiment at different time points after the initial incubation B-technique of 4 h . the fluorescence B-property of the medium steadily increases over time after the removal of cy5 - snas , thus adding credence to the notion that there is net exocytosis of free cy5 moieties or processed dna fragments to the medium ( figure s5 ) . [SEP]
[CLS] from our studies , we have shown that snas enter into the endocytic pathway after entry into the cell B-material . [SEP]
[CLS] snas progress largely into late endosomes , which is their final intracellular location over the 24 h time frame considered . [SEP]
[CLS] a small , unquantifiable portion of these particles escape the endosome and are found in the cytosol . [SEP]
[CLS] these are the entities that are likely responsible for knockdown in both antisense and rnai mediated gene regulation pathways . [SEP]
[CLS] both immunofluorescence and tem ultrastructural analysis support this conclusion . [SEP]
[CLS] moreover , snas traffic along this route and reach the late endosomes , independent of the surface oligonucleotide sequences and the core B-material compositions studied here . [SEP]
[CLS] our pulse - chase experiments also show that the intracellular fate of snas is guided only by the single - entity agent alone and seems invariant to the quantity of snas uptaken . [SEP]
[CLS] finally , we show that snas are partially broken down by nucleases within the late endosome , and degradation products are differentially processed by the cellular transport machinery : the core B-material is retained in the late endosome , while the dye or oligonucleotide fragments are cleared from the cell B-material , at least for this cell line . [SEP]
[CLS] the sna architecture can function successfully as a gene regulation construct , in part due to the increased stability of the oligonucleotides on snas as compared to their free forms . [SEP]
[CLS] this allows them a longer intracellular residency time . [SEP]
[CLS] the observation that the vast majority of snas are tied up in the endosome suggests that they particularly potent gene regulation agents . [SEP]
[CLS] indeed , a small amount escapes the endosome , which accounts for their activity in antisense and likely sirna pathways . [SEP]
[CLS] any increased availability of snas to the cytosol will further boost the therapeutic activity of snas , and we pose the design and synthesis of snas capable of more efficient endosomal escape as a challenge to the community . [SEP]
[CLS] the work also has several additional implications . [SEP]
[CLS] it brings to light the importance of realizing next generation snas that can take advantage of their location inside late endosomes , which may include introducing functionalities to modulate processes such as immune activation and exosome packaging . [SEP]
[CLS] it also underscores the importance of designing synthesizing hollow snas or structures with biodegradable B-property cores B-material to avoid the unanticipated consequences of the core B-material materials on cellular function . [SEP]
[CLS] synthesis of oligonucleotides . [SEP]
[CLS] dnas were synthesized on an mm48 oligonucleotide synthesizer ( bioautomation ) using standard solid - phase synthesis and reagents ( glen research ) . [SEP]
[CLS] all dnas were purified using a prostar high - performance liquid B-technique chromatography I-technique ( hplc ) instrument ( varian ) with a microsorb c18 column ( varian ) . [SEP]
[CLS] s1 contains detailed sequence information on the dnas . [SEP]
[CLS] preparation of sna nanoconjugates . [SEP]
[CLS] for gold B-material - sna nanoconjugates , thiolated dnas were added to 13 nm citrate - capped aunps B-nanoparticle at a concentration of 1 od of dna per ml of 10 nm aunps B-nanoparticle supplemented with 0 . 5 % tween - 20 . [SEP]
[CLS] after stirring at rt for 1 h , the solution was aged with gradual additions of nacl over 6 h to bring the final nacl concentration to 0 . 5 m . functionalized aunps B-nanoparticle were separated from free dna strands via dialysis against nanopure water B-material using a 50 kda amicon molecular weight cutoff membrane ( millipore ) . [SEP]
[CLS] aunp B-nanoparticle and dna concentrations were determined by measuring their extinction at 524 and 260 nm , respectively , on a cary 5000 uv−vis spectrophotometer ( agilent ) . [SEP]
[CLS] hollow snas were prepared based on published methods . [SEP]
[CLS] quantum dot sna nanoconjugates were also prepared as detailed previously using cdse - zns quantum B-nanoparticle dots I-nanoparticle ( ocean nanotech ) . [SEP]
[CLS] cell B-material culture and sna treatment . [SEP]
[CLS] all cell B-material experiments described in this work employ c166 cells B-material ( mouse endothelial ) , which were cultured at 37 °c and 5 % co 2 in dmem supplemented with 10 % fbs and 1 % streptomycin / penicillin . [SEP]
[CLS] to measure the extent of cellular association by icp - ms , cells B-material were first seeded in a 24 - well plate at a population of 5 × 10 4 cells B-material per well 24 h in advance and incubated B-technique with 0 . 3 ml of snas ( 10 nm in dmem ) per well . [SEP]
[CLS] to visualize the extent of cellular uptake by tem , cells B-material were seeded in a 6 - well plate at a population of 5 × 10 5 cells B-material per well and then incubated B-technique with 1 . 5 ml of snas ( 10 nm in dmem ) per well . [SEP]
[CLS] for both icp - ms and tem studies , snas were removed at different time points , followed by dmem rinses , trypsinization for counting using a hemacytometer and centrifugation at 8000 rpm for 5 min to form a cell B-material pellet . [SEP]
[CLS] for pulsechase experiments , cells B-material were first treated with 10 nm sna for either 1 or 4 h , washed twice with dmem , and replenished with fresh dmem . [SEP]
[CLS] the cells B-material were then incubated B-technique for the designated duration of time before harvesting them for icp - ms and tem studies . [SEP]
[CLS] icp - ms [SEP]
[CLS] cell B-material pellets were digested with 0 . 3 ml of 3 % hcl in concentrated hno 3 at rt overnight . [SEP]
[CLS] after adding 5 μl of 5 ppm indium B-material ( internal standard ) and 5 ml of matrix solution ( 2 % hcl and 2 % hno 3 ) , the au - 197 content of the resultant solution was measured by an x series ii icp - ms ( thermo fisher ) after subtracting the background gold B-material content of untreated cells B-material . [SEP]
[CLS] unless otherwise mentioned , reported values represent mean ± se from the average of three independent experiments . [SEP]
[CLS] tem [SEP]
[CLS] cell B-material pellets were fixed by resuspension in 3 . 7 % paraformaldehyde ( pfa ) in pbs for 15 min . [SEP]
[CLS] cells B-material were then pelleted again by centrifugation at 6000 rpm for 5 min and enrobed in molten 2 % agarose at 37 °c . [SEP]
[CLS] molten agarose cell B-material mixtures were expressed into water B-material at rt to produce " noodle - shaped " gels for ease of processing . [SEP]
[CLS] following this , the cell - containing noodle gels were fixed in 2 . 5 % glutaraldehyde in 100 mm sodium B-material cacodylate buffer ( ph = 7 . 4 ) , stained by 1 % oso 4 and by 0 . 9 % oso 4 and 0 . 3 % k 4 fe ( cn ) 6 , with all steps carried out at 4 °c for 2 h . [SEP]
[CLS] after gradual dehydration with ethanol and propylene oxide B-material , the cell - containing noodle gels were embedded in epon 812 resins ( electron B-technique microscopy I-technique sciences ) and further polymerized . [SEP]
[CLS] we deposited 80 nm - thick sections on 200 - mesh copper B-material grids ( electron B-technique microscopy I-technique sciences ) and stained with 2 % uranyl acetate ( spi supplies ) and reynolds lead citrate for visualization under a jem 1230 microscope ( jeol ) using a beam voltage of 80 kv . an orius sc 1000 ccd camera ( gatan ) was used to record the images . [SEP]
[CLS] endosome and luminal vesicle diameter measurements were taken using imagej and freehand blob identification . [SEP]
[CLS] diameter is defined as the average of xand y - direction feret diameter . [SEP]
[CLS] data were binned into bins [UNK] % of average measurement . [SEP]
[CLS] confocal microscopy B-technique and immunofluorescence . [SEP]
[CLS] seeded in a 35 mm fluorodish ( world precision instruments ) , cells B-material were incubated B-technique with 10 nm of cy5 - snas or cy5 - hollow - sna in complete dmem for different time points . [SEP]
[CLS] cells B-material were rinsed with pbs , fixed in 3 . 7 % pfa in pbs for 15 min , and imaged under a zeiss lsm 510 inverted confocal scanning microscope . [SEP]
[CLS] the excitation wavelength of cy5 - snas was 633 nm , and the corresponding emission filter was 660−710 nm . [SEP]
[CLS] to track the colocalization of snas with intracellular proteins B-material , after incubation B-technique with 10 nm cy5 - snas for different durations of time , cells B-material were rinsed with pbs , fixed in 3 . 7 % pfa in pbs , and permeated with 0 . 1 % triton x - 100 for 10 min . [SEP]
[CLS] after blocking with 2 % bsa in pbs for 1 h , cells B-material were stained with a primary antibody B-material against the protein B-material marker of interest at 5 μg / ml ( 1 % bsa in pbs ) overnight at 4 °c . [SEP]
[CLS] if necessary , after rinses with 0 . 05 % tween - 20 in pbs , cells B-material were stained with an alexafluor 488 - labeled secondary antibody B-material ( invitrogen alexa fluor 488 goat anti - rabbit igg ( h + l ) ) at 1 μg / ml ( 1 % bsa in pbs ) for 1 h at rt . [SEP]
[CLS] the excitation wavelength of the secondary antibody B-material was 488 nm , and the corresponding emission filter was 500−550 nm . [SEP]
[CLS] the primary antibodies B-material include rabbit against eea1 ( abcam ab2900 ) , rabbit against rab9 ( santa cruz biotechnology fl - 201 ) , rabbit against lamp1 ( abcam ab24170 ) , and rabbit against giantin ( abcam ab24586 ) . [SEP]
[CLS] to measure the extent of colocalization between the fluorescence B-property signals of snas and protein B-material markers , the zen digital imaging ( zeiss ) software allows for the calculation of the manders overlap coefficient . [SEP]
[CLS] an overlap coefficient higher than 0 . 6 indicates strong colocalization . [SEP]
[CLS] to probe the intracellular fate of the individual components of qd - snas ( i . e . , the qd ) core B-material and the fluorescein - labeled oligonucleotides ) , cells B-material were incubated B-technique with 10 nm qd - snas in dmem for different durations of time . [SEP]
[CLS] following the same rinsing , fixation , and permeation procedures as listed above , the cells B-material were imaged under a confocal scanning microscope . [SEP]
[CLS] the excitation wavelengths for qd and fluorescein are 633 and 488 nm , respectively . [SEP]
[CLS] the corresponding emission filters for qd and fluorescein are 660−710 nm and 500−550 nm , respectively . [SEP]
[CLS] oligonucleotide quantification . [SEP]
[CLS] 10−12 nmoles of cy5 - labeled snas were treated with dnase i ( new england biolabs m0303s ) or dnase ii ( sigma d4138 ) ( 5u / rxn ) for set time points . [SEP]
[CLS] 1 % sds was added to denature the enzymes and stop degradation . [SEP]
[CLS] all snas were pelleted at 10 000 × g and washed with water B-material . [SEP]
[CLS] pellets were resuspended in a cleaving buffer ( 100 mm kcn and 100 mm dtt ) to dissolve the gold B-material core B-material . [SEP]
[CLS] after solution had cleared , the cy5 fluorescence B-property of the oligonucleotides was read on a synergy h4multimode microplate reader ( bio - tek ) to determine the quantity of oligo lost due to dnase activity . [SEP]
[CLS] free oligos were assayed in a similar manner in which a quencher ( dabycl ) was conjugated to the opposite ( 3 ′ ) end of the oligo to suppress fluorescence B-property . [SEP]
[CLS] oligo cleavage was calculated through increase of fluorescence B-property as quencher and fluorophore are separated . [SEP]
[CLS] for qd - sna , 0 . 5 % hcl was used to dissolve the qd core B-material and dismantle the sna . [SEP]
[CLS] following acid treatment , ph was normalized to neutral using naoh before reading for fluorescein fluorescence B-property . [SEP]
[CLS] ■ associated content * s supporting information synthesized dna sequences in this work , additional tem images , cell - free sna stability assays , and recycling of fluorescent B-property sna oligonucleotide fragments . [SEP]
[CLS] this material B-material is available free of charge via the internet at http : / / pubs . acs . org . [SEP]
[CLS] corresponding author chadnano @ northwestern . edu present address † department of electronic engineering ( biomedical engineering ) , the chinese university of hong kong , shatin , new territories , hong kong , china . [SEP]
[CLS] 1 . confocal microscopy B-technique of cy5 - labeled au - snas ( red ) and immunofluorescence staining of organelle markers ( green ) . [SEP]
[CLS] markers are eea1 ( early endosome ) , rab9 ( late endosome ) , lamp1 ( lysosomes ) , and giantin ( trans - golgi network . [SEP]
[CLS] ) most snas colocalize with late endosomes during continuous incubation B-technique in c166 cells B-material . [SEP]
[CLS] some sna colocalization with early endosomes is observed . [SEP]
[CLS] snas do not colocalize at any time with lysosomes or the trans - golgi network . [SEP]
[CLS] mander ' s colocalization coefficients are displayed in yellow ( > 0 . 6 indicates substantial colocalization . [SEP]
[CLS] tem micrographs show intracellular au - snas collect inside increasingly larger and more perinuclear compartments over time . [SEP]
[CLS] nu = nucleus , ex = extracellular space . [SEP]
[CLS] ( a ) most au - snas traffic through increasingly larger , membrane - bound vesicles and remain inside these compartments over 24 h of continuous incubation B-technique in c166 cells B-material . [SEP]
[CLS] arrows indicate clear membrane boundaries . [SEP]
[CLS] the bottom panel contains magnified images of the boxed area of the top panel . [SEP]
[CLS] ( b ) high magnification of large vesicles inside cells B-material after 16 h incubation B-technique . [SEP]
[CLS] numerous luminal vesicles and compartment size ranges suggest these are late endosomes . [SEP]
[CLS] the right panel shows the magnified image of the boxed area of the left panel . [SEP]
[CLS] ( c ) size distribution of ( top ) endosomes containing au - snas and ( bottom ) luminal vesicles in these endosomes after long incubations B-technique ( 16−24 h ) . [SEP]
[CLS] sizes of these features align with literature values for late endosomes . [SEP]
[CLS] 1 . diagrams of sna variant constructs a [SEP]
[CLS] location analysis for sna variant constructs and treatment variant in c166 cells B-material . [SEP]
[CLS] immunofluorescence rab9 staining ( green ) of cells B-material treated with different sna constructs ( red ) for 4 h shows that ( a ) hollow snas ( no core B-material ) , qd - snas with a cdse - zns core B-material , ( b ) snas consisting of a repeated thymidine sequence ( sna : t 30 ) , and snas consisting of a sequence antisense to the transcript B-event that encodes the survivin oncogene ( sna : sv ) also colocalize strongly with late endosomes . [SEP]
[CLS] scale bar = 10 μm for all confocal images . [SEP]
[CLS] ( c ) pulse - chase experiments show cy5 - labeled au - snas that have already entered the cell B-material 1 h postincubation progress in the endocytic cycle . [SEP]
[CLS] immunofluorescence staining shows that snas colocalize with early endosomes ( eea - 1 ) but not late endosomes ( rab9 ) after 1 h incubation B-technique . [SEP]
[CLS] they then colocalize with late endosomes but not early endosomes 24 h after the initial incubation B-technique of 1 h . [SEP]
[CLS] mander ' s colocalization coefficients are displayed in yellow . [SEP]
[CLS] ( d ) representative tem micrographs show rare occurrences of au - snas within small compartments as isolated entities after 1 h incubation B-technique . [SEP]
[CLS] ( e ) large clusters of au - snas ( > 200 particles per cluster ) are found abundantly in large , perinuclear compartments 24 h after the initial incubation B-technique of 1 h . for ( d ) and ( e ) , the bottom panel contains magnified images of the boxed area of the top panel . [SEP]
[CLS] nu = nucleus , ex = extracellular space . [SEP]
[CLS] buffer tests of ( a ) au - snas and ( b ) qd - snas show that both structure variants on the sna architecture offer protection against dnase i and ii compared to free , single - stranded dna ( ssdna ) . [SEP]
[CLS] dnase degradation profiles for both au - sna and qd - sna are sufficiently similar to suggest that they behave similarly under intracellular enzymatic environments . [SEP]
[CLS] note that , in buffer , the sna architecture is more prone to degradation by dnase ii , an enzyme commonly found in late endosomes or lysosomes , than dnase i , which is usually found in extracellular fluids or the cytosol . [SEP]
[CLS] snas composed of a cdse / zns qd core B-material and fluoresceintagged dna strands ( fl - qd - snas ) are used as a proxy to monitor and visualize the degradation of snas in c166 cells B-material . [SEP]
[CLS] by confocal imaging , the qd core B-material ( red ) and the fluorescein - tagged oligonucleotides ( green ) have visibly separated at 16 h and beyond , likely due to enzymatic cleavage . [SEP]
[CLS] before 16 h , overlapping signals of qd and fl - dnas ( yellow ) indicate that the sna architecture is largely intact . [SEP]
[CLS] mander ' s coefficients are indicated in yellow for merged images , showing gradual loss of colocalization between the qd and the fl - dna over time . [SEP]
[CLS] analysis of material B-material retention inside c166 cells B-material as a function of growth time post - 4 h incubation B-technique with cy5 - labeled au - snas . [SEP]
[CLS] ( a ) gold B-material content in a single cell B-material population ( pellet ) remains fairly constant over time . [SEP]
[CLS] slight increases may be due to uptake of residual snas loosely adhered to the plastic of the tissue culture plate . [SEP]
[CLS] ( b ) fluorescence B-property in a single population of cells B-material sharply decreases after the incubation B-technique , indicating the expulsion of cy5 or dna fragments into the extracellular space . [SEP]
[CLS] error bar indicates the standard deviation from triplicate experiments . [SEP]
[CLS] due to their large size , charged surfaces , and environmental sensitivity , proteins B-material do not naturally cross cell B-material - membranes in intact form and , therefore , are difficult to deliver for both diagnostic and therapeutic purposes . [SEP]
[CLS] based upon the observation that clustered oligonucleotides can naturally engage scavenger receptors that facilitate cellular transfection , nucleic acid−metal organic framework nanoparticle B-nanoparticle ( mof np B-nanoparticle ) conjugates have been designed and synthesized from nu - 1000 and pcn - 222 / mof - 545 , respectively , and phosphate - terminated oligonucleotides . [SEP]
[CLS] they have been characterized structurally and with respect to their ability to enter mammalian cells B-material . [SEP]
[CLS] the mofs act as protein B-material hosts , and their densely functionalized , oligonucleotide - rich surfaces make them colloidally stable and ensure facile cellular entry . [SEP]
[CLS] with insulin as a model protein B-material , high loading and a 10 - fold enhancement of cellular uptake ( as compared to that of the native protein B-material ) were achieved . [SEP]
[CLS] importantly , this approach can be generalized to facilitate the delivery of a variety of proteins B-material as biological probes or potential therapeutics . [SEP]
[CLS] p roteins play key roles in living systems , and the ability to deliver active proteins B-material to cells B-material is attractive for both diagnostic and therapeutic purposes . [SEP]
[CLS] potential uses involve the evaluation of metabolic pathways , 2 regulation of cellular B-event processes I-event , and treatment of disease involving protein B-material deficiencies . [SEP]
[CLS] during the past decade , a series of techniques have been developed to facilitate protein B-material internalization by live cells B-material , including the use of complementary transfection agents , nanocarriers , and protein B-material surface modifications . [SEP]
[CLS] although each strategy has its own merit , none are perfect solutions ; they can cause cytotoxicity B-property , reduce protein B-material activity , and suffer from low delivery payloads . [SEP]
[CLS] for example , we have made the observation that one can take almost any protein B-material and functionalize its surface with dna to create entities that will naturally engage the cell B-material - surface I-material receptors involved in spherical nucleic B-material acid I-material ( sna ) uptake . [SEP]
[CLS] while this method is extremely useful in certain situations , it requires direct modification of the protein B-material and large amounts of nucleic B-material acid I-material , on a per - protein basis , to effect transfection . [SEP]
[CLS] ideally , one would like to deliver intact , functional proteins B-material without the need to chemically modify them , and to do so in a nucleic - acid efficient manner . [SEP]
[CLS] metal organic frameworks ( mofs ) have emerged as a class of promising materials for the immobilization and storage of functional proteins B-material . [SEP]
[CLS] their mesoporous structures allow for exceptionally high protein B-material loadings , and their framework architectures can significantly improve the thermal and chemical stabilities of the encapsulated proteins B-material . [SEP]
[CLS] however , although mof nps B-nanoparticle have been recognized as potentially important intracellular delivery vehicles B-material for proteins B-material , their poor colloidal stability and positively charged surfaces , inhibit their cellular uptake and have led to unfavorable bioavailabilities B-property . [SEP]
[CLS] therefore , the development of general approaches for reducing mof np B-nanoparticle aggregation , minimizing positive charge ( which can cause cytotoxicity B-property ) , and facilitating cellular uptake is desirable . [SEP]
[CLS] herein , we report a new method for the intracellular delivery of proteins B-material that relies on nucleic acid−mof np B-nanoparticle conjugates ( scheme 1a ) . [SEP]
[CLS] in this protocol , two water stable zirconium mesoporous mofs , nu - 1000 ( zr 6 ( μ 3 - o ) 4 ( μ 3 - oh ) 4 ( oh ) 4 ( h 2 o ) 4 ( tbapy ) 2 , h 4 - tbapy = tetraethyl 4 , 4 ′ , 4 ″ , 4 ″ ′ - ( pyrene - 1 , 3 , 6 , 8 - tetrayl ) tetrabenzoic acid ) and p c n - 2 2 2 / m o f - 5 4 5 ( z r 6 ( μ 3 - o ) 4 ( μ 3 - oh ) 4 ( oh ) 4 ( h 2 o ) 4 ( tcpp - h 2 ) 2 , h 4 - tcpp - h 2 = tetrakis ( 4carboxyphenyl ) porphyrin ) , were synthesized in nanoparticle B-nanoparticle form and used to encapsulate insulin , a model protein B-material for the studies described herein ( scheme 1b ) . [SEP]
[CLS] next , via modification of literature procedures , these insulin @ mof nps B-nanoparticle were surface functionalized with terminal phosphate - modified dna to yield insulin @ dna - mof nps B-nanoparticle ( scheme 1c ) . the 3d oligonucleotide shell B-material creates a steric and electrostatic barrier B-property to stabilize mof nps B-nanoparticle in high dielectric media and renders them functional with respect to cellular entry . [SEP]
[CLS] in principle , this strategy can be generalized to mofs with different pore sizes and topologies , thereby creating an arsenal of nucleic acid−mof - based delivery vehicles B-material for transporting functional enzymes across cellular membranes with high payloads . [SEP]
[CLS] mof np B-nanoparticle syntheses and insulin encapsulations were realized via literature protocols . [SEP]
[CLS] specifically , nu - 1000 mof nps B-nanoparticle [ 180 ( 20 ) × 70 ( 10 ) nm ] were synthesized via a solvothermal reaction of zirconium chloride B-material ( zrcl 4 ) with h 4 - tbapy ligands , modulated by acetic acid in n , n - dimethylformamide ( dmf ) at 90 °c ( figure 1a ) . [SEP]
[CLS] similarly , pcn - 222 nps B-nanoparticle [ 210 ( 30 ) × 50 ( 10 ) nm ] were synthesized via a solvothermal reaction between zirconyl chloride B-material octahydrate ( zrocl 2 • 8h 2 o ) and h 4 - tcpp - h 2 ligands , modulated by dichloroacetic acid in dmf at 130 °c ( figure 1b ) . [SEP]
[CLS] next , the thermally activated crystals of nu - 1000 were treated with a bis - tris - propane buffer ( btp , ph = 7 ) solution of insulin ( 0 . 4 mg / ml ) . [SEP]
[CLS] the mof np B-nanoparticle insulin encapsulation efficiencies were determined by measuring the s [ for insulin ] and zr ( for mofs ) contents by inductively coupled plasma - optical emission B-technique spectroscopy I-technique . [SEP]
[CLS] the maximum insulin loadings of 34 and 63 wt % were determined for nu - 1000 and pcn - 222 nps B-nanoparticle , respectively , which are consistent with our previous report . [SEP]
[CLS] the excess insulin in the supernatant was removed by sequential washing steps with di water B-material . [SEP]
[CLS] the insulin @ mof nps B-nanoparticle were functionalized with nucleic B-material acids I-material by coordinating the terminal phosphate - modified oligonucleotides to the surface zr sbus . [SEP]
[CLS] the sequence used here , 5 ′ ( dggt ) 10 - phosphate 3 ′ , was chosen because it is known with snas that a g - rich shell B-material , relative to poly dt shells B-material , facilitates higher cellular uptake . [SEP]
[CLS] in a typical np B-nanoparticle functionalization experiment , excess oligonucleotides were added to a colloidal dispersion of mof nps B-nanoparticle and incubated B-technique for 4 h ( supporting information ) . [SEP]
[CLS] particle dna coverage was quantitively determined by measuring the p to zr ratio by icp - oes ( 8 ± 1 nmol / mg for nu - 1000 nps B-nanoparticle and 10 ± 1 nmol / mg for pcn - 222 nps B-nanoparticle ) . [SEP]
[CLS] powder x - ray diffraction ( pxrd ) and scanning electron B-technique microscopy I-technique ( sem ) confirmed that the crystallinity and morphologies of the mof nps B-nanoparticle were maintained , post - dna functionalization ( figures s2 and s3 ) . [SEP]
[CLS] importantly , dynamic B-technique light I-technique scattering I-technique ( dls ) verified that dna surface functionalization significantly increases mof np B-nanoparticle colloidal stability in cellular media ( 90 % dmem buffer + 10 % fetal bovine serum ) for at least 24 h ; for comparison , unfunctionalized nu - 1000 nps B-nanoparticle aggregated in less than 1 h , hampering further in vitro use ( figure 1c , d , em image of aggregated nps B-nanoparticle : figure s5 ) . [SEP]
[CLS] in addition to colloidal stability , the intra - and extracellular stability of protein B-material delivery vehicles B-material in serum and serum free but biologically relevant matrices is important . [SEP]
[CLS] indeed , the ability to control degradation could be useful in the development of temporally controlled drug delivery applications . [SEP]
[CLS] under physiological conditions , intracellular fluid exhibits significantly higher inorganic phosphate concentration ( 5−10 mm ) as compared to that of serum ( [UNK] mm ) . [SEP]
[CLS] therefore , the degradation profiles of insulin @ dna - nu - 1000 nps B-nanoparticle and insulin @ dna - pcn - 222 nps B-nanoparticle were evaluated by exposing them to solutions designed to emulate both extracellular and intracellular conditions ( supporting information ) . [SEP]
[CLS] to simulate serum , mof nps B-nanoparticle were incubated B-technique with 90 % dmem buffer + 10 % blood serum ( ph = 7 . 0 ) at 37 °c with gentle shaking ( 400 rpm ) , where less than 5 % of degradation occurred within 12 h for both vehicles B-material , and less than 20 % within 96 h , suggesting dna - mof nps B-nanoparticle exhibit excellent stability and may be compatible with blood ( figure 2c , dashed ) . [SEP]
[CLS] in contrast , when the same mof nps B-nanoparticle were incubated B-technique in an intracellular medium simulant ( 1 × phosphate buffered saline , ph = 7 . 0 ) at 37 °c with gentle shaking , the particles degrade at much faster rates ( figure 2c , solid ) due to the high phosphate content , which competitively binds to zr clusters . [SEP]
[CLS] interestingly , dna - pcn - 222 nps B-nanoparticle exhibit a faster degradation rate ( half - life = 1 h ) when compared to that of dna - nu - 1000 nps B-nanoparticle ( half - life = 40 h ) . [SEP]
[CLS] such degradation kinetics could be useful for in vivo purposes by providing a means to control the temporal release of proteins B-material from particles , once inside cells B-material . [SEP]
[CLS] to directly visualize nucleic B-material acid I-material - modified , insulin encapsulated mof nps B-nanoparticle , we employed confocal B-technique laser I-technique scanning I-technique microscopy I-technique to image them . [SEP]
[CLS] due to the resolution limits of confocal microscopy B-technique , larger particles ( 2 . 8 μm × 10 μm for nu - 1000 ) , alexafluor 647 dye ( af647 ) - labeled insulin , and tamra - labeled dna were used . [SEP]
[CLS] with such particles , the colocalization of af647 and tamra signals can be clearly observed , verifying the encapsulation of insulin and dna surface functionalization of the mof ( figure 2a ) . [SEP]
[CLS] to obtain detailed information regarding relative distribution of insulin and dna , z - stack images of a single mof particle were taken , where tamra signal ( dna ) was observed to preferentially occupy the periphery while af647 ( insulin ) was present throughout the particle ( figure 2b and figure s7 ) . [SEP]
[CLS] brighter af647 signals were observed at both ends of the particle as compared to the center section of the mof , consistent with the previous observation that proteins B-material diffuse into nu - 1000 through its 1d channels . [SEP]
[CLS] due to the large diameter of the mof pores ( 3 . 2 nm for nu - 1000 and 3 . 7 nm for pcn - 222 ) , single stranded dna was also expected to penetrate through the mof pores and functionalize the internal surface , leading to fluorescence B-property signal inside the particles . [SEP]
[CLS] as verified by n 2 adsorption isotherms , reduced n 2 uptake capacity was observed postinsulin encapsulation for both mofs , and further loss of porosity was observed post - dna functionalization ( figures s9 and s10 ) . [SEP]
[CLS] furthermore , an enzyme - linked immunosorbent assay ( elisa ) was employed to determine whether insulin would leach from the mof np B-nanoparticle pores and / or lose catalytic activity during the dna functionalization process . [SEP]
[CLS] in both cases , no appreciable leaching and / or insulin activity loss was observed for insulin @ dna - nu - 1000 and insulin @ dna - pcn - 222 constructs ( figure s8 ) . [SEP]
[CLS] as previously stated , a key characteristic of sna - np B-nanoparticle conjugates is their ability to effectively enter cells B-material . [SEP]
[CLS] therefore , we tested whether insulin @ dna - mof nps B-nanoparticle exhibited enhanced cellular uptake . [SEP]
[CLS] specifically , nu - 1000 and pcn - 222 nps B-nanoparticle were encapsulated with af647 - labeled insulin and functionalized with tamra - labeled dna and incubated B-technique with human ovarian adenocarcinoma cells B-material , skov - 3 , for 0 . 5 , 2 , 6 , and 24 h ( supporting information ) . [SEP]
[CLS] as a control group , a mixture of free tamra - labeled dna and af - 647 - labeled insulin was incubated B-technique with cells B-material at the same concentration . [SEP]
[CLS] confocal B-technique laser I-technique scanning I-technique microscopy I-technique confirms the enrichment of insulin in cellular vesicles , as evidenced by strong colocalization of af647 and tamra signals in cellular vesicles ( figures 3a−c ) . [SEP]
[CLS] the z - stack images confirm that the insulin @ dna - mof nps B-nanoparticle are internalized by the cells B-material , as opposed to attached to their membranes . [SEP]
[CLS] consistent with this conclusion , flow B-technique cytometry I-technique showed a 10 - fold increase in fluorescence B-property in cells B-material treated with insulin @ dna - mof nps B-nanoparticle as compared to those treated with the free insulin + dna control group ( figure 3d ) . [SEP]
[CLS] the insulin @ dna - mof nps B-nanoparticle exhibits similar levels of enhancement in cellular uptake , as compared to that of conventional sna - np B-nanoparticle conjugates . [SEP]
[CLS] finally , mtt B-technique assays I-technique show that the particles result in no apparent cytotoxicity B-property or antiproliferative effects ( figures 3e ) . [SEP]
[CLS] in conclusion , we have developed a facile strategy for using nucleic - acid modified mof nps B-nanoparticle to deliver proteins B-material across cell B-material membranes at high payloads and negligible cytotoxicity B-property . [SEP]
[CLS] this work is important since it highlights how clustered surface oligonucleotides on these modular materials can be used to make them colloidally stable in physiological environments and useful for intracellular biological applications . [SEP]
[CLS] future design iterations will allow for encapsulating various proteins B-material by tuning the mof pore sizes , and potentially codelivery of protein B-material and nucleic B-material acid I-material targets that are important for many purposes , including in vivo imaging , 2 gene regulation , therapeutics , and the study of fundamental cellular B-event processes I-event . [SEP]
[CLS] ■ associated content [SEP]
[CLS] ( a ) schematic illustration of insulin encapsulation in the mesoporous channels of mof nps B-nanoparticle followed by dna surface functionalization ; ( b ) crystal structures of two mesoporous zr mofs : nu - 1000 and pcn - 222 / mof - 545 and their respective organic linkers ; ( c ) dna functionalization of insulin encapsulated mof nps B-nanoparticle using 3 ′ terminal phosphate modified nucleic B-material acids I-material [SEP]
[CLS] ( a ) representative confocal fluorescence B-property micrographs of 10 μm insulin @ dna - nu - 1000 particles verified the colocalization of insulin ( af647 channel ) and dna ( tamra channel ) . [SEP]
[CLS] ( b ) z - stack image of a single 10 μm insulin @ dna - nu - 1000 crystal . [SEP]
[CLS] ( c ) degradation profiles of dna - nu - 1000 nps B-nanoparticle and dna - pcn - 222 nps B-nanoparticle incubated B-technique in extracellular medium ( dashed lines ) and in simulated intracellular medium ( solid lines ) at 37 °c with 400 rpm shaking . [SEP]
[CLS] ( a−c ) flow B-technique cytometry I-technique plots and confocal fluorescence B-property micrographs of sk - ov cells B-material after treatment with free insulin + dna ( a ) , insulin @ dna - nu - 1000 ( b ) , and insulin @ dna - pcn - 222 ( c ) . [SEP]
[CLS] ( d ) cellular uptake of insulin delivered in different constructs as determined by flow B-technique cytometry I-technique . [SEP]
[CLS] fluorescence B-property at 647 nm was measured in sk - ov cells B-material after treatment with insulin at various incubation B-technique time ( 0 . 5 and 2 h ) . [SEP]
[CLS] ( e ) mtt B-technique assay I-technique verifies no appreciable cytotoxicity B-property induced by insulin @ dna - pcn - 222 and insulin @ dna - nu - 1000 nps B-nanoparticle . [SEP]
[CLS] scale bar = 10 μm . [SEP]
[CLS] we directly measure the dynamics of the hiv trans - activation response ( tar ) −dna hairpin with multiple loops using single - molecule forster resonance energy transfer ( smfret ) methods . [SEP]
[CLS] multiple fret states are identified that correspond to intermediate B-property melting states of the hairpin . [SEP]
[CLS] the stability of each intermediate B-property state is calculated from the smfret data . [SEP]
[CLS] the results indicate that hairpin unfolding obeys a " fraying and peeling " mechanism , and evidence for the collapse of the ends of the hairpin during folding is observed . [SEP]
[CLS] these results suggest a possible biological function for hairpin loops serving as additional fraying centers to increase unfolding rates in otherwise stable systems . [SEP]
[CLS] the experimental and analytical approaches developed in this article provide useful tools for studying the mechanism of multistate dna hairpin dynamics and of other general systems with multiple parallel pathways of chemical reactions . [SEP]
[CLS] the melting and annealing of dna hairpins are essential in many biological processes such as replication , transcription B-event , recombination , gene expression , and dna transposition for both prokaryotic and eukaryotic systems . [SEP]
[CLS] furthermore , hairpins with multiple loops are known to play specific roles in viral replication . [SEP]
[CLS] an important example is the human immunodeficiency virus - 1 ( hiv - 1 ) trans - activation response region ( tar ) hairpin [SEP]
[CLS] the tar sequence is remarkably well conserved among hiv isolates , indicating a strong selection pressure to maintain its structure . [SEP]
[CLS] thus , the tar hairpin is of therapeutic interest . [SEP]
[CLS] the tar−rna hairpin and its complement , tar− dna hairpin , are involved in several crucial steps in the viral life cycle . [SEP]
[CLS] the tar hairpin has four bulges , which have been found to be critical to the biological function of the tar sequence because they determine the hairpin unfolding / folding dynamics . [SEP]
[CLS] as a general topic , understanding hairpin dynamics is further motivated by the advent of therapeutics with aptamers , which are small rna and dna molecules that often form single or multiloop hairpin conformations . [SEP]
[CLS] in order to understand the molecular - scale dynamics of dna / rna hairpins , hairpins have been studied using technologies such as temperature - jump , optical trap , single - molecule fluorescence B-property resonance energy transfer ( smfret ) , and combinations of spectroscopic techniques . [SEP]
[CLS] however , these hairpin structures usually have one single loop connecting a stem region of several base pairs ( figure 1a ) . [SEP]
[CLS] it is generally understood that the unfolding / folding rates of such simple dna hairpins are dependent on the binding energy of the hairpin , the diffusion rate of the two ends of the stem followed by nucleation , and the propagation of base pairing . [SEP]
[CLS] this process yields folding times that range from milliseconds to microseconds , depending on the sequence length and base composition . [SEP]
[CLS] smfret is particularly suited to this study due to its wide applications in studying the single - molecule dynamics of nucleic B-material acids I-material . [SEP]
[CLS] for dna melting ( unfolding ) , a " fraying and peeling mechanism " has been predicted , and for annealing ( folding ) a " collapsing mechanism " has been proposed . [SEP]
[CLS] molecular dynamics simulations of the unfolding of short double - stranded dna have suggested that dna is opened via untwisting and then peeling . [SEP]
[CLS] this rapid " fraying " at the end of the helix has been experimentally observed for simple model dna molecules . [SEP]
[CLS] this mechanism suggests that the unfolding of the dna helix starts from one end of the stem and progresses dynamically to the other end of the dna , similar to unzipping a zipper ( figure 1a ) . [SEP]
[CLS] during the folding process of dna hairpins , end - toend contact ( collapse ) has been observed using temperaturejump measurements . [SEP]
[CLS] this mechanism suggests that the unfolded dna stalks are extremely flexible and end to end closing is common ( figure 1b ) . [SEP]
[CLS] this flexibility is consistent with the molecular dynamics simulations where multiple intermediate B-property states and trap states have been observed . [SEP]
[CLS] it remains an open question as to whether the general conclusions discussed above can be extended to describe the dynamics of more complex biologically relevant dna hairpins that include loops and bulges . [SEP]
[CLS] we hypothesize that a possible biological function for a hairpin loop / bulge is to serve as an additional fraying center to increase unfolding rates in otherwise stable systems . [SEP]
[CLS] this has been explained thermodynamically using a free energy penalty in hairpin pairing , and the effect of the bulges on folding / unfolding dynamics of the hairpin has been predicted . [SEP]
[CLS] however , it has been difficult to experimentally measure the stability of intermediate B-property states for complicated structures because of the coexistence of multiple states . [SEP]
[CLS] in this article , we carried out single - molecule fret experiments to study the complex dynamics of hiv tar−dna hairpin . [SEP]
[CLS] in order to tune the lifetime of the tar−dna folding / unfolding dynamics to our measuring time scale , we introduced two additives , urea and poly ( ethylene glycol ) ( peg ) , to the buffer solution . [SEP]
[CLS] after the smfret data were obtained , we performed a state analysis algorithm and derived a statistical analysis model to calculate the stabilities of the intermediate B-property states . [SEP]
[CLS] ■ experimental section sample preparation . [SEP]
[CLS] purified and labeled single - stranded dna ( ssdna ) tar and a mutant with the bulges removed ( figure 2 ) were acquired from trilink biotechnologies . [SEP]
[CLS] the ssdnas were modified with functional groups : biotin was used for surface immobilization ; cy3 - amidite was directly coupled to the 5 ′ end and cy5 - succinimidyl ester was coupled to a c6 amino linker at the 3 ′ end of the dna ; dt spacers B-material were designed at the end of the sequences to reduce unwanted photophysical effects . [SEP]
[CLS] the ssdnas were immobilized on glass substrates using the biotin−streptavidin interaction . [SEP]
[CLS] briefly , plasma cleaned glass coverslips were functionalized with aminosilane ( vectabond , - vector laboratories ) . [SEP]
[CLS] the slides were then grafted in a aqueous solution of 25 % ( m / m , mass fraction ) methoxypoly ( ethylene glycol ) 5000 propionic acid n - succinimidyl ester ( > 80 % , sigma - aldrich ) , 0 . 25 % ( m / m ) sunbright bi - 050ts ( biotin - peg - coo - mal , m w 5000 , nof corporation , japan ) , and 0 . 8 % ( m / m ) nahco 3 ( sigma - aldrich ) . [SEP]
[CLS] custom hybriwell chambers ( grace bio - labs ) which had a volume of [UNK] μl , secure seal spacers B-material ( grace bio - labs ) , tube connectors ( grace bio - labs ) , and teflon tubing ( western analytical products ) were used to construct a flow chamber that was attached to each biotin - pegylated slide . [SEP]
[CLS] the biotin - pegylated slide was incubated B-technique with 2 mg ml −1 streptavidin ( invitrogen ) in 25 mm hepes ( sigma - aldrich ) and 40 mm nacl ( sigma ) buffer solution for 10 min followed by dna ( 200 pm ) adsorption for 20 min . [SEP]
[CLS] before the dnas were attached to the streptavidin - labeled substrates , the dna samples were denatured at 80 °c in buffer solution for 2 . 5 min and annealed at 60 °c for 2 . 5 min , and then 2 mm mgcl 2 ( ambion ) was added and the solution was reannealed at 0 °c for 5 min to homogenize the samples . [SEP]
[CLS] fret measurements [SEP]
[CLS] single - molecule images were acquired by a home - built sample scanning confocal microscope based on a zeiss axiovert 200 microscope . [SEP]
[CLS] raster scanning of the sample coverslip was achieved by a closed - loop xyz piezo stage ( p - 517 . 3cl ; physik instrumente ) with 100 × 100 × 20 μm travel range and a minimum resolution of [UNK] nm ( spm 1000 ; rhk technology ) . [SEP]
[CLS] a 532 nm diode - pumped solid - state laser ( coherent , compass 315m - 100 sl ) was used as the excitation source . [SEP]
[CLS] the light was expanded to overfill the back aperture of a fluar 100× 1 . 3 na oil immersion microscope objective lens ( carl zeiss , gmbh ) which focused the laser light to a spot with a full width at half - maximum ( fwhm ) beam radius and height of [UNK] nm and [UNK] μm , respectively . [SEP]
[CLS] the intensity of the laser was controlled with a neutral density filter to be [UNK] μw before the objective , yielding an estimated total power density at the sample of [UNK] w cm −2 . [SEP]
[CLS] the fluorescence B-property signal was collected and refocused by the same objective and was separated from the excitation light using a dichroic mirror ( z532rdc ; chroma technology ) . [SEP]
[CLS] scattered laser light was removed by the use of notch and emission long - pass filters ( nhpf - 532 . 0 , kaiser optical ; et585 and et685 , chroma technology ) . [SEP]
[CLS] the refocused signal was then further separated by a beam splitter ( chroma 640 dcxr ) into donor emission and acceptor emission fluorescence B-property and then finally directed to two avalanche photodiodes detectors ( spcm - aqr - 15 ; perkinelmer ) . [SEP]
[CLS] the smfret experiments were carried out at room temperature ( 20 ± 1 °c ) . [SEP]
[CLS] into the flow cell B-material , a buffer solution was flowed at 1 μl min −1 for the duration of the measurements . [SEP]
[CLS] the hepes buffer solution containing an oxygen B-material - scavenging system to extend the lifetime of the fluorophores was used in all experiments , and was prepared according to an established protocol : 45 3 % ( w / w ) β - d - ( + ) - glucose ( sigma - aldrich ) , 0 . 1 mg ml −1 of glucose oxidase ( sigma ) , 0 . 02 mg ml −1 of catalase ( sigma - aldrich ) , 40 mm nacl , 25 mm hepes buffer , and saturated trolox solution ( 6 - hydroxy - 2 , 5 , 7 , 8 - tetramethylchroman - 2 - carboxylic B-material acid ; sigma - aldrich ) . [SEP]
[CLS] in addition , cosolute 2 mm mgcl 2 , urea ( sigma - aldrich ) and / or peg - 6000 ( sigma - aldrich ) were added to the solution from stock solutions of 10 m urea and 60 % peg respectively when needed . [SEP]
[CLS] smfret analysis . [SEP]
[CLS] all analysis programs were written in matlab ( r2009b ) except for the hidden - markov models ( hmms ) analysis methods for fret efficiency trajectories , which were provided by the hammy gui ( http : / / bio . physics . uiuc . edu / hammy . html , accessed 09 / 2013 ) and vbfret ( http : / / vbfret . sourceforge . net , accessed 09 / 2013 ) . [SEP]
[CLS] the emission intensity trajectories were collected at 1 ms resolution and later binned to 10 ms time steps to improve signal - to - noise ratio . [SEP]
[CLS] the corrected fluorescence B-property signal trajectories were used directly to calculate the fret efficiency ( e fret ) , as the fraction of the fluorescence B-property signal of the acceptor dye over the total signal of acceptor dye and the donor dye : [SEP]
[CLS] where i acceptor and i donor correspond respectively to cy5 and cy3 fluorescence B-property intensity with background and crosstalk correction and blinking removed . [SEP]
[CLS] the fitting processes and algorithms can be found in the original literature . [SEP]
[CLS] briefly , trajectories of all the molecules are combined into a single data file without further modification , and then the file is fed to the two software packages for fitting . [SEP]
[CLS] during the fitting , the number of states is varied and the other fitting parameters are kept at the software defaults . [SEP]
[CLS] simulation of wormlike chain ( wlc ) model . [SEP]
[CLS] the average fret efficiency , ⟨ e ⟩ , within any long - enough bin time is calculated with wlc : [SEP]
[CLS] where r is a unitless value representing the end - to - end distance r over the maximum possible distance l of the ends - labeled polymer B-material ; r 0 is the constant forster radius ; and p ( r ) is the probability factor : [SEP]
[CLS] where a is a normalization constant : [SEP]
[CLS] and t is related to another constant called persistence length l p , a basic mechanical property quantifying the stiffness of a polymer B-material : t = l / l p . [SEP]
[CLS] the established wlc model can be applied to our smfret data of tar−dna . [SEP]
[CLS] the maximum possible length of the ssdna l = 0 . 63n nm , where n is the number of unpaired nucleotides ( nt ) between the two ends with 0 . 63 nm / nt length . [SEP]
[CLS] the forster radius r 0 for cy3−cy5 dye has been measured to be [UNK] nm when attached to dna . [SEP]
[CLS] the persistence length of tar− dna in urea is estimated from comparing the histogram of smfret data and simulated fret values . [SEP]
[CLS] the smfret values are simulated with metropolis monte carlo simulations of the time trajectory of the end - to - end distance r . [SEP]
[CLS] in every time step ( 10 ps ) , r is allowed to randomly walk between 0 and l with a gaussian distributed distance step centered at 0 . 55 nm and a standard deviation 0 . 2 nm according to the above probability function ( representing a 1d diffusion coefficient of [UNK] . 5 × 10 −4 cm 2 s −1 = ( 0 . 55 nm ) 2 / 2 / 0 . 01 ns ) . [SEP]
[CLS] at each step , the donor will be excited at a probability of 1 / 5 ns −1 , [UNK] times slower than its fluorescence B-property decay rate . [SEP]
[CLS] if the donor is excited , then it has a decay lifetime of τ d [UNK] 1 ns into donor fluorescence B-property or ( r / r 0 ) 6 τ d into a nonexcited acceptor molecule . [SEP]
[CLS] if the acceptor is excited , it has a fluorescence B-property decay lifetime 1 . 3 ns as measured ( 1 . 3 ± 0 . 1 ns , see supporting information ) . [SEP]
[CLS] the total simulation time for each number of nucleotide is 1 ms . [SEP]
[CLS] the fret efficiency is the fraction of the number of steps of acceptor emission over sum of the steps of acceptor emission and donor emission . [SEP]
[CLS] photophysics of the dye molecules . [SEP]
[CLS] blinking and bleaching of the dyes , as well as the dye−dna interaction , were confirmed to have little influence on our smfret measurements of the hairpin dynamics . [SEP]
[CLS] we labeled the two ends of the dna hairpin with cy3 and cy5 and immobilized the hairpin on pegylated glass slides via biotin−streptavidin interaction , as shown in figure 2 . [SEP]
[CLS] one potential issue with smfret experiments is that the photophysical stability of the dyes can change depending on the solution as well as the dye− dna interaction . [SEP]
[CLS] these conditions can affect the quantum yields of the dyes and thus affect the fret efficiency between the donor dye and the acceptor dye . [SEP]
[CLS] when covalently attached to dna , cyanine dyes are well - known to bend and attach to dna basepairs with hydrophobic B-property interactions I-property , varying the dyes ' quantum yields via conformational confinement and charge transfer . [SEP]
[CLS] the average quantum yields of the dyes are dependent on the dna sequences they are attached to ; however , the variation of single - dye quantum yield is not observed during our smfret measurement , probably because the above - mentioned dynamics are too fast to be observed on the time scale of milliseconds to seconds common for single - molecule measurements . [SEP]
[CLS] as each of our smfret data points is calculated from the total photon counts of the two dyes during 10 ms , the variations at shorter time scale are time - averaged . [SEP]
[CLS] stable photon counts with shot noise were observed for the smfret time trajectory of the bulge - removed mutant dna hairpin in hepes buffer solution ( figure 3a ) , for which no unfolding dynamics are expected at room temperature and a stable fret value is expected . [SEP]
[CLS] this stability of photon counts ( representing the quantum yield ) confirms that any dye−dna interactions are ( 1 ) minimal and ( 2 ) faster than the dynamics measured in our experiments . [SEP]
[CLS] the single - step bleaching profile confirms that we are measuring single - molecule events . [SEP]
[CLS] the stability of the dyes are also observed in the presence of different cosolutes ( figure 3b−d ) , which is consistent with the unchanged lifetimes of the dyes under the different solutions ( see supporting information for time - resolved fluorescence B-property data ) . [SEP]
[CLS] this stability of smfret at the millisecond time scale is consistent with other smfret studies of dna hairpins labeled with the same two dyes . [SEP]
[CLS] tuning the folding / unfolding lifetime . [SEP]
[CLS] two challenges arise when measuring the dynamics of the tar−dna hairpins : the equilibrium lies strongly toward the folded state of the hairpin , and some of the kinetic processes are too fast to be observed by our millisecond time resolution . [SEP]
[CLS] on the basis of the dynamics established from model hairpins , we calculate that the folded - state and unfolded - state lifetimes of the states of tar−dna hairpin are at [UNK] ms and [UNK] μs , respectively ( see supporting information ) . [SEP]
[CLS] these values indicate that , at equilibrium , the tar−dna hairpin effectively remains folded at room temperature , with brief explorations of the unfolded state that are too fast to be resolved with typical smfret experiments carried out at the 1−100 ms time scale . [SEP]
[CLS] therefore , our ability to characterize even two - state folding kinetics of the hairpin is limited by the fast folding rate ( or unstable unfolded state ) . [SEP]
[CLS] in the retroviral replication process , the unfolding / folding dynamics are altered by the nucleocapsid ( nc ) protein B-material , which destabilizes the two break points near the open end of the tar hairpin and allows for the characterization of the proteininduced hairpin unfolding / folding dynamics of the outermost bulge by smfret , which has a minimum time resolution at [UNK] ms level . [SEP]
[CLS] in order to understand the mechanism of multiloop hairpin unfolding / folding dynamics , alternative methods were pursued to shift the equilibrium toward the unfolded states and to slow down the dynamics to our experimental time resolution . [SEP]
[CLS] to this effect , we introduced two additives , poly ( ethylene glycol ) ( peg ) and urea , to the buffer solution . [SEP]
[CLS] it is well - known that crowding agents such as peg , sucrose , and glycerol increase the viscosity B-property of aqueous solutions , and studies have shown that peg solutes can destabilize dna at small weight values of peg but do not significantly affect the stability of dna if the peg is larger than 1 kda . [SEP]
[CLS] thus , peg - 6000 is used in this study . urea , a commonly used destabilizer of dna and proteins B-material , was used to induce helix unfolding and to shift the hairpin folding equilibrium away from the folded state at room temperature . [SEP]
[CLS] although a general consensus on the biological relevance of urea as a denaturant has not been reached , there has been recent evidence in support of urea to perturb conformational changes of nucleic B-material acids I-material and proteins B-material . [SEP]
[CLS] this conclusion is consistent with the successful application of urea in studying human telomerase rna pseudoknot folding / unfolding dynamics using smfret . [SEP]
[CLS] the smfret efficiency distribution of the tar - dna hairpin is broadened when peg - 6000 is added to the buffer solution ( figure 4f ) , as the standard deviation increases to 0 . 12 fret efficiency compared to 0 . 02 in hepes buffer . [SEP]
[CLS] under the same conditions , the standard deviation of the bulge - removed mutant only slightly increases to 0 . 04 ( figure 4b ) . [SEP]
[CLS] we confirmed that peg - 6000 has negligible effects on the time - averaged secondary structure of dna and the dye ' s photophysical response using circular dichroism ( cd ) and fluorescence B-property anisotropy decay measurements for both the standard tar−dna and the mutant construct in the presence and absence of peg ( see supporting information ) . [SEP]
[CLS] therefore , we consider peg - 6000 a suitable crowding agent to slow down the dynamics of the tar−dna and mutant constructs and that the broadening of the smfret distributions depicted in figure 4 can be attributed primarily to conformational broadening . [SEP]
[CLS] further analysis of the distribution of the fret efficiencies of the two dna hairpins in peg solution suggests that the unfolding of the hairpins by thermoagitation , known as " fraying " , 42 stops after each loop , as long as there are sufficient base pairs between loops to provide a barrier B-property to further unfolding . [SEP]
[CLS] we compared the distribution of the fret efficiencies ( figure 4 ) with previous reported distributions of end - labeled tar− dna . [SEP]
[CLS] the fret efficiency distribution of tar−dna is consistent with the hairpin unfolding to the second bulge from the opening ( figure 2 , bulge 2 ) . [SEP]
[CLS] this is expected because only two base pairs connect bulges 1 and 2 in tar−dna , making it the weakest of the bulge connecting sections . [SEP]
[CLS] this behavior has also been observed previously in the presence of nc proteins B-material . [SEP]
[CLS] by analyzing the dwell times for transitions between the two observed smfret states of tar−dna with 24 % peg , as identified by hammy , we can confirm that the addition of peg slows down the unfolding / folding dynamics of the tar−dna hairpin . [SEP]
[CLS] the unfolded - state and folded - state lifetimes of the tar−dna hairpin are 142 and 353 ms , respectively ( supporting information figure s6 ) , and are slower by 3 orders and 1 order of magnitude , respectively , when in the presence of peg , shifting them well within the resolvable time frame of smfret observations . [SEP]
[CLS] slowing down the fraying dynamics , however , does not allow us access to all of the possible open hairpin states . [SEP]
[CLS] thus further perturbation is required to accomplish this goal . [SEP]
[CLS] by tuning the concentration of a denaturant , urea , we can shift the tar−dna hairpin equilibrium to more opened states to observe each of the distinct loop unfolding / folding transitions . [SEP]
[CLS] the ensemble smfret histograms for each condition are included in figure 5 , and short pieces of smfret trajectories for each condition are shown in figure 6 . [SEP]
[CLS] the trajectories in figure 6 shift to more open states as the urea concentration is increased , referred to as s 1 , s 2 , s 3 , s 4 , and s 5 . [SEP]
[CLS] because fluorescence B-property measurements have suggested that the photophysical properties of the dyes are not changed by the presence of urea ( see supporting information ) , the primary explanation for the broadening of the fret distribution of the fret efficiencies in figure 5 is urea - induced hairpin unfolding . [SEP]
[CLS] single - molecule time trajectories ( figure 6 ) suggest that the broadening is due to transitions among newly observable fret states . [SEP]
[CLS] in the presence of 1 and 2 m urea , the fret efficiency distributions of the dna hairpin ( figure 5a , b ) are almost the same as the distributions of those with no urea ( figure 4 ) , and thus only one state is observed in the time trajectories ( figure 6f ) . [SEP]
[CLS] when the urea concentration increases to around 3 m , the fret efficiency distribution ( figure 5c ) becomes more broad and tails toward a fret efficiency value of 0 . 8 , in the direction of the state between 0 . 6 and 0 . 8 observed in the single - molecule fret time trajectories ( figure 6g ) . [SEP]
[CLS] more states are observed at successively higher urea concentrations ( figures 5d , e and 6h , i ) , until , in the presence of 6 m urea ( figures 5f and 6j ) , all states become observable , including those with fret efficiencies at [UNK] . 4 and [UNK] . 2 . [SEP]
[CLS] qualitatively , the smfret data change the ensemble steadystate view of urea denaturation of dna to a more dynamic picture . [SEP]
[CLS] the ability of urea to control the equilibrium of the tar−dna hairpins is more obvious in the average values of the ensemble fret efficiency ( figure 6k ) . [SEP]
[CLS] just like the results one would get from ensemble measurements , urea reduces the average fret values . [SEP]
[CLS] at the single - molecule level , however , the larger the urea concentration , the longer the dwell times of more opened states ( figure 6l ) . [SEP]
[CLS] according to figures 4−6 , as well as previous studies on smfret of tar−dna , we assigned the fret efficiency 1 . 0−0 . 9 to state s 1 of tar−dna ; [UNK] . 8 to state s 2 ; [UNK] . 6 to state s 3 ; [UNK] . 4 to state s 4 ; and 0 . 3−0 . 0 to the completely unfolded state s 5 , all associated with opening through the sequential bulge regions . [SEP]
[CLS] the assignment is consistent with our hypothesis that a hairpin with four bulges and a loop should yield five resolvable states ( figure 7 ) . [SEP]
[CLS] because the states are defined by the bulges that are connected with the breaking points ( bp n ) ( figure 7a ) , quantitatively , the equilibrium constant of each breaking point can be calculated from the probabilities of the states ( figure 7b ) . [SEP]
[CLS] according to the ergodic principle , the probability of each state measured at the single - molecule level represents its concentration in ensemble experiments . [SEP]
[CLS] state s 1 represents the eight microstates that have bp 1 closed but can have bp 2−4 either opened or closed ( figure 7c ) ; state s 2 contains four microstates ; state s 3 has two microstates ; and states s 4 and s 5 have only the one microstate . [SEP]
[CLS] as such , a statistical approach is proposed to obtain the equilibrium constants k closed , n from the probabilities of the five fret states : [SEP]
[CLS] the equilibrium constant is defined by closed probability f n : k closed , n = f n / ( 1 − f n ) , and the free energy can be calculated via δg n = −rt ln k closed , n , where r is gas constant and t is temperature . [SEP]
[CLS] the measured probability of each state can be expressed as a function of f n , and the following expressions can be written : [SEP]
[CLS] therefore , f n can be calculated from the measured state probabilities p n , and δg n can be calculated from f n . [SEP]
[CLS] in order to obtain the probabilities of the states p n under 6 m urea when all the states are observed , the hidden markov model ( hmm ) and the wormlike chain ( wlc ) model were used to analyze and refine the fret states of tar−dna in the next two sections . the rate constants of the transitions among different fret states are extractable from the time trajectories , but the process is complicated by measurement noise , state - blur induced by fast transitions within each binned time , variation among molecules , the breakdown of ergodicity for individual molecules , and the complexity of the transitions among the five states . many methods have been developed to analyze or assist in the analysis of these kinds of complicated time trajectories including the widely used hmm , 46 , 48 , 50−52 , 75−79 which has recently been successfully used to analyze very complicated hairpin smfret data . [SEP]
[CLS] using hhm to obtain the probabilities of the five states . [SEP]
[CLS] first we use hmm to fit the trajectories for fret states and extract the dwell - time distributions of the different states using two hmm packages hammy and vbfret . [SEP]
[CLS] both packages are well - established programs that use the hmm principle but implement it differently , with hammy using maximum likelihood ( ml ) and vbfret using maximum evidence ( me ) as a measurement of the goodness of fit . [SEP]
[CLS] the ml and me scores increase with the number of states and does not reach the maximum even at 10 states ( figure 8a ) . [SEP]
[CLS] this is expected because noise and state - blur caused by the fast dynamics among the states make the data better fitted with more parameters , which is consistent with the analysis results of simulated traces with five preset states , designed to have similar noise levels and fast dynamics on the order of the bin time . [SEP]
[CLS] the five states identified by hammy are 0 . 92 , 0 . 76 , 0 . 57 , 0 . 42 , and 0 . 33 and by vbfret are 0 . 94 , 0 . 89 , 0 . 74 , 0 . 54 , and 0 . 37 . [SEP]
[CLS] the fitting results from both packages are compared to the simulation results of wormlike chain ( wlc ) model to check which result is more consistent with our model of the hairpin dynamics . [SEP]
[CLS] wlc model is a theory that can be used to explain the average separation distance of the two ends of a polymer B-material chain under fast diffusion , where the most important parameter is the persistence length of the polymer B-material that characterizes the softness of the polymer B-material chain . [SEP]
[CLS] we are particularly interested in this model because smfret of cy3 - and cy5 - labeled ssdna has been used to measure the persistence lengths of ssdna under specific solutions ; thus , all parameters except for the persistence length have been established in the literature . [SEP]
[CLS] because the persistence length varies with salt B-material conditions , and is affected by ph value and urea , too , different persistence lengths are used to simulate the fret curves vs the number of the bases between the two ends of the unfolded hairpin ( figure 8b ) . [SEP]
[CLS] from these curves , the five states identified by hammy follow the trend well , while the five states identified from vbfret follow the trend only if the first two states are combined together . [SEP]
[CLS] as such , the results from hammy are adapted for further analysis in this case . [SEP]
[CLS] the probabilities of each state are obtained from the fitted time trajectories . [SEP]
[CLS] the time trajectories and the five - state hammy result are shown in figure 9a . [SEP]
[CLS] the distributions of these states are further extracted from the fitted data ( figure 9b ) . [SEP]
[CLS] when the states are assigned to each molecule , some " inactive " molecules turn out to stay only in a single state during the entire observation period . [SEP]
[CLS] these molecules might stay fixed due to the strong molecule−substrate interaction and are thus not counted for the statistical distribution of the states to reduce the effect of immobilization [SEP]
[CLS] the dwell times of the first state and the last state of each molecule are also excluded from the histogram to remove the edge effect . [SEP]
[CLS] after these treatment , the probabilities for the five states 0 . 92 , 0 . 76 , 0 . 57 , 0 . 42 , and 0 . 33 are 0 . 09 , 0 . 27 , 0 . 33 , 0 . 20 , and 0 . 11 , respectively . [SEP]
[CLS] free energy of each break point under 6 m urea . [SEP]
[CLS] equations 6−10 can be used to calculate the free energy change of each bp n that is independent of other breaking points and the microstates , although the overall free energy of all the bp n should be 0 . [SEP]
[CLS] our calculations produce the probability of the tar−dna hairpin staying folded f 1 = 0 . 09 , f 2 = 0 . 30 , f 3 = 0 . 52 , and f 4 = 0 . 65 . [SEP]
[CLS] the equilibrium constant k closed , n = f n / ( 1 − f n ) yields k closed , 1 = 0 . 10 , k closed , 2 = 0 . 42 , k closed , 3 = 1 . 1 , and k closed , 4 = 1 . 8 for the four bp n . [SEP]
[CLS] as such , the free energies , δg n = −rt ln ( k closed , n ) , for the base pairs in the presence of 6 m urea are 5 . 6 , 2 . 1 , −0 . 15 , and −1 . 5 kj mol −1 for the four breaking points at 20 °c with 6 m urea . [SEP]
[CLS] the order of the free energies is not consistent with the order of the hybridization energy ( supporting information figure s2 ) or the number of hydrogen B-material bonds 14 , 10 , 13 , and 11 for bp 1 to bp 4 , respectively , but rather suggests that in addition to the hydrogen B-material binding strength ( enthalpy control ) , the closer a base pair is to the anchoring point at the end loop , the easier it is for it to remain hydrogen B-material bonded ( entropy control ) . [SEP]
[CLS] in summary , our experimental observations are consistent with the hypothesis that the bulges are the fraying centers of hairpin folding / unfolding . [SEP]
[CLS] in addition , we developed an approach to extract the equilibrium constants of the folding / unfolding of each breaking point to estimate its relative stability . [SEP]
[CLS] experimentally , we have successfully demonstrated the method to slow down the dynamics and to open more conformational states for the zipped hairpin by using peg and urea as cosolutes . [SEP]
[CLS] the quantitative data analysis is consistent with our model ; however , our data analysis is based on the assumptions and models , which will certainly affect the results if they are further optimized . [SEP]
[CLS] in addition , because the noise and the fast transitions blur our binned data , the existing methods have difficulties to fit the states without specifying the number of states . [SEP]
[CLS] thus , we are developing new methods hopefully to analyze the data more objectively or even in a model - free fashion . [SEP]
[CLS] the results of this study support a " fraying and peeling " mechanism for the unfolding and " collapse " mechanism for the folding of dna hairpins . [SEP]
[CLS] our quantitative analysis of the free energy of each breaking point suggests that the stability of the paired region is a function of both the pairing sequence and its distances to the anchoring / loop positions . [SEP]
[CLS] the method developed in this paper will be very useful to study the mechanism for the inhibition of the hiv ' s tar−dna transcription B-event with short dna oligomers or rna aptamer and for studying other systems , such as tar−rna hairpin , with multiple interconverting states that might be otherwise unresolvable . [SEP]
[CLS] * s supporting information additional experimental methods , labeling strategy , theoretical calculation of folding / unfolding lifetimes for the dna hairpin loops , photophysics of cy3 and cy5 , full time trajectories , and tar−dna dynamics under peg . [SEP]
[CLS] this material B-material is available free of charge via the internet at http : / / pubs . acs . org . [SEP]
[CLS] corresponding author * phone + 1 - 713 - 348 - 4232 ; e - mail cflandes @ rice . edu ( c . f . l . ) . [SEP]
[CLS] 1 . schematic of proposed examples of unfolding / folding routes of ( a , b ) model dna hairpins and ( c ) the tar−dna hairpin with two dyes cy3 and cy5 labeled to the ends . [SEP]
[CLS] urea molecules within the solution are shown , and the double helix is not shown for easier demonstration . [SEP]
[CLS] structures of the dna hairpins used in the smfret studies . [SEP]
[CLS] predicted secondary structure of the ( a ) tar−dna with four bulges and a loop and ( b ) tar−dna mutant with the bulges removed . [SEP]
[CLS] cy3 and cy5 were used as the donor and acceptor dye molecules which were coupled to the 5 ′ - dt and 3 ′ - dt of the dna , respectively . [SEP]
[CLS] the dnas were attached to the surface via a biotin linker attached to a - dt in the hairpin loop region . [SEP]
[CLS] representative photon trajectories show stable photon counts of cy3 and cy5 attached to the ends of mutant dna hairpin in ( a ) hepes buffer with 2 mm mg 2 + , ( b ) hepes buffer with 2 mm mg 2 + and 24 % peg , ( c ) hepes buffer with 2 mm mg 2 + and 6 m urea , and ( d ) hepes buffer with 6 m urea ( full trajectory shown in the supporting information ) . [SEP]
[CLS] these are raw data for typical molecules binned at 10 ms with bleaching of either dye shown as the transition point of the signals . [SEP]
[CLS] the fret histograms of over 50 molecules / each are shown in figure 4a−d , respectively . [SEP]
[CLS] global ensemble histogram of the mutant and tar−dna in ( a , e ) hepes buffer with 2 mm mg 2 + ; ( b , f ) hepes buffer with 2 mm mg 2 + and 24 % peg ; ( c , g ) hepes buffer with 2 mm mg 2 + and 6 m urea ; ( d , h ) hepes buffer with 6 m urea . [SEP]
[CLS] the total counts of the histograms are normalized to unity . [SEP]
[CLS] insets show the mean fret efficiency , μ , ( error is the standard deviation of three independent measurements ) representing the average conformational structure of the dna ; the standard deviation , σ , of the histogram that represents the variation of the fret distribution and thus the range of conformations ; and the number of single molecules measured for each sample , # . [SEP]
[CLS] insets indicate the different concentrations of urea , the mean fret efficiency ( μ ) , the standard deviation ( σ ) of the fret efficiency , and the number of molecules measured ( # ) . [SEP]
[CLS] proposed structures and smfret trajectories with their fret efficiencies showing five different states of tar−dna in its folded form s 1 ( a , f ) , 0−2 m urea ( scheme showing as an example structure of the state ) ; s 2 ( b , g ) , 3 m urea ; s 3 ( c , h ) , 4 m urea ; s 4 ( d , i ) , 5 m urea ; and unfolded hairpin form s 5 ( e , j ) , 6 m urea in the absence of mg 2 + ( full trajectories shown in the supporting information ) . [SEP]
[CLS] ( k ) the mean fret efficiency as a function of urea concentration represents the denaturing ( unfolding ) of the dna . [SEP]
[CLS] error bars are standard deviation of three measurements , > 20 molecules for each urea concentration at different days for three different samples . [SEP]
[CLS] relatively small error bars indicate that the number of molecules is large enough to represent ensemble average . [SEP]
[CLS] ( l ) [SEP]
[CLS] number of states observed under our specific experimental conditions . [SEP]
[CLS] ( a ) scheme of the breaking points of tar−dna . [SEP]
[CLS] the four breaking points ( bp n ) define five states s n , and each state has probability of p n to be observed in smfret experiments . [SEP]
[CLS] each breaking point has a closed probability f n . [SEP]
[CLS] bp 1a and bp 1b are jointed together as has been discussed earlier in the main text . [SEP]
[CLS] ( b ) scheme of smfret trajectory with random transitions between states . [SEP]
[CLS] the histogram summarizes the random trajectory with the bars representing the probabilities of observing the states . [SEP]
[CLS] ( c ) scheme of the microstates and the transitions between the five fret states . [SEP]
[CLS] ( a ) fitting probability as a function of number of states with hammy ( maximum likelihood , ml ) and vbfret ( maximum evidence , me ) . [SEP]
[CLS] five - state is picked based on the hairpin unfolding model explained in the text . [SEP]
[CLS] ( b ) average fret efficiencies vs the number of unpaired bases between the donor dye and the acceptor dye predicted by wormlike chain model assuming 0 . 8 , 1 . 2 , and 1 . 5 nm persistence length of the ssdna . [SEP]
[CLS] the curves represent the simulated data ( see experimental section ) . [SEP]
[CLS] the blue dots are the states identified by hammy , and the magenta squares are the states identified by vbfret where the first two points are combined together . [SEP]
[CLS] ( a ) summary of the smfret time trajectories of tar−dna under 6 m urea . [SEP]
[CLS] the blue line is the binned data , and the red line is the fitted result from hammy at five states . [SEP]
[CLS] ( b ) fret distributions of the five states identified by hammy . [SEP]
[CLS] present addressesj . c . : department of chemistry and biochemistry , ohio university , athens , oh 45701 . [SEP]
[CLS] n . k . p . : department of biotechnology , iiet , invertis university , bareilly 243123 , india . [SEP]
[CLS] this work presents molecular - level investigations of how wellcharacterized silica - supported phospholipid bilayers formed from either pure dopc or a 9 : 1 mixture of dopc : dotap interact with positively and negatively charged 4 nm gold B-material metal B-nanoparticle nanoparticles I-nanoparticle at ph 7 . 4 and nacl concentrations ranging from 0 . 001 to 0 . 1 m . [SEP]
[CLS] second harmonic generation ( shg ) charge screening measurements indicate the supported bilayers carry a negative interfacial potential . [SEP]
[CLS] resonantly enhanced shg measurements probing electronic transitions within the gold B-material core B-material of the nanoparticles B-nanoparticle show the particles interact irreversibly with the supported bilayers at a range of concentrations . [SEP]
[CLS] at 0 . 1 m nacl , surface coverages for the particles functionalized with the negatively charged ligand mercaptopropionic acid ( mpa ) or wrapped in the cationic B-material polyelectrolyte poly ( allylamine ) hydrochloride ( pah ) are estimated from a joint analysis of qcm - d , xps , afm , and tof - sims to be roughly 1 × 10 7 and 1 × 10 11 particles cm −2 , respectively . [SEP]
[CLS] results from complementary shg charge screening experiments point to the possibility that the surface coverage of the mpa - coated particles is more limited by interparticle coulomb repulsion due to the charges within their hydrodynamic volumes than with the pah - wrapped particles . [SEP]
[CLS] yet , shg adsorption isotherms indicate that the interaction strength per particle is independent of ionic strength and particle coating B-material , highlighting the importance of multivalent interactions . [SEP]
[CLS] 1 h nmr spectra of the lipids B-material within vesicles suspended in solution show little change upon interaction with either particle type but indicate loosening of the gold - bound pah polymer B-material wrapping upon attachment to the vesicles . [SEP]
[CLS] the thermodynamic , spectroscopic , and electrostatic data presented here may serve to benchmark experimental and computational studies of nanoparticle B-nanoparticle attachment processes at the nano−bio interface . [SEP]
[CLS] supported lipid B-material bilayers I-material serve as important models for probing binding interactions without the need for competitive binding assays . [SEP]
[CLS] they have proven particularly useful for elucidating interactions between nanoparticles B-nanoparticle and cell B-material membranes in interfacial biological environments that are now collectively referred to as the nano−bio interface , an emerging area of research spanning nanotechnology , biology , environmental science , and chemistry . [SEP]
[CLS] while the interactions at aqueous / lipid B-material bilayer I-material interfaces are often driven by electrostatics , experimental quantification of interfacial charge densities and potentials is difficult , even when using scanning probes , labels , or conducting substrates . [SEP]
[CLS] this problem is compounded if one wishes to probe how lipid B-material bilayers I-material interact with nanoparticles B-nanoparticle . [SEP]
[CLS] quartz crystal microbalance ( qcm ) , scanning probe , or surface plasmon resonance approaches have been used to monitor changes in total mass coupled to a surface or dielectric constants in the interfacial region , but direct labelfree methods that probe either a nanoparticle B-nanoparticle - or bilayerspecific property have largely been restricted to microscopic techniques that require ultrahigh - vacuum conditions , [SEP]
[CLS] i . introductionunder which volatile species are lost and drying effects may dominate the experimental outcome [SEP]
[CLS] given that a single " silver B-material bullet " method for probing the nano−bio interface is not available at this time , combinations of complementary experimental techniques are needed if one wishes to understand , control , and predict binding interactions at biological membranes . [SEP]
[CLS] here , we take a step in that direction by synchronizing nanoparticle B-nanoparticle and bilayer synthesis with second harmonic and vibrational sum frequency generation ( shg and sfg ) measurements with qcm with dissipation monitoring ( qcm - d ) , fluorescence B-property , nuclear magnetic B-property resonance ( nmr ) spectroscopy B-technique , x - ray photoelectron spectroscopy B-technique ( xps ) , atomic B-technique force I-technique microscopy I-technique ( afm ) , and time - of - flight secondary ion B-material mass spectrometry ( tof - sims ) measurements across multiple laboratories . [SEP]
[CLS] first , we show that well - characterized phosphatidylcholine ( pc ) - rich lipid B-material bilayers I-material supported on fused silica carry a sizable negative interfacial potential at ph 7 . 4 . [SEP]
[CLS] second , we probe how the supported lipid B-material bilayers I-material , as well as vesicles suspended in solution , interact with spherical 4 nm gold B-material metal B-nanoparticle nanoparticles I-nanoparticle ( aunps B-nanoparticle ) , which are chosen because of chemical stability , ease of functionalization , and their high potential for use in biomedical applications . [SEP]
[CLS] we provide estimates for absolute surface coverages and interaction strengths for particles that are either functionalized with the negatively charged ligand mercaptopropionic acid ( mpa ) or wrapped in the cationic B-material polyelectrolyte poly ( allylamine ) hydrochloride ( pah ) . [SEP]
[CLS] finally , we discuss the results in the context of a mechanism in which the attached mpa - aunps B-nanoparticle contain a higher charge density and are therefore subject to increased interparticle charge−charge repulsion within their hydrodynamic volume than the attached pah - aunps B-nanoparticle . [SEP]
[CLS] the overall energetics of attaching an individual aunp B-nanoparticle to the bilayer are found to be driven by multivalent interactions that appear to be largely independent of ionic strength and particle coating B-material . [SEP]
[CLS] ii . a . [SEP]
[CLS] bilayer preparation . [SEP]
[CLS] in the experiments , we used 1 , 2dimyristoyl - sn - glycero - 3 - phosphocholine ( dmpc ) , 1 , 2 - dimyristoyl - sn - glycero - 3 - phospho - ( 1 - rac - glycerol ) ( dmpg ) , 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ( dopc ) , and 1 , 2 - dioleoyl - 3 - trimethylammonium - propane ( dotap , all from avanti polar lipids B-material , figure 1 ) , chosen for their contrasting phase transition temperatures , their differing charge states , and the biological relevance of the pc headgroup . [SEP]
[CLS] as described in detail in the supporting information , we prepared lipid B-material bilayers I-material from small B-material unilamellar I-material vesicles I-material of pure dopc as well as from lipid B-material mixtures containing 90 % dopc or dmpc and 10 % positively or negatively charged lipids B-material . [SEP]
[CLS] small B-material unilamellar I-material lipid B-material vesicles , prepared by extrusion though a 0 . 05 μm membrane filter ( avanti , 610000 ) , exhibited ζ potentials of −4 ± 3 , −5 ± 1 , and −2 ± 1 mv for pure dopc in 0 . 1 , 0 . 01 , and 0 . 001 m nacl buffered to ph 7 . 4 with 0 . 01 m tris . [SEP]
[CLS] ζ potentials of + 10 ± 2 , + 22 ± 4 , and + 31 ± 4 mv were obtained for the 9 : 1 mixtures of dopc : dotap at 0 . 1 , 0 . 01 , and 0 . 001 m nacl , respectively , at ph 7 . 4 . [SEP]
[CLS] corresponding values for the 9 : 1 mixtures of dmpc : dmpg were −12 ± 1 , −19 ± 2 , and −18 ± 2 mv for 0 . 1 , 0 . 01 , and 0 . 001 m nacl at ph 7 . 4 . [SEP]
[CLS] substrate supported lipid B-material bilayers I-material were assembled by vesicle fusion , and experiments were carried out at room temperature ( 24−26 °c ) . [SEP]
[CLS] all bilayers were formed at 0 . 1 m nacl ( in the presence of 0 . 005 m cacl 2 for dmpc : dmpg bilayers ) . [SEP]
[CLS] for the particle adsorption experiments conducted at 0 . 001 and 0 . 01 m nacl , we lowered the salt B-material concentration to the desired value prior to introduction of nanoparticles B-nanoparticle . [SEP]
[CLS] as detailed in the supporting information , the mass of the supported lipid B-material bilayers I-material was calculated using a voigt−kelvin viscoelastic model from changes in frequency and energy dissipation measured by qcm - d . the topography of the bilayer was assessed by tapping mode afm . [SEP]
[CLS] the electrostatic and structural properties of the bilayers were probed by second harmonic and vibrational sum frequency generation ( shg and sfg ) using our previously published approach , with modifications as detailed in the supporting information . [SEP]
[CLS] to assess the fluidity of the supported lipid B-material bilayers I-material , fluorescence B-property recovery after photobleaching ( frap ) was carried out using pc lipids B-material tagged with topfluor on an inverted laser scanning confocal fluorescence B-property microscope ( lsm 710 , zeiss ) using 488 nm laser excitation and acquiring emission images at 500−550 nm at a 230 ms frame rate . [SEP]
[CLS] lipid B-material diffusion coefficients within the bilayers were assessed at 0 . 1 m nacl and ph 7 . 4 ( 0 . 01 m tris ) . [SEP]
[CLS] ii . b . gold B-nanoparticle nanoparticle I-nanoparticle synthesis , functionalization , and characterization . [SEP]
[CLS] for our experiments , we synthesized , purified , and characterized 4 nm aunps B-nanoparticle functionalized with poly ( allylamine ) hydrochloride ( pah ) or mercaptopropionic acid ( mpa ) . [SEP]
[CLS] briefly , 4 nm citrate - stabilized aunps B-nanoparticle were prepared by direct reduction in aqueous solution , as were 4 nm mpa - aunps B-nanoparticle . [SEP]
[CLS] pah - aunps B-nanoparticle were prepared by wrapping citrate - stabilized aunps B-nanoparticle using a polyelectrolyte deposition approach . [SEP]
[CLS] the pah - aunps B-nanoparticle were subsequently purified by centrifugation and washing ( 7500g , 90 min ) . [SEP]
[CLS] the mpa - aunps B-nanoparticle were purified by diafiltration using 20 volume equivalents of nanopure deionized B-material water I-material . [SEP]
[CLS] we characterized the purified aunps B-nanoparticle by uv−vis absorption B-technique spectroscopy I-technique , transmission B-technique electron I-technique microscopy I-technique ( tem ) , dynamic B-technique light I-technique scattering I-technique ( dls ) , laser - doppler microelectrophoresis , and xps ; the complete characterization data are provided in the supporting information , specifically figures s1 and s2 . [SEP]
[CLS] the diameters of the aunp B-nanoparticle cores B-material were determined in solution by uv−vis to be [UNK] . 5 nm , and tem size analysis indicated that the pah - aunps B-nanoparticle possessed a core B-material diameter ( d core B-material ) of 4 . 5 ± 1 . 4 nm ( n = 1085 ) . [SEP]
[CLS] ζ potentials of the pah - aunps B-nanoparticle determined at ph 7 . 4 ( 0 . 01 m tris buffer ) and at 0 . 001 and 0 . 1 m nacl were + 38 ± 3 and + 32 ± 2 mv , respectively , and their number - averaged hydrodynamic diameter at 0 . 001 m nacl was found to be 6 ± 2 nm and ranged between 20 and 80 nm ( figure s2d ) at 0 . 1 m nacl . [SEP]
[CLS] xps analysis indicated that about four pah polymer B-material strands are associated with each aunp B-nanoparticle . [SEP]
[CLS] with [UNK] kda / polymer B-material we find that each particle is associated with [UNK] ammonium groups , along with a number of counterions that depends on the ionic strength . [SEP]
[CLS] for the mpa - aunps B-nanoparticle , d core B-material was 4 . 2 ± 1 . 2 nm ( n = 613 ) . [SEP]
[CLS] ζ potentials of the negatively charged mpa - aunps B-nanoparticle determined at ph 7 . 4 ( 0 . 01 m tris ) and 0 . 001 and 0 . 1 m nacl were −29 ± 1 and −27 ± 2 mv , respectively , and their number - averaged hydrodynamic diameter at 0 . 001 m nacl was found to be 9 ± 2 nm . [SEP]
[CLS] at 0 . 1 m nacl , the number - averaged hydrodynamic diameter of the mpa - aunps B-nanoparticle increased from between 100 to 400 nm to between 420 and 450 nm over the course of 2 h ( figure s2c ) . [SEP]
[CLS] uv−vis spectra ( figure s2 ) indicate that for 10 nm mpa - or pah - aunps B-nanoparticle maintained at 0 . 1 m nacl and ph 7 . 4 ( 0 . 01 m tris ) , the electronic properties of the particles do not change much over the course of 150 min , whereas measurements of the hydrodynamic diameters indicate that the mpa - aunps B-nanoparticle aggregate . [SEP]
[CLS] ii . c . nmr spectroscopy B-technique of vesicles and aunps B-nanoparticle . [SEP]
[CLS] 1 h nmr spectra were acquired in d 2 o using a varian unity inova 500 mhz narrow bore spectrometer . [SEP]
[CLS] for samples containing residual water B-material , the large water B-material peak was suppressed using presaturation . [SEP]
[CLS] unless stated otherwise , the presaturation delay for aqueous samples was 2 s and the acquisition time was 1 s , with a four - step purge . [SEP]
[CLS] the number of scans for each sample was 128 or 256 , depending on the signal - to - noise ratio . [SEP]
[CLS] all nmr spectra were processed using mnova nmr software . [SEP]
[CLS] lipid B-material vesicles were prepared by extrusion as described above , except that lipids B-material were rehydrated in tris buffer solution in d 2 o ( 0 . 1 m nacl , 0 . 01 m tris , pd 7−8 ) . [SEP]
[CLS] lipid B-material vesicles ( 12 mm ) and pah - aunp B-nanoparticle ( 10 nm particle concentration ) or free pah polymer B-material ( 10 nm ) were agitated in an nmr sample tube for 10 s and then allowed to interact at room temperature for 2 h before nmr acquisition . [SEP]
[CLS] iii . a . supported lipid B-material bilayers I-material are in a fluid state as probed by frap measurements [SEP]
[CLS] iii . resultsand [SEP]
[CLS] structurally invariant with ionic strength as probed by sfg . [SEP]
[CLS] 2a shows that the final acoustic masses obtained following formation of each bilayer type are invariant with [ nacl ] and correspond to lipid B-material surface coverages that range from [UNK] . 4 to 4 . 1 × 10 14 cm −2 , in agreement with reported values for wellformed supported lipid B-material bilayers I-material . [SEP]
[CLS] 2b shows that the change in acoustic mass detected by the qcm - d sensor follows the well - established vesicle - rupture mechanism that involves a large mass gain until a critical vesicle coverage is attained , followed by a partial mass loss as vesicles rupture and fuse to form a supported lipid B-material bilayer I-material . [SEP]
[CLS] afm images ( figure 2c , d ) of the surfaces after bilayer formation show smooth surfaces with some topographic height variations mirroring those of the underlying sio 2 film ( consistent with prior reports ) and confirm that the vesicles have fused to form lipid B-material bilayers I-material . [SEP]
[CLS] frap measurements indicate diffusion coefficients at 26 °c for topfluorpc of 1 . 7 ± 0 . 3 μm 2 / s for the bilayers formed from dmpc / dmpg , 1 . 8 ± 0 . 4 μm 2 / s for the bilayers formed from dopc / dotap , and 2 . 1 ± 0 . 6 μm 2 / s for those formed from pure dopc . [SEP]
[CLS] these results are consistent with the presence of well - formed bilayers in the fluid state . [SEP]
[CLS] moreover , the mobile B-property fraction for all three systems exceeded 85 % , in agreement with previous reports for fluid solid - supported lipid B-material bilayer I-material systems . [SEP]
[CLS] we then applied vibrational sfg spectroscopy B-technique to probe for structural changes along the carbon B-material backbone and the methyl groups of the headgroups of phospholipids comprising the supported lipid B-material bilayers I-material as they were subjected to salt B-material concentrations of 0 . 001 , 0 . 01 , and 0 . 1 m at ph 7 . 4 in the presence of 0 . 01 m tris buffer , as those electrolyte concentrations were applied in the studies aimed at quantifying the interfacial electrostatics ( vide inf ra ) . [SEP]
[CLS] as shown in figure 2e , we find that the sfg spectra obtained from both types of bilayers are largely invariant with salt B-material concentration over the range studied , indicating that structural rearrangements within the lipid B-material alkyl chains , as probed by sfg in the c−h stretching region , are negligible under these experimental conditions . [SEP]
[CLS] these spectra were recorded using the ssp polarization combination , which is sensitive to those components of vibrational transition dipole moments oriented perpendicular to the interface . [SEP]
[CLS] sfg spectra shown in the supporting information indicate variations of ±30 % in the intensities of the various vibrational modes . [SEP]
[CLS] details regarding spectroscopic assignments , and the role of tris buffer , are presented in the supporting information as well . [SEP]
[CLS] iii . b . [SEP]
[CLS] supported lipid B-material bilayers I-material surveyed produce a negative interfacial potential . [SEP]
[CLS] having verified the integrity of the lipid B-material bilayers I-material using a variety of experimental techniques , we characterized the interfacial potential , [UNK] 0 , and charge densities , σ 0 , associated with the supported lipid B-material bilayers I-material using the eisenthal χ ( 3 ) method ( figure 3 ) . [SEP]
[CLS] this variant of shg is based on the second - order polarization of interfacial water B-material molecules in the presence of an interfacial potential , [UNK] 0 . [SEP]
[CLS] intuitively , the method can be thought of as an optical voltmeter in which the shg response is proportional to [UNK] 0 , 24 which is obtained by integrating the electrostatic potential , [UNK] ( ζ ) , from the interface ( z = 0 ) to infinity ( z = ∞ ) . [SEP]
[CLS] in other words , the interfacial potential we report here is for zero distance from the silica surface and referenced to the potential in the bulk solution ( 0 v ) . [SEP]
[CLS] before presenting the experimental results , we briefly discuss some considerations that are important when applying the eisenthal χ method to supported lipid B-material bilayers I-material . [SEP]
[CLS] first , changes in the salt B-material concentration lead to changes in the ion B-material diffusion potential across a supported lipid B-material bilayer I-material in ≥2 min . [SEP]
[CLS] given that we collect our shg data at a given salt B-material concentration for approximately 15 min following a 5 min waiting period after changing the salt B-material concentration , the interfacial potential we report here accounts for changes in the diffusion potential with changes in the salt B-material concentration . [SEP]
[CLS] 2e shows that the sfg spectra of the bilayers studied here are invariant with salt B-material concentration . [SEP]
[CLS] this finding is consistent with the notion 2 that the magnitude of the permanent dipoles formed predominantly by the lipid B-material alkyl chains within the bilayer is independent of salt B-material concentration . [SEP]
[CLS] while the field due to the permanent dipoles should sum to zero under ideal conditions , we caution that any residual nonzero bilayer potential due to bilayer asymmetry induced in the presence of the underlying charged fused silica substrate may contribute to [UNK] 0 in a minor fashion . [SEP]
[CLS] second , the shg signal intensity ( i shg ) has been shown experimentally to decline when [UNK] 0 at negatively charged surfaces becomes more positive and to increase when [UNK] 0 decreases at positively charged surfaces . [SEP]
[CLS] this experimental observation is generally interpreted as follows : changes in [UNK] 0 cause changes in the i shg according to the polarization ( or , intuitively , the up or down alignment , even though we caution that dipolar alignment is not the only determinant in the χ effect ) of the water B-material dipole moments within the shg active interfacial region that are subject to [UNK] 0 . [SEP]
[CLS] while computational approaches have indicated that quadrupole contributions may be important for shg signals produced at uncharged air / liquid interfaces , those contributions are independent of the direction of the water B-material dipole moments and would not produce the experimental outcomes observed at charged aqueous / solid interfaces . [SEP]
[CLS] third , recent nonlinear optical studies have shown qualitatively that the polarization of interfacial water B-material molecules depends on the chemical identity and charge of the headgroups of lipid B-material monolayers I-material at the air / water B-material interfaces . [SEP]
[CLS] specifically , heterodyne - detected sfg spectra and molecular dynamics simulations [SEP]
[CLS] indicate that , on average , water B-material molecules interacting with zwitterionic headgroups of phospholipid monolayers at the air−water interface point their oxygen B-material atoms I-material toward the bulk aqueous phase as if they interacted with a negatively charged silica surface ( albeit to a smaller extent ) . [SEP]
[CLS] preferential hydroxide B-material ion B-material adsorption has been invoked to rationalize the observation of negative ζ potentials over bilayers prepared from zwitterionic lipids B-material at ph [UNK] 4 . [SEP]
[CLS] however , similar to what has been reported for the case of cl − ions B-material , 9 oh − may not have a large enough binding interaction with the pc headgroup , and the effect could be due to the orientation and the binding strengths of water B-material molecules to the phosphate and trimethylammonium ends of the of the permanent dipole of the zwitterionic headgroup . [SEP]
[CLS] in our system , the pure bilayers are formed from zwitterionic dopc , and lipids B-material with pc headgroups are the majority component in the bilayers formed from the lipid B-material mixtures , making these literature precedents particularly relevant for interpreting our data . [SEP]
[CLS] if [UNK] 0 emanating from the largely zwitterionic bilayers over the sio 2 support were indeed negative , we would expect shg signal intensities from the bilayers studied here to decrease with increasing [ nacl ] . [SEP]
[CLS] 3 shows that this expectation is indeed met at ph 7 . 4 in 0 . 01 m tris between 10 −5 and 0 . 1 m nacl . [SEP]
[CLS] the interfacial charge density , and thus any apparent charge on the zwitterionic headgroups , is then obtained by fitting a suitable double - layer model to the shg e - field vs [ nacl ] plots . [SEP]
[CLS] recent spin - labeled and scanning probe studies as well as molecular dynamics simulations have confirmed the appropriateness of gouy−chapman theory for describing electrostatics within the electrical double layer over supported lipid B-material bilayers I-material that was put forth in earlier work by macdonald and bangham . [SEP]
[CLS] while taking care to not overinterpret the model , we apply gouy−chapman theory here in a fashion that accounts for the charges associated with the silica surface and the presence of tris buffer , as detailed below . [SEP]
[CLS] we recently reported shg salt B-material screening data for the fused silica / water B-material interface maintained at ph 7 that resulted in an interfacial charge density of −0 . 013 ± 0 . 002 c / m 2 . [SEP]
[CLS] as shown in the supporting information , we fit the gouy− chapman model to the fused silica data shown in figure 3 , where 0 . 01 m tris is present , and find an additional charge density of + 0 . 009 ( + 0 . 003 / −0 . 001 ) c / m 2 due to adsorbed trish + cations B-material that must be taken into account . [SEP]
[CLS] assuming the interfacial species is trish + , we calculate that 6 ( + 2 / −1 ) × 10 12 buffer cations B-material are present per cm 2 at the fused silica / water B-material interface . [SEP]
[CLS] taking the sum of the charge densities for the fused silica and the trish + ( i . e . , assuming that the trish + cations B-material remain in the water B-material layer between the fused silica substrate and the proximal leaflet of the bilayer ) , we then apply the gouy− chapman model , with the interfacial potential referenced to that of the bulk aqueous phase ( 0 v , vide supra ) , to obtain the charge density of the bilayers as the average of the distal and proximal leaflets of the bilayer . [SEP]
[CLS] this analysis yields charge densities of −0 . 011 ( + 0 . 003 / −0 . 005 ) c / m 2 for pure dopc bilayers , −0 . 015 ( + 0 . 003 / −0 . 004 ) c / m 2 for bilayers formed from the 9 : 1 mixture of dopc / dotap , and −0 . 024 ( + 0 . 003 / −0 . 005 ) c / m 2 for bilayers formed from the 9 : 1 mixture of dmpc / dmpg . [SEP]
[CLS] we conclude that following the subtraction of the interfacial charge density of the bare fused silica / water B-material interface and that of trish + at ph 7 . 4 , the bilayers studied here are associated with apparent negative interfacial charge densities that are largely indistinguishable from one another . [SEP]
[CLS] given that the majority component of the bilayer is a zwitterionic lipid B-material , this finding is not surprising . [SEP]
[CLS] given the evidence from sfg spectroscopy B-technique ( bottom spectrum in figure 2e ) , it is likely that trish + is present at the surface in our dopc / dotap bilayer experiments . [SEP]
[CLS] yet , the trish + surface coverage may be less when compared to the case of the dmpc / dmpg bilayers because of coulombic repulsion between trish + and the positively charged tap headgroup . [SEP]
[CLS] given that both bilayers contain 90 % zwitterionic pc headgroups , the changes in trish + surface coverage for the various bilayer systems studied here are probably minor [SEP]
[CLS] provided that each lipid B-material occupies an area of 0 . 72 nm 2 [SEP]
[CLS] , we calculate that each zwitterionic headgroup of the bilayer formed from pure dopc is associated with an apparent charge of −0 . 028 ( + 0 . 008 / −0 . 007 ) × 10 −19 c , corresponding to 1 . 8 ± 0 . 5 % of an elementary negative charge . [SEP]
[CLS] our estimate depends on the headgroup size but is in reasonable agreement with that reported using scanning probes , given the differences in ionic strength , bilayer composition , and substrate . [SEP]
[CLS] interact with supported lipid B-material bilayers I-material rich in dopc [SEP]
[CLS] the apparent negative interfacial potentials carried by sio 2supported bilayers formed from the largely zwitterionic phospholipids led us to test their interaction with aunps B-nanoparticle functionalized with oppositely charged ligands . [SEP]
[CLS] we chose 4 nm positively and negatively charged spherical B-nanoparticle gold I-nanoparticle nanoparticles I-nanoparticle because of their stability , their potential biomedical applications , and well - established methods for their synthesis , characterization , and functionalization . [SEP]
[CLS] as described in section ii , positively and negatively charged particles were prepared by functionalizing them with the polyelectrolyte poly ( allylamine ) hydrochloride ( pah ) and mercaptopropionic acid ( mpa ) , respectively . [SEP]
[CLS] we monitor nanoparticle B-nanoparticle interactions with supported lipid B-material bilayers I-material first by tracking changes in acoustic mass using qcm - d ( figure 4a ) . [SEP]
[CLS] the positively charged pah - aunps B-nanoparticle show substantial and irreversible attachment ( 210 ± 20 hz ) to the dopc / dotap bilayer , corresponding to acoustic masses of roughly 3 . 8 μg / cm 2 ( or roughly 6 × 10 12 particles attached per cm 2 ) at 0 . 1 m nacl over 60 min and large changes in dissipation ( ( 28 ± 2 ) × 10 −6 ) when compared to the 0 . 5 × 10 −6 dissipation factors of both bilayers ( changes in frequency and dissipation are shown in the supporting information ) . [SEP]
[CLS] in contrast , the hour long exposure of supported lipid B-material bilayers I-material formed from the 9 : 1 mixture of dopc / dotap to the mpacoated particles at 12 . 8 nm concentration leads to acoustic masses at or near the detection limit ( 5 ng / cm 2 , which corresponds to 5 × 10 9 aunps B-nanoparticle per cm 2 ) . [SEP]
[CLS] likewise , dissipation factors show a small change of 0 . 5 × 10 −6 . [SEP]
[CLS] complementary xps experiments show no signal in the gold B-material spectral region for this particle / bilayer combination , even at increased pass energy and signal averaging ( detection limit on the order of 0 . 01 ng / cm 2 or 1 × 10 7 aunps B-nanoparticle per cm ) . [SEP]
[CLS] yet , figure 4b , c shows afm images of bilayers formed from a 9 : 1 mixture of dopc and dotap before and after exposure to 12 . 8 nm of the negatively charged mpa - functionalized particles , at 0 . 1 m nacl and 0 . 01 m tris buffer , that indicate a sparse coverage of bright spherical features which are distinguished from intact vesicles attached to the bilayer by their heights of up to 15 nm above the bilayer . [SEP]
[CLS] complementary tof - sims experiments carried out on the same system after rinsing with buffer and then drying shows ample signal intensity from gold B-material at 196 . 95 amu ( figure 4d ) . [SEP]
[CLS] iii . d . [SEP]
[CLS] evaluating bilayer integrity upon nanoparticle B-nanoparticle attachment . [SEP]
[CLS] to assess the quality of the supported lipid B-material bilayers I-material after exposure to the nanoparticles B-nanoparticle , we carried out frap measurements using topfluorpc . [SEP]
[CLS] the diffusion coefficients for topfluorpc were statistically indistinguishable before and after exposure to pah - or mpa - aunps B-nanoparticle at concentrations of 0 . 1 and 10 nm ( p > 0 . 05 ; table s4 ) . [SEP]
[CLS] these results are again consistent with what we reported ( vide supra ) in the absence of the nanoparticles B-nanoparticle and indicate , again , the presence of well - formed bilayers that are in the fluid state following exposure to both types of nanoparticles B-nanoparticle at two different concentrations . [SEP]
[CLS] iii . e . reversibility of attachment . [SEP]
[CLS] we then probed the interaction of the positively and negatively charged nanoparticles B-nanoparticle with the bilayer formed from a 9 : 1 mixture of dopc / dotap by shg with the expectation of probing the shgactive transitions within the metal B-material core B-material of nanoparticles B-nanoparticle directly at the aqueous / supported lipid B-material bilayer I-material interface . [SEP]
[CLS] after rinsing away excess lipid B-material vesicles with tris buffer at 0 . 1 m nacl , the shg intensity was recorded at 300 ± 5 nm in the absence of nanoparticles B-nanoparticle . [SEP]
[CLS] we typically obtain an average of 80 ± 10 counts / s for a bilayer at 0 . 1 m nacl , 0 . 01 m tris , and ph 7 . 4 when using pulse energy of 0 . 4 μj , and this intensity level serves as a reference point for our experiments . [SEP]
[CLS] then , tris buffer solutions containing increasing concentrations of gold B-nanoparticle nanoparticles I-nanoparticle in 0 . 1 m nacl were injected into the sample cell B-material , without rinsing with buffer between successive increases in particle concentration , and the shg intensity was recorded near 300 nm after each injection until a steady signal was attained . [SEP]
[CLS] 5a shows that the shg intensity increases near 300 nm when the bilayers are exposed to pah - and mpa - nps B-nanoparticle at concentrations as low as 1 × 10 −14 m , suggesting resonance enhancement of the shg signal upon particle adsorption to the interface as an important shg signal generation process in both cases . [SEP]
[CLS] studies described below verify the signals to be due to shg and not fluorescence B-property . [SEP]
[CLS] the increases in the shg intensity obtained upon addition of gold B-nanoparticle nanoparticles I-nanoparticle to the supported lipid B-material bilayers I-material may be attributable to the electronic structure , quadrupole modes , or local field enhancement of the nanoparticles B-nanoparticle . [SEP]
[CLS] the relatively featureless absorption spectra in the 300 ± 20 nm tuning range that is readily accessible with the tunability of the optical parametric amplifier used in our setup make it difficult to verify unambiguously whether the shg intensity increases are attributable to shg resonance enhancement . [SEP]
[CLS] yet , while the interfacial potential produced in the presence of charged adsorbates B-property , in this case the charged ligands , lipids B-material , and counterions , may contribute to the shg process , the observation that the shg signals increase for the positively charged and for negatively charged particles suggests that resonant enhancement contributes strongly to the shg process in both cases . [SEP]
[CLS] our comprehensive set of afm , xps , qcm - d , tof - sims , and shg experiments indicate that much fewer mpa - aunps B-nanoparticle attach to the supported lipid B-material bilayers I-material studied here than pah - aunps B-nanoparticle . [SEP]
[CLS] indeed , we estimate from xps that at 0 . 1 m nacl concentration less than 0 . 01 ng / cm 2 particles , or just 1 × 10 7 4 nm aunps B-nanoparticle per cm 2 , are attached to the supported lipid B-material bilayer I-material formed from the 9 : 1 mix of dopc : dotap , whereas the surface coverage is approximately 3 . 8 μg / cm 2 ( or roughly 6 × 10 12 particles attached per cm 2 ) for the pah - coated particles under the conditions of the qcm - d experiments . [SEP]
[CLS] yet , the shg signals produced by the attached mpa - coated particles are larger than those obtained from the attached pah - coated particles , pointing to an in - phase , or constructive , interference between the resonantly enhanced and potential - dependent shg terms . [SEP]
[CLS] destructive interference between the two terms probably suppresses shg signal intensities in the case of the pah - aunps B-nanoparticle , which produce less shg signal intensity even though their surface coverages are several orders of magnitude above those we estimate for the mpa - aunps B-nanoparticle . [SEP]
[CLS] this interpretation of the data is subject to the provision that the electronic properties relevant for the shg process of nanoparticles B-nanoparticle , which can be assessed from their uv−vis absorption spectra , are largely invariant during the experiments , which control experiments shown in figure s2 indicate to be the case . [SEP]
[CLS] yet , we caution that the subtle changes at 240 and 570 nm that are seen in the uv−vis spectra may influence the extent of shg resonance enhancement . [SEP]
[CLS] 5b shows that the interaction of the particles is largely irreversible at all particle concentrations studied in shg experiments , at least over the time frames surveyed . [SEP]
[CLS] trends in the shg intensity may be attributed to slight systematic drifts in the laser pulse energy , which were minimized by maintaining the incident pulse energy to within 2 . 5 % of 0 . 4 μj before and after recording each shg vs time trace and monitored by recording the shg intensity from a nonlinear crystal , potassium B-material dihydrogen phosphate ( kdp ) turned to its phasematching angle , simultaneously with the experiments . [SEP]
[CLS] iii . f . controls in the presence of aunps B-nanoparticle . [SEP]
[CLS] given that the measurements we report here are the first shg studies tracking aunps B-nanoparticle interacting with supported lipid B-material bilayers I-material , we briefly discuss control experiments to assess whether the signals recorded at 300 nm were indeed due to second harmonic generation , as opposed to fluorescence B-property or other processes produced by the particles . [SEP]
[CLS] 5c shows no evidence for fluorescence B-property entering the photomultiplier , and the spectral bandwidth corresponds to second harmonic of a 120 fs input pulse at a fundamental wavelength of 612 nm . [SEP]
[CLS] any departure from a symmetric line shape is attributed to pulse chirping . [SEP]
[CLS] 5d shows that the shg intensity depends quadratically on the incident pulse energy , as expected for shg . [SEP]
[CLS] finally , figure 5e shows that the shg signal is well polarized along the surface normal under our experimental conditions . [SEP]
[CLS] additional control experiments ( figure s14 ) show that the free pah ligands held at concentrations equivalent to those expected to be representative of the nanoparticles B-nanoparticle do not produce appreciable changes in the shg or sfg intensity when they interact with the bilayers ( or the plain fused silica substrate ) at 0 . 1 m nacl and 0 . 01 m tris buffer . [SEP]
[CLS] iii . g . nanoparticles B-nanoparticle interact with suspended vesicles . [SEP]
[CLS] prior to carrying additional experiments on the supported lipid B-material bilayers I-material , we wanted to evaluate whether the positively and negatively charged particles interacted similarly in the absence of the sio 2 substrate and whether any changes occurred in the chemical environment of the lipids B-material and the ligands upon attachment . [SEP]
[CLS] we therefore carried out a set of h nmr experiments in which we exposed gold B-material metal B-nanoparticle nanoparticles I-nanoparticle to suspensions of vesicles prepared from the lipids B-material used to prepare the supported lipid B-material bilayers I-material . [SEP]
[CLS] while nmr is highly sensitive to changes in the chemical environment through the chemical shift tensor , it requires fairly high concentrations , which is why we used vesicles in solution . [SEP]
[CLS] the resonances of surface - bound ligands have characteristic features : relative to free ligands , bound ligands typically display broadened line widths ( a result of decreased relaxation times ) and upfield chemical shifts . [SEP]
[CLS] from the presence or absence of these features , we extracted molecular - level information about changes in surface chemistry as the particles interact with the lipid B-material vesicles . [SEP]
[CLS] lipid B-material vesicle chemical shifts were assigned as shown in figure 6 . [SEP]
[CLS] the lipid B-material vesicles feature three prominent broad peaks for the headgroup ( −n−ch 3 , 3 . 15 ppm ) and aliphatic ( −ch 2 − , 1 . 20 ppm , and −r−ch 3 , 0 . 80 ppm ) protons . [SEP]
[CLS] the mpafunctionalized aunps B-nanoparticle exhibit narrow nmr peaks that are typical for small molecules freely tumbling in solution , such as mpa detached from the nps B-nanoparticle . [SEP]
[CLS] mpa is a short enough ligand ( and all its protons are located sufficiently close to the np B-nanoparticle core B-material ) that any resonances from attached mpa are almost certainly broadened beyond detection , and we would not expect to see them in the spectrum . [SEP]
[CLS] given that we expect some free ligand to be in solution , it is likely that the narrow resonance peaks in the mpa - aunp B-nanoparticle spectrum arise from this small amount of detached mpa ( the low signal - to - noise ratio points to how small this amount is ) . [SEP]
[CLS] it is clear that mpa is attached to the gold B-material because the particles would not be stable or well - dispersed without that passivating layer . [SEP]
[CLS] finally , the pah - wrapped aunps B-nanoparticle show citrate signatures near 2 . 5 ppm and broadened nmr features consistent with the presence of bound pah ligand . [SEP]
[CLS] the 9 : 1 dopc : dotap vesicles ( 12 mm ) were first allowed to interact with free pah ( 10 nm ) for 2 h in tris buffer in d 2 o ( 0 . 1 m nacl , 0 . 01 m tris , pd 7−8 ) , at which point nmr spectra were acquired . [SEP]
[CLS] if the particle or polymer B-material were to attach to the outer surface of the liposome B-nanoparticle , one would expect the lipid B-material signalsparticularly those of the headgroup protonsto broaden or shift . [SEP]
[CLS] alternatively , if the liposome B-nanoparticle were to fragment , mobilization of the aliphatic chains should give rise to sharper peaks . [SEP]
[CLS] yet , the chemical shifts and line widths of the lipid B-material peaks remain the same , suggesting that the liposome B-nanoparticle does not undergo substantial changes in morphology or surface chemistry upon interaction with the free pah ligand . [SEP]
[CLS] the 9 : 1 dopc : dotap vesicles ( 12 mm ) were then allowed to interact with pah - aunps B-nanoparticle ( 10 nm ) and mpa - aunps B-nanoparticle ( 10 nm ) particles for 2 h in tris buffer in d 2 o ( 0 . 1 m nacl , 0 . 01 m tris , pd 7−8 ) , at which point the nmr spectra were acquired . [SEP]
[CLS] 6 shows that the nmr response of the dopc : dotap vesicles barely changes upon interaction with the mpa - aunps B-nanoparticle . [SEP]
[CLS] in contrast , when the pah - aunps B-nanoparticle interact with dopc : dotap vesicles , the nmr signals from the chain group methylene group ( 1 . 38 ppm ) , chain group methine group ( 1 . 85 ppm ) , and side group methylene group ( 2 . 91 ppm ) shift downfield by 0 . 02−0 . 03 ppm and appear between those of free pah ( shown in the supporting information ) and the pah - aunps B-nanoparticle . [SEP]
[CLS] this response is consistent with the notion that the surface - bound pah loosens its attachment to the particle upon contact with the lipid B-material vesicles , more resembling free pah , at least in terms of what is measured in nmr . [SEP]
[CLS] meanwhile , we find that the lipid B-material vesicle itself is not structurally affected . [SEP]
[CLS] iii . h . apparent free energies of particle attachment . [SEP]
[CLS] having verified qualitatively similar behavior for mpa - and pah - coated particles interacting with vesicle suspensions and supported lipid B-material bilayers I-material , we returned to the supported lipid B-material bilayers I-material , as that system enables us to probe binding interactions without the need for quantitative separations or competitive binding assays . [SEP]
[CLS] specifically , we proceeded to assess the role of ionic strength on the interaction of the pah - and mpa - aunps B-nanoparticle with supported lipid B-material bilayers I-material prepared from 9 : 1 mixtures of dopc / dotap by recording shg adsorption isotherms at 0 . 001 , 0 . 01 , and 0 . 1 m nacl . [SEP]
[CLS] as shown in figure 7a , ionic strength plays a role in this interaction in terms of shg signal intensities : we observed larger increases in i shg with increasing concentrations of pah - aunps B-nanoparticle at lower vs at higher ionic strength , indicative of a larger amount of attachment of the pah - aunps B-nanoparticle to the bilayer at low ionic strength . [SEP]
[CLS] this result points to the importance of the hydrodynamic particle diameter in determining particle surface coverages at a given ionic strength . [SEP]
[CLS] 7 also shows that the inflection points in the adsorption isotherms occur , within an order of magnitude , at similar particle concentrations for all ionic strengths and ligand−particle combinations surveyed . [SEP]
[CLS] this finding indicates that the apparent free energy of attachment per mole of particles is largely independent of ionic strength and ligand identity , again for the conditions surveyed . [SEP]
[CLS] to provide a rough estimate of the interaction energy , we fit our shg data for the mpa - aunps B-nanoparticle interacting with the 9 : 1 dopc : dotap supported lipid B-material bilayer I-material at 0 . 1 m salt B-material to the standard langmuir adsorption model [SEP]
[CLS] the shg adsorption isotherms indicate leveling off of the shg signal intensity at higher particle concentrations , i . e . , surface coverage saturation , thereby satisfying a key model assumption . [SEP]
[CLS] another key assumption in the langmuir model , however , namely reversibility , may not be met as seen by shg ( figure 5b ) , at least over the time scale of our experiments . [SEP]
[CLS] despite the fact that this and other assumptions of the langmuir model are probably not met , even under conditions of thermal averaging of the heterogeneous nature of a fluid lipid B-material bilayer I-material , the model has been used previously to describe the attachment of nanoparticles B-nanoparticle and proteins B-material to lipid B-material bilayers I-material with the rationale that the desorption rate is too slow to be measured over reasonable time scales . [SEP]
[CLS] in our preliminary analysis , we fit the langmuir adsorption isotherm either to the e shg values computed by taking the square root of the shg intensity at various nanoparticle B-nanoparticle concentrations , subtracting the nonresonant background , and normalizing to the largest e shg value at monolayer coverage ( figure 7b , c ) or to the shg intensities ( data not shown ) . [SEP]
[CLS] this latter fit was performed to account for the possibility that resonantly enhanced shg signals from 4 nm aunps B-nanoparticle adsorbed to supported lipid B-material bilayers I-material that are detected at the photon multiplier may be the incoherent portion of the shg signal produced at the interface . [SEP]
[CLS] just like in solution , the aunps B-nanoparticle should be randomly attached to the bilayer , in which case the phase relationship among the shg e - field produced by each particle should be random , provided that the particles are separated over distances approaching or exceeding the shg coherence length ( which is likely given the particle surface coverage estimates discussed above ) . [SEP]
[CLS] the fit using e - fields yielded an apparent equilibrium constant , k l app , of ( 1 . 8 ± 0 . 5 ) × 10 12 m −1 for 0 . 1 m nacl , corresponding to apparent adsorption free energies of −80 ± 0 . 7 kj / mol of particles attached to the supported lipid B-material bilayer I-material formed from the 9 : 1 mix of dopc : dotap when using the 55 . 5 molarity of water B-material as a standard state for adsorption from solution . [SEP]
[CLS] fits obtained from the shg intensity , i shg , as opposed to e - fields , e shg , produced equilibrium constants that are 10 % smaller , with comparable confidence intervals . [SEP]
[CLS] these energies are the observed apparent adsorption free energies , which include coulombic and non - coulombic interactions as well as entropic contributions from water B-material molecules and counterions being released into the bulk during the attachment process . [SEP]
[CLS] to put these apparent free energies into perspective , we first consider hydrogen B-material bonding : if we assume the strength of an average hydrogen B-material bond to be between 15 and 25 kj / mol , 3−5 hydrogen B-material bonds would be involved in the interaction , if it were to be indeed langmuirian and dominated by h - bonding . [SEP]
[CLS] alternatively , if van der waals interactions , such as those between two methylene groups ( [UNK] . 5 kj / mol ) of the lipids B-material and ligands , were to dominate the interaction , then 30−35 such groups would be involved . [SEP]
[CLS] these estimates represent a first step toward quantifying how many interactions are involved when nanoparticles B-nanoparticle interact with supported lipid B-material bilayers I-material under the conditions of our experiments . [SEP]
[CLS] departures of the recorded shg responses from the standard langmuir adsorption model are likely due to the irreversibility of attachment and lateral particle−particle interactions . [SEP]
[CLS] an analysis of such interactions requires high - quality adsorption isotherms , which are unfortunately not produced by this system , even from triplicate measurements . [SEP]
[CLS] we emphasize again that the inflection points in the recorded adsorption isotherms occur at similar particle concentrations for all ionic strengths and ligand−particle combinations surveyed , indicating that the apparent free energy of attachment per mole of particles is largely independent of ionic strength and ligand identity for the conditions surveyed . [SEP]
[CLS] iii . j . [SEP]
[CLS] estimating the charge per attached nanoparticle B-nanoparticle . [SEP]
[CLS] to further assess whether the mpa - and the pah - aunps B-nanoparticle are charged when they attach to the lipid B-material bilayer I-material and to learn about the importance of coulombic interactions in addition to a possible role of h - bonding , we carried out charge screening experiments on the attached particles . [SEP]
[CLS] in these experiments , we change the nacl concentration while monitoring the shg signal intensity at constant nanoparticle B-nanoparticle concentration , which should maintain conditions of constant particle coverage according to the irreversible attachment of aunps B-nanoparticle to the bilayers ( figure 5b ) . [SEP]
[CLS] 8 shows the saltdependent shg response obtained from a supported lipid B-material bilayer I-material formed from a 9 : 1 mixture of dopc / dotap maintained at 0 . 1 m nacl and 0 . 01 m tris buffer and subsequently exposed to 1 nm mpa - and pah - aunps B-nanoparticle relative to the signal intensity obtained from the bilayer at the same salt B-material and buffer conditions . [SEP]
[CLS] decreases in the salt B-material concentration result in shg intensity gains , consistent with the χ effect . [SEP]
[CLS] assuming the aggregation state and the optical properties of the particles attached to the bilayers are invariant with ionic strength over the course of the experiment , we fit a gouy−chapman model to the shg field vs nacl concentration data . [SEP]
[CLS] when expressing the total charge density additively by summing the fused silica charge density , the trish + charge density , the dopc / dotap bilayer charge density , and the nanoparticle B-nanoparticle charge density , fit parameters with errors that were 2−10 times larger than the point estimates were obtained for the pah - aunps B-nanoparticle , indicating the total charge density ( bilayer plus particles ) ranges somewhere between −0 . 06 and + 0 . 02 c / m 2 . [SEP]
[CLS] the gouy−chapman model analysis is based on mean field theory , and the charge density term for the nanoparticles B-nanoparticle yields the net number of coulombs per m 2 associated with all particles present , irrespective of how many particles are present ( e . g . , not the charge per nanoparticle B-nanoparticle ) . [SEP]
[CLS] computing the charge density per nanoparticle B-nanoparticle requires knowledge of how many particles are present per m 2 . [SEP]
[CLS] qcm - d experiments conducted at 1 nm pah - aunp B-nanoparticle indicate roughly 1 × 10 11 particles attached per cm 2 , which is an upper bound as the mass sensed by qcm - d includes hydrodynamically coupled water B-material . [SEP]
[CLS] after taking into account the bilayer charge density ( −0 . 015 ( + 0 . 003 / −0 . 004 ) c / m 2 ) , the total charge density per pah - aunp B-nanoparticle is estimated from the gouy−chapman model fit to be between −4 . 5 × 10 −17 and + 3 . 5 × 10 −17 c ; i . e . , they are associated with anywhere between approximately 200 positive or 200 negative charges . [SEP]
[CLS] to understand this finding , we consider the following : the independent measurements of adsorption isotherms recorded at varying salt B-material concentrations ( section iii . h ) and of charge screening experiments ( section iii . j ) highlight how important counterions are for the pah - aunp B-nanoparticle system studied here . [SEP]
[CLS] specifically , the [UNK] + 30 mv zeta B-property potentials I-property we obtain for the pah - aunps B-nanoparticle in solution indicate some amount of net positive charge associated with the particles at the zeta plane . [SEP]
[CLS] the pah molecular mass is [UNK] 000 g / mol , with the allylamine repeat B-material unit I-material having a molecular mass of 57 g / mol . [SEP]
[CLS] xps analysis indicated an average of four pah polymers B-material per particle . [SEP]
[CLS] from this result we calculate that approximately 1000 positive charges are available on average at the particle surface , assuming full ionization B-property of the ammonium group at ph 7 . 4 . [SEP]
[CLS] similar to reports that micelles B-material formed at [UNK] mm ionic strength from cetyltrimethylammonium bromide B-material and chloride B-material carry 71 % and 55 % of their cationic B-material charge as counterions , and similar to reports that condensed counterions are key to forming dna duplexes , 70−78 origami , colloidal crystals , and even spherical nucleic B-material acids I-material from otherwise highly charged single strands , the pah - aunps B-nanoparticle studied here are likely to contain condensed ( i . e . , strongly coordinated ) counterions that substantially reduce the effective charge density . [SEP]
[CLS] upon attachment to the supported lipid B-material bilayer I-material , interaction with the negatively charged phosphate groups in the pc headgroups , and / or any negatively charged counterions associated with the electrical double layer above them , appears to reduce the net charge of the attached particles even further to somewhere between 200 hundred positive or 200 negative charges per particle . [SEP]
[CLS] an estimate of the number of lipid B-material headgroups that may provide , either by themselves or by means of associated counterions , negative charge to the attached particle is possible by calculating , from the lipid B-material density reported in section iii . a , the number of lipids B-material located within the footprint of the attached particle , which we assume to be 26 nm 2 based on the hydrodynamic radii for these particles at 0 . 001 m nacl ( section ii . b ) . [SEP]
[CLS] this calculation yields 100−200 lipids B-material and / or associated counterions with them that are in close proximity to the attached particle , clearly in the range to allow for nearly quantitative charge neutralization of the particle . [SEP]
[CLS] we emphasize that the range of charges per attached particle includes charge contributions from any charged species located within the hydrodynamic volume , provided they are in the shg active region of the interface . [SEP]
[CLS] in contrast to the positively charged pah - aunps B-nanoparticle , the surface coverage of the negatively charged mpa - aunps B-nanoparticle is estimated to be less than 0 . 01 ng / cm 2 particles , or just 1 × 10 7 aunps B-nanoparticle per cm 2 , the lower end of our estimated xps detection limit . [SEP]
[CLS] yet , the interfacial charge density we obtain from fitting the gouy− chapman model to the charge screening data shown in figure 8 ranges between −0 . 05 and −0 . 01 c / m 2 for the mpa - aunpcontaining supported lipid B-material bilayer I-material , which is largely indistinguishable from the −0 . 06 and + 0 . 02 c / m 2 charge density obtained from the pah - aunp B-nanoparticle - containing supported lipid B-material bilayer I-material . [SEP]
[CLS] with a detection limit of better than 3 × 10 −4 c / m 2 under similar aqueous phase conditions , the shg χ method should be appropriate for determining 10 9 charged mpa - coated particles per cm 2 . [SEP]
[CLS] ( this estimate assumes the mpa particles carry the same net 200 elementary charges as the pah - coated particles , which is reasonable , at least within an order of magnitude , given the similar magnitudes in zeta B-property potential I-property of the two particles types studied here at 0 . 1 m nacl concentration . [SEP]
[CLS] ) yet , the mpa - aunp B-nanoparticle coverages is estimated to be just 1 × 10 7 particles / cm 2 , pointing to the possibility that the attached mpa - aunps B-nanoparticle carry a much larger net negative charge than the attached pah - aunps B-nanoparticle or that the charge per particle is too small to be evaluated using gouy−chapman theory . [SEP]
[CLS] the small surface coverage we estimate for the mpa - aunps B-nanoparticle appears to support , at least given the current set of data , the former case , i . e . , much larger charge density per attached mpa - coated particles than per pah - coated particle . [SEP]
[CLS] this outcome of our analysis is readily understood when considering that the mpaand pah - aunps B-nanoparticle exhibit hydrodynamic diameters of 250 and 70 nm , respectively , at 0 . 1 m nacl concentration , corresponding to a [UNK] - fold increase in volume for the mpa - coated particles to carry condensed , i . e . , strongly bound , ions B-material for repelling like charges . [SEP]
[CLS] our findings thus points toward a mechanism in which the attachment of mpa - and pah - aunps B-nanoparticle is controlled by the hydrodynamic volume and the number of charges within it . [SEP]
[CLS] in summary , we have presented the first comprehensive , multiprobe molecular - level investigation of the nano−bio interface that focuses on the lipids B-material of the bilayer , the metal B-material core B-material of the nanoparticles B-nanoparticle , and the role of molecular structure , electrostatics , and ionic strength in mediating the interactions between bilayers and nanoparticles B-nanoparticle . [SEP]
[CLS] first , we prepared wellcharacterized supported lipid B-material bilayers I-material . [SEP]
[CLS] next , we carried out the first label - free determination of the interfacial charge densities and potentials of supported lipid B-material bilayers I-material by means of nonlinear optics . [SEP]
[CLS] third , we demonstrated the applicability of second harmonic generation for following the interaction of positively and negatively charged aunp B-nanoparticle cores B-material with supported lipid B-material bilayers I-material under varying nanoparticle B-nanoparticle concentrations , while paying particular attention to the role of bilayer order and disorder , as well as electrostatics of the bilayers and the particles . [SEP]
[CLS] special attention was paid to control experiments ruling out , within the constraints of our experiments , exogenous effects and to carrying out complementary studies using nmr , qcm - d , afm , xps , frap , and tof - sims . [SEP]
[CLS] while avoiding overinterpretation of our experimental data and the models we apply to analyze them , the data set we have presented is consistent with an apparent negative charge density of 1 . 8 ± 0 . 5 % of an elementary negative charge for zwitterionic pc head groups in supported lipid B-material bilayers I-material . [SEP]
[CLS] depending on the salt B-material concentration , we found that pc - rich supported lipid B-material bilayers I-material are associated with sizable negative interfacial potentials , irrespective of if they contained minority components of positively or negatively charged lipids B-material . [SEP]
[CLS] structural changes within the alkyl tails of the lipids B-material constituting the bilayers , probed in the c−h stretching region using vibrational sum frequency generation , were found to be negligible for salt B-material concentrations between 0 . 001 and 0 . 1 and 0 . 01 m tris buffer . [SEP]
[CLS] we then quantified the extent and reversibility of nanoparticle−bilayer interactions by recording resonantly enhanced shg signals at 0 . 1 m nacl and 0 . 01 m tris buffer as a function of aunp B-nanoparticle concentration between 10 −14 and 10 −9 m . [SEP]
[CLS] a combination of nonlinear optics , xps , qcm - d , afm , and tof - sims indicates that the supported lipid B-material bilayers I-material interact with positively and negatively charged particles at 0 . 1 m nacl . [SEP]
[CLS] complementary studies of dopc / dotap vesicles suspended in 0 . 1 m nacl and 0 . 01 m tris buffer showed their nmr spectra change little , if at all , upon interaction with the mpa - aunps B-nanoparticle . [SEP]
[CLS] in contrast , nmr spectra are consistent with loosening of the gold B-material - bound pah polymer B-material wrapping upon attachment to the lipid B-material vesicles , while the lipid B-material vesicles themselves are not structurally affected . [SEP]
[CLS] from pah - aunp B-nanoparticle adsorption isotherms recorded using dopc / dotap supported lipid B-material bilayers I-material we calculated apparent adsorption free energies of approximately −80 kj / mol at 0 . 1 m salt B-material concentration . [SEP]
[CLS] the adsorption isotherms appear to be independent of ionic strength , i . e . , interfacial potential . [SEP]
[CLS] qcm - d indicates 1 × 10 11 pah - aunps B-nanoparticle are attached per cm 2 at 1 nm particle concentration and 0 . 1 m salt B-material concentration . [SEP]
[CLS] resonantly enhanced shg signals are larger at 0 . 001 m vs 0 . 1 m salt B-material concentration . [SEP]
[CLS] shg charge screening experiments carried out on the attached pah - aunps B-nanoparticle indicate the presence of about 200 negative or positive charges , which are likely to be distributed over the hydrodynamic volume of each attached particle . [SEP]
[CLS] we propose that the two main contributors to determining the thermodynamics and coverage of pah - coated , 4 nm aunps B-nanoparticle are ( 1 ) multivalent van der waals or hydrogen B-material bonding interactions , which contribute an apparent free energy gain of −80 kj per mol of adsorbing B-property particles that seem to be largely invariant with ionic strength , ( 2 ) the hydrodynamic diameter of the adsorbing B-property particles , which is smaller at low ionic strength than at high ionic strength , a result that reflects the higher shg signal intensities observed at low vs high ionic strength , and ( 3 ) charge−charge repulsion among the attached pah - aunps B-nanoparticle , which limits the surface coverage to the saturation levels observed in the shg isotherms . [SEP]
[CLS] unlike the pah - aunps B-nanoparticle , the attached mpa - aunps B-nanoparticle are likely to be associated with larger charge densities at 0 . 1 m nacl , which includes counterions and any other charged species present within the hydrodynamic volume . [SEP]
[CLS] even though only [UNK] × 10 7 mpa - aunps B-nanoparticle attach to the supported lipid B-material bilayer I-material , the shg signals produced by the former are sizable , pointing to an in - phase , or constructive , interference between the resonantly enhanced and potential - dependent shg terms . [SEP]
[CLS] deconstructive interference between the two terms probably suppresses shg signal intensities in the case of the pah - aunps B-nanoparticle , which produce less shg signal intensity even though their surface coverages are several orders of magnitude above those we estimate for the mpa - aunps B-nanoparticle . [SEP]
[CLS] the quantitative results presented here may serve as important benchmarks for computational and experimental studies of lipid B-material bilayers I-material and may serve to calibrate interaction potentials and spectrochemical labels . [SEP]
[CLS] as an example , the apparent independence of the δg obs app value reported here for the mpa - aunps B-nanoparticle interacting with the dopc : dotap bilayer indicates it is in fact the chemical interaction free energy , with any influences from coulombic interactions being largely negligible . [SEP]
[CLS] this result will be useful in atomistic simulations of how pah - aunps B-nanoparticle may interact with bilayers . [SEP]
[CLS] the findings presented here may also allow predictions of how charged species interact with lipid B-material membranes I-material rich in phosphatidylcholine headgroups . [SEP]
[CLS] the results from our study open the possibility for future work aimed at elucidating the interplay between resonant and nonresonant shg from charged aunps B-nanoparticle , testing how applicable our findings are to other particle / ligand / lipid B-material combinations , surveying particle−bilayer interactions for bilayers containing not just lipids B-material but also other components known to be important in biological membranes , such as lipopolysaccharides , and focusing on the effects of particle size and core B-material composition on the interaction of nanoparticles B-nanoparticle with supported lipid B-material bilayers I-material . [SEP]
[CLS] 1 . molecular structure of the lipids B-material used in this work . [SEP]
[CLS] c−h stretches ( orange , green , and purple ) are probed by sfg . [SEP]
[CLS] interfacial potentials set up by positive ( blue ) and negative ( red ) charges are probed by shg . [SEP]
[CLS] ( a ) total acoustic mass gain , evaluated by qcm - d , for conditions indicated . [SEP]
[CLS] ( b ) formation of supported lipid B-material bilayers I-material from vesicles formed from 9 : 1 mixtures of dmpc / dmpg ( solid line ) and dopc / dotap ( dashed line ) probed by qcm - d at 0 . 1 m nacl . [SEP]
[CLS] the small decrease in mass in the 9 : 1 dmpc : dmpg bilayer commencing at 1000 s is most likely due to the removal of loosely attached intact vesicles during rinsing with ca 2 + - free solution . [SEP]
[CLS] ( c , d ) afm ( 5 × 5 μm 2 ) images of bilayers formed from dmpc / dmpg and dopc / dotap at 0 . 1 m nacl . [SEP]
[CLS] ( e ) normalized ssp - polarized sfg spectra of supported lipid B-material bilayers I-material at ph 7 . 4 and in the presence of 0 . 01 m tris ( gray ) for 0 . 001 m ( green ) and 0 . 1 m ( red ) nacl using vesicles formed from 9 : 1 dmpc / dmpg ( top pair ) and dopc / dotap ( bottom pair ) mixtures . [SEP]
[CLS] 3 . normalized shg response ( symbols ) and gouy−chapman fits ( lines ) as a function of nacl concentration at ph 7 . 4 and 0 . 01 m tris buffer for plain fused silica ( black [UNK] ) and bilayers prepared from pure dopc ( green [UNK] ) and 9 : 1 mixtures of dopc / dotap ( blue ⊕ ) and dmpc / dmpg ( red [UNK] ) . [SEP]
[CLS] ( a ) acoustic mass gains determined by qcm - d upon 60 min exposure of a supported lipid B-material bilayer I-material formed from a 9 : 1 mixture of dopc and dotap to 12 . 8 nm positively charged pah - coated and negatively charged mpa - coated 4 nm spherical aunps B-nanoparticle in 0 . 01 m tris buffer and 0 . 1 m nacl at ph 7 . 4 . [SEP]
[CLS] ( b , c ) topography image of bilayers formed from a 9 : 1 mixture of dopc and dotap in 0 . 01 m tris , 0 . 1 m nacl , ph 7 . 4 , on an ultraflat thermal sio 2 substrate wafer before ( b ) and after ( c ) exposure to mpa - aunps B-nanoparticle ( 10 nm ) . [SEP]
[CLS] images from the same experiment carried out on a freshly prepared bilayer and adjusted to the same scale . [SEP]
[CLS] ( d ) normalized tof - sims spectra of 9 : 1 dopc : dotap bilayer before ( bottom ) and after ( top ) interaction with 1 nm mpa - aunps B-nanoparticle at room temperature and in 0 . 1 m nacl and ph 7 . 4 ( 0 . 01 m tris buffer ) and subsequently rinsed with buffer [SEP]
[CLS] bilayer samples were prepared in and then removed from the flow cell B-material following rinsing and allowed to dry prior to analysis . [SEP]
[CLS] ( a ) shg intensity as a function of nanoparticle B-nanoparticle concentration for a supported lipid B-material bilayer I-material formed from a 9 : 1 mixture of dopc and dotap upon exposure to negatively charged mpa - aunps B-nanoparticle ( red [UNK] ) and positively charged pah - aunps B-nanoparticle ( blue ⊕ ) . [SEP]
[CLS] figure 5 . p - in / all - out polarization combination , λ shg = 306 nm , 0 . 1 m nacl and 0 . 01 m tris buffer . [SEP]
[CLS] uncertainties on each shg e - field is below 1 % as given by the poisson statistics of photon counting . [SEP]
[CLS] reproducibility of the experiments is assessed by performing the experiments in duplicate for the pah - aunps B-nanoparticle and in triplicate for the mpa - aunps B-nanoparticle . [SEP]
[CLS] see text for details . [SEP]
[CLS] ( b ) shg intensity as a function of time at 305 nm in the presence of a bilayer formed from a 9 : 1 mixture of dopc and dotap in the presence of 10 −9 m mpa ( red , offset by 1 . 5 ) , 10 −11 m mpa ( pink , offset by 1 . 25 ) , and 10 −11 m pah ( blue ) . [SEP]
[CLS] at t = 500 s nanoparticles B-nanoparticle were introduced to the bilayer at 0 . 1 m nacl and 0 . 01 m tris . [SEP]
[CLS] a buffer rinse was introduced to the bilayer system at t = 1500 s . [SEP]
[CLS] ( c ) shg intensity as a function of monochromator wavelength collected using the p - in / all - out polarization combination and gaussian fit resulting in 3 . 2 ± 0 . 1 nm bandwidth for 0 . 1 nm concentration of pah - coated ( blue ⊕ ) and 3 . 1 ± 0 . 1 nm for 0 . 1 nm concentration of 4 nm mpa - aunps B-nanoparticle ( red [UNK] , offset by 140 ) interacting with a supported lipid B-material bilayer I-material formed from a 9 : 1 mixture of dopc and dotap at 0 . 1 m nacl and 0 . 01 m tris buffer . [SEP]
[CLS] any departure from the gaussian fit function is due to chirping of the pulse . [SEP]
[CLS] ( d ) shg intensity collected using the p - in / all - out polarization combination as a function of pulse energy at 612 nm for 1 nm concentration of pah - coated 4 nm aunps B-nanoparticle interacting with a supported lipid B-material bilayer I-material formed from a 9 : 1 mixture of dopc and dotap at 0 . 1 m nacl and 0 . 01 m tris buffer . [SEP]
[CLS] blue curve is a power function of the form y = a + bx p , where p = 2 . 28 ± 0 . 06 . [SEP]
[CLS] ( e ) shg intensity at 306 nm collected as a function of polarizer analyzer angle while probing with p - polarized fundamental light for 0 . 1 nm concentration of pah - coated ( blue ⊕ , bottom ) and 10 pm ( pink [UNK] , offset by 80 ) and 1 nm ( red [UNK] , offset by 80 ) concentrations of 4 nm mpa - aunps B-nanoparticle interacting with a supported lipid B-material bilayer I-material formed from a 9 : 1 mixture of dopc and dotap at 0 . 1 m nacl and 0 . 01 m tris buffer . [SEP]
[CLS] 6 . normalized proton nmr spectra of ( bottom to top ) vesicles formed from a mixture of 9 : 1 dopc : dotap , mpa - aunps B-nanoparticle , mpa - aunps B-nanoparticle interacting with vesicles formed from a mixture of 9 : 1 dopc : dotap , pah - aunps B-nanoparticle , and pah - aunps B-nanoparticle interacting with vesicles formed from a mixture of 9 : 1 dopc : dotap . [SEP]
[CLS] all measurements carried out at room temperature and in 0 . 1 m nacl and ph 7 . 4 ( 0 . 01 m tris ) . [SEP]
[CLS] see text for details . [SEP]
[CLS] shg intensity as a function of nanoparticle B-nanoparticle concentration for a supported lipid B-material bilayer I-material formed from a 9 : 1 mixture of dopc and dotap upon exposure to positively charged pah - aunps B-nanoparticle ( a ) and negatively charged mpa - aunps B-nanoparticle ( b ) at 0 . 001 m ( dark blue and red ) , 0 . 01 m ( blue and red ) , and 0 . 1 m ( light blue and red ) nacl . [SEP]
[CLS] figure 7 . p - in / all - out polarization combination , λ shg = 306 nm , and 0 . 01 m tris buffer . [SEP]
[CLS] uncertainties on each shg e - field is below 1 % as given by the poisson statistics of photon counting . [SEP]
[CLS] ( c ) shg e - field normalized to the maximum e - field at high particle concentration and referenced to zero particle concentration for 4 nm aunps B-nanoparticle coated with negatively charged mpa ligands recorded at 0 . 01 m tris buffer , ph 7 . 4 , 298 k , and 0 . 1 m nacl . [SEP]
[CLS] the solid lines represent a fit of the langmuir adsorption model to the experimental data , specifically θ = k l c / ( 1 + k l c ) , where k l is the apparent equilibrium attachment constant , c is the particle concentration , and θ is the relative shg e - field . [SEP]
[CLS] the thin lines indicate the upper and lower bounds of the langmuir adsorption isotherm fit based on the uncertainty in the equilibrium constant . [SEP]
[CLS] normalized shg response ( symbols ) as a function of nacl concentration at ph 7 . 4 and 0 . 01 m tris buffer for bilayers prepared from 9 : 1 mixtures of dopc and dotap ( green [UNK] ) in the presence of 1 nm 4 nm aunps B-nanoparticle coated with pah ( blue ⊕ ) ligands or mpa ( red [UNK] ) , referenced to the shg e - field obtained at 0 . 1 m nacl and in the absence of nanoparticles B-nanoparticle . [SEP]
[CLS] solid lines are best fit of a gouy−chapman model to the data . [SEP]
[CLS] each value is calculated as described in the supporting information . [SEP]
[CLS] herein , we describe a rapid , divergent method for using spherical nucleic B-material acids I-material ( snas ) as a universal platform for attaching rna to dna - modified nanoparticles B-nanoparticle using enzymemediated techniques . [SEP]
[CLS] this approach provides a sequence - specific method for the covalent attachment of one or more in vitro transcribed rnas to a universal sna scaffold , regardless of rna sequence . [SEP]
[CLS] the rnaananoparticle constructs are shown to effectively knock down two different gene targets using a single , dual - ligated nanoparticle B-nanoparticle construct . [SEP]
[CLS] nature has engineered rapid , highly sequence - specific enzymes capable of repairing spliced rna and dna molecules . [SEP]
[CLS] these enzymes , known as ligases , catalyze the covalent attachment of the 3 0 hydroxyl B-material group I-material of one oligonucleotide to the 5 0 phosphate of another 1a3 with remarkable specificity . [SEP]
[CLS] this specificity is made possible by the sequence information encoded in the terminal ends of oligonucleotide strands prior to their attachment to a given nucleic B-material acid I-material sequence . [SEP]
[CLS] indeed , ligation reactions have become powerful tools in various recombinant dna - based technologies , 4a6 in genome sequencing , and for synthesizing dnaarna heterostructures . [SEP]
[CLS] certain nucleic B-material acid I-material functionalized nanostructures have emerged as effective new tools for molecular diagnostics and intracellular gene regulation . [SEP]
[CLS] specifically , nanoparticles B-nanoparticle conjugated to small interfering rna ( sirna ) have gained significant attention due to their ability to target mrna for degradation in a sequence - specific manner . [SEP]
[CLS] despite their promising regulatory role in gene expression , sirnas suffer from their inherent chemical instability in cellular environments . [SEP]
[CLS] many researchers have therefore developed methods for conjugating sirna to the surface of nanoscale materials to improve the chance of successful delivery into cells B-material . [SEP]
[CLS] chemical strategies including disulfide modification , pegylation , and the condensation of sirna using polycations have been used to facilitate the assembly of sirnas on nanoparticles B-nanoparticle . [SEP]
[CLS] such constructs are typically synthesized by first functionalizing an oligonucleotide with an unnatural binding group ( e . g . , alkylthiols or alkylamines ) that can be adsorbed onto a particle of interest . [SEP]
[CLS] this approach requires a specialty oligonucleotide to be synthesized for every nanoconstruct that one envisions . [SEP]
[CLS] a potentially more appealing approach would be to prepare a universal construct that in a divergent manner can be used with readily accessible synthons to prepare a large set of constructs with the nucleic B-material acids I-material of interest . [SEP]
[CLS] in this regard , it would be extremely useful to be able to utilize ligases to interface relatively high cost rna sequences with a readily available , low - cost dna - nanoparticle B-nanoparticle construct . [SEP]
[CLS] herein , we describe a method for assembling sirna on dna - based spherical nucleic B-material acid I-material ( sna ) gold B-nanoparticle nanoparticle I-nanoparticle conjugates via a t4 dna ligase - catalyzed reaction ( figure 1 ) . [SEP]
[CLS] this is done in the context of a universal dna - based sna gold nanoparticle I-nanoparticle conjugate . [SEP]
[CLS] snas often consist of a gold B-nanoparticle nanoparticle I-nanoparticle core B-material that has been densely functionalized with dna or sirna oligonucleotides modified with terminal alkylthiols . [SEP]
[CLS] snas have been shown to exhibit many desirable qualities as cellular transfection and gene regulation materials , including high cellular uptake and resistance to certain enzymatic degradation pathways . [SEP]
[CLS] importantly , we find here that the sterically limiting sna structure and its high local salt B-material concentration , which can impede certain enzymatic systems , does not prohibit enzymatic ligation . [SEP]
[CLS] moreover , the directionality of the dna afforded by the sna template allows one to attach rna to specific ends of a nucleic B-material acid I-material sequence and avoids nonproductive cyclization reactions . [SEP]
[CLS] enzyme - mediated assembly of sirna on snas has two key advantages over the direct attachment of a chemically modified rna to the surface of a nanoparticle B-nanoparticle . [SEP]
[CLS] the first advantage is the practical reduction in cost and time it takes to assemble in vitro transcribed sirna onto nanoparticles B-nanoparticle as opposed to synthesizing chemically modified rna on an automated rna synthesizer . [SEP]
[CLS] extensive purification and post processing of the modified rna sequence over several days is required prior to use in assembly of an rnaa nanoparticle construct . [SEP]
[CLS] in the case of in vitro rna transcription B-event , the reaction requirements are t7 rna polymerase , a high - yielding rna synthesizing enzyme , a short double - stranded dna ( dsdna ) template , and millimolar solutions of atp , gtp , ctp , utp , and gmp . [SEP]
[CLS] these individual nucleotide solutions in the presence of the dsdna template and t7 rna polymerase can generate high micromolar quantities of rna in solution in under 2 h . [SEP]
[CLS] in addition , the direct 5 0 modification of the rna with gmp during its enzymatic synthesis removes the need for post synthetic chemical modification of the rna . [SEP]
[CLS] the second major advantage is programmability , as one can design the ligation reaction components to preassemble specific rna sequences at an sna surface based on the sequence information encoded in 2a , the dna bridge ( figure 1 ) . [SEP]
[CLS] the initial step of the ligation reaction involves two oligonucleotides ( one dna 1 , the other rna 3a ) that are to be covalently attached through the generation of a new phosphodiester bond . [SEP]
[CLS] these oligonucleotides are assembled end - to - end ( 3 0 oh of dna to 5 0 po of rna ) through hybridization to 2a . [SEP]
[CLS] the dna bridges therefore provide a powerful way to add one or more unique rna sequences to the surface of an sna in a single one - step enzymatic reaction . [SEP]
[CLS] once assembled , ligase can be added to the solution , resulting in the covalent attachment of the dna anchor to the adjacent rna oligonucleotide . [SEP]
[CLS] it is important to note that one key requirement for the ligation of rna to dna is that the rna 5 0 end must have a guanosine monophosphate ( gmp ) as opposed to the native triphosphate ( gtp ) . [SEP]
[CLS] this difference in phosphate units is a requirement for compatibility with the enzyme ' s mechanism of attachment and the need for atp when sealing the phosphodiester bond between the different oligonucleotide strands ( figure 1 ) . [SEP]
[CLS] incorporation of a 5 0 gmp modification is achieved via the in vitro transcription B-event of rna , wherein an excess of gmp relative to gtp ( 30 : 1 ) results in the end - labeling of the rna transcript B-event . [SEP]
[CLS] once modified [SEP]
[CLS] ( 1 ) [SEP]
[CLS] a second dna oligonucleotide , the dna " bridge " ( 2a ) , is complementary to the 3 0 end of 1 . [SEP]
[CLS] upon hybridization with 1 , the sticky end generated by the dna bridge ( 2a ) is used to guide the hybridization and assembly of the 5 0 end of an rna ( 3a ) to the sna surface . [SEP]
[CLS] for sirna sequences , this strand is the rna sense strand of an sirna duplex . [SEP]
[CLS] ( b ) by changing the sequence of the sticky end of the dna bridge at its 3 0 end ( 2b ) , it can be made complementary to the 5 0 end of a different rna oligonucleotide ( 3b ) , thus allowing different rna sequences to be assembled on an sna passivated with the same dna anchor ( 1 ) . [SEP]
[CLS] articlewith a terminal gmp residue , the rna is purified and ligated to the sna . [SEP]
[CLS] in order to investigate the ligation efficiency of t4 dna ligase for attaching rna to a dna - functionalized nanoparticle B-nanoparticle , snas were prepared using a 5 0 hexyl dithiol dna anchor ( 1 ) ( supporting information , table s1 ) . [SEP]
[CLS] note that oligonucleotides may also be immobilized using readily available cyclic disulfides . [SEP]
[CLS] to accurately determine the average number of rna strands ligated to the surface of the sna , a new photocleavable assay was developed . [SEP]
[CLS] in this assay , a second dna anchor was synthesized in which a photocleavable ( pc ) linker was incorporated near the 3 0 end of dna anchor ( figure s1 , supporting information ) . [SEP]
[CLS] upon irradiation with 365 nm light , the pc linker was cleaved , freeing the hybrid rnaadna oligonucleotides from the nanoparticle B-nanoparticle surface . [SEP]
[CLS] the freed oligonucleotides were then isolated from the nanoparticles B-nanoparticle in solution via centrifugation and quantified using a fluorescence B-property intercalation assay . [SEP]
[CLS] for 13 nm gold B-nanoparticle nanoparticles I-nanoparticle initially functionalized with 80 dna anchor strands , there were 65 ( 5 rna strands ligated per particle . [SEP]
[CLS] post ligation , the complementary antisense ( as ) sirna oligonucleotide was hybridized to the covalently attached sense strand ( 3a ) in the presence of 1a pbs at 37 °c for 1 h . [SEP]
[CLS] the total number of sirna duplexes formed was 35 ( 5 , illustrating that this approach can yield comparable coverages to that of conventional sirna sna architectures . [SEP]
[CLS] we also assessed the increase in average particle size accompanying the enzymatic ligation reaction using dynamic B-technique light I-technique scattering I-technique ( dls ) and agarose B-technique gel I-technique electrophoresis I-technique ( figure 2 ) . [SEP]
[CLS] after ligation , the particles were washed with 8 m urea and centrifuged to remove any excess dna bridge that may remain after the ligation reaction was completed . [SEP]
[CLS] dls measurements showed the average size and polydispersity of the particles increased after ligation ( table s2 , supporting information ) . [SEP]
[CLS] after characterization of the ligated sirna snas , it was necessary to ensure that the biochemical recognition and therapeutic function of the sirna was maintained after undergoing the enzymatic ligation strategy . [SEP]
[CLS] to evaluate this , ligated rna snas containing a sirna sequence targeting green fluorescent B-property protein B-material ( gfp ) mrna were fluorophore labeled with cyanine 5 ( cy5 ) to track the constructs intracellularly . [SEP]
[CLS] incubation B-technique of the cy5 - labeled sirnaasnas with a gfp - expressing cell B-material line allows for simultaneous assays of uptake and biological function using flow B-technique cytometry I-technique . [SEP]
[CLS] gfp fluorescence B-property is then used as a proxy to infer relative gfp protein B-material levels . [SEP]
[CLS] the ligated gfp sirna sna constructs were readily taken up into mouse endothelial cells B-material ( figure 3aad ) and able to reduce the expression of gfp protein B-material by up to 53 % relative to untreated cells B-material ( figure 3e ) . [SEP]
[CLS] after establishing that the ligated sirna snas maintain their biological activity , a second , dual sirna construct consisting of two different sirna sequences was assembled using the hybrid rnaadna ligation reaction . [SEP]
[CLS] the ability to attach two different sirna sequences to the same nanoparticle B-nanoparticle would enable a single construct to simultaneously target two different mrnas in the same cell B-material . [SEP]
[CLS] an sirna targeting gapdh ( a ubiquitous housekeeping gene ) was chosen as a second sequence to ligate alongside gfp sirna on the sna . [SEP]
[CLS] the sense strand for each sirna sequence was in vitro transcribed and ligated to the universal sna scaffold . [SEP]
[CLS] all sequences used in the construction of the dual sirna snas are shown in table s1 ( supporting information ) . [SEP]
[CLS] each sense strand ( 3a and 3b ) was added to the ligation reaction in equal concentrations , allowing the 5 0 end of the individual rna sense strands ( gfp or gapdh sirna ) to assemble at the 3 0 end of the sna ' s dna anchor ( 1 ) via hybridization with its respective dna bridge ( 2a and 2b ) ( table s1 , supporting information ) . [SEP]
[CLS] after ligation of the sirna sense strands to the sna scaffold , the constructs were incubated B-technique with both gfp and gapdh antisense strands ( 4a and 4b ) to generate the final duplexed version of the particle . [SEP]
[CLS] in order to track the ability of the as sirnas to hybridize to the same sna as well as to visualize the constructs ' post to investigate the sequence - specific rnai capability of the dual sirna sna nanoparticle B-nanoparticle construct , the duplexed sirna particles were incubated B-technique with c166 gfp - expressing cells B-material ( cells which also express the ubiquitous protein B-material gapdh ) overnight . [SEP]
[CLS] after treatment , the cells B-material were lysed and the individual protein B-material expression levels of gfp and gapdh proteins B-material were evaluated . [SEP]
[CLS] under this dual treatment , there was substantial knockdown of both gfp and gapdh proteins B-material as shown by western blots , indicating both ligated sirna sequences are biologically active ( figure 5 ) . [SEP]
[CLS] as shown by densitometry quantification of averaged triplicate western blots ( right panel , figure 5 ) , the effects of the dual sirna sna were comparable to those produced by traditional polymer - assisted sirna transfections . [SEP]
[CLS] in summary , we have developed a robust and straightforward biochemical strategy for assembling rna at the surface of an sna while preserving its biological function . [SEP]
[CLS] using this method , two or more in vitro transcribed rnas can be attached to an sna through the use of programmable , sequence - specific dna bridges . [SEP]
[CLS] this enzyme - mediated approach opens up a rapid route for preparing single rnaananoparticle constructs capable of targeting multiple sites on a single mrna transcript B-event , as well as the ability to study the synergistic effects of targeting two different mrna transcripts B-event in related biochemical pathways . [SEP]
[CLS] as the sna assembly method has recently been expanded to include myriad nanoscale starting materials , the assembly of rna onto a variety of different materials can be realized quickly and efficiently , accelerating the study of rnaanp - based delivery platforms for a wide array of biological applications . [SEP]
[CLS] sna assembly and characterization [SEP]
[CLS] au nps B-nanoparticle ( 13 nm ) were functionalized with a 5 0 hexyl dithiolated dna anchor ( table s1 , supporting information ) purchased from trilink biotechnologies . [SEP]
[CLS] the dna anchor was first treated with dtt to activate the terminal thiol B-material ( c6 sas phosphoramidite ) and washed over sephadex g - 25 to remove any excess dtt . [SEP]
[CLS] the dna anchor is then incubated B-technique with 13 nm citrate - capped au nps B-nanoparticle and treated with 0 . 1 % sds buffered in 10 mm sodium B-material phosphate buffer . [SEP]
[CLS] particles were salted to a final nacl concentration of 0 . 3 m over the course of 5 h and washed 3a at 21130 rcf for 15 min to remove excess salts B-material and dna . [SEP]
[CLS] quantification of rna loading onto sna . [SEP]
[CLS] the average number of dna anchor molecules at the surface of each aunp B-nanoparticle was determined by dissolving the aunp B-nanoparticle with 40 mm kcn in 1a pbs . [SEP]
[CLS] once the gold B-material is dissolved by kcn , the average number of dna oligonucleotides per np B-nanoparticle left in solution was determined using sybr green , a dna intercalator , and monitoring total emission at 520 nm on a fluorescence B-property plate reader . [SEP]
[CLS] to determine the average number of rna strands ligated on the dna anchorfunctionalized snas , a photocleavable ( pc ) phosphoramidite was incorporated into a second dna anchor molecule for analytical studies . [SEP]
[CLS] the pc - modified dna anchor ( 5 0 - sh - t30 - pc - tttttaattcacccaac - 3 0 ) was ordered from trilink biotechnologies and used as received without further purification . [SEP]
[CLS] using the same kcn method for dissolving the aunps B-nanoparticle , the pc dnaasnas were salted to 0 . 3 m nacl and analyzed for total dna anchor loading . [SEP]
[CLS] post ligation of gfp sense rna ( 3a ) and removal of excess bridge by washing 3a with 8 m urea and centrifuging at 21130 rcf for 6 min , the particles were irradiated [SEP]
[CLS] articlewith 365 nm light for 10 min with mixing to initiate photocleavage [SEP]
[CLS] the particles were then centrifuged , and the supernatant was removed and analyzed using sybr green and a fluorescence B-property plate reader to determine the total number of ligated sirna strands . [SEP]
[CLS] a 1 % agarose gel showing the sna mobility B-property pre - and postirradiation with a pc dna anchor is shown in figure s1 ( supporting information ) . [SEP]
[CLS] rna in vitro transcription B-event and ligation [SEP]
[CLS] all rna sense strands used in the ligation reactions and for assembly of sirna snas were generated through in vitro transcription B-event . [SEP]
[CLS] the antisense strands later hybridized to the sense sirna were synthesized using an automated rna synthesizer ( mermaid ) as no chemical modifications were needed for assembly on the nanoparticle B-nanoparticle surface . [SEP]
[CLS] the dsdna template for the transcription B-event of the gfp sense rna strand ( 3a ) was transcribed using the ssdna template 5 0 - gctaatacgactcactatagggag ggcaagctgaccct - gaag ttc - 3 0 ( sequence design based on a validated sirna gfp sequence from ambion ) . [SEP]
[CLS] this ssdna template was pcr amplified using the corresponding 3 0 and 5 0 primers , 5 0 - biotin - gaacttcagggtcagctt - 3 0 and 5 0 - gctaatacgactcacta - tagggagac - 3 0 , respectively . [SEP]
[CLS] the italicized half of the ssdna template can be changed depending on the sirna sequence of interest . [SEP]
[CLS] all ssdna templates and the forward and reverse primers were purchased from integrated dna technologies ( idt ) . [SEP]
[CLS] through primer extension , the dsdna was biotinylated by the 3 0 primer , providing a facile route for immobilization on streptavidin - coated polystyrene beads and purification from the pcr reaction components . [SEP]
[CLS] these dsdna templated beads were then used for transcription B-event with t7 rna polymerase ( invitrogen ) . [SEP]
[CLS] transcription B-event reactions were run for 2 h at 37 °c and contain the dsdna template , t7 rna polymerase , 1 mm atp , ctp , gtp , utp , dtt , and 30 mm gmp . [SEP]
[CLS] after the reaction was complete , the solution was centrifuged , and the rna remaining in the supernatant was removed and run through a size - exclusion column ( sephadex - 25 ) to remove any excess nucleotides and residual dtt . [SEP]
[CLS] enzymatic ligation reactions containing the sirna sense strand for gfp ( 3a ) , the corresponding dna bridge molecule ( 2a ) , and dna snas were then incubated B-technique at 37 °c overnight in a ratio of 1 : 2 : 5 . [SEP]
[CLS] the ligation reaction mixture included 5 mm atp and 1a ligase buffer ( invitrogen ) for maintaining enzyme stability . [SEP]
[CLS] after ligation , the ligated sirna snas were washed with 8 m urea 3a by centrifugation at 21130 rcf for 12 min intervals . [SEP]
[CLS] this is to ensure removal of the enzyme and any residual nucleic B-material acids I-material that were not ligated to the surface of the sna . [SEP]
[CLS] the same procedure was performed for the gapdh sequence ( 3b ) using bridge 2b ( table s1 , supporting information ) . [SEP]
[CLS] to generate the duplexed sirna structure at the sna surface , the corresponding antisense sirna sequences were hybridized to the ligated sirna snas by incubation B-technique with 10 μm solutions of the respective as sirna sequences ( 4a and 4b for gfp and gapdh respectively ) at 37 °c in 1a pbs for 1 h , followed by centrifugation at 21130 rcf 3a to remove excess as sirna that did not hybridize to the sna . [SEP]
[CLS] the same procedure was followed to prepare the fluorophorelabeled versions of the sirna snas for cellular uptake studies . [SEP]
[CLS] dynamic B-technique light I-technique scattering I-technique ( dls ) . [SEP]
[CLS] snas pre - and postligation were analyzed using a malvern zetasizer instrument . [SEP]
[CLS] sna samples were washed 3a in h 2 o and centrifuged at 21130 rcf prior to dls measurements . [SEP]
[CLS] all samples were run in triplicate with five scans averaged per sample analyzed . [SEP]
[CLS] agarose B-technique gel I-technique electrophoresis I-technique [SEP] B-technique
[CLS] ligated rna - snas were analyzed using 1 % agarose gels run at 90 v for 30 min in 0 . 5a tae buffer . [SEP]
[CLS] gels were imaged on a fujifilm gel imager using 1a sybr gold B-material stain . [SEP]
[CLS] confocal microscopy B-technique and cellular uptake studies . [SEP]
[CLS] for the single sirna sequence sna , cell B-material uptake studies were evaluated through the use of a cy5 - dna probe backfilled onto the sna surface during the initial aunp B-nanoparticle functionalization step . [SEP]
[CLS] the cy5 - dna probe was incorporated on to the ligated gfp sirnaasna nanoparticles B-nanoparticle by initially functionalizing the aunps B-nanoparticle with a 5 : 1 ratio of 5 0 - thiolated dna anchor ( 1 ) to a 5 0 - thiolated polyt 20 - cy5 dna oligonucleotide . [SEP]
[CLS] these particles were then ligated with the gfp sirna sense strand ( 3a ) through the methods described above . [SEP]
[CLS] the particles were added to the media of c166 gfp - expressing cells B-material . [SEP]
[CLS] all cells B-material were cultured at 37 °c and 5 % co 2 in dmem supplemented with 10 % fbs and 1 % streptomycin / penicillin unless specified otherwise . [SEP]
[CLS] seeded sparsely in a 35 mm fluorodish ( world precision instruments ) , c166 - gfp cells B-material were grown overnight before being incubated B-technique with 5 nm of cy5 - labeled sirna - snas in optimem for 8 h . cells B-material were then rinsed with pbs , fixed in 3 . 7 % pfa in pbs for 15 min , and imaged under a zeiss lsm 510 inverted confocal microscope . [SEP]
[CLS] the excitation wavelength of cy5 was 633 nm , and the corresponding emission filter was 660a710 nm . [SEP]
[CLS] the dual - ligated gfp / gapdh sirna snas were analyzed by confocal microscopy B-technique through fluorophore labeling of the as sirna strands ( 4a and 4b ) with cy3 and cy5 . [SEP]
[CLS] their uptake was evaluated in hela cells B-material using a lecia sp5 multilaser confocal microscope . [SEP]
[CLS] the lecia confocal microscope was used for observing multiple fluorescent B-property probes ( cy3 and cy5 channels ) and tracking the colocalization of fluorescence B-property within the cells B-material . [SEP]
[CLS] hela cells B-material were treated with 1 nm dually ligated snas for 24 h in optimem , followed by one wash with pbs , and resuspension in dmem . [SEP]
[CLS] flow B-technique cytometry I-technique studies [SEP] B-technique
[CLS] total gfp protein B-material level was analyzed using a guava benchtop flow cytometer , using fluorescence B-property intensity to proxy for protein B-material level . [SEP]
[CLS] cells B-material were seeded at 25 % confluency in 96 - well plate format 24 h prior to sna treatment . [SEP]
[CLS] ligated sirna snas and control constructs were incubated B-technique with these cells B-material in optimem for 16 h before media exchange into fresh , complete dmem for an additional 48 h of recovery time . [SEP]
[CLS] after treatment , cells B-material were thoroughly washed with pbs and detached using trypsin before directly assayed live by flow B-technique cytometry I-technique ( guava 8ht , millipore ) . [SEP]
[CLS] western B-technique blot I-technique analysis I-technique . [SEP]
[CLS] c166 cells B-material were plated in a 6 - well cell B-material culture dish and incubated B-technique with 1 ml of particles ( 5 nm of nanoparticles B-nanoparticle in optimem medium ) or transfected with 200 nm of sirnas using commercially available dharmafect reagent overnight . [SEP]
[CLS] cells B-material were washed three times with pbs and homogenized in 0 . 1 ml of ice - chilled mammalian cell B-material lysis buffer containing 1a protease and phosphatase inhibitor ( thermo scientific ) . [SEP]
[CLS] the homogenate were cleared by centrifugation at 18406 rcf for 5 min , and the supernatant was kept as protein B-material lysis . [SEP]
[CLS] total protein B-material amount in the lysis was quantified using pierce bca protein B-material assay kit . [SEP]
[CLS] the lysis with same amount of total protein B-material was transferred to a 1 . 5 ml microcentrifuge tube and incubated B-technique with an equal volume of dithiothreitol ( dtt ) containing loading buffer ( 54 mg / ml ) . [SEP]
[CLS] after being boiled for 5 min , samples with equal amounts of total proteins B-material were fractionated by 4a20 % precast gradient gel ( bio - rad ) . [SEP]
[CLS] the intact gel was then transferred to nitrocellulose membranes ( thermo scientific ) and blocked in odyssey blocking buffer ( li - cor biosciences ) . [SEP]
[CLS] proteins B-material were detected with primary antibodies B-material against gapdh ( 1 : 500 ) ( santa cruz biotechnology ) , gfp ( 1 : 1000 ) ( clontech laboratories inc . ) , and β - actin ( 1 : 1000 ) ( cell B-material signaling technology ) , followed by irdye 680 / 800 secondary antibodies B-material ( 1 : 10000a1 : 5000 ) ( li - cor biosciences ) diluted in pbst containing 5 % nonfat milk . [SEP]
[CLS] the desired bands were visualized using the odyssey clx infrared imaging system ( li - cor biosciences ) . [SEP]
[CLS] conflict of interest : the authors declare no competing financial interest . [SEP]
[CLS] supporting information available : rna and dna sequence information , photocleavable gel shift assay , and tabulated dls measurements . [SEP]
[CLS] this material B-material is available free of charge via the internet at http : / / pubs . acs . org [SEP]
[CLS] enzyme - mediated assembly of rna onto a universal sna scaffold . [SEP]
[CLS] ( a ) sna consisting of a gold B-nanoparticle nanoparticle I-nanoparticle core B-material modified with a dna " anchor " oligonucleotide ( 1 ) . [SEP]
[CLS] a second dna oligonucleotide , the dna " bridge " ( 2a ) , is complementary to the 3 0 end of 1 . [SEP]
[CLS] upon hybridization with 1 , the sticky end generated by the dna bridge ( 2a ) is used to guide the hybridization and assembly of the 5 0 end of an rna ( 3a ) to the sna surface . [SEP]
[CLS] for sirna sequences , this strand is the rna sense strand of an sirna duplex . [SEP]
[CLS] ( b ) by changing the sequence of the sticky end of the dna bridge at its 3 0 end ( 2b ) , it can be made complementary to the 5 0 end of a different rna oligonucleotide ( 3b ) , thus allowing different rna sequences to be assembled on an sna passivated with the same dna anchor ( 1 ) . [SEP]
[CLS] size determination of ligated sirna snas . [SEP]
[CLS] ( a ) dls data showing the increase in size between the citrate - capped 13 nm au nanoparticles B-nanoparticle ( pink ) , the snas modified with the dna ligation anchor ( red ) and the final rna - dna chimera formed at the surface of the au nps B-nanoparticle ( blue ) . [SEP]
[CLS] ( b ) electrophoretic B-property mobility I-property of the ligated sirna sna and its intermediates B-property . [SEP]
[CLS] dna anchor functionalized sna ( lane one ) , dna anchor modified sna hybridized to the rna specific dna bridge ( lane 2 ) , fully ligated sirna construct post treatment with t4 dna ligase and removal of dna bridge ( lane 3 ) . [SEP]
[CLS] 3 . cellular uptake and gene knockdown of gfp by sirna ligated snas . [SEP]
[CLS] ( a ) confocal microscopy B-technique of c166 cells B-material treated with 5 nm cy5 - labeled snas . [SEP]
[CLS] ( b ) same image as ( a ) without cy5 filter . [SEP]
[CLS] ( c ) individual cell B-material showing the diffuse uptake of the ligated snas into the cell B-material as indicated by cy5 fluorescence B-property . [SEP]
[CLS] ( d ) corresponding bright field image of cell B-material shown in ( c ) . [SEP]
[CLS] ( e ) comparison of c166 gfp - expressing cells B-material by flow B-technique cytometry I-technique showing relative changes in the amount of gfp protein B-material expression post treatment with gfp sirna ligated snas . [SEP]
[CLS] compared to the untreated cells B-material , 10 nm treatment of the cells B-material with the ligated snas resulted in 53 % decrease in gfp expression . [SEP]
[CLS] the ligated snas also follow a dose - dependent knockdown of gfp protein B-material over the concentrations investigated . [SEP]
[CLS] scale bar is 50 μm in a , b and 10 μm in c , d . [SEP]
[CLS] assembly and localization of two sirnas on a single sna scaffold . [SEP]
[CLS] ( a ) schematic representation of the differential hybridization of fluorophore - labeled antisense rna oligonucleotides on the same dually ligated sna batch ( particles ligated with both gfp 3a and gapdh 3b sirna ) . [SEP]
[CLS] the sna ligated to the gfp and gapdh sirna sense strands is then hybridized to either the gfp - cy3 as , gapdh - cy5 as sequence , or both fluorophore - labeled as sequences . [SEP]
[CLS] ( b ) dual hybridized snas viewed under cy3 emission channel , pseudocolored as green . [SEP]
[CLS] ( c ) dual hybridized snas viewed under cy5 emission channel . [SEP]
[CLS] ( d ) dual hybridized snas viewed under both cy3 and cy5 emission channels where colocalization of the fluorescence B-property from the gfp and gapdh as strands is evident . [SEP]
[CLS] scale bar is 15 μm [SEP]
[CLS] articlecellular uptake , fluorophore - labeled versions of the gfp and gapdh as sequences ( cy3 and cy5 , respectively ) were prepared and hybridized to the particles . [SEP]
[CLS] the results of their uptake into hela cells B-material are shown in figure4 , in which fluorescence B-property from both the gfp - cy3 as rna and the gapdh - cy5 as rna are seen colocalized throughout the cells B-material . [SEP]
[CLS] 5 . targeted gene knockdown by dual - ligated sirna snas . [SEP]
[CLS] compared with gene expression levels in untreated cells B-material , both gfp and gapdh protein B-material expression levels are significantly reduced in gfp - c166 mouse cells B-material treated with dual - ligated gfp / gapdh snas . [SEP]
[CLS] error bars indicate standard deviation of the mean from three independent experiments . [SEP]
[CLS] the same sirna sequences transfected using the commercially available transfection agent , dharmafect , served as a positive control . [SEP]
[CLS] soft micellar nanoparticles B-nanoparticle can be prepared from dna conjugates designed to assemble via base pairing such that strands containing a polymer B-material corona and a cholesterol tail generate controlled supramolecular architecture . [SEP]
[CLS] functionalization of one dna conjugate strand with a biorecognition ligand results in shielding of the ligand when in the micelle B-material , while encoding of the dna sequences with overhangs allows supramolecular unpacking by addition of a complementary strand and sequence - specific unshielding of the ligand . [SEP]
[CLS] the molecular assembly / disassembly and ' on - off ' switch of the recognition signal is visualized by fret pair signalling , page and a facile turbidimetric binding assay , allowing direct and amplified readout of nucleic B-material acid I-material sequence recognition . [SEP]
[CLS] the encoding of information into materials through monomer B-material sequence is a central process in nature , utilising components with angstrom - scale dimensions to build devices that operate over nanometre and micron lengthscales . [SEP]
[CLS] natural materials of this type are essentially ' so ' nanomachines , with structures that are exible enough to assemble , dissociate and recombine rapidly , enabling functions such as replication , transcription B-event and translation . [SEP]
[CLS] nucleic B-material acid I-material components of this cellular machinery have evolved primarily as biological information storage and transfer materials , but increasingly their potential as synthetic operators and actuators is being realized as chemists exploit new dna and rna sequences for functions not previously seen in nature . [SEP]
[CLS] these include molecular computers , 4 motors , nanoreactors , [SEP]
[CLS] introductionas well as carriers , sensors and diagnostics . [SEP]
[CLS] in addition , the ability to encode ' dormant ' information in dna sequences , i . e . structures generated from paired nucleic B-material acids I-material that are stable until exposed to a complementary sequence , enables dna assemblies to be used for logic operations important in a biomedical context . [SEP]
[CLS] a common obstacle in translating a drug candidate from in vitro efficacy to clinical applicability is ensuring that the active compound reaches the disease site while minimising off - target effects . [SEP]
[CLS] for most anti - cancer drugs this is particularly problematic as they are oen hydrophobic B-property , leading to indiscriminate diffusion into tissue when administered systemically ( intravenously ) . [SEP]
[CLS] combined with the inherent cytotoxicity B-property of these drugs this leads to severe side effects that limit the administrable dose and thus clinical efficacy . [SEP]
[CLS] encapsulation of a drug within nanoparticles B-nanoparticle reduces its ability to permeate into tissues resulting in increased circulation times . [SEP]
[CLS] additionally , the enhanced permeation and retention ( epr ) effect , where macromolecules and nanoparticles B-nanoparticle migrate across the ' leaky ' vasculature of tumour sites and are retained there , allows for passive targeting of tumours . [SEP]
[CLS] a more advanced class of delivery vehicles B-material are those which that respond to an additional stimulus so that , once accumulated at the tumour site , they may be activated to either release the drug or promote cellular uptake . [SEP]
[CLS] for example , salmaso et al . demonstrated that gold B-nanoparticle nanoparticles I-nanoparticle coated with a temperature - responsive polymer B-material and non - temperature responsive polymer B-material bearing folate were only taken up by folate - receptor positive cells B-material when the responsive segments were collapsed . [SEP]
[CLS] a similar deshielding approach has recently been demonstrated by mirkin et al . these examples used temperature as a stimulus , however temperature is a ' crude ' stimulus and difficult to control in vivo , thus replacement of the responsive elements with nucleic B-material acid I-material assemblies should allow for greater specicity . [SEP]
[CLS] many elegant studies have reported the use of dna - assemblies and logic operators on solid nanoparticle B-nanoparticle supports , such as gold B-material , silver B-material , metal B-material oxides I-material and quantum B-nanoparticle dots I-nanoparticle . [SEP]
[CLS] while highly effective in diagnostic applications , ' hard ' nanoparticles B-nanoparticle of this type are less attractive from the viewpoint of combined signalling and therapeutic delivery applications , as the solid core B-material limits their loading with bioactive molecules as well as reducing the inherent exibility of the nanoparticle B-nanoparticle framework . [SEP]
[CLS] furthermore , recent reports have indicated that some metal B-material - based nanoparticles B-nanoparticle can induce pro - inammatory and pro - apoptotic effects . [SEP]
[CLS] accordingly there are advantages in the biomedical eld for nanoparticles B-nanoparticle based solely on organic components . [SEP]
[CLS] here we aim to demonstrate that ' so ' , and serum - stable micellar nanoparticles B-nanoparticle with a nucleic B-material acid I-material reporter function can be self - assembled from polymer - dna conjugates . [SEP]
[CLS] the design motif utilises a strand - matching architecture to project a shielding polymer B-material corona that hides a biological recognition signal ( biotin ) . [SEP]
[CLS] the system was designed to include toehold sequences in the nucleic B-material acid I-material segments such that the micelles B-material may be selectively unshielded by addition of a competing complementary strand resulting in presentation of the signal for binding ( fig . 1 ) . [SEP]
[CLS] oligonucleotides ( hplc puried ) except strand a2 were purchased from biomers . net gmbh ( ulm , germany ) and were used without purication . [SEP]
[CLS] strand a2 ( hplc puried ) was purchased from sigma - aldrich and used without further purication . [SEP]
[CLS] for sequences and modications see table 1 . poly ( ethylene glycol ) monomethyl ether B-material ( m n 1900 da ) was purchased from polysciences europe gmbh ( eppelheim , germany ) . [SEP]
[CLS] n , n 0 [SEP]
[CLS] - disuccinimidyl carbonate B-material ( 95 % ) , tris - borate - edta buffer ( tbe , 10a concentrate ) , acrylamide / bis - acrylamide 29 / 1 ( 40 % solution ) , ammonium persulfate ( $ 98 % ) , n , n , n 0 , n 0tetramethylethylenediamine ( temed , 99 % ) , orange g , formamide ( > 95 . 5 % , bioreagent ) , ethylenediaminetetraacetic acid disodium salt B-material ( edta , > 99 % ) , urea ( electrophoresis B-technique grade ) , tris ( hydroxymethyl ) aminomethane hydrochloride ( tris $ hcl , > 99 % ) , acetic acid ( > 99 % ) , triethylamine ( tea , > 99 % ) , glycerol ( > 98 % ) , 3 - hydroxypicolinic acid ( 3 - hpa ) , diammonium hydrogen B-material citrate ( dahc ) , water B-material ( bpc grade ) , stains - all ( 95 % ) , methylene blue hydrate ( > 97 % ) , fetal bovine serum ( fbs ) , avidin ( bioultra , 10 - 15 units per mg protein B-material ) and sodium B-material phosphotungstate tribasic hydrate ( puriss . p . a . ) were purchased from sigma - aldrich ( gillingham , uk ) . [SEP]
[CLS] dulbecco ' s phosphatebuffered saline ( dpbs , without ca 2 + and mg 2 + ) was purchased from lonza . [SEP]
[CLS] anhydrous dmso ( > 99 . 5 % , < 50 ppm water B-material ) was purchased from acros . [SEP]
[CLS] all other solvents were fisher hplc or analytical grade and used without further purication . [SEP]
[CLS] poly [ ethylene glycol ] monomethyl ether B-material ( m n 1900 g mol a1 , 1 . 9 g , 1 mmol ) was dried by azeotropic distillation with toluene and then dissolved in dichloromethane / triethylamine / acetonitrile ( 4 ml , 2 . 5 / 0 . 5 / 1 ) . [SEP]
[CLS] n , n 0 - disuccinimidyl carbonate B-material ( 640 mg , 2 . 5 mmol ) was added and the reaction mixture stirred at room temperature overnight ( 16 h ) . [SEP]
[CLS] the resulting solution was precipitated with diethyl ether B-material / petrol and the resulting solid isolated by centrifugation . [SEP]
[CLS] the product was puried by dissolution in dcm and reprecipitation in diethyl ether B-material / petrol for two further times . [SEP]
[CLS] oligo a1 ( 10 mg , 1 . 42 mmol ) was resuspended in 2 ml of dulbecco ' s pbs ( dpbs ) at ph 7 . 5 . [SEP]
[CLS] synthesis of peg - a1mpeg - nhs ( 14 . 5 mg , 7 . 1 mmol ) was dissolved in 200 ml of dmso and added dropwise to the oligonucleotide solution . [SEP]
[CLS] the conjugation was allowed to proceed for 24 hours . [SEP]
[CLS] aer 24 hours the coupling efficiency was examined using high performance liquid B-technique chromatography I-technique ( hplc , see below for details ) . [SEP]
[CLS] hplc analysis revealed that only around 50 % of the starting oligonucleotide had coupled to the peg in 24 hours , another 2 . 5 equivalents of mpeg - nhs ( 7 . 25 mg , 3 . 55 mmol ) in 100 ml of dmso were therefore added to the reaction and it was allowed to proceed for a further 24 hours . [SEP]
[CLS] the reaction was again monitored by hplc aer 48 hours , hplc analysis showed 66 % conversion . [SEP]
[CLS] another 5 equivalents of mpeg - nhs ( 14 . 5 mg , 7 . 1 mmol ) in 200 ml of dmso were added to the reaction and le to react for a further 24 hours . [SEP]
[CLS] analysis aer 72 hours revealed that conjugation had achieved roughly 80 % coupling efficiency . [SEP]
[CLS] at this time point the reaction was stopped and lyophilized . [SEP]
[CLS] the crude mixture was resuspended in dnase free water B-material ( 1 ml ) and puried by semi - preparative hplc ( see conditions below ) . [SEP]
[CLS] the volume collected was concentrated under reduced pressure ( to remove organic solvent ) and then subsequently lyophilized to remove the water B-material . [SEP]
[CLS] peg - a1 was collected as a white powder , the powder was dissolved in dnase free water B-material and quantied using optical density at 260 nm . [SEP]
[CLS] in total , 251 od ( 7 . 5 mg , 75 % yield ) of peg - a1 were recovered as the pure product . [SEP]
[CLS] the product was aliquoted into vials , freeze dried and then kept at a20 c prior to being used . [SEP]
[CLS] the pure product was analysed by maldi - tof . [SEP]
[CLS] mass expected : 8965 g mol a1 . [SEP]
[CLS] mass found : 9320 g mol a1 . [SEP]
[CLS] oligo a3 ( 0 . 5 mg , 0 . 066 mmol ) was resuspended in 188 ml of dulbecco pbs ( dpbs ) at ph 7 . 5 . [SEP]
[CLS] synthesis of peg - a3mpeg - nhs ( 0 . 67 mg , 0 . 328 mmol ) was dissolved in 60 ml of dmso and added dropwise to the oligonucleotide solution . [SEP]
[CLS] the conjugation was allowed to proceed for 24 hours . [SEP]
[CLS] aer 24 hours the coupling efficiency was examined using hplc ( see below for details ) . [SEP]
[CLS] hplc analysis revealed that only around 6 % of the starting oligonucleotide had coupled to the peg in 24 hours , consequently another 5 equivalents of mpeg - nhs ( 1 . 34 mg , 0 . 656 mmol ) in 100 ml of dmso were added to the reaction and it was allowed to proceed for a further 24 hours . [SEP]
[CLS] the reaction was again monitored by hplc aer 48 hours , hplc analysis showed 44 % conversion . [SEP]
[CLS] another 2 . 5 equivalents of mpeg - nhs ( 0 . 67 mg , 0 . 328 mmol ) in 100 ml of dmso along with 100 ml of thf were added to the reaction and le to react for another 96 hours . [SEP]
[CLS] analysis aer 144 hours revealed that conjugation had achieved roughly 80 % coupling efficiency . [SEP]
[CLS] at this time the reaction was stopped , the organic solvent was removed under reduced pressure and the water B-material removed using lyophilization . [SEP]
[CLS] the crude mixture was aerwards resuspended in dnase free water B-material ( 1 ml ) and puried by semi - preparative hplc ( see conditions below ) . [SEP]
[CLS] the solution collected was concentrated under reduced pressure ( to remove organic solvent ) and then subsequently lyophilized to remove the water B-material . [SEP]
[CLS] peg - a3 was collected as a purple powder , which was dissolved in dnase free water B-material and quantied using optical density at 260 nm . [SEP]
[CLS] peg - a3 ( 6 . 3 od , 180 mg , 36 % yield ) was recovered as a pure product . [SEP]
[CLS] this conjugate was aliquoted into vials , freeze dried and then kept at a20 c prior to being used . [SEP]
[CLS] the identity of the product was determined by maldi - tof . [SEP]
[CLS] mass expected : 9520 g mol a1 . [SEP]
[CLS] mass found : 9840 g mol a1 . [SEP]
[CLS] reverse phase high performance liquid B-technique chromatography I-technique ( rp - hplc ) was performed on a shimadzu prominence uplc system tted with a dgu - 20a5 degasser , lc - 20ad low - pressure gradient pump , cbm - 20a lite system controller , sil - 20a autosampler and an spd - m20a diode array detector . [SEP]
[CLS] analytical separations were performed on a phenomenex clarity 3 mm oligo - rp c18 column ( 4 . 6 a 50 mm ) with a gradient of meoh ( 10 - 70 % for peg - a1 and 35 - 70 % for peg - a3 over 20 min ) in 0 . 1 m triethylammonium acetate ( teaa , ph 7 . 5 ) / mecn ( 95 / 5 ) at a ow rate of 1 . 0 ml min a1 as a mobile B-property phase . [SEP]
[CLS] semi - preparative separations were performed on a phenomenex clarity 3 mm oligo - rp c18 column ( 10 a 50 mm ) under the same conditions at a ow rate of 5 . 0 ml min a1 . [SEP]
[CLS] matrix - assisted laser desorption / ionization B-property time - of - ight ( maldi - tof ) mass spectrometry was performed on a bruker maldi - tof ultraex iii spectrometer operated in linear , positive ion B-material mode . [SEP]
[CLS] 3 - hpa containing dahc was used as the matrix for the oligonucleotide analysis . [SEP]
[CLS] briey , a saturated solution of 3 - hpa ( 50 mg ml a1 ) was prepared by adding 25 mg of 3 - hpa to 500 ml of 50 % acn / water B-material . [SEP]
[CLS] 25 ml of dahc solution ( 100 mg ml a1 ) was added to 225 ml of the 3 - hpa solution , to give a nal dahc concentration of 10 mg ml a1 . [SEP]
[CLS] equal volumes of matrix solution and odn solution ( 0 . 2 mm ) were mixed and 2 ml of the mixture was spotted onto the maldi plate and allowed to dry . [SEP]
[CLS] denaturing page preparation of samples . [SEP]
[CLS] samples were prepared by dilution in denaturing loading buffer and heating to 95 c for 2 minutes before rapid cooling ice . [SEP]
[CLS] preparation of gels . [SEP]
[CLS] gel casting solution was prepared by mixing the components described in table s2 † and carefully pipetted into preassembled plate . [SEP]
[CLS] a 10 - well comb was inserted and the gel allowed to set for 45 min . [SEP]
[CLS] electrophoresis B-technique [SEP] B-technique
[CLS] gels were pre - run for 20 min at 200 v in 1a tbe . [SEP]
[CLS] wells were then washed with the same buffer and samples loaded at approximately 200 pmoles per well . [SEP]
[CLS] gels were run at 200 v until the orange g loading dye had migrated off the bottom of the gel ( approximately 80 min ) . [SEP]
[CLS] staining [SEP]
[CLS] gels were washed with water B-material then stained with 0 . 02 % methylene blue in 1a tbe for 20 minutes . [SEP]
[CLS] excess stain was removed by destaining with distilled B-material water I-material until the bands were clear against the background . [SEP]
[CLS] images were recorded using a standard at bed scanner ( hp scanjet 2710 ) . [SEP]
[CLS] native page preparation of samples . [SEP]
[CLS] samples were prepared by dilution in native loading buffer . [SEP]
[CLS] loading dye ( orange g ) was added only to the ladder . [SEP]
[CLS] preparation of gels . [SEP]
[CLS] gel casting solution was prepared by mixing the components described in table s2 † and carefully pipetted into preassembled plate . [SEP]
[CLS] a 10 - well comb was inserted and the gel allowed to set for 45 min . [SEP]
[CLS] electrophoresis B-technique [SEP] B-technique
[CLS] gels were pre - run for 20 min at 200 v in 1a tbe . [SEP]
[CLS] wells were washed with the same buffer and samples loaded at approximately 200 pmoles per well . [SEP]
[CLS] gels were run at 200 v until the orange g loading dye was approximately 1 cm from the bottom of the gel ( approximately 80 min ) . [SEP]
[CLS] staining . [SEP]
[CLS] gels were washed with water B-material then stained with stains - all staining solution for 20 minutes in the dark . [SEP]
[CLS] staining solution was removed and gels destained until bands were clearly visible against background . [SEP]
[CLS] gels were imaged as before . [SEP]
[CLS] strands to be hybridized were mixed in equimolar quantities at a concentration of 50 - 150 mm in hybridization buffer and placed in a 95 c water B-material bath . [SEP]
[CLS] the water B-material bath was allowed to cool to room temperature over a period of 2 hours . [SEP]
[CLS] dynamic B-technique light I-technique - I-technique scattering I-technique was performed using a viscotek 802 dls instrument tted with an internal laser ( l 830 ae 5 nm , pmax 60 mw ) . [SEP]
[CLS] hybrid peg - a1 : b1 ( 50 mm in annealing buffer ) was ltered through a 0 . 45 mm membrane ( whatman spartan , regenerated cellulose ) prior to analysis . [SEP]
[CLS] laser power was adjusted to until detection rate of at least 300 kcps was achieved . [SEP]
[CLS] a series of 10 a 3 seconds experiments was recorded and hydrodynamic radii distributions calculated with viscotek / malvern omnisize3 soware . [SEP]
[CLS] transmission B-technique electron I-technique microscopy I-technique ( tem ) was performed on an fei tecnai 12 biotwin microscope . [SEP]
[CLS] samples were prepared by rst treating a holey carbon B-material - coated 400 mesh copper B-material tem grid with graphene oxide B-material solution ( 0 . 1 mg ml a1 ) for 30 min . [SEP]
[CLS] excess graphene oxide B-material solution was wicked away with the aid of lter paper and the grids dried for 30 min . [SEP]
[CLS] oligonucleotide solution ( 10 ml , 5 mm ) was then placed on the grid ; aer 2 minutes the excess liquid was wicked away with the aid of lter paper and the sample allowed to dry for 5 min . [SEP]
[CLS] samples were stained by addition of 5 ml of a 3 % solution of sodium B-material phosphotungstate for 1 min before wicking away as before . [SEP]
[CLS] samples were then dried overnight prior to imaging . [SEP]
[CLS] hybrid peg - a1 : b1 ( 50 mm ) or a2 : c in dpbs were mixed with fbs at a ratio of 9 / 1 and incubated B-technique at 37 c . [SEP]
[CLS] aliquots were removed at 0 , 24 , 48 and 72 h . and frozen until later analysis . [SEP]
[CLS] samples were analyzed by native page as described previously . [SEP]
[CLS] by page [SEP]
[CLS] the hybrid to be analyzed was combined with either complementary ( strand c ) or scrambled ( strand d ) dna at equimolar quantities in hybridization buffer at a nal concentration of 10 mm for both hybrid and displacement strand . [SEP]
[CLS] samples were incubated B-technique at room temperature for 1 h and then analysed by native page as described previously . [SEP]
[CLS] by fret / uorimetry [SEP]
[CLS] fluorescence B-property experiments were performed using a varian cary eclipse spectrophotometer ( l ex 494 nm , excitation slit width 2 . 5 mm ; l em 519 nm , emission slit width 5 mm ) . [SEP]
[CLS] hybrids peg - a1 : b1 and peg - a3 : b3 were combined at a 4 / 1 mole ratio in hybridization buffer at a total concentration of 5 mm ( i . e . 1 mm uorophore ) . [SEP]
[CLS] 500 ml aliquots were added to a uorescence cuvette and the uorescence measured over a period of 10 minutes . [SEP]
[CLS] aer this time 25 ml of 100 mm ( 1 mole equiv . ) of oligonucleotide c or d , or an equivalent volume of buffer , was added to the cuvette and the uorescence measured for a further 30 min . [SEP]
[CLS] emission intensities were baseline corrected to the emission intensity at t ¼ 0 min . [SEP]
[CLS] percentage emission was calculated using background uorescence prior to strand addition as zero and the uorescence of a solution containing 1 mm strand b3 as 100 % . [SEP]
[CLS] full emission spectra were recorded prior to addition of dna and 30 min post addition . [SEP]
[CLS] avidin was dissolved in dpbs at a concentration of 0 . 2 mg ml a1 protein B-material ( 2 . 5 units per ml ) and aliquots ( 100 ml ) were transferred to the wells of a 96 - well plate . [SEP]
[CLS] 25 ml of peg - a1 : b2 ( 50 mm ) was added to each well followed by 75 ml of oligo c ( 100 mm , 6 mol equiv . ) , oligo d ( 100 mm , 6 mol equiv . ) , or buffer alone . [SEP]
[CLS] additional control wells were prepared by combining avidin ( 100 ml ) , buffer ( 25 ml ) and oligo c ( 75 ml ) . [SEP]
[CLS] samples were incubated B-technique for 30 min before the absorbance at 550 nm was measured using a plate reader ( tecan innite m200 ) . [SEP]
[CLS] the use of nanoparticles B-nanoparticle in diagnosis and therapy requires the solving of numerous synthetic and biological challenges . [SEP]
[CLS] systems of this type may need to encapsulate a drug or a signal molecule , traverse complex biological barriers B-property and then report a disease event or release a drug at a determined time point and / or a specic cellular or external cue . [SEP]
[CLS] a ' so ' nanoparticle B-nanoparticle structure is advantageous in this environment because biological membranes are inherently exible and a number of studies have shown that micelle - and vesicle - like nanoparticles B-nanoparticle exhibit better biodistribution and cell B-material uptake properties than more rigid analogues . [SEP]
[CLS] in addition , a more uid - like structure at a nanoparticle B-nanoparticle surface facilitates macromolecular exchange interactions , such as ligand - receptor B-material interactions or nucleic B-material acid I-material strand switching . [SEP]
[CLS] a particularly important example of a switching operation in the drug delivery context is the need to keep a recognition ligand on a polymer B-material or particle hidden / dormant but to expose the specic functionality in response to a biological trigger . [SEP]
[CLS] viruses carry out operations of this type to sequentially expose cell B-material entry ligands and endosomal escape functionality , and this ' hide - reveal ' concept has been used in synthetic viralmimetic polymers B-material and thermo - responsive nanoparticles B-nanoparticle to enhance drug delivery and selective cell B-material entry . [SEP]
[CLS] accordingly , we set out a design concept for ' hide - reveal ' nanoparticles B-nanoparticle , utilizing dna - strand recognition to encode for sequence specic assembly and derivitisation at the respective 3 0 and 5 0 ends of each to direct functionality to the interior and exterior of the particle as shown schematically in fig . 1 . [SEP]
[CLS] the oligonucleotide sequences and the modications required ( a ) to assemble the structures , ( b ) to incorporate an orthogonal biological recognition signal ( biotin ) , ( c ) to provide a shielding polymer B-material at the exterior ( peg ) , and ( d ) to unveil the ligand via competitive strand displacement are given in table 1 . [SEP]
[CLS] the primary chain of oligo a is 22 bases long and 5 0 - modied with a poly ( ethylene glycol ) chain to provide protection and shielding . [SEP]
[CLS] oligo b is 5 0 - modied with cholesterol and the 3 0 - terminus of oligo b is modied with a biotin moiety . [SEP]
[CLS] oligo b is complementary to the 12 bases at the 3 0 terminus of oligo a allowing hybridization via watson - crick base - pairing to yield a hybrid that shields the biotin moiety within the micelle B-material . [SEP]
[CLS] the 10 base toehold allows disruption of the hybrid upon addition of oligo c which is complementary to the full 22 bases of oligo a . [SEP]
[CLS] displacement of oligo a results in unshielding of the micelle B-material exposing the biotin moieties and making them accessible to bind avidin . [SEP]
[CLS] pegylation of amino - modied oligos a1 and a3 was achieved by reaction with succinimidyl carbonate - activated peg ( scheme s1 † ) followed by purication by high pressure liquid B-technique chromatography I-technique ( hplc ) . [SEP]
[CLS] successful conjugation was conrmed by page , hplc and maldi - tof mass spectrometry ( fig . 2a , s2 and s3 † respectively ) . [SEP]
[CLS] subsequent assembly of the conjugates into supramolecular structures was achieved by a simple mixing / annealing process . [SEP]
[CLS] hybridization of strand peg - a1 with strand cholesterol oligo b1 to produce hybrid peg - a1 : b1 led to the formation of discrete objects , as apparent in dynamic lightscattering ( dls , fig . 2c and s4 † ) and transmission B-technique electron I-technique microscopy I-technique ( tem ) . [SEP]
[CLS] the dls intensity distribution ( fig . 2c , black line ) is dominated by a population centered at r h $ 100 nm with small populations at 26 and 2 nm . [SEP]
[CLS] the number distribution ( fig . 2c , red line ) , calculated from intensity distribution in the range 2 < r h < 10 3 nm , shows a main population with r h 25 nm and small population at 60 nm . [SEP]
[CLS] this agrees well with tem where objects approximately 40 nm diameter are observed although dispersity is higher ( fig . 2d ) . [SEP]
[CLS] in addition , labeled hybrids could be formed by hybridization of peg - a1 with chol - biotin oligo b2 ( hybrid peg - a1 : b2 ) , and peg - a3 ( 3 0 - quencher ) with chol - uorescein oligo b3 ( hybrid peg - a3 : b3 , labeled for fret ) . [SEP]
[CLS] if dna conjugates are to be used in vitro / vivo then they need to be stable under physiological conditions . [SEP]
[CLS] consequently , the stability of micellar hybrid peg - a1 : b1 towards degradation by serum enzymes ( including nucleases ) was determined by incubation B-technique with 10 % fetal calf serum ( fcs ) at 37 c and analysis by native page . [SEP]
[CLS] hybrid peg - a1 : b1 was considerably more stable to degradation compared to unmodied ( i . e . no 3 0 or 5 0 modications ) hybrid a2 : c ( fig . s6 † ) with little evident degradation over 72 h ; however , the band - broadening imparted by the pegylation makes quantitative analysis difficult . [SEP]
[CLS] the comparative stability is expected as " spherical nucleic B-material acids I-material " have been previously demonstrated to be more stable than the linear form and the additional peg coating B-material should further reduce the accessibility of the dna segments to nucleases . [SEP]
[CLS] the " programmable " responsive unshielding was investigated by page and fret analysis . [SEP]
[CLS] for page experiments , hybrids were incubated B-technique with 1 molar equivalent of either oligonucleotide c ( complementary ) or d ( scrambled ) at room temperature . [SEP]
[CLS] samples were then analyzed by native page ( fig . 3a and s7 † ) . [SEP]
[CLS] when incubated B-technique with complementary oligo c ( lane 7 ) the band for hybrid peg - a1 : b1 is lost ( cf . lane 4 ) and bands for chol oligo b1 and hybrid peg - a1 : c found in its place ( cf . lanes 3 and 6 respectively ) . [SEP]
[CLS] conversely , when incubated B-technique with scrambled oligo d the peg - a1 : b1 band still remains and no new bands are observed . [SEP]
[CLS] ‡ equivalent bands were observed for the hybrids peg - a1 : b2 and peg - a3 : b3 when analysed in the same manner ( fig . s7a and b † ) . [SEP]
[CLS] for fret experiments hybrids peg - a1 : b1 and peg - a3 : b3 were mixed at a 4 / 1 ratio . [SEP]
[CLS] peg - a3 : b3 has a uorescein and a quencher moiety ( black hole quencher 1 , bhq - 1 , biosearch technologies ) attached to the 3 0 ends of the strands forming the duplex . [SEP]
[CLS] while the duplex is intact , fret from the uorescein to bhq - 1 results in minimal uorescence and an ' off ' state . [SEP]
[CLS] if the double helix is disrupted fret is lost resulting in switching ' on ' of the uorescence ( fig . 3b ) . [SEP]
[CLS] samples were incubated B-technique for 10 min to determine a baseline then 1 equivalent of complementary oligo c or scrambled oligo d , or an equivalent volume of buffer , was added and emission monitored for a further 30 minutes . [SEP]
[CLS] addition of either oligo d or buffer alone results in a minimal decrease in emission intensity due to a small dilution of the solution . [SEP]
[CLS] as expected , addition of oligo c results in a rapid increase in emission before the uorescence reached a plateau value . [SEP]
[CLS] these data agree with that from the page experiments and conrm that the micelle B-material corona may be removed by addition of a complementary dna strand . [SEP]
[CLS] comparison of these data to those for an equivalent concentration of the chol - uorescein oligo b3 alone conrms restoration of approximately 45 % of the expected uorescence ( fig . s8b † ) . [SEP]
[CLS] although this is lower than expected it likely results from incomplete unshielding due to steric hindrance resulting in some quencher remaining in close enough proximity to the uorescein to maintain fret . [SEP]
[CLS] finally , to determine if unpacking could be used for the presentation of the targeting B-material ligand I-material for binding , micelles B-material of hybrid peg - a1 : b2 were evaluated in a biotin - avidin binding assay . [SEP]
[CLS] briey , peg - a1 : b2 was mixed with avidin in the presence of oligo c , oligo d or buffer alone . [SEP]
[CLS] if unpacking is successful the biotin will bind to the avidin resulting in crosslinking , and subsequent precipitation , that may be quantied by measuring the solution turbidity ( absorbance at 550 nm , fig . 3d ) . [SEP]
[CLS] turbidity 3a and s6 there is an extra band observed with a molecular weight between that of oligo d and hybrid peg - a1 : b1 . [SEP]
[CLS] as it also appears in the lane where only oligo d was added we conclude that this is due to self - assembly of oligo d . [SEP]
[CLS] measurements demonstrate addition of complementary oligo c results in a signicant increase in absorbance compared to the avidin treated with peg - a1 : b2 alone ( p < 0 . 0001 , 1 - way anova ) ; addition of scrambled oligo d had no effect on absorbance conrming sequence specic binding . [SEP]
[CLS] additionally , to conrm that turbidity was caused by unpacking and not a sequence specic interaction between oligo c and avidin , the same measurement was performed in the absence of peg - a1 : b2 and no effect on turbidity was observed . [SEP]
[CLS] in conclusion we have demonstrated that oligonucleotide sequences may be used to direct the supramolecular assembly and unshielding of micellar nanoparticles B-nanoparticle in a pre - programmable manner . [SEP]
[CLS] the dna conjugate micelles B-material formed rapidly and displayed high stability to serum enzymes as well as securely shielding a recognition ligand in the absence of a specic nucleic B-material acid I-material strand . [SEP]
[CLS] unveiling of the ligand by addition of the complementary strand was easily detected by a simple turbidimetry assay in which the protein B-material , avidin , bound to the exposed biotin signals . [SEP]
[CLS] experiments to evaluate the ability of dna conjugates to switch in response to specic sequences in vitro and in vivo are continuing in our laboratories . [SEP]
[CLS] programmable control over ligand presentation may have important applications in drug - delivery , e . g . controlled targeting in the presence of a therapeutic nucleic B-material acid I-material sequence . [SEP]
[CLS] in addition , sensing applications can be envisioned for systems that , like the fret pair shown herein , can modulate their spectral properties in a programmable way allowing for in vitro and in vivo monitoring of unshielding . [SEP]
[CLS] finally , the exibility in the design and synthesis offered by nucleic acid based materials combined with the opportunity to tailor polymers B-material and ligands for specic biomedical tasks suggests materials of this type may prove useful in the personalized diagnostics and patient - group stratied therapeutics . [SEP]
[CLS] 1 schematic representation of the structure of dna conjugates and the unshielding of a ligand via strand displacement . [SEP]
[CLS] oligo a holds a peg chain to provide shielding , oligo b is complementary to part of oligo a and is modified with cholesterol and biotin . [SEP]
[CLS] when annealed the resulting hybrid forms a micellar structure with the ligand shielded . [SEP]
[CLS] oligo c is a dna sequence complementary to oligo a resulting in strand displacement and unshielding of the ligand ( biotin ) . [SEP]
[CLS] 3 ( a ) strand displacement analyzed by page . [SEP]
[CLS] lanes : ( 1 ) ladder , ( 2 ) peg - a1 , ( 3 ) b1 , ( 4 ) peg - a1 : b1 , ( 5 ) c , ( 6 ) peg - a1 : c , ( 7 ) peg - a1 : b1 + c , ( 8 ) d , ( 9 ) peg - a1 : b1 + d . ( b ) schematic of specific oligonucleotide detection via disruption of f orster resonance energy transfer ( fret ) . [SEP]
[CLS] when complementary oligonucleotide is added hybrid peg - a3 : b3 is disrupted resulting in loss of fret and a consequential increase in fluorescence B-property . [SEP]
[CLS] ( c ) baseline corrected fluorescence B-property intensity of mixed micelles B-material of peg - a1 : b1 and peg - a3 : b3 ( 4 / 1 mole ratio ) before and after addition of complementary oligo c ( blue ) , scrambled oligo d ( red ) , or an equivalent volume of buffer ( black ) . [SEP]
[CLS] ( d ) turbidimetry measurements ( absorbance at 550 nm ) of avidin solutions treated with combinations of peg - a1 : b2 , complementary oligo c and scrambled oligo d . [SEP]
[CLS] circles represent individual measurements , lines represent mean and bars represent standard deviation . [SEP]
[CLS] there is only a detectable increase in absorbance when both peg - a1 : b2 and oligo c are present . [SEP]
[CLS] 2 ( a ) denaturing page of oligonucleotides before and after pegylation . [SEP]
[CLS] lanes : 1 . ladder , 2 . a1 , 3 . peg - a1 , 4 . a3 , 5 . peg - a3 . [SEP]
[CLS] ( b ) native page of oligonucleotides and hybrids . [SEP]
[CLS] lanes : 1 . ladder , 2 . peg - a1 , 3 . peg - a3 , 4 . b1 , 5 . b2 , 6 . b3 , 7 . hybrid peg - a1 : b1 , 8 . hybrid peg - a1 : b2 , 9 . hybrid peg - a3 : b3 . [SEP]
[CLS] ( c ) dynamic B-technique light I-technique - I-technique scattering I-technique of hybrid peg - a1 : b1 . [SEP]
[CLS] intensity distribution ( black line ) and number distribution ( red line ) . [SEP]
[CLS] ( d ) transmission electron micrograph of hybrid peg - a1 : b1 stained with sodium B-material phosphotungstate . [SEP]
[CLS] sequences and modifications of oligonucleotides used [SEP]
[CLS] multivalent interactions between deformable mesoscopic units are ubiquitous in biology , where membrane macromolecules mediate the interactions between neighbouring living cells B-material and between cells and solid substrates . [SEP]
[CLS] lately , analogous artificial materials have been synthesised by functionalising the outer surface of compliant brownian units , for example emulsion droplets and lipid B-material vesicles , with selective linkers , in particular short dna sequences . [SEP]
[CLS] this development extended the range of applicability of dna as a selective glue , originally applied to solid nano and colloidal particles . [SEP]
[CLS] on very deformable lipid B-material vesicles , the coupling between statistical effects of multivalent interactions and mechanical deformation of the membranes gives rise to complex emergent behaviours , as we recently contributed to demonstrate [ parolini et al . , nat . commun . , 2015 , 6 , 5948 ] . [SEP]
[CLS] several aspects of the complex phenomenology observed in these systems still lack a quantitative experimental characterisation and a fundamental understanding . [SEP]
[CLS] here we focus on the dna - mediated multivalent interactions of a single liposome B-nanoparticle adhering to a flat supported bilayer . [SEP]
[CLS] this simplified geometry enables the estimate of the membrane tension induced by the dna - mediated adhesive forces acting on the liposome B-nanoparticle . [SEP]
[CLS] our experimental investigation is completed by morphological measurements and the characterisation of the dna - melting transition , probed by in situ fo ¨rster resonant energy transfer spectroscopy B-technique . [SEP]
[CLS] experimental results are compared with the predictions of an analytical theory that couples the deformation of the vesicle to a full description of the statistical mechanics of mobile B-property linkers . [SEP]
[CLS] with at most one fitting parameter , our theory is capable of semi - quantitatively matching experimental data , confirming the quality of the underlying assumptions . [SEP]
[CLS] due to both fundamental and practical interest , the complex phenomenology of multivalent selective interactions has recently been a hot research topic in soft matter and physical chemistry . [SEP]
[CLS] one of the main driving forces behind this effort has been the development of self - assembly strategies based on dna - mediated multivalent interactions . [SEP]
[CLS] introduced by the seminal works of mirkin 2 and alivisatos , who nearly two decades ago demonstrated the selective dna - mediated selfassembly of gold B-nanoparticle nanoparticles I-nanoparticle , this approach has been optimised to the point of mastering the structure of multicomponent crystal lattices and amorphous materials . applications of these self - assembly strategies span from photonics and plasmonics , to biosensing , and gene therapy . [SEP]
[CLS] an abundance of experimental studies called for the development of models to understand the complex statistical mechanics of dna - mediated multivalent interactions between solid particles . [SEP]
[CLS] more recently , the same selfassembly strategy has been applied to compliant brownian units , including emulsion droplets , and lipid B-material vesicles . [SEP]
[CLS] especially for the case of liposomes B-nanoparticle , which are significantly more deformable than emulsion droplets , dna - mediated adhesion plays at a similar magnitude as other forces , leading to rich coupling . [SEP]
[CLS] moreover , on these liquid surfaces , dna linkers can freely diffuse , and the confinement induced by binding results in significant entropic contributions to the hybridisation free energy . [SEP]
[CLS] we recently demonstrated that the coupling between mechanical deformability of giant - unilamellar B-material - vesicles I-material ( guvs ) and dna - mediated interactions results in unexpected emergent response to temperature changes , leading to negative thermal expansion and tuneable porosity of dna - guv assemblies . [SEP]
[CLS] by means of an analytical theory , that jointly describes the geometrical features of the guvs and the statistical mechanics of dna tethers , we rationalised experimental evidence and highlighted the importance of translational entropy in coupling the dna B-event - I-event binding I-event free energy to the morphology of the substrates . [SEP]
[CLS] nonetheless , a further experimental investigation is necessary to assess all the aspects of the complex phenomenology of dna - guv conjugates , and test the accuracy of the theoretical framework applied to their description . [SEP]
[CLS] in particular , a direct measurement of the dna - mediated adhesive forces and the quantitative characterisation of the melting transition would represent new strong tests of the current understanding . [SEP]
[CLS] in this article , we present experiments aimed at measuring the temperature - dependence of the membrane tension induced on giant liposomes B-nanoparticle by dna - mediated adhesion . [SEP]
[CLS] membrane tension is accessed by flickering measurements , in which we reconstruct the spectrum of thermal fluctuations of the liposomes B-nanoparticle at the equatorial cross - sections . [SEP]
[CLS] this is a classic technique widely used to study vesicle tension and bending rigidity . [SEP]
[CLS] to make these measurements possible , we adopt an experimental geometry in which liposomes B-nanoparticle do not interact with each other , instead they adhere to supported lipid B-material bilayers I-material ( sbls ) fabricated on rigid glass substrates , as demonstrated in fig . 1 . [SEP]
[CLS] this allows imaging of the equator of the vesicle through confocal microscopy B-technique . [SEP]
[CLS] various morphological observables are also precisely assessed via 3 - dimensional confocal imaging . [SEP]
[CLS] finally , the use of suitably labelled dna tethers enables the direct assessment of the relative number of formed dna bonds by means of in situ fo ¨rster resonant energy transfer ( fret ) spectroscopy B-technique . [SEP]
[CLS] experimental results are compared to predictions from our analytical theory , extended from ref . 30 to apply to this new geometry . [SEP]
[CLS] using at most one fitting parameter , we find semi - quantitative agreement for all the measured quantities , confirming the accuracy of the underlying assumptions of our model . [SEP]
[CLS] the remainder of this article is structured as follows . [SEP]
[CLS] first we describe the experimental setup , including sample preparations protocols , materials , imaging and image analysis techniques . [SEP]
[CLS] then we outline our theoretical model focusing on the novel aspects introduced here for the guv - plane geometry . [SEP]
[CLS] finally we discuss the experimental results and compare them with theoretical predictions . [SEP]
[CLS] 1 shows a schematic of a dna - functionalised guv adhering to a dna - functionalised sbl . [SEP]
[CLS] guvs and sbls are prepared and separately functionalised with cholesterol labeled dna constructs . [SEP]
[CLS] the hydrophobic B-property cholesterol inserts into the lipid B-material bilayer I-material allowing dna tethers to freely diffuse . [SEP]
[CLS] connected to the cholesterol anchor there is a section of length l = 9 . 8 nm ( 29 base pairs ) of double - strands dna ( dsdna ) , terminating in a single - stranded dna ( ssdna ) sticky end , which mediates the attractive interactions . [SEP]
[CLS] to further facilitate the pivoting motion of the dna tethers , 4 unpaired adenine bases are left between the cholesterol anchor and the dsdna spacer B-material . [SEP]
[CLS] one unpaired adenine is left between the dsdna spacer B-material and the sticky end . [SEP]
[CLS] two mutually complementary sticky ends are used : a and a 0 , of 7 bases each ( see fig . 1 ) . [SEP]
[CLS] guvs and sbls are functionalised with equal molar fractions of both a and a 0 . [SEP]
[CLS] tethers can therefore form intra - membrane loops and intermembrane bridges . [SEP]
[CLS] the latter are responsible for the observed adhesion and are confined within the contact area between the guvs and the sbls . [SEP]
[CLS] we use fret spectroscopy B-technique to estimate the fraction of formed dna bonds . [SEP]
[CLS] to enable these measurements the termini of the sticky ends a and a 0 are functionalised with a cy3 and a cy5 molecules respectively , spaced by an unpaired adenine base . [SEP]
[CLS] note that the sticky ends used here are shorter than those adopted in our previous study ( 7 basepairs instead of 9 ) , this choice was made to lower the melting temperature of the dna to within an experimentally accessible temperature , and reduce the overall strength of the dna - mediated adhesion , making it easier to measure by flickering spectroscopy B-technique . [SEP]
[CLS] guvs electroformation [SEP]
[CLS] 1 , 2 - dioleoyl - sn - glycero - 3 - phosphocholine ( dopc , avanti polar lipids B-material ) guvs are prepared by standard electroformation in 300 mm sucrose ( sigma aldrich ) solution in double - distilled B-material water I-material . [SEP]
[CLS] full details can be found in ref . 30 . [SEP]
[CLS] supported bilayers . [SEP]
[CLS] dopc supported bilayers ( sbls ) are formed by rupture of small B-material unilamellar I-material vesicles I-material ( suvs , b100 nm ) on the hydrophilised glass bottom of sample chambers . [SEP]
[CLS] suvs are prepared by standard extrusion . [SEP]
[CLS] briefly , 200 ml of 25 mg ml a1 dopc solution in chloroform are left to dry in a glass vial , then hydrated by adding 500 ml of 300 mm sucrose solution and mixed by vortexing for at least 5 minutes . [SEP]
[CLS] the solutions are then transferred in plastic vials and treated with 5 cycles of rapid freezingunfreezing by alternatively immersing the vial in baths of liquid nitrogen B-material and warm water B-material . [SEP]
[CLS] extrusion is carried out using a handdriven mini - extruder ( avanti polar lipids B-material ) with a polycarbonate track - etched membrane ( 100 nm pores , whatman ) . [SEP]
[CLS] to facilitate rupture of the suvs and bilayer formation , the extruded solution is diluted in a 1 : 9 ratio in iso - osmolar solution containing te buffer ( 10 mm tris ( hydroxymethyl ) aminomethane , 1 mm ethylenediaminetetra acetic acid , sigma aldrich ) , 5 mm mgcl 2 , and 272 mm glucose ( sigma aldrich ) . [SEP]
[CLS] sample chambers are obtained by applying adhesive siliconerubber multi - well plates ( 6 . 5 mm a 6 . 5 mm a 3 . 2 mm , flexwell , grace biolabs ) on glass coverslips ( 26 mm a 60 mm , no . 1 , menzel - glsa ¨er ) , cleaned following a previously reported protocol . [SEP]
[CLS] to form sbls , the glass bottom of the cells B-material is hydrophilised by plasma cleaning on a plasmochemical reactor ( femto , diener electronic , germany ) , operated at frequency of 40 khz , pressure of 30 pa , and power input of 100 w for 5 minutes . [SEP]
[CLS] within 5 minutes from plasma cleaning , each cell B-material is filled with 100 ml of diluted suv solution and incubated B-technique for at least 30 minutes at room temperature to allow bilayer formation . [SEP]
[CLS] to remove excess lipid B-material and magnesium B-material , the cells B-material are repeatedly rinsed with the experimental solution ( te buffer , 100 mm nacl , 87 mm glucose ) . [SEP]
[CLS] care is taken in keeping the bilayers covered in buffer to avoid exposure to air . [SEP]
[CLS] for experiments not involving fret spectroscopy B-technique guvs and sbls are stained with 0 . 8 - 1 . 2 % molar fraction of texas red 1 , 2 - dihexadecanoyl - sn - glycero - 3 - phosphoethanolamine , triethylammonium salt B-material ( texas red dhpe , life technologies ) . [SEP]
[CLS] dna preparation . [SEP]
[CLS] the dna tethers are pre - assembled from two ssdna strands , one of them ( i ) functionalized with a cholesterol molecule and the second ( ii ) carrying the sticky end : [SEP]
[CLS] the bold letters indicate the segments forming the dsdna spacer B-material , the italic letters the inert flexible spacers B-material . [SEP]
[CLS] dna is purchased lyophilized from integrated dna technologies , reconstituted in te buffer , aliquoted , and stored at a20 1c . [SEP]
[CLS] for assembling the constructs , we dilute equal amounts of the two single strands , ( i ) and ( ii ) , to 1 . 6 mm in te buffer containing 100 mm nacl . [SEP]
[CLS] hybridization is carried out by ramping down the temperature from 90 1c to 20 1c at a rate of a0 . 2 1c min a1 on a pcr machine ( eppendorf mastercycler ) . [SEP]
[CLS] membrane functionalisation . [SEP]
[CLS] fuctionalisation of the supported bilayers is carried out by injecting 90 ml of iso - osmolar experimental solution ( te buffer , 87 mm glucose , 100 mm nacl ) containing x moles of dna constructs into each of the siliconerubber cells B-material , with equal molarity of a and a 0 strands . [SEP]
[CLS] similarly , guvs are functionalised by diluting 10 ml of electroformed vesicle solution in 90 ml of iso - osmolar experimental solution containing x moles of dna constructs . [SEP]
[CLS] guvs and slbs are incubated B-technique for at least 1 hour at room temperature to allow grafting . [SEP]
[CLS] after incubation B-technique , 10 ml of the liposome B-nanoparticle solution are injected into the sample chambers , which are immediately closed with a second clean coverslip and sealed using rapid epoxy glue ( araldite ) . [SEP]
[CLS] care is taken to prevent the formation of air bubbles . [SEP]
[CLS] sedimentation of the guvs results in the formation of an adhesion patch between guvs and supported bilayer . [SEP]
[CLS] in a typical sample a fraction of the guvs is found to form clusters . [SEP]
[CLS] we limit our analysis to isolated guvs . [SEP]
[CLS] we tested samples at different dna concentrations , obtained by setting x to x low = 0 . 05 , x med = 0 . 5 and x high = 1 . 5 pmoles . [SEP]
[CLS] we have previously quantified the surface coverage of guvs functionalised with x med in r med dna = 390 ae 90 mm a2 . [SEP]
[CLS] here we proportionally assume r low dna = 39 ae 9 mm a2 , r high dna = 1200 ae 300 mm a2 . [SEP]
[CLS] having estimated that the overall surface of the sbl is approximately equal to the surface of the guvs used for each sample , we assume equal dna coverages for the sbl . [SEP]
[CLS] this assumption is confirmed by fluorescence B-property emission measurements : at sufficiently high temperature , when no dna bridges are formed between guvs and sbl , dna is uniformly distributed on both interfaces . [SEP]
[CLS] in this regime the fluorescence B-property emission from dna located within the contact area between a guv and the sbl approximately equals twice the intensity measured on the free sbl ( 2 . 1 ae 0 . 1 ) , confirming that guvs and sbl have , within experimental errors , the same dna coverage . [SEP]
[CLS] imaging and temperature cycling . [SEP]
[CLS] the samples are imaged on a leica tcs sp5 laser - scanning confocal microscope . [SEP]
[CLS] texas red dhpe is excited with a he - ne laser ( 594 nm ) . [SEP]
[CLS] for fret spectroscopy B-technique measurements , cy3 is excited with an ar - ion B-material laser line ( 514 nm ) . [SEP]
[CLS] the temperature of the sample is regulated with a home - made peltier device controlled by a pid ( proportionalintegral - derivative ) controller , featuring a copper B-material plate to which the sample chamber is kept in thermal contact . [SEP]
[CLS] two thermocouples are used as temperature sensors . [SEP]
[CLS] the first sensor , kept in contact with the copper B-material plate , serves as a feedback probe for the pid controller . [SEP]
[CLS] the second thermocouple is inserted in a dummy experimental chamber , filled with water B-material , and used to precisely probe the temperature of the sample . [SEP]
[CLS] for all the temperature - dependent experiments imaging is carried out using a leica hcx pl apo cs 40 a 0 . 85 na dry objective , to prevent heat dissipation . [SEP]
[CLS] the temperature - dependent morphology of adhering guvs is captured via confocal z - stacks and reconstructed using a custom script written in matlab . [SEP]
[CLS] briefly : each z - stack contains an adhering guv . [SEP]
[CLS] the plane of the sbl / adhesion patch is identified as the one with maximum average intensity , then the adhesion patch is reconstructed by thresholding , following the application of a bandpass filter to flatten the background and remove pixel - level noise . [SEP]
[CLS] from the area a p of the adhesion patch we extract the patch - radius as [SEP]
[CLS] the portion of the z - stack above the sbl is then scanned , and in each plane the contour of the vesicle , suitably highlighted by filtering and thresholding , is fitted with a circle . [SEP]
[CLS] the slice featuring the largest circle is identified as the equatorial plane , determining the vesicle radius r . [SEP]
[CLS] the contact angle is derived as y = sin a1 ( r p / r ) ( see fig . 1 ) . [SEP]
[CLS] for flickering experiments , movies are recorded across the equatorial plane . [SEP]
[CLS] details of the flickering analysis are reported in the next section . [SEP]
[CLS] to improve the quality of the signal , imaging for morphological characterisation and flickering analysis is carried out on samples stained with texas red dhpe . [SEP]
[CLS] fret spectroscopy B-technique measurements are carried out by performing a spectral emission scan ( l - scan ) of the contact area between an adhering guv and the surrounding free sbl . [SEP]
[CLS] while exciting this journal is © the owner societies 2015 the donor ( cy3 ) , the emission of donor and acceptor is reconstructed by scanning the acquisition window from 530 to 785 nm , with intervals of 6 . 375 nm . [SEP]
[CLS] similarly to the case of z - stacks , the adhesion patch is identified in each image by filtering and thresholding . [SEP]
[CLS] for fret imaging , non - fluorescent B-property lipids B-material are used in order to prevent undesired energy transfer between the bilayer and the dna tethers . [SEP]
[CLS] control experiments . [SEP]
[CLS] control experiments are performed to measure temperature - dependent membrane tension of nonadhering guvs . [SEP]
[CLS] plain , non - functionalised , guvs are imaged while laying on a glass substrate passivated with bovine serum albumin ( bsa , sigma aldrich ) . [SEP]
[CLS] imaging is either carried out in confocal microscopy B-technique , as described above , or in epifluorescence microscopy B-technique using a nikon eclipse ti - e inverted microscope , a nikon plan apo 40 a 0 . 95 n . a . dry objective and a iidc point grey research grasshopper - 3 camera . [SEP]
[CLS] for control experiments guvs are diluted in a 1 : 9 ratio in iso - osmolar glucose solution to enable sedimentation . [SEP]
[CLS] flickering analysis [SEP]
[CLS] typically , in case of phase - contrast imaging , the contour of fluctuating guvs is reconstructed by finding the inflection point in the radial intensity profile , as in ref . 34 . [SEP]
[CLS] in case of fluorescence B-technique imaging I-technique , confocal or epifluorescence , the maximum of the intensity profile is commonly used to mark the membrane position . [SEP]
[CLS] here , we designed a matlab algorithm to reconstruct the contour of guvs from their equatorial cross section with sub - pixel precision . [SEP]
[CLS] the algorithm proceeds following these steps : [SEP]
[CLS] ( 1 ) the position of the centre , and a rough - guess value of the radius r of the guv are automatically detected on the first frame of the video by processing of the thresholded image ( see fig . 2a ) . [SEP]
[CLS] ( 2 ) the image of the radial profile c ( r , j ) , where r is the distance from the centre and j the azimuthal angle , is selected within an annular region of user - defined width that contains the membrane . [SEP]
[CLS] the annular region is then mapped onto a rectangular stripe using a cubic interpolation ( see fig . 2b ) . [SEP]
[CLS] ( 3 ) for each j , the position of the membrane is roughly located as the maximum of c ( r , j ) . [SEP]
[CLS] the precision of this estimate is limited by the pixel size and highly susceptible to noise . [SEP]
[CLS] ( 4 ) for each j , the contour location is refined by evaluating the centroid of c ( r , j ) within an interval of 6 pixel centred around the maximum found in step 3 . [SEP]
[CLS] this interval is chosen as about 3 times the spatial resolution of the microscope to include the entire radial section of the membrane . [SEP]
[CLS] the centroid calculation is iterated 5 times , each time by re - centring the 6 - pixel interval around the centroid found in the previous step . [SEP]
[CLS] this procedure allows for sub - pixel resolution . [SEP]
[CLS] ( 5 ) the algorithm cycles three times from point 2 to 4 , each time refining the estimate of the vesicle ' s centre and mean radius using the obtained contour . [SEP]
[CLS] the resulting radial profile r ( j ) is shown in fig . 2b . [SEP]
[CLS] the procedure is repeated for every frame in a video of the fluctuating membrane , each time using the center and radius values of the previous frame as the starting point of the algorithm . [SEP]
[CLS] an example of the temporal evolution of the membrane radial profile r ( j ) is shown in fig . 2c . [SEP]
[CLS] the spectrum of the thermal fluctuations of the contour is calculated using matlab fast fourier transform algorithm , using the formula [SEP]
[CLS] where m is the number points used to map the contour , and then averaged over the ensemble of frames . [SEP]
[CLS] we present a quantitative model describing the dna - mediated adhesion of guvs on flat supported bilayers . [SEP]
[CLS] the model is adapted from reference , where we treated the case of two identical adhering guvs . [SEP]
[CLS] let us consider the interaction between an infinite sbl and a guv adhering to it where u membrane accounts for the mechanical deformation of the guv , u dna encodes for dna - mediated adhesion , and u 0 is the reference energy , calculated for isolated guv and sbl . [SEP]
[CLS] in eqn ( 1 ) , u membrane summarises three contributions : stretching energy , bending energy , and the entropic cost of suppressing thermal fluctuations of the contact area between the guv and the sbl . [SEP]
[CLS] in the limit of strong adhesion the stretching energy dominates over the other two contributions , which we can neglect . [SEP]
[CLS] as discussed later , for the case of dna - mediated interactions , this condition is generally verified at low enough temperature . [SEP]
[CLS] we can rewrite [SEP]
[CLS] where k a is the stretching modulus of the membrane , a ( y ) is the overall ( stretched ) area of the guv , and a [UNK] the reference , unstretched , area . [SEP]
[CLS] in the limit of strong adhesion the guv will take the shape of a truncated sphere with contact angle y ( fig . 1 ) , which we take as the independent variable of our model . [SEP]
[CLS] within the assumption of constant inner volume v = 4pr 0 3 / 3 , where r 0 is a reference radius , the total area a of the guv and the adhesion patch area a p can be expressed as a function of the contact angle y ( see appendix , section a . 1 ) . [SEP]
[CLS] the un - stretched vesicle area a [UNK] a strong temperature - dependence [SEP]
[CLS] where a is the area thermal expansion coefficient and t 0 is the neutral temperature of the guvs , at which its reduced volume equals unity and a = 4pr 0 2 ( see appendix , section a . 1 ) . [SEP]
[CLS] we now focus on the dna - mediated contribution to the interaction energy in eqn ( 1 ) : u dna . [SEP]
[CLS] given that the persistence length of dsdna is b50 nm c l = 9 . 8 nm , we can model the dsdna spacers B-material as rigid rods that , thanks to the fluidity of the membrane , can freely diffuse on the surface of the bilayers . [SEP]
[CLS] free pivoting motion is guaranteed by the flexibility of the joint between the cholesterol anchor and the dsdna spacer B-material ( fig . 1 ) . [SEP]
[CLS] as demonstrated in ref . 30 , we can regard the sticky ends as point - like reactive sites , neglecting their physical dimensions . [SEP]
[CLS] moreover we can safely assume that the distance between the adhering membranes within the contact area is equal to l . [SEP]
[CLS] finally , we neglect excluded volume interactions between unbound dna tethers . [SEP]
[CLS] the last two assumptions guarantee a uniform distribution of unbound dna tethers and loops over the guv and sbl surfaces . [SEP]
[CLS] the free energy change associated to the formation of a single bridge ( b ) or a loop ( l ) within the guv is [SEP]
[CLS] where dg 0 = dh 0 a tds 0 is the hybridisation free energy of untethered sticky ends , which can be calculated from the nearest - neighbour thermodynamic model , eventually corrected to account for neighbouring non - hybridised bases . [SEP]
[CLS] in eqn ( 4 ) , the term atds conf b / l accounts for the confinement entropic loss taking place when tethered sticky ends hybridise , and can be estimated as ( see appendix , section a . 2 ) [SEP]
[CLS] where r 0 = 1 m is a reference concentration , and we highlighted the coupling with the geometry of the guv via the y - dependence . [SEP]
[CLS] note that , contrary to the case of two adhering guvs , here [SEP]
[CLS] the local roughness of the membranes could also influence dg ( ref . 46 ) - this effect will be studied elsewhere . [SEP]
[CLS] we indicate with n the total number of a and a 0 tethers on the guvs , and model the sbl as an infinite reservoir of tethers . [SEP]
[CLS] a combinatorial calculation detailed in the appendix ( sections a . 3 and a . 4 ) allows the derivation of the overall hybridisation energy for the system of linkers [SEP]
[CLS] in eqn ( 6 ) , x b and x l are the fraction of tethers involved in bridges / loops , given by [SEP]
[CLS] where [SEP]
[CLS] in eqn ( 9 ) and ( 10 ) the hybridisation free energy is re - defined as dg * = dg a k b t log n . [SEP]
[CLS] the combinatorial contribution ak b t log n , typically estimated in ba10k b t , has a stabilising effect . [SEP]
[CLS] the quantity a n f appearing in eqn ( 6 ) indicates the average number of unbound dna tethers anchored to the sbl available within the adhesion patch . [SEP]
[CLS] the concentration c f of unbound tetheres of each type ( a or a 0 ) on the sbl , such that [SEP]
[CLS] where c 0 = r dna / 2 is the total concentration of a and a 0 tethers . [SEP]
[CLS] the full derivation of eqn ( 11 ) is provided in the appendix ( section a . 4 ) . [SEP]
[CLS] note that the concentration of loops on the sbl is c l = c 0 a c f , and the fraction of loops is x sbl l = c l / c 0 . [SEP]
[CLS] eqn ( 6 ) generalises the expression found in ref . 30 to the case in which vesicles are in contact with an infinite reservoir of tethers . [SEP]
[CLS] the overall dna contribution to the interaction energy in eqn ( 1 ) is [SEP]
[CLS] where the term on the right - hand side accounts for the change in overall confinement entropy following the area - change of the guv . [SEP]
[CLS] by combining eqn ( 2 ) , ( 6 ) , and ( 12 ) into eqn ( 1 ) , we obtain an analytical expression for the interaction energy of the guv + sbl system as a function of the only independent variable y [SEP]
[CLS] note that u depends on y though the adhesion area a p ( see eqn ( a . 22 ) ) and the total area a of the guv . [SEP]
[CLS] on the other hand , the reference energy u 0 is y - independent ( see derivation in the appendix , section a . 5 ) . [SEP]
[CLS] the interaction energy can be minimised to calculate all the morphological observables ( e . g . the contact angle y , area of the adhesion patch a p , area of the spherical section of the guv a ) , as well as the fraction of formed bridges and loops ( x b / l ) . [SEP]
[CLS] the model features seven input parameters : the thermal expansion coefficient a , the stretching modulus k a , the length of the dsdna tether l , the hybridisation enthalpy dh 0 and entropy ds 0 of the sticky ends , the dna coating B-material density r dna ( used to calculate the overall number of strands per guv : 2n ) , the neutral temperature t 0 and radius r 0 . [SEP]
[CLS] the stretching modulus is estimated from literature data as k a = 240 ae 90 mn m a1 , with the error bar covering the entire range of reported values . [SEP]
[CLS] the thermal expansion coefficient has been experimentally estimated as a = 1 . 3 ae 0 . 7 k a1 . [SEP]
[CLS] the hybridisation enthalpy and entropy are estimated according to the nearest neighbours thermodynamic rules as dh 0 = a54 ae 5 kcal mol a1 and ds 0 = a154 ae 13 cal mol a1 k a1 . [SEP]
[CLS] it is not clear whether the stabilising effect of the non - hybridised dangling bases neighbouring the sticky ends , in the present case the adenine bases present at both sides of the sequences ( see fig . 1 ) , should be taken into account . [SEP]
[CLS] it is indeed possible that their attractive contribution is compensated or overwhelmed by the repulsive effect of the long inert dna connected to the sticky ends . [SEP]
[CLS] the errorbars in dh 0 and ds 0 are included to cover both these scenarios . [SEP]
[CLS] the length l = 9 . 8 nm of the dsdna spacers B-material can be precisely estimated from the contour length of dsdna ( 0 . 388 nm per base - pair ) . [SEP]
[CLS] the dna coating B-material density is estimated for different samples as explained in the experimental section . [SEP]
[CLS] the neutral temperature t 0 changes widely from vesicle to vesicle due to the polydispersity of electroformed samples . [SEP]
[CLS] we generally use t 0 as a fitting parameter . [SEP]
[CLS] for a known t 0 , the ' ' neutral radius ' ' r 0 is experimentally determined as r 0 = r | t = t 0 . [SEP]
[CLS] if t 0 falls outside the experimentally accessible temperature range , we extrapolate [SEP]
[CLS] where t 1 is the minimum experimentally accessible temperature . [SEP]
[CLS] errors in the input parameters are numerically propagated to the theoretical predictions . [SEP]
[CLS] briefly , we sample the results of the model using random values of the input parameters x ae dx extracted from a gaussian distribution with mean equal to x and standard deviation equal to dx . [SEP]
[CLS] from a sample of size of 10 000 , we estimate the theoretical prediction of each observable as the median of the sampled distribution . [SEP]
[CLS] errorbars cover the interval between the 16th and the 84th percentile . [SEP]
[CLS] in fig . 3 we can visually compare confocal images of a dnafunctionalised guv adhering to a sbl ( a ) and a non - adhering guv on a passivated glass surface ( b ) . [SEP]
[CLS] both guvs and sbl are stained with fluorescent B-property lipids B-material . [SEP]
[CLS] it is clear from both the 3d reconstruction and the vertical cross section that the adhering guv takes the shape of a truncated sphere , with a flat and circular contact region . [SEP]
[CLS] this evidence confirms the assumption that , at low enough temperature , dna - mediated adhesion is strong enough to guarantee the dominance of stretching over bending and other contributions to the deformation energy . [SEP]
[CLS] the fluorescence B-property intensity measured within the adhesion patches is almost exactly equal to twice the value measured on the sbl outside the adhesion region ( 1 . 95 times for the vesicle shown in fig . 3a ) . [SEP]
[CLS] this evidence confirms the presence of two lipid B-material bilayers I-material in close contact within the adhesion area and excludes the possibility of dna - mediated fusion of the two membranes . [SEP]
[CLS] the non - adhering guv displayed in fig . 3b does not exhibit a flat adhesion patch , as clear from the vertical cross - section . [SEP]
[CLS] note that for both the adhering and the non - adhering guvs , the bottom part of the stacks appears brighter due to the z - dependent response of the instrument . [SEP]
[CLS] in this section we discuss the temperature - dependence of the morphology of adhering guvs . [SEP]
[CLS] in fig . 4a - c we show the experimentally determined contact angle y , adhesion - patch radius r p , and vesicle radius r for a typical adhering guv in a sample with dna concentration equal to r med dna = 390 ae 90 mm a2 . [SEP]
[CLS] the contact angle displays a non - monotonic behaviour as a function of temperature , with a positive slope at low t , followed by a sudden decay for t than b30 1c . [SEP]
[CLS] the adhesion radius follows the same trend , as does the vesicle radius , which however displays much smaller relative variations . [SEP]
[CLS] the solid lines in fig . 4a - c represent theoretical predictions calculated using the input parameters in table 1 , and using the neutral temperature t 0 as a fitting parameter . [SEP]
[CLS] greyshaded regions indicate propagated uncertainty in the theoretical predictions . [SEP]
[CLS] the agreement between theory and experiments is quantitative at low temperatures . [SEP]
[CLS] at high t , the theory fails to predict the drop in contact angle observed in experiments . [SEP]
[CLS] this behaviour is expected since our theoretical description is valid in the limit of strong adhesion , where the attractive forces are sufficient to suppress bending contributions to the interaction energy . [SEP]
[CLS] at high temperature the dna , which in the present experiment features relatively short sticky ends , starts to melt , causing the loosening of the adhesive forces and a change in the guv shape , detected as a shrinkage of the adhesion area . [SEP]
[CLS] the temperature - dependence of the fraction of dna bonds is quantified and discussed in the following sections . [SEP]
[CLS] in fig . 4d - f we show the relative deviations of the experimentally - determined morphological observables from the theoretical predictions , defined as ( x exp a x th ) / x th , for x = y , r p , and r . [SEP]
[CLS] the data , collected from various vesicles are consistent : the experimental data fall within theoretical errorbars at low t , deviating at higher temperature due to the failure of the strong adhesion assumption . [SEP]
[CLS] in fig . 5 we show experimental , and theoretically predicted morphological observables for the case of low dna concentration , r low dna = 39 ae 9 mm a2 . [SEP]
[CLS] similarly to the case of higher dna concentration , experimental data are backed by theoretical predictions . [SEP]
[CLS] in this case , however , the high - temperature deviation of the experiments from the theoretical predictions appears to be less evident , and shifted towards higher temperatures . [SEP]
[CLS] this suggests the presence of an ( however small ) adhesive force hindering the partial detachment of the guvs . [SEP]
[CLS] we ascribe this behaviour to the effect of non - specific membrane - membrane adhesion , e . g . dispersion attraction , that for higher dna coverage is suppressed by steric repulsion . [SEP]
[CLS] in the bottom row we summarise the results for 4 vesicles . [SEP]
[CLS] points represent the relative deviation of experimental data from theoretical predictions ( x exp a x th ) / x th , with x = y ( d ) , r p ( e ) , and r ( f ) . [SEP]
[CLS] grey - shaded regions mark the uncertainty interval of the theory . [SEP]
[CLS] model parameters are reported in table 1 . [SEP]
[CLS] 1 input parameters of the model a = 1 . 3 ae 0 . 7 k a1 k a = 240 ae 90 mn m a1 l = 9 . 8 nm dh 0 = a54 ae 5 kcal mol a1 ds 0 = a154 ae 13 cal mol a1 k a1 in the bottom row we summarise the results for 4 vesicles . [SEP]
[CLS] points represent the relative deviation of experimental data from theoretical predictions ( x exp a x th ) / x th for x = y ( d ) , r p ( e ) , and r ( f ) . [SEP]
[CLS] grey - shaded regions mark the uncertainty interval of the theory . [SEP]
[CLS] model parameters are reported in table 1 . [SEP]
[CLS] this journal is © the owner societies [SEP]
[CLS] 2015 [SEP]
[CLS] we investigate the temperature - dependence of the fraction of formed dna bonds via in situ fret measurements . [SEP]
[CLS] cyanine fluorophores , cy3 ( donor ) and cy5 ( acceptor ) , are connected to the 3 0 termini of a and a 0 sticky ends , as sketched in fig . 1 . [SEP]
[CLS] fret efficiency is described by [SEP]
[CLS] where d is the distance between the fluorophores and r f is the forster radius , equal to 5 . 4 nm for the case of cy3 - cy5 . [SEP]
[CLS] when sticky ends are bound to form a loop or a bridge , the distance between cy3 and cy5 is approximately equal to the length of the hybridised sticky ends , b2 . 4 nm , therefore we can assume a very high energy transfer efficiency for bound linkers . [SEP]
[CLS] the average distance between unbound linkers is sufficiently high to guarantee a comparatively very low transfer efficiency between unpaired tethers . [SEP]
[CLS] note that for fret experiments the lipid B-material membrane I-material is not stained with texas red to avoid spurious signal ( energy transfer between texas red and cy5 ) . [SEP]
[CLS] in fig . 6a we show the confocal image of an adhesion patch ( top ) , segmented to separate the actual adhesion area from the surrounding free sbl . [SEP]
[CLS] this enables an efficient characterisation of fret efficiency in situ . [SEP]
[CLS] the emission spectra , collected within the patch and on the sbl while exciting the donor at 514 nm , are shown in fig . 6b and c respectively . [SEP]
[CLS] colours from blue to red indicate low to high temperature . [SEP]
[CLS] spectra are normalised to the emission peak of cy3 , correctly found at b568 nm . [SEP]
[CLS] note that for this experiment we used high dna concentration r high dna to strengthen the signal that otherwise would be too weak for a wavelength scan . [SEP]
[CLS] as expected , the emission of the acceptor , peaked at b665 nm , visibly drops at high temperature . [SEP]
[CLS] we quantify this effect in fig . 6d , where we plot the normalised acceptor emission intensity i = i cy5 / ( i cy5 + i cy3 ) , where the i cy5 / cy3 are the peak - intensities estimated through a local gaussian fit . [SEP]
[CLS] although qualitatively similar , the i - curves measured within and outside the adhesion patch exhibit some differences . [SEP]
[CLS] for the case of free sbl , the emission ratio remains constant or slightly decreases upon heating , up to b40 1c , then it gradually drops down to b0 . 15 . [SEP]
[CLS] this decay is ascribed to the melting transition of dna loops formed within the sbl . [SEP]
[CLS] the fret signal measured within the patch is higher at low temperatures . [SEP]
[CLS] this effect is probably due to the higher dna density found within the patch at low temperature , which increases the probability of energy transfer between unpaired strands . [SEP]
[CLS] indeed we find that the overall fluorescence B-property intensity measured within the patch at t o 20 1c is between 6 and 13 times higher than the intensity measured on the sbl . [SEP]
[CLS] the fret signal measured within the patch is found to increase upon heating , before suddenly decreasing at t b 45 1c . [SEP]
[CLS] the increase in fret efficiency cannot be explained by an increase in dna density within the patch , since the local dna density decreases as the adhesion area becomes larger upon heating . [SEP]
[CLS] a possible explanation of this behaviour could be radiative cross - excitation between the two fluorophores , that becomes more efficient as the adhesion patch gets less crowded upon heating . [SEP]
[CLS] at high temperatures the fret signal measured within the patch plateaus at b0 . 15 , in line with what we measure on the sbl . [SEP]
[CLS] this confirms that at high enough temperature , when no bonds are formed , the dna concentration is uniform across all the surfaces . [SEP]
[CLS] the curves i ( t ) can be used to semi - quantitatively estimate the temperature dependence of the overall fraction of dna bonds . [SEP]
[CLS] we fit the low temperature plateaus ( t o 35 1c ) in fig . 6d with linear baselines b ( t ) and assume that i ( t ) plateaus to a constant value for t 4 57 1c . [SEP]
[CLS] the fraction of formed dna bonds is thereby estimated as [SEP]
[CLS] a better estimate of f ( t ) could be obtained by measuring i ( t ) up to higher temperatures , and fitting the hightemperature plateau with a second linear baseline . [SEP]
[CLS] however , temperatures higher than 65 1c cannot be safely probed due to the risk of destabilisation of the dsdna spacers B-material . [SEP]
[CLS] the experimental f ( t ) data in fig . 6e indicate that the dna melting transition is relatively broad , spanning more than 30 1c . [SEP]
[CLS] moreover , the melting seems to occur at a higher temperature ( by about 5 1c ) within the adhesion patch . [SEP]
[CLS] the experimentally estimated fraction of dna bonds can be compared with theoretical predictions . [SEP]
[CLS] on the free sbl , only loops can form , and therefore f ( t ) should be compared to the fraction of loops x sbl l , calculated according to eqn ( 11 ) , ( a . 17 ) and ( a . 18 ) ( appendix ) . [SEP]
[CLS] within the adhesion patch we count contributions from bridges , loops formed on the guv , and loops formed on the sbl . [SEP]
[CLS] by assuming evenly distributed loops , the overall number of dna bonds found within the patch is [SEP]
[CLS] where x b / l are the fractions of loops and bridges on the guv , given by eqn ( a . 28 ) and ( a . 29 ) ( appendix ) . [SEP]
[CLS] the overall number of dna tethers of each species ( a or a 0 ) , bound and unbound , found within the patch is [SEP]
[CLS] where we assume that also unbound dna is evenly distributed across the surfaces . [SEP]
[CLS] the theoretically predicted fraction of bonds within the patch is thus n bound / n tot . [SEP]
[CLS] in fig . 6e we compare theoretical f ( t ) with experimental estimates . [SEP]
[CLS] since the choice of the neutral temperature t 0 and radius r 0 do not noticeably affect the melting curves , theoretical predictions are calculated using a fixed t 0 = a20 1c and r 0 = 10 mm , with no fitting parameters . [SEP]
[CLS] our model captures the width of the dna transition as well as the difference in melting temperature between the patch and the free sbl . [SEP]
[CLS] however the theory underestimates the average melting point by 5 - 10 1c . [SEP]
[CLS] this deviation is , at least partially , ascribable to the attractive effect of cy3 and cy5 molecules on the stability of dna . [SEP]
[CLS] for duplexes labeled with either of the molecules , the stabilisation has been quantified in a positive melting - temperature shift of 1 . 4 - 1 . 5 1c . [SEP]
[CLS] the presence of both molecules is expected to cause a greater shift . [SEP]
[CLS] another explanation could be an underestimation of the dna concentration r high dna . [SEP]
[CLS] the impossibility of probing the hightemperature baselines could also play a role . [SEP]
[CLS] the temperature dependence of the membrane tension is measured by flickering analysis of the equatorial cross sections of guvs . [SEP]
[CLS] the tension is extracted by fitting the power spectra of the thermal fluctuations , determined as explained in the experimental section , with the function [SEP]
[CLS] where l is the contour length of the equatorial cross - section of the guv , s is the membrane tension , k the bending modulus , and q x the wave vector evaluated along the contour . [SEP]
[CLS] eqn ( 19 ) is derived from the original work of helfrich , describing the fluctuations of an infinite 2d membrane , and corrected to account for the fact that , by imaging an equatorial crosssections , only modes propagating along the horizontal direction should be considered . [SEP]
[CLS] of the discrete set of wave vectors q x ( n ) = 2pn / l , modes with n o 6 are excluded from the analysis . [SEP]
[CLS] mode n = 0 and n = 1 correspond to size changes and translations of the guv . [SEP]
[CLS] modes with n 4 2 describe thermal fluctuations . [SEP]
[CLS] however , eqn ( 19 ) is derived for a planar membrane , and should not be used to describe modes with n o 6 , which are influenced by the spherical geometry of the guv . [SEP]
[CLS] for our analysis we fit the spectra for modes 6 o n o 16 . [SEP]
[CLS] at higher q we approach the resolution limits of the current method . [SEP]
[CLS] in fig . 7a blue circles mark the tension measured as a function of temperature for adhering guvs . [SEP]
[CLS] in fig . 7b we show examples of power spectra fitted by eqn ( 19 ) . [SEP]
[CLS] the tension typically lies in the interval 2 a 10 a7 - 2 a 10 a6 n m a1 , with clear variations between different guvs . [SEP]
[CLS] in the tested range , the tension consistently displays a weak dependence on temperature changes . [SEP]
[CLS] for comparison , the membrane tension is measured on non - adhering guvs supported by a passivated glass substrate . [SEP]
[CLS] the values of s measured for non - adhering guvs ( red lozenges in fig . 7a ) are significantly lower than those measured on adhering vesicles , falling within the range 10 a8 - 5 a 10 a7 n m a1 , and being clustered around 2 - 3 a 10 a8 n m a1 . [SEP]
[CLS] furthermore , membrane tension of non - adhering guvs typically displays a strong decrease upon increasing temperature . [SEP]
[CLS] the large variability observed in the tension of adhering and , in particular , of free guvs , is ascribed to the polydispersity of electroformed samples , which produces vesicle populations with very different excess areas ( t 0 ) . [SEP]
[CLS] with the present technique we cannot access the tension of vesicles adhering to sbl for the case of higher dna concentrations . [SEP]
[CLS] indeed , for values of s in the grey - shaded region on fig . 7a , the relevant portions of the fluctuation power spectra are masked by experimental noise deriving from the finite resolution of the contour - tracking procedure . [SEP]
[CLS] the membrane tension can be evaluated within the framework of our model . [SEP]
[CLS] at equilibrium , the derivative of the interaction energy in eqn ( 1 ) , taken with respect to the guv area , is [SEP]
[CLS] where we used [SEP]
[CLS] eqn ( 20 ) suggests that by measuring s we can directly probe the dna - mediated forces . [SEP]
[CLS] the blue - shaded region in fig . 7a marks the model predictions for s , calculated using the parameters in table 1 , r low dna = 39 ae 9 mm a2 , and values of the neutral temperature covering the experimentally observed range ( a60 o t 0 o 0 1c ) . [SEP]
[CLS] the size of the vesicles does not impact the predictions of s , therefore we fix r 0 = 10 mm . [SEP]
[CLS] solid blue lines mark the uncertainty interval propagating from the errorbars of the model parameters ( table 1 ) . [SEP]
[CLS] with no fitting parameters , we observe a semi - quantitative agreement between theory and experiments . [SEP]
[CLS] in particular , the theory predicts the weak temperaturedependence of s observed in the experiments . [SEP]
[CLS] the green - shaded region in fig . 7a indicates the theoretical prediction calculated using r med dna = 390 ae 90 mm a2 . [SEP]
[CLS] the predicted tension falls within the non - accessible region . [SEP]
[CLS] in this work we experimentally investigated temperaturedependent adhesion of giant - unilamellar - vesicles I-material on supported lipid B-material bilayers I-material mediated by mobile B-property dna linkers . [SEP]
[CLS] the simple geometry of the problem allows for an accurate characterisation of the morphology of adhering guvs and the temperature dependent fraction of bound dna tethers by means of confocal microscopy B-technique . [SEP]
[CLS] for the first time to our knowledge , we quantify the temperature - dependent membrane tension induced by dna bonds by analysing the thermal fluctuations of the guvs imaged across their equatorial plane . [SEP]
[CLS] the experimental results are compared to theoretical predictions from our recently developed model , which we here extend to the case of vesicle - plane adhesion . [SEP]
[CLS] the model takes into account both the elastic deformation of the guv and the statistical - mechanical details of the dna - mediated interactions . [SEP]
[CLS] for sufficiently high dna coverage , the adhesion contact angle exhibits a re - entrant temperature dependence . [SEP]
[CLS] upon heating from low temperature the contact angle increases , reaching a maximum at t c 30 - 40 1c . [SEP]
[CLS] upon further temperature increase , the contact angle drops . [SEP]
[CLS] the re - entrance is less pronounced or absent for lower dna coverage . [SEP]
[CLS] with a single fitting parameter , the model is capable of quantitatively predicting the low temperature regime and ascribes the increase in contact angle to the interplay between the temperature - dependent excess area of the guv and the entropic coupling between the hybridisation free - energy of the mobile B-property tethers and the adhesion area . [SEP]
[CLS] the theory is developed in the limit of strong adhesion , therefore it fails to predict the re - entrant behaviour of the adhesion area , caused by the weakening of the dna bonds . [SEP]
[CLS] the less - pronounced re - entrance observed for low dna concentrations is ascribable to non - specific adhesive interactions that kick - in at high temperature , and are suppressed by steric repulsion for samples with high dna coverage . [SEP]
[CLS] the melting of dna bonds is investigated in situ by fret measurements . [SEP]
[CLS] we observe a broad melting transition and find that bonds formed within the guv - plane adhesion patch are more stable than in - plane bonds formed on free bilayers . [SEP]
[CLS] with no fitting parameters our model can semi - quantitatively reproduce these features , although an underestimation of the melting temperature is observed . [SEP]
[CLS] membrane tension measurements performed on adhering guvs demonstrate a weak temperature dependence . [SEP]
[CLS] in a similar range of temperatures , non - adhering guvs exhibit significantly lower tension , rapidly decreasing upon heating . [SEP]
[CLS] the differences in magnitude and trend demonstrates the role played by dna in mediating membrane adhesion . [SEP]
[CLS] experimental results are in semi - quantitative agreement with theoretical prediction , which further demonstrates the accuracy of the model used to describe hybridisation free - energy of tethered ) . [SEP]
[CLS] green lines mark the corresponding errorbars . [SEP]
[CLS] model parameters are reported in table 1 . [SEP]
[CLS] mobile B-property linkers , and in particular the translational - entropic contributions that couples it to the adhesion area . [SEP]
[CLS] our experimental observations and the agreement with the theoretical predictions help to clarify the complex mechanisms controlling adhesion of soft units mediated by multiple linkers . [SEP]
[CLS] besides the fundamental interest for the still poorly understood physics of multivalent interactions , our findings can help the design of functional , responsive , tissue - like materials with promised applications in biosensing , encapsulation - release mechanisms , and filtration . [SEP]
[CLS] finally , the conclusions drawn for our model system can be adapted to the quantitative description of cell B-event adhesion I-event and spreading on solid substrates , with possible biomedical implications in prosthetics and scaffolds for tissue regeneration . [SEP]
[CLS] a details on model derivation [SEP]
[CLS] in the limit of strong adhesion the guv will take the shape of a truncated sphere with contact angle y ( fig . 1 ) , which we take as the independent variable of our model . [SEP]
[CLS] in this simple geometry , the contact ( patch ) area , total area , and volume of the guv are respectively a p = pr 2 sin 2 y ( a . 1 ) [SEP]
[CLS] in the limit of water - impermeable membranes , the internal volume of the guvs can be taken as a constant [SEP]
[CLS] where we introduce a reference radius r 0 . [SEP]
[CLS] by using eqn ( a . 3 ) and ( a . 4 ) we obtain [SEP]
[CLS] the reference temperature t 0 is therefore defined as the temperature at which a guv has reduced volume equal to 1 . [SEP]
[CLS] for t o t 0 , when v 4 1 , an isolated vesicle resembles a turgid sphere , with non - zero membrane tension whereas for t 4 t 0 , v o 1 , it assumes the features of a ' ' floppy ' ' balloon , with excess area . [SEP]
[CLS] at t = t 0 an isolated vesicle is a perfect sphere with zeromembrane tension and radius equal to the reference radius r 0 . [SEP]
[CLS] by combining eqn ( a . 2 ) , ( a . 3 ) and ( a . 5 ) we obtain an explicit , y - dependent expression for the stretching energy in eqn ( a . 2 ) . [SEP]
[CLS] note that eqn ( a . 5 ) has been derived under a constant - volume assumption ( eqn ( a . 4 ) ) . [SEP]
[CLS] alternatively , an equivalent relation can be derived for water permeable - solute impermeable - guvs , in which the volume is set by the balance between the osmotic pressure drop across the membrane and the laplace pressure . [SEP]
[CLS] in relevant experimental conditions the two assumptions lead to very similar results . [SEP]
[CLS] the rotational contribution takes the same expression for loop and bridge formation , and encodes for the reduction of configurational entropy following the hybridisation of two rigid tethers with fixed grafting sites ds rot ¼ k b log 1 4pr 0 l 3 ! ; [SEP]
[CLS] ( a . 9 ) [SEP]
[CLS] where r 0 = 1 m is a reference concentration . [SEP]
[CLS] the translational contribution encodes for the lateral confinement following the binding of two mobile B-property tethers . [SEP]
[CLS] for the case of loops , upon binding , two tethers initially capable of exploring the entire guv surface area a , are confined to within a region bl 2 from each other ds trans [SEP]
[CLS] for the case of bridge formation , we consider a free linker on the sbl , initially located within the contact area a p , and a second linker on the guv , which is free to explore the entire surface area a . [SEP]
[CLS] upon binding , the area available to the pair is reduced to 4pl 2 a p , resulting in . [SEP]
[CLS] by combining eqn ( a . 8 ) - ( a . 11 ) with eqn ( 4 ) , we obtain the hybridisation free energy of bridge formation on the loops formation on the guv in eqn ( 5 ) . [SEP]
[CLS] we now focus on the description of the tethers anchored to the sbl , and calculate the equilibrium fraction of formed loops in the absence of an adhering guv . [SEP]
[CLS] this information is needed for the calculation of the guv - sbl adhesive interaction as well as for a direct comparison with experimental data . [SEP]
[CLS] from the saddle - point equations eqn ( a . 27 ) we obtain eqn ( 7 ) and ( 8 ) in the text , where we find x b1 = x b2 = x b . [SEP]
[CLS] by solving eqn ( 7 ) and ( 8 ) we obtain [SEP]
[CLS] ( a . 29 ) [SEP]
[CLS] note that for simplicity the fraction of bridges and loops are indicated as x b / l . [SEP]
[CLS] the saddle point equations for x f ( x f1 = x f2 ) read x f a x b = a n f / n , which confirms that the density of the free tethers in the patch region is equal to that of the reservoir , as expected . [SEP]
[CLS] by inserting the saddle - point solutions for x l , x b , and x f in eqn ( a . 24 ) and ( a . 25 ) we can calculate the free energy u hyb ( eqn ( 6 ) ) . [SEP]
[CLS] the reference energy u 0 in eqn ( 1 ) is calculated for isolated guv and sbl and can be written as [SEP]
[CLS] ( a . 30 ) [SEP]
[CLS] the stretching term is 30 [SEP]
[CLS] note that the stretching contribution is only present for prestretched vesicles , i . e . if the reduced volume is v 4 1 ( i . e . t o t 0 ) . [SEP]
[CLS] the dna contribution is calculated for a guv of area equal to the unstretched area a [UNK] , in which only loops can form . [SEP]
[CLS] by following the steps outlined in section a . 4 and in ref . 30 we calculate the fraction of tethers involved in loops [SEP]
[CLS] where q l is given by eqn ( 10 ) . [SEP]
[CLS] the dna part of the reference energy is [SEP]
[CLS] where n [UNK] = c f a [UNK] is the number of free tethers present within area a [UNK] on the sbl . [SEP]
[CLS] a [UNK] is the zero - stretching adhesion area , which the guv - sbl system would form for negligibly small attractive forces when t 4 t 0 , as derived in ref . 30 . [SEP]
[CLS] note that u 0 does not depend on the contact angle y therefore its form does not influence the equilibrium features of the system . [SEP]
[CLS] 1 not - to - scale schematics of a dna - functionalised guv adhering to a supported bilayer and details of the functionalisation . [SEP]
[CLS] 3 from left to right : 3d reconstruction from confocal z - stack , and confocal cross section of an adhering guv ( a ) and a non - adhering guv on a glass substrate ( b ) . [SEP]
[CLS] for 3d rendering , confocal images have been acquired with a leica hcx pl apo cs 63 a 1 . 4 na oil immersion objective for better resolution , and deconvolved using the experimental pointspread function . [SEP]
[CLS] scale bars : 10 mm . [SEP]
[CLS] 4 experimental and theoretical temperature - dependent vesicle adhesion for samples with intermediate B-property dna coating B-material density r med dna = 390 ae 90 mm a2 ( see experimental section ) . [SEP]
[CLS] in the top row we demonstrate the temperature - dependence of the contact angle y ( a ) , adhesion - patch radius r p ( b ) , and vesicle radius r ( c ) for a typical adhering guv . [SEP]
[CLS] points indicate experimental data , solid lines mark theoretical predictions , with errorbars visualised as grey - shaded regions . [SEP]
[CLS] fitting parameter t 0 = a10 1c . in the bottom row we summarise the results for 4 vesicles . [SEP]
[CLS] points represent the relative deviation of experimental data from theoretical predictions ( x exp a x th ) / x th , with x = y ( d ) , r p ( e ) , and r ( f ) . [SEP]
[CLS] grey - shaded regions mark the uncertainty interval of the theory . [SEP]
[CLS] model parameters are reported in table1 . [SEP]
[CLS] 5 experimental and theoretical temperature - dependent vesicle adhesion for samples with low dna coating B-material density r low dna = 39 ae 9 mm a2 ( see experimental section ) . [SEP]
[CLS] in the top row we demonstrate the temperature - dependence of the contact angle y ( a ) , adhesion - patch radius r p ( b ) , and vesicle radius r ( c ) for a typical adhering guv . [SEP]
[CLS] points indicate experimental data , solid lines mark theoretical predictions , with errorbars visualised as grey - shaded regions . [SEP]
[CLS] fitting parameter t 0 = a40 1c . in the bottom row we summarise the results for 4 vesicles . [SEP]
[CLS] points represent the relative deviation of experimental data from theoretical predictions ( x exp a x th ) / x th for x = y ( d ) , r p ( e ) , and r ( f ) . [SEP]
[CLS] grey - shaded regions mark the uncertainty interval of the theory . [SEP]
[CLS] model parameters are reported in table1 . [SEP]
[CLS] 6 in situ fret spectroscopy B-technique characterisation of the temperature - dependence of the fraction of dna bonds . [SEP]
[CLS] ( a ) confocal image of the adhesion patch of a guv . [SEP]
[CLS] at the bottom we show the same image segmented with our software to separate the adhesion patch ( blue ) from the free supported bilayer ( green ) . [SEP]
[CLS] scale bar : 15 mm . [SEP]
[CLS] ( b ) fluorescence B-property emission spectra measured within the adhesion patch for a typical vesicle ( blue area in panel ( a ) ) . [SEP]
[CLS] colours from blue to red mark low to high temperatures in the interval 14 . 5 r t r 62 . 9 1c . [SEP]
[CLS] the curves are normalised to the emission peak of cy3 ( 568 nm ) . [SEP]
[CLS] in panel ( c ) we show the emission spectra measured on the free sbl ( green area in panel ( a ) ) . [SEP]
[CLS] ( d ) relative intensity of the cy5 emission peak ( 665 nm ) measured within ( blue circles ) and outside ( green lozenges ) the adhesion patch . [SEP]
[CLS] the curves are relative to 3 different vesicles . [SEP]
[CLS] the amplitude of the cy5 and cy3 emission peaks is determined through a gaussian fit of the 5 data points closest to the maximum . [SEP]
[CLS] ( e ) experimental ( symbols ) and theoretically predicted ( solid lines ) fraction of formed dna bonds within ( blue circles ) and outside ( green lozenges ) the adhesion patch [SEP]
[CLS] experimental data are extracted from the curves in panel ( d ) as described in the text . [SEP]
[CLS] theoretical curves are calculated using the parameters in table 1 , dna - coating B-material density r high dna = 1200 ae 300 mm a2 , t 0 = a20 , and r 0 = 10 mm . [SEP]
[CLS] note that the value of t 0 does not significantly affect these quantities . [SEP]
[CLS] blue and green shaded regions indicate propagated uncertainties of the blue and green solid lines . [SEP]
[CLS] cyan shaded region marks their overlap . [SEP]
[CLS] 7 experimental and theoretical temperature - dependent membrane tension . [SEP]
[CLS] ( a ) , membrane tension s measured from filckering experiments . [SEP]
[CLS] blue circles indicate adhering guvs with low dna coverage ( r low dna = 39 ae 9 mm a2 ) . [SEP]
[CLS] red lozenges indicate non - adhering guvs on a bsa - coated glass surface . [SEP]
[CLS] grey - shaded regions mark regimes in which membrane tension is currently inaccessible to our technique . [SEP]
[CLS] the blue - shaded band indicates the theoretical prediction for s calculated with the fitting parameter a60 r t 0 r 0 1c and low dna coverage . [SEP]
[CLS] blue lines mark the corresponding errorbars . [SEP]
[CLS] the green - shaded band indicate the theoretical prediction for s for higher dna coverage ( r med dna = 390 ae 90 mm a2 ) . [SEP]
[CLS] green lines mark the corresponding errorbars . [SEP]
[CLS] model parameters are reported in table1 . [SEP]
[CLS] ( b ) fluctuation - amplitude spectra for adhering guvs with low dna coverage and various temperatures . [SEP]
[CLS] symbols indicate experimental data and solid lines indicate fits according to eqn ( 19 ) . [SEP]
[CLS] from top to bottom the fitted values of the membrane tension are s = 3 . 6 ae 0 . 3 , 2 . 6 ae 0 . 2 , 1 . 7 ae 0 . 1 a 10 a7 n m a1 . [SEP]
[CLS] 7 experimental and theoretical temperature - dependent membrane tension . [SEP]
[CLS] ( a ) , membrane tension s measured from filckering experiments . [SEP]
[CLS] blue circles indicate adhering guvs with low dna coverage ( r low dna = 39 ae 9 mm a2 ) . [SEP]
[CLS] red lozenges indicate non - adhering guvs on a bsa - coated glass surface . [SEP]
[CLS] grey - shaded regions mark regimes in which membrane tension is currently inaccessible to our technique . [SEP]
[CLS] the blue - shaded band indicates the theoretical prediction for s calculated with the fitting parameter a60 r t 0 r 0 1c and low dna coverage . [SEP]
[CLS] blue lines mark the corresponding errorbars . [SEP]
[CLS] the green - shaded band indicate the theoretical prediction for s for higher dna coverage ( r med dna = 390 ae 90 mm a2 ) . [SEP]
[CLS] green lines mark the corresponding errorbars . [SEP]
[CLS] model parameters are reported in table1 . [SEP]
[CLS] ( b ) fluctuation - amplitude spectra for adhering guvs with low dna coverage and various temperatures . [SEP]
[CLS] symbols indicate experimental data and solid lines indicate fits according to eqn ( 19 ) . [SEP]
[CLS] from top to bottom the fitted values of the membrane tension are s = 3 . 6 ae 0 . 3 , 2 . 6 ae 0 . 2 , 1 . 7 ae 0 . 1 a 10 a7 n m a1 . [SEP]
[CLS] cos yþ 2 ð2 a cos yþ ! 1 = 3 ; ( a . 5 ) which can be inserted into eqn ( a . 1 ) and ( a . 2 ) to make the y - dependence of a p and a explicit . [SEP]
[CLS] let us now recall the definition of reduced volume of a vesicleby combining eqn ( a . 6 ) with expression for the temperaturedependent unstretched area in eqn ( 3 ) , we obtain [SEP]
[CLS] free energy for bridge and loop formation for the case of mobile B-property linkers , the configurational entropic contribution to the bridge / loop formation free energy ds conf b / l ( eqn ( a . 4 ) ) can be split into a rotational and translational contribution ds conf b / l = ds rot + ds trans b / l . [SEP]
[CLS] ( a . 8 ) [SEP]
[CLS] we notice that , contrary to the case of two adhering guvs , here ds trans b = ds trans l [SEP]
[CLS] this journal is © the owner societies 2015 phys . chem . chem . phys . , 2015 , 17 , 15615 - - 15628 | 15627 [SEP]
[CLS] by exploiting the exquisite selectivity of dna hybridization , dna - coated colloids ( dnaccs ) can be made to self - assemble in a wide variety of structures . [SEP]
[CLS] the beauty of this system stems largely from its exceptional versatility and from the fact that a proper choice of the grafted dna sequences yields fine control over the colloidal interactions . [SEP]
[CLS] theory and simulations have an important role to play in the optimal design of self assembling dnaccs . [SEP]
[CLS] at present , the powerful model - based design tools are not widely used , because the theoretical literature is fragmented and the connection between different theories is often not evident . [SEP]
[CLS] in this perspective , we aim to discuss the similarities and differences between the different models that have been described in the literature , their underlying assumptions , their strengths and their weaknesses . [SEP]
[CLS] using the tools described in the present review , it should be possible to move towards a more rational design of novel self - assembling structures of dnaccs and , more generally , of systems where ligand - receptor B-material are used to control interactions . [SEP]
[CLS] colloidal suspensions are well described by the same statistical mechanical equations as systems of atoms B-material or small molecules . [SEP]
[CLS] as a consequence , colloids behave in many respects as ' scaled - up ' models of atoms B-material , and like atoms B-material , colloidal suspensions can be found in phases that resemble gases , liquids or crystals . [SEP]
[CLS] there are , however , important differences . [SEP]
[CLS] one is that colloids move in a solvent , rather than in vacuum . [SEP]
[CLS] the other is that , through modification of the colloid or the solvent , we can tune the this journal is © the owner societies 2016 interactions between colloids in a way that is not possible for atoms B-material or small molecules . [SEP]
[CLS] due to our ability to control colloidal interactions , colloids exhibit a much richer phase behaviour than atomic systems . [SEP]
[CLS] over the past two decades , our ability to design colloidal interactions has undergone a quantum jump with the development of so - called dna - coated colloids ( dnaccs ) , colloidal particles functionalised with short sequences of singlestranded dna ( ssdna ) . [SEP]
[CLS] dna allows the colloids to bind selectively , either directly or through single - stranded linkers , to other particles or surfaces coated with complementary ssdna sequences . [SEP]
[CLS] dna - coated colloids were developed by the groups of mirkin and alivisatos in the 1990s . [SEP]
[CLS] over the past decades , dnaccs ( sometimes referred to as ' ' spherical nucleic B-material acid I-material ' ' ) have been studied intensively because of their promise for the development of new classes of ordered colloidal materials ( see e . g . ref . 4 - 7 ) . [SEP]
[CLS] furthermore , this system has interesting biomedical applications as ultra - sensitive biomarkers B-property , detectors or efficient drug - and gene - delivery carriers . [SEP]
[CLS] in all cases , applications exploit the selectivity of dna - dna hybridization between a complementary pair , as well as specific features of the physics of multivalent interactions . [SEP]
[CLS] several reviews on dnaccs have appeared in the literature , both from an experimental and from a theoretical perspective . [SEP]
[CLS] very recently , jones have provided a general overview describing the use of dna - mediated interactions for the programmable self - assembly of nanomaterials B-material , highlighting the connections , and the differences , between dnaccs and dna - based nanotechnology . [SEP]
[CLS] the focus of the present perspective is different . [SEP]
[CLS] it is not our aim to provide a comprehensive overview of all simulation and modelling results in the field , but rather , to present a critical assessment of the various theoretical and computational approaches that have been proposed to describe dnaccs . [SEP]
[CLS] however , this perspective is not just a review : to illustrate the points that we make , we also include new results . [SEP]
[CLS] we aim to highlight the connections between different theoretical models , and explain the differences between various coarse - graining strategies used to describe dnaccs . [SEP]
[CLS] we believe that the present perspective is timely , as the rational design of novel dnaccbased materials will require a complete integration of theory and experiment . [SEP]
[CLS] at present , theory and computational work are mostly used to explain experimental observations a posteriori . [SEP]
[CLS] yet these experiments are still largely driven by empirical or semi - empirical rules . [SEP]
[CLS] even when model predictions preceded experiments , the link between theory and experiments is often less than clear . [SEP]
[CLS] at present , there is a bewildering variety of dnacc models in the literature , and this very fact makes it difficult for the nonexpert to decide what approach to use . [SEP]
[CLS] the net result is that most models are never used by experimentalists . [SEP]
[CLS] more worrying is the fact that theoretical models that have been shown to be based on questionable premises continue to be used in the literature , thus carrying the risk that analysis of the experimental data is flawed . [SEP]
[CLS] for these reasons , the aim of this review is to discuss the various models in terms of their underlying assumptions , their range of applicability and their relative strengths and weaknesses , so that experimentalists can use them in an informed way . [SEP]
[CLS] the remainder of the paper will be structured as follows . [SEP]
[CLS] in section ii , we describe the general principles common to the various analytical theoretical models , and then proceed to discuss the connections between them . [SEP]
[CLS] in particular , we aim to establish a ' hierarchy of accuracy ' in terms of the effects included . [SEP]
[CLS] these analytical models contain input parameters that depend on the molecular structure of the system , e . g . the specific dna - sequence grafted on the colloids , or details of the spacer B-material used for grafting . [SEP]
[CLS] when a systematic study of the effect of these parameters is required , and in order to relax some simplifying assumptions necessarily included in the analytical models , one should resort to computer experiments , i . e . simulations . [SEP]
[CLS] we will deal with these ' ' experiments ' ' in section iii , where we will focus on the various coarse - graining strategies that have been proposed in the literature . [SEP]
[CLS] in particular , we discuss which microscopic features are retained in the various models , and which are ignored . [SEP]
[CLS] in particular , we will focus on the question if the model can yield meaningful predictions of the relevant experiments . [SEP]
[CLS] in the concluding section ( section iv ) , we will identify possible new directions where the existing theoretical and computational framework could be profitably integrated with experiments to yield a more rational design of dnacc - based systems . [SEP]
[CLS] the common idea behind basically all theoretical models to describe dna - mediated interactions in dnaccs is to account correctly for the interactions induced by the dna strands that coat B-material the colloids ( a schematic of the system is depicted in fig . 1 ) [SEP]
[CLS] on the one hand , as in general for all colloidal systems stabilised by ( non - absorbing ) grafted polymers B-material , a non - specific repulsion arises from compression of the dna strands trapped between the colloidal cores B-material . [SEP]
[CLS] on the other hand , a highly specific interparticle attraction occurs because of the decrease in free - energy due to bond formation ( fig . 2 provides a schematic illustration ) . [SEP]
[CLS] that bond - mediated interactions are the driving force leading to aggregation is also evident from experimental studies that show no aggregation between dnaccs grafted with noncomplementary strands . [SEP]
[CLS] in fact , other ( non - specific ) attractive interactions , in particular those arising from the van der waals forces between the colloidal cores B-material , are typically much weaker than those arising from bond formation on the lengthscales at which bond - mediated binding occurs . [SEP]
[CLS] this is mainly due to the fact that the non - specific steric repulsive forces due to dna compression prevent the colloids to approach at short distances where dispersion forces are strong . [SEP]
[CLS] for similar reasons , the length - scale for binding , i . e . the typical distance between colloidal cores B-material between bound particles , is dictated by the length of the spacer B-material used to graft dna on the surface of fig . 1 a schematic representation of a typical dna - coated colloid , and the working principle behind their induced attraction . [SEP]
[CLS] in dnaccs , singlestranded dna , here represented as a red or white circle , is grafted onto a colloidal core B-material via a spacer B-material , typically another polymer B-material or double - stranded dna ( dsdna ) . [SEP]
[CLS] upon recognition of a complementary sequence , a singlestranded dna can hybridise with it to form a joining dsdna helix , leading to a highly - specific ligand - receptor B-material bond . [SEP]
[CLS] the figure presents two possible , mutually exclusive binding configurations , f a and f b . [SEP]
[CLS] considering all possible binding configurations and their energy provides the binding thermodynamics of the system . [SEP]
[CLS] 2 schematic representation of the origin of attractive and repulsive forces , here shown for a simple system of strands considered as rigid rods and in the case where only a single bond can be formed . [SEP]
[CLS] ( a ) upon binding , the system acquires an additional energy dg bond ij , which depends on both the chemical identity of the active strands ( white and red circles ) as well as on details of the spacer B-material ( black sticks ) and position of the tethering points ( see eqn ( 3 ) ) . [SEP]
[CLS] in particular , note that whereas in the unbound state ( left ) the active strands can freely move on a hemisphere , upon binding ( right ) they are forced to stay on a circle : this reduction in configurational space is at the origin of dg cnf , ( see eqn ( 4 ) ) . [SEP]
[CLS] ( b ) when all colloids are far apart from each other , the active strands can span a whole hemisphere . [SEP]
[CLS] however , this space is again reduced once colloids start to get closer . [SEP]
[CLS] this reduction is at the origin of the dominant repulsive forces f rep between dnaccs . [SEP]
[CLS] the colloids , which is typically in the range of nanometers to tens of nanometers . [SEP]
[CLS] in various practical realisations of dnaccs , other polymer B-material chains than dna are grafted to the colloidal surface . [SEP]
[CLS] using an additional different polymer B-material for steric stabilisation makes it possible to tune repulsion and attraction separately . [SEP]
[CLS] both theory and experiments have shown that tuning the relative strength of attraction and repulsion can lead to self - assembly of aggregates of different types . [SEP]
[CLS] it is crucial to note that the interaction between two grafted colloid is due to both energy and entropy , the latter accounting for the fact that often not just one but several binding configurations are possible ( see fig . 1 ) . hence , to compute the interaction strength , we need to evaluate a free energy , rather than an energy , as would be the case for atoms B-material in vacuum . [SEP]
[CLS] to compute the free energy of interaction of dnaccs , we start from the statistical mechanical relation between the free energy f of a system and its partition function z . [SEP]
[CLS] we consider an arbitrary number of colloids , each of which has an arbitrary number of strands grafted on its surface . [SEP]
[CLS] we choose a reference state where all colloids are at infinite distance from each other , and calculate the free - energy with respect to this state : [SEP]
[CLS] where z = z 0 + z bound is the partition function of all strands in the system ( i . e . a sum over boltzmann weights ) , which depends on their grafting points { r } , as well as on the positions of all colloids { r } . z 0 is the partial sum over all spacer B-material states where no bond forms , z bound the partial sum over all states with at least one bond , and finally z n z ( { r = n } ) is the partition function when all colloids are at infinite separation . [SEP]
[CLS] with this splitting , we can identify two terms , f att and f rep , i . e . the attractive and repulsive component of the interaction , respectively . [SEP]
[CLS] note that when binding between strands on the same particle can occur , this identification cannot be made , although the general approach to calculate f interaction ( see eqn ( 1 ) ) still holds . [SEP]
[CLS] we stress that no approximation has been made in the derivation of eqn ( 1 ) . [SEP]
[CLS] since z = z 0 + z bound , f att is always negative ( and , hence , attractive ) . [SEP]
[CLS] instead f rep is the ratio between two partition functions where bonds are never allowed , although in one case ( z 0 ) strands can feel the excluded volume due to all colloids , including the one on which they are grafted , whereas in the other ( z n ) strands can only feel the latter . [SEP]
[CLS] if we assume that only repulsive interactions between the colloidal cores B-material and the strands occur , i . e . we consider non - adsorbing B-property polymers B-material , a good approximation for most type of polymers B-material used in dnaccs , then z 0 / z n o 1 , and f rep is always positive ( i . e . repulsive ) [SEP]
[CLS] use of eqn ( 1 ) would allow one to calculate the interaction between dnaccs including all type of many - body interactions arising from bond formations , and for arbitrary position of the colloids . [SEP]
[CLS] this expression does not assume that dna - mediated interactions are pairwise additive - and , in fact , often they are not . [SEP]
[CLS] for this reason , the general expression is needed to compute the interaction free energy of dense phases of dnaccs . [SEP]
[CLS] let us next consider how f att and f rep can be calculated . [SEP]
[CLS] one route is both obvious and pointless : ' brute - force ' atomistic simulations . [SEP]
[CLS] simulations that would describe the colloids , the dna and the solvent in atomistic detail are simply much too expensive to be used to predict , say , phase diagrams . [SEP]
[CLS] fully atomistic simulations can certainly be used to study certain aspects of dnaccs , but not to study a suspension containing hundreds or thousands of such particles . [SEP]
[CLS] in what follows , some degree of coarse - graining is therefore essential . [SEP]
[CLS] typically , this means that we will use an implicit solvent model and that we will assume that the binding free energy between complementary dna strands is known . [SEP]
[CLS] in other words : models to describe systems consisting of many dnaccs are always coarse - grained . [SEP]
[CLS] to arrive at an analytically tractable coarse - grained model , the following assumptions are commonly made : ( 1 ) we usually ignore the spatial extent of the nucleotide sequences that can hybridise to bind two strands together . [SEP]
[CLS] the hybridising strand is then represented as a point - like attractive site , also called ' active site ' or ' sticky end ' , tethered to the colloidal surface by a long chain - the spacer B-material or linker ( see also fig . 1 ) . [SEP]
[CLS] representing the ' sticky ' binding sequence as a point is a reasonable approximation as long as the spacer B-material is much longer than the sticky end , a situation typically encountered in experimental systems . [SEP]
[CLS] considering extended sticky ends results in slightly different binding affinities between constructs . [SEP]
[CLS] accounting for the finite spatial extent for sticky end does not qualitatively change the theoretical treatment of dnaccs , and hence we limit our discussion to point - like sticky ends . [SEP]
[CLS] ( 2 ) we ignore steric interactions between different strands and , more generally , any non - selective strand - strand interaction . [SEP]
[CLS] this approximation is very common , but it is likely to break down for dense dna ' brushes ' . [SEP]
[CLS] with the above assumptions , f rep can be expressed as : [SEP]
[CLS] where the index i runs over all strands in the system , and o i and o n i are , respectively , the partition function of a single linker when the colloids are in positions { r } and the partition function for the case where all colloids are at infinite separation . [SEP]
[CLS] for specific geometries and simple polymer B-material models for the linker , such as rigid rods or gaussian chains , there are either exact or very accurate approximate expressions to calculate f rep . [SEP]
[CLS] for more complex cases , f rep can be obtained using standard monte carlo techniques to sample polymer B-material conformations . [SEP]
[CLS] rogers and crocker showed that a simple algorithm that assumes that polymers B-material can be described as ideal , non - interacting chains that cannot intersect the colloids gave essentially quantitative agreement with the repulsive interaction determined in experiments . [SEP]
[CLS] this agreement suggests that in this specific system , the neglect of chain - chain and attractive chain - colloid interactions is justified . [SEP]
[CLS] it also suggests that electrostatic interactions due to the dense clouds of counter ions B-material around dna can be included in an effective way via their influence on the single - chain conformations . [SEP]
[CLS] these depend on the chosen length of the segments in the freely - jointed chain , which is the quantity directly affected by electrostatics . [SEP]
[CLS] in section iii we discuss non - specific interactions between spacers B-material in more detail . [SEP]
[CLS] the calculation of f att is more challenging , since here we need to account for all possible binding configurations with an arbitrary number of bonds ( see fig . 1 ) . [SEP]
[CLS] in order to do that , it is useful to split the problem of calculating f att into two parts . [SEP]
[CLS] the first stage involves calculating the bond formation energy for all individual pairs of ( i , j ) strands , and the second stage accounts for the fact that there may be many ways in which the dna strands on adjacent colloids may be connected ( as illustrated in fig . 1 ) . [SEP]
[CLS] if the spacer B-material does not influence the structure of the sticky end , one can split the bond formation free energy into two terms : [SEP]
[CLS] the first term on the r . h . s . of eqn ( 3 ) is the free - energy of binding for untethered , complementary dna strands i and j in solution : exp [ abdg 0 ij ] = k eq ij r 0 , where k eq ij denotes the equilibrium binding constant , and r 0 the standard concentration of 1 m . [SEP]
[CLS] the second term represents the configurational entropic cost associated with linking two tethered strands i and j : the number of configurations of a linked ij strand is less than that of the unlinked i and j strands ( see fig . 2a ) . [SEP]
[CLS] dg cnf ( r i , r j , { r i } ) depends on the exact positions of the grafting points , and on the position of all nearby colloids in the system , since these exclude all strand configurations that would intersect them . [SEP]
[CLS] ref . 22 and 23 give an explicit expression that can be used to calculate this quantity for arbitrary linkers and colloids positions , which in its most general case reads : [SEP]
[CLS] where o ij is the partition function for two bound linkers i and j , whereas o i , j is the partition function for the unbound case . [SEP]
[CLS] both terms depend on r i , r j and { r i } , and can be calculated using coarse - grained models for the linkers . [SEP]
[CLS] when strand - strand interactions can be neglected , an equivalent and possibly more illuminating form of eqn ( 4 ) useful for calculations is : [SEP]
[CLS] where p ee ( d ) denotes the fraction of all random configurations of the two chains tethered at two grafting points at separation d , that have overlapping sticky ends , i . e . it is the probability that two non - interacting tethers will form a ' bridging ' configuration . [SEP]
[CLS] the terms w ij and w i ( j ) account for the effect of hard - core B-material overlaps with the colloids : they denote the number of i , j or ij configurations that do not overlap with the colloidal cores B-material ( see fig . 2 ) . [SEP]
[CLS] note that , w i ( j ) for the unbound linkers is equal to the o i ( j ) s appearing in the definition of f rep , eqn ( 2 ) . [SEP]
[CLS] dg cnf ( r i , r j , { r i } ) can be either calculated analytically for simple polymer B-material models , or computed via monte carlo simulations , as we discuss in section iii . [SEP]
[CLS] in a small number of ( important ) cases , it can be approximated analytically . [SEP]
[CLS] the different theories that have been proposed to estimate f att make different approximations to calculate this quantity . [SEP]
[CLS] however , all models assume that strands are non - interacting ( although the interaction can still be included in calculating the bond energy ) . [SEP]
[CLS] this approximation is reasonable when different chains are not close to each other . [SEP]
[CLS] at higher grafting densities ( see e . g . ref . 31 and 32 ) , the simple analytical approaches break down , but the simple theories can still be used to gain insight in the effect of the structure of the spacer B-material ( e . g . rigid rods or freely jointed chains ) on the interactions between dnaccs . [SEP]
[CLS] for example , ' ' theory accounts ' ' quite well for the experimentally observed trends in the melting properties of dnaccs . [SEP]
[CLS] under the assumption that the binding free energy dg bond ij between i and j does not depend on the presence of other strands ( i . e . the non interaction assumption ) , knowledge of dg bond ij for all possible pairs of constructs ( i , j ) is sufficient to write down an exact expression for the partition function z : [SEP]
[CLS] where the sum runs over all binding configuration f of the system ( see fig . 1 ) , i . e . z / z 0 is simply equal to the sum of all boltzmann weights due to bonds energies over all possible binding configurations . [SEP]
[CLS] note that dg bond ij depends on the position of the strands via dg cnf , see eqn ( 3 ) , and hence depends on the spatial configuration of the colloids . [SEP]
[CLS] an exact enumeration of the terms in eqn ( 6 ) becomes rapidly intractable since the number of binding configurations increases very rapidly with the number of strands in the system . [SEP]
[CLS] hence eqn ( 6 ) is typically simplified under some assumptions . [SEP]
[CLS] it is at this level of approximation that differences between the various analytical theories show up . [SEP]
[CLS] short of exact enumeration , z / z 0 , and hence f att , can be calculated to any given accuracy via monte carlo simulations using thermodynamic integration , providing a route to fully include all possible competitive effects between formation of different binding configurations , as well as about the spatial distribution of the strands . [SEP]
[CLS] the exact solution reads : bf att bdg 0 a a ¼ [SEP]
[CLS] where p ij is the probability that a bond between strands i and j is observed , as sampled by mc simulations . [SEP]
[CLS] in ref . 23 and 27 we have shown that the ' exact ' monte carlo results are approximated extremely well by a very simple expression : [SEP]
[CLS] and [SEP]
[CLS] where the indexes i and j run over all possible strands in the system . [SEP]
[CLS] in eqn ( 8 ) , p i is the probability that strand i is not bound , [SEP]
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[CLS] found by solving the set of coupled self - consistent equations represented by eqn ( 9 ) . [SEP]
[CLS] eqn ( 8 ) and ( 9 ) can be derived using a recursion formula to count all possible binding configurations ( see ref . 27 ) . [SEP]
[CLS] in our derivation of eqn ( 8 ) and ( 9 ) we used an approximate yet very accurate formula for counting the degeneracy of each binding configuration , accounting for the fact that no two linkers can bind to the same target . [SEP]
[CLS] from a combinatorial point of view , it turns out that our procedure can be mapped onto wertheim ' s theory for the equilibrium properties of fluids with highly directional intermolecular forces . [SEP]
[CLS] wertheim ' s solution is a basic ingredient of the widely used saft theory . [SEP]
[CLS] in practice , each reactive dna strand ( or , more generally , each binder B-property ) must be considered in the wertheim picture as a spatially and orientationally fixed particle with a single binding site , but each having a unique bond energy , which in our case must also be defined as in eqn ( 3 ) . [SEP]
[CLS] in our system , we also have the additional benefit that some of the approximations wertheim used are always exactly satisfied conditions , in particular the fact that if two strands are bound a third one cannot bind at the same time . [SEP]
[CLS] once the problem is cast in these terms , it becomes mathematically equivalent to that of wertheim . [SEP]
[CLS] the approximations underlying eqn ( 8 ) and ( 9 ) become exact in the case of mobile B-property constructs , see ref . 28 and section iii for more details . [SEP]
[CLS] finally , we stress that although these equations provide the f att induced by the interaction between grafted strands . [SEP]
[CLS] it is easy to show that they remain the same when binding between these strands is not direct , but is mediated by ( or compete with ) , free complementary strands in solution , another widely used bonding scheme in dnaccs . [SEP]
[CLS] in practice , eqn ( 8 ) and ( 9 ) remain valid , as well as the configurational part of the bond free - energy dg cnf , whereas the solution hybridisation free - energy dg 0 ij between strands must be shifted with a density dependent factor . [SEP]
[CLS] since eqn ( 8 ) and ( 9 ) agree extremely well with the corresponding mc simulations , they also properly reproduce all the ( important ) correlations introduced by the competition between strands for the same binding partner . [SEP]
[CLS] moreover , they are completely general in that they can be used to treat an arbitrary number of different binding pairs , with any spatial distribution . [SEP]
[CLS] hence , they can be used to calculate colloid - colloid interactions also when highly non - homogeneous dna coatings B-material are present , as in the experimental system reported by pine and coworkers mimicking patchy particles . [SEP]
[CLS] earlier expressions that have been proposed in the literature can be derived as approximations of eqn ( 8 ) and ( 9 ) under different assumptions . [SEP]
[CLS] in particular , the aforementioned models all shared a mean - field approximation for binding , i . e . unlike eqn ( 8 ) and ( 9 ) they do neglect correlations that are due to the fact that two strands cannot bind simultaneously to the same binding partner ( the ' valence constraint ' ) . [SEP]
[CLS] furthermore , strands were not only treated as if they were completely independent from each other , but also as if they were all statistically equivalent . [SEP]
[CLS] the first approximation becomes progressively worse as the binding strength of bonds increases ( relative to k b t ) . [SEP]
[CLS] this drawback can be heuristically corrected by a self - consistent mean field treatment , which yields the mean - field version of eqn ( 9 ) . [SEP]
[CLS] the assumption that all bonds are statistically equivalent gets worse as the grafting of chains becomes more heterogeneous , since in this case each strand will experience a different environment . [SEP]
[CLS] however , if we assume that both approximations are justified , then the expression for f att simplifies : f att e ak b thni ( 10 ) where hni is the number of bonds formed in the system . [SEP]
[CLS] this expression for f att is called the ' ' weak binding ' ' or the ' ' poisson ' ' approximation . [SEP]
[CLS] it provides a lower bound for the real interaction energy , as it always overestimates the number of possible bonds : any correlation would reduce the number of bonds that can form . [SEP]
[CLS] it has been pointed out that models that are based on the poisson approximation may overestimate the attractive interaction by as much as two orders of magnitude , corresponding to approximately e5k b t per dna bridge . [SEP]
[CLS] the ' ' weak binding ' ' approximation can be obtained formally from the expression given by eqn ( 7 ) by a simple assumption . [SEP]
[CLS] the integrand in eqn ( 7 ) is nothing but the expected number of bound pairs in the system . [SEP]
[CLS] if this number can be approximated by a poisson distribution ( an approximation that breaks down at higher binding strength ) , then eqn ( 10 ) follows from eqn ( 7 ) . [SEP]
[CLS] it can be also shown that eqn ( 10 ) corresponds to a first - order approximation of eqn ( 8 ) and ( 9 ) ( the second - order correction is positive ) , and its validity is limited to a region where each strand has very few binding partners or small binding energy ( hence the ' ' weak binding regime ' ' ) . [SEP]
[CLS] another possible drawback of eqn ( 10 ) is that it cannot be generalised easily to treat an arbitrary number of binding partners with different binding energies , since eqn ( 10 ) was derived considering not just independent , but also equivalent strands . [SEP]
[CLS] this is a serious limitation , as the phase diagram of dnaccs can be ' designed ' by making colloids with more than one type of strand ( see e . g . ref . 29 ) . [SEP]
[CLS] a possible way to estimate f att for an arbitrary number of types of strands was proposed by rogers and crocker . [SEP]
[CLS] heuristically keeping eqn ( 10 ) as the expression for the attractive contribution , they proposed to use mass balance equations valid for chemical reactions in solution to describe binding between grafted strands , an approach dubbed ' ' local chemical equilibrium ' ' ( lce ) . [SEP]
[CLS] in ref . 25 it is assumed that the binding for grafted strands can be treated as an equilibrium binding reaction in solution with a non - homogeneous distribution of active sites . [SEP]
[CLS] this distribution is obtained by monte carlo sampling the position of the end - point of polymer B-material chains . [SEP]
[CLS] f att is then approximated as : strands with the same active site are considered equivalent , whereas in eqn ( 8 ) and ( 9 ) all strands have different identities ) c 0 a ( b ) ( r ) , the initial concentration of active sites when no binding occurs , and c ab ( r ) the concentration of bound a - b pairs once binding is allowed . [SEP]
[CLS] note that eqn ( 13 ) represents a very strong constraint : in general , mass conservation makes it valid when concentrations are integrated over the whole volume . [SEP]
[CLS] eqn ( 12 ) and ( 13 ) instead assume that this is true at each point in space , a much stronger statement . [SEP]
[CLS] in fact , the approximation in eqn ( 13 ) is in general not justified , as we discuss in more detail below . [SEP]
[CLS] in order to derive the ( lce ) expression for dna B-event binding I-event , it is important to realise that tethered dna strands are distinguishable . [SEP]
[CLS] the derivation of the condition for local chemical equilibrium is therefore different from the case where the reactants and products comprise molecules that are indistinguishable . [SEP]
[CLS] as an illustration , we will assume that the tethering of dna strands results in a confinement of the ' reactive ' ends , but that the freeenergy cost of tethering can be ignored ( this assumption can subsequently be refined ) . [SEP]
[CLS] we consider the hybridisation reaction a + b " ab . [SEP]
[CLS] as we can ignore the tethering free energy , we can treat the mixture of a , b and ab strands as an ideal gas of distinguishable particles . [SEP]
[CLS] we assume that , a , b and ab are confined to a volume v that is small , but large enough to contain many molecules . [SEP]
[CLS] the partition function of a system with n a ( n b ) monomers B-material of type a ( b ) and n ab complexes is then : [SEP]
[CLS] where n 0 a ( n 0 b ) denotes the original ( i . e . pre - reaction ) concentration of a ( b ) , and n 0 n 0 a + n 0 b , and q int ( x ) is the internal partition function of complex x ( x = a , b , ab ) . [SEP]
[CLS] note that the combinatorial factors in eqn ( 14 ) are not related to indistinguishability but to the number of ways to select n ab reaction partners from n a and from n b monomers B-material . [SEP]
[CLS] the factor n ab ! accounts for the number of ways in which n ab distinguishable particles of type a and the same number of type b can be paired . [SEP]
[CLS] the partition function is a function of n ab . [SEP]
[CLS] it is maximised for [SEP]
[CLS] hence , for large enough numbers of reactive particles in the volume v , the hybridisation of tethered strands obeys the same equilibrium expression as a binary gas mixture . [SEP]
[CLS] however , for small particle numbers this expression fails even qualitatively ( see appendix a ) . [SEP]
[CLS] this means that the lce is reasonable in the ' mean - field ' limit where any given dna strand can bind to many others , but it fails badly when this number is o ( 1 ) or less . [SEP]
[CLS] this is typically the case when the average distance between grating points is not small compared with the length of the tether . [SEP]
[CLS] it may seem surprising that the approach of ref . 25 yields results that were compatible with the experimental data , given the fact that eqn ( 12 ) is based on an approximation , and that the mean - field assumption is not expected to be particularly good in the case studied in ref . 25 . [SEP]
[CLS] it is probably more likely that the accuracy of the experiments reported in ref . 25 was insufficient to discriminate between different models . [SEP]
[CLS] for this reason , the best way to discriminate between various approximate models is to use computer ' experiments ' in which ' exact ' monte carlo simulations of a specific model are used to compare between different approximations . [SEP]
[CLS] in particular , if a model cannot reproduce the behaviour of a well - characterised model system , its agreement with experiments must be fortuitous . [SEP]
[CLS] in ref . 24 , we compared the different analytical theories with mc calculations to test the validity of mass balance equations and found that lce can give results that differ substantially from the ' exact ' simulation results , especially at high binding strengths ( see also fig . 3 ) . [SEP]
[CLS] it should come as no surprise that the agreement worsens at high binding energies as eqn ( 10 ) is only recovered from our analytical theory eqn ( 8 ) in the weakbinding limit p i - 1 . [SEP]
[CLS] a more positive finding is that , in the weak - binding limit eqn ( 10 ) remains still valid for an otherwise arbitrary distribution of binding strengths . no such good news is in store for the lce : it fails , even in the weak - binding limit because mass - balance equations can only be used to calculate the bond densities when considering the binding between free - strands in solution , not for grafted strands , where the local mass - conservation implicit in eqn ( 12 ) does not hold . however , starting from our self - consistent equations , eqn ( 8 ) and ( 9 ) we can show that a better lce approximation for the binding densities is given by : [SEP]
[CLS] together with the self - consistent condition : [SEP]
[CLS] n a being the number of strands of type a in the system . [SEP]
[CLS] once corrected , the lce treatment gives essentially exact results , at least for weak binding where eqn ( 10 ) is valid , when compared with full monte carlo simulations , unless the number of binding partners per linker is very small ( see appendix a and fig . 3 ) . [SEP]
[CLS] the advantage of eqn ( 8 ) and ( 9 ) is that these equations agree with the mc simulations over a wide range of binding regimes , not just in the weak binding limit ( see fig . 3 ) . [SEP]
[CLS] in this figure , we also show a comparison with a form of lce that was recently proposed by rogers and manoharan . [SEP]
[CLS] the latter theory assumes local mass balance but uses a different approximation for f att ( ref . 44 , esi ) . [SEP]
[CLS] the theoretical work presented thus far is based on various assumptions , as summarised in table 1 . [SEP]
[CLS] the validity of the approximations involved in the theory should be tested against numerical simulations for the same model system , whereas the quality of the underlying molecular model ( flexibility , binding strength , inter B-property - I-property molecular I-property interactions I-property ) should be checked against experiments . [SEP]
[CLS] the most stringent experimental test to date is the ability to reproduce the inter - colloidal potentials as measured this journal is © the owner societies 2016 in optical - tweezer experiments . [SEP]
[CLS] probably even more importantly than reproducing quantitative data , the presented theoretical framework also provides important qualitative insight : it allows one to rationalize various important properties of dnaccs , such as the fact the dna - mediated binding of dnaccs has a much sharper temperature response than free dna in solution . [SEP]
[CLS] as a consequence , dnaccs show higher selectivity in discriminating between slightly different target dna strands as used in biosensing applications . [SEP]
[CLS] moreover , the theory explains why dnaccs have a higher dissociation ( ' melting ' ) temperature than free dna in solution and it accounts for the dependence of the melting temperature of dnaccs on the type of spacer B-material used for fig . 3 f att as a function of distance between dna - functionalized planes at various temperatures and coating B-material densities , calculated via different approximate analytical theories ( dashed lines ) and monte carlo data ( shaded region representing the uncertainty determined in the simulations ) . [SEP]
[CLS] different panels represent different combinations of bdg 0 = [ a10 , a15 ] from left to right ( roughly proportional to temperature ) , and strands grafting densities r graft e [ 0 . 006 , 0 . 03 ] strands per nm 2 from top to bottom . [SEP]
[CLS] for reproducibility of these results , we mention that planes have a surface area of 14 884 nm 2 and either 93 or 19 ( randomly grafted ) strands in the high and low density regime , respectively . [SEP]
[CLS] color code : red = self - consistent theory with added spatial averaging of receptors , 23 blue = self - consistent theory , green = lce 0 as in the original version , orange = lce 1 , as modified by rogers and manoharan [SEP]
[CLS] note that only the self - consistent theory properly describe the monte carlo data in all ranges of densities and bdg 0 . [SEP]
[CLS] both forms of lce provide a decent comparison at high dg 0 ( = high temperatures ) and high grafting densities , but fail outside this regime . [SEP]
[CLS] grafting dna [SEP]
[CLS] within the model , all these effects follow from either multivalent effects due to the large number of binding configurations , or from the entropic penalty dg cnf arising due to grafting constrains upon bond formation . [SEP]
[CLS] the availability of an accurate and predictive theory ( eqn ( 8 ) and ( 9 ) ) made it possible to explore how the competition between different strands for binding would affect the phase behaviour of dnaccs . [SEP]
[CLS] examples are the prediction of the design rules for mixed - strand dnaccs that can undergo re - entrant melting , and the possibility to achieve temperature independent interactions due to purely entropy - driven binding . [SEP]
[CLS] although the subsequent experiments that reported both effects used a slightly different system than the example discussed in ref . 29 , the physical basis of the observed effects was the same . [SEP]
[CLS] the easiest way to see this is by considering the predicted temperature dependence of the inter - colloidal potential of the systems of ref . 29 and 44 . [SEP]
[CLS] the underlying cause of the reentrant melting is simply that the effective attraction first increases with decreasing temperature , and then drops again , whereas the reason for a widened solidfluid coexistence stems from purely entropy - driven ( and hence temperature independent , considering f att / k b t ) interactions , see fig . 4 . the first behaviour is due to a thermal control of the dominant type of bond in systems featuring competing linkages B-property , while the second arises from purely combinatorial factors between configurations featuring the same number of hybridised strands , but a different number of bonds between colloids ( since some of the hybridise strands do not contribute to inter - particle binding ) . the fact that seemingly different systems can be described by the same theory and exhibit the same behaviour , allows us to make an important general point : as long as we have quantitative information about the strength of the ligandreceptor interactions , the nature of the linkers is immaterial . [SEP]
[CLS] ssdna is a very convenient molecule to link different colloids together , but there are many other candidates ( for example , the cucurbit [ 8 ] uril system , or sialic acid / hemagglutinin complexes when nanocolloids / virus interactions are concerned ) : the underlying physics remains exactly the same . [SEP]
[CLS] we stress this fact because the development of applications non - dnabased multivalent binding is expanding rapidly , and such applications can be rationalized , and designed , using the presented framework . [SEP]
[CLS] in the context of dna - mediated interaction , cooperativity is often mentioned as a crucial aspect . [SEP]
[CLS] in the theoretical description above , cooperativity plays no role ; we assume explicitly that the formation of one bond does not change the binding strength of others , and yet our theoretical predictions agree very well with experiment . [SEP]
[CLS] to avoid all confusion , we stress that , of course , the fact that one bond between two colloids has formed , makes it easier for subsequent bonds to form , simply because the loss of translational entropy of the binding partner is incurred in the first binding event only . [SEP]
[CLS] however , that phenomenon has nothing to do with a phenomenon where , at a microscopic level , the strength of subsequent bonds is enhanced by a change in the local environment . [SEP]
[CLS] in fact , the latter type of cooperativity is very hard to demonstrate , and it would seem that all experiments where special , cooperative effects were invoked ( in the context of an overly simplistic theory ) can , in fact , be explained within the theoretical framework that we discuss here , without cooperativity . [SEP]
[CLS] this does not mean that there cannot be cooperative effects but simply that the evidence is lacking , if agreement with a simpler theory can be viewed as occam ' s razor . [SEP]
[CLS] having said this , there are definitely cases where cooperativitybut , in this case almost certainly negative cooperativity - could play a role : in nano - sized colloids , and possibly for the recently developed micron - size colloids , grafted strands can be close enough to each other to feel the local change in the environment upon binding , be it entropic or electrostatic in origin . our key message is that the sharper melting behaviour of dnaccs , the key experimental finding that is often cited as evidence in support of the cooperativity model , is perfectly well explained by our simple model that ignores cooperativity ( and , in fact , by even much simpler , non - cooperative models ) . [SEP]
[CLS] on the experimental front , a sharper melting is also clearly observed for systems of micron - sized dnaccs where grafting densities were too low for any cooperativity to occur . [SEP]
[CLS] it is appealing that a simple theory that needs not invoke specific , system - dependent cooperative effects can provide a unified explanation of dnacc melting for a wide range of systems . [SEP]
[CLS] 4 schematic representation of the variation of the strength of the effective pair - attraction between dna - coated colloids . [SEP]
[CLS] panel a shows the case of a non - monotonic temperature dependence of the interaction strength . [SEP]
[CLS] such behaviour can be realised in dna coated colloids and results in a re - entrant phase diagram in which the solid phase melts upon both heating and cooling ( see text and ref . 29 and 46 ) . [SEP]
[CLS] dna - mediated reentrant melting has been observed in subsequent experiments . [SEP]
[CLS] in ref . 29 , the dna - mediated potentials were induced via inter - and intra - particle hybridisation between grafted strands , whereas in ref . 44 an indirect hybridisation via free - strands in solution was used . [SEP]
[CLS] panel b shows the case where attraction at low temperatures is entropy - dominated . [SEP]
[CLS] in that case , the attraction strength is approximately df att e atds , which , when multiplied by bdf att , becomes temperature independent . [SEP]
[CLS] a temperature - independent ( scaled ) interaction results in a temperature - density phase diagram with a constant width of the gas - aggregate coexistence region . [SEP]
[CLS] this journal is © the owner societies 2016 [SEP]
[CLS] any numerical model of dnacc systems must account for the multi - scale nature of the problem . [SEP]
[CLS] the reason is that the physical properties of dnaccs depend on both microscopic and mesoscopic features , such as the sticky - end sequences , the structure of the spacers B-material , and the grafting density of the dna units , that enter into the design of the system at different length scales . [SEP]
[CLS] in fact , the differences between various types of dnaccs are substantial . [SEP]
[CLS] for instance , whilst nano - sized dnaccs may be coated with only a few dozen dna strands , micron - sized dnaccs may be coated with tens of thousands of dna strands , although typically the coating B-material density in this case is much lower than for nanocolloids . [SEP]
[CLS] not surprisingly , particles that are that different require different coarse - grained modelling and simulation strategies . [SEP]
[CLS] the modelling approaches for micron and nano - sized colloids will be reviewed in sections iiia and iiic respectively . [SEP]
[CLS] in section iiib we extend the discussion to micron - sized particles with mobile B-property rather than grafted strands , e . g . suspensions of functionalised lipid B-material - bilayers I-material and emulsions . [SEP]
[CLS] although , in principle , fully atomistic modelling of dnaccs is conceivable , the cost of such an approach would be prohibitive and , as in other areas of colloid science , there is no need for such a brute - force approach . [SEP]
[CLS] however , it should also be clear that microscopic details matter as the interaction between ssdna strands depends sensitively on their nucleotide sequence . [SEP]
[CLS] any model , no matter how coarse - grained , must take this specificity into account . [SEP]
[CLS] roughly speaking , there are three classes of coarse - grained models that account for the dna specificity in different ways . [SEP]
[CLS] the most detailed approaches explicitly account for the pairing of the individually - resolved nucleotides ( see part 1 of fig . 5 ) [SEP]
[CLS] recently , more portable but expensive models , also capturing the double stranded structure of dna , have been introduced . [SEP]
[CLS] a more coarse - grained approach uses empirical rules for the binding free - energy of dna sequences . [SEP]
[CLS] in this description , the binding sequences are represented as structureless points ( see part 2 of fig . 5 ) , and the binding free - energy is given by eqn ( 3 ) , as discussed in section ii . [SEP]
[CLS] finally , a less well - founded , but nevertheless frequently used approach is based on the ' chemical equilibrium ' approximation ( see part 3 of fig . 5 ) . [SEP]
[CLS] the chemical equilibrium theory treats binding reaction as a dimerisation reaction in a confined gas of sticky end groups . [SEP]
[CLS] the advantage of this approach is that it is simple and , when the balance between free and bound ideal constructs is properly implemented ( see appendix b in ref . 23 ) , it becomes equivalent to rigorous treatments . [SEP]
[CLS] however these methods are practical only in the ' mean field limit ' , where the spatial distributions of sticky ends are not strongly dependent on the exact position of the grafting points . [SEP]
[CLS] micron - sized colloids may be coated with several thousands of constructs per particle . [SEP]
[CLS] a detailed representation of such a large number of constructs is usually not computationally feasible even when the aim is only to study the effective interaction between two colloids . [SEP]
[CLS] the problem is , however , not as serious as it seems , because large particles tend to have a radius of curvature that is much larger than the length of the binding strands . [SEP]
[CLS] under these conditions , one can use the derjaguin approximation ( see e . g . ref . 67 ) to calculate effective pair potentials between colloids from knowledge of the distance - dependence of the interaction between two flat , parallel surfaces . [SEP]
[CLS] if this approximation is not used , a local chemical equilibrium approach ( or a ' ' spatially averaged ' ' version of the self - consistent equations , see ref . 23 and 27 , and fig . 3 ) is still tractable , but for all approaches based on the explicit calculation of interaction between complementary strands , use of the derjaguin approximation becomes imperative . [SEP]
[CLS] with the derjaguin approximation , the fact that micron - sized colloids can be coated with very large numbers of dna strands does not create a technical challenge . [SEP]
[CLS] as discussed section ii , most theoretical approaches developed to study dnaccs are based on the assumption that non - binding strand - strand interactions can be ignored . [SEP]
[CLS] we stress that this assumption is not essential ( and questionable for nano - sized colloids : see ref . 31 and 32 and section iiic ) , but in many cases , the neglect of non - binding strand - strand interactions is justified , in which case an accurate estimate of dg cnf is all that is required to obtain quantitatively accurate potentials from computer simulations . [SEP]
[CLS] in practice , we calculate the partition function of unbound strands and the configurational part of the binding free energy of two strands , dg cnf , by simulation . [SEP]
[CLS] the strand - strand binding free energy dg 0 is not obtained from simulation but from the empirical santalucia rules . [SEP]
[CLS] again , more sophisticated rules could be used where necessary . [SEP]
[CLS] once these two ingredients are known , and if strand - strand interactions can be neglected , the theoretical approaches described in section ii can be used to calculate accurate inter - particle potentials . [SEP]
[CLS] as was demonstrated in experiments of bracha for dna - chips et al . strand conformations are weakly affected by dna - dna interaction up to thousand strands per mm 2 . [SEP]
[CLS] moreover , a rough estimate suggests that the contribution of non - bonded spacer B-material interactions to the free energy of the system is of the order of one k b t unit per strand , which is much smaller in absolute terms than the typical values of the configurational and combinatorial free - energy that are around e10k b t for typical spacers B-material . [SEP]
[CLS] a notable exception of a micron - size system where nonbonded strand interactions may be significant is provided by the densely grafted micron - sized colloids studied by pine and co - workers . [SEP]
[CLS] the reason why higher grafting densities could be reached in ref . 55 and 58 is that in these experiments , grafting was achieved by using click chemistry , rather than via the use of rather bulky streptavidin constructs that anchor the biotinylated spacers B-material . [SEP]
[CLS] having sketched the various theoretical approaches to predict the binding strength of dnaccs , we next consider how to estimate the change in the configurational free energy dg cnf of the grafted strands as the distance between colloids is varied . [SEP]
[CLS] in general , dg cnf can be computed using monte carlo simulations . [SEP]
[CLS] practical aspects of these calculations for ssdna constructs are discussed in appendix b . [SEP]
[CLS] in this modelling , it is important to account for intra - chain coulomb interactions . [SEP]
[CLS] for instance , ssdna constructs have been modelled as uniformly charged , freely - jointed chains ( fjc ) with screened debye - hu ¨ckel interaction ( see , e . g . ref . 73 ) . [SEP]
[CLS] such an approach has been used to model micron - sized and , as we will discuss later , nanosized dnaccs . [SEP]
[CLS] when accounting for electrostatic interactions in this way , the electrostatic contribution has to be removed from the phenomenological dna hybridisation free energies , to avoid double counting . [SEP]
[CLS] the numerical approach to compute the configurational contribution to the dnacc interaction described in appendix b is efficient for short to medium length linkers that can be coarsegrained into at most a few dozen kuhn segments . [SEP]
[CLS] however , the same methods would be computationally unfeasible when applied to very long chains . [SEP]
[CLS] numerical studies of colloids functionalised by very long dna strands such as l - dna require a further simplification of the description of the dna degrees of freedom . [SEP]
[CLS] bozorgui and martinez employed a numerical approach based on the ' blob ' representation of long polymer B-material chains in which entire dna constructs are lumped into sites interacting via gaussian potentials . [SEP]
[CLS] multi - blob models have been used by mladek et al . , but the approach , whilst fairly accurate , quickly becomes cumbersome . [SEP]
[CLS] the potentials between the blob and the colloids have been parametrised by using explicit ' all - monomer ' simulations , while the hybridisation reactions between complementary strands is simulated using a dedicated mc move that attempts to join two complementary blobs with a cost equal to dg bond ij , where dg cnf is given by the energy of an harmonic spring connecting the centres of the two blobs of the form bdg cnf = 0 . 534 ( r / r g a 0 . 730 ) , where r g is the gyration radius of the strand and the numerical values 61 that introduced flanking beads to avoid binding between more than two bases . [SEP]
[CLS] although the molecular mechanism for binding is retained in these models , none of them tries to match the real thermodynamics of dna hybridisation but simply use an effective nucleotide - nucleotide interaction which should be considered as a tunable model parameter . [SEP]
[CLS] ( c ) nucleotide level models , such as snp3 or oxdna , have been parametrised using thermodynamic experiments . [SEP]
[CLS] ( 2 ) models that feature an implicit dna hybridisation rely on empirical estimates of the hybridisation free energy of the sticky ends ( dg 0 ij ) . [SEP]
[CLS] ( a ) chains , interacting by non selective hamiltonians ( h ns ) , hybridise by coalescing the two reactive end - elements according to dg 0 ij and to a configurational cost ( dg cnf ij ) , that depends on the algorithm that generates the hybridised chain . [SEP]
[CLS] notice that the non selective hamiltonians of the free and bound states are not the same . [SEP]
[CLS] ( b ) a similar scheme was previously proposed by mladek et al . that also considered the case in which more reactive sites bind resulting in a rigid dsdna segment . [SEP]
[CLS] ( c ) for long l - dna ref . 74 - 76 used a blob representation in which reactive blobs are chained according to an hybridisation free energy dg bnd . [SEP]
[CLS] ( 3 ) for ideal spacers B-material a local chemical equilibrium balance can be used to calculate the fraction of hybridised strands ( ab ) . [SEP]
[CLS] the concentrations of the sticky ends around the colloids c ( r ) are calculated by the end - to - end distribution functions of the tethered spacers B-material , while the equilibrium constant between sticky ends is dg 0 ij . [SEP]
[CLS] have also been parametrised using all monomer B-material simulations ( see part 2c of fig . 5 ) . [SEP]
[CLS] the ultimate aim of coarse - grained modelling of dnaccs is to predict their phase behaviour . [SEP]
[CLS] once the tools are in place to compute the dnacc interactions , standard free - energy calculations can be used to compute phase diagrams . [SEP]
[CLS] examples of such studies can be found in ref . 74 - 76 . [SEP]
[CLS] these simulations showed that dnaccs exhibit very interesting phase behaviour . [SEP]
[CLS] for example , the simulations showed that the liquid - crystal - vapour triple point disappears as the number of grafted chains drops below a threshold of around 12 . [SEP]
[CLS] in that case , the ' ground - state ' at zero pressure but high densities is a liquid , not a crystal . [SEP]
[CLS] recent simulations on ' patchy ' colloids , showed similar behaviour in such systems . [SEP]
[CLS] recent experiments have studied dnaccs where the dna strands are mobile B-property , rather than grafted in fixed positions . [SEP]
[CLS] examples include emulsions and lipid B-material bilayers I-material functionalised by hydrophobised B-property dna constructs terminated by reactive sticky end sequences . [SEP]
[CLS] it is straightforward to extend the theoretical tools to model dnaccs with grafted dna strands to the case where these strands are mobile B-property : due to the translational degrees of freedom of the binders B-property , the hybridisation free - energy needs to include an extra configurational term ( dg trn ) that accounts for the constraint on the relative position of two tethering points when bound [SEP]
[CLS] this confining entropic cost dg trn ij destabilises dna pairing . [SEP]
[CLS] however , the combinatorial gain is higher in system of mobile B-property tethers because each binder B-property can potentially bind more complementary partners than in a system with fixed tethering points . [SEP]
[CLS] a quantitative analysis of all the entropic and configurational terms of eqn ( 18 ) for a system containing both bridges ( i . e . bonds between strands residing on different particles ) and loops ( i . e . between strands on the same particle , when this bears complementary sequences ) has been given in ref . 33 . [SEP]
[CLS] interestingly , in these systems the self - consistent theory developed in ref . 23 , 27 and 42 ( eqn ( 8 ) and ( 9 ) ) becomes exact . [SEP]
[CLS] this was demonstrated in ref . 28 , starting from an exact free energy functional f fng ; dg bond e e a a calculated as a function of the number of bridges between bound colloids ( { n } ) and the matrix of the hybridisation free energies dg bond ij ( where i and j now represents a strand type , not a single specific strand ) . [SEP]
[CLS] taking the large number of binder B-property per particle limit , a saddle - point approximation provided the most probable number of bridges between colloids ( % n ) df ðfng ; fdg bond gþ dn [SEP]
[CLS] remarkably eqn ( 19 ) is equivalent to eqn ( 9 ) . [SEP]
[CLS] using { % n } one can calculate the attractive potential between colloids f att ¼ fðf ng ; fdg bond gþ ( 20 ) and recover exactly the expression given in ( 8 ) ( once the number of bridges % n are expressed in term of probability of making bridges between colloids ) . [SEP]
[CLS] it is possible to generalise this discussion to systems featuring different types of bonds including not only bridges but also loops . [SEP]
[CLS] slightly different expressions are obtained in presence of infinite reservoirs of binders B-property as shown in the study of a vesicle interacting with a large supported lipid B-material bilayer I-material . [SEP]
[CLS] eqn ( 19 ) and ( 20 ) were used to simulate rigid colloids functionalised by mobile B-property strands ( a possible experimental realisation being that of ref . 86 ) in which colloidal suspensions are sampled using an effective interaction free energy f att ( { r i ( t ) } ) + f rep ( { r i ( t ) } ) that only depends on the positions of the colloids { r i ( t ) } . [SEP]
[CLS] we note that the behaviour of dnaccs with mobile B-property linkers can be qualitatively different from that of dnaccs with grafted linkers , in other words : mobile B-property linkers introduces new physics in the system . [SEP]
[CLS] for example , ref . 28 showed that the inter - colloidal potential induced by dna - hybridisation of mobile B-property linkers is intrinsically a multi - body interaction . [SEP]
[CLS] this is due to the fact that each construct on a given particle can bridge all the neighbouring particles . [SEP]
[CLS] hence , different neighbouring colloids may compete for the same linkers . [SEP]
[CLS] as the number of bonds with different neighbours are now correlated , the free - energy cannot be decomposed into the sum of pair - interactions . [SEP]
[CLS] the fact that dnaccs with mobile B-property linkers have intrinsically multi - body interactions can be exploited used to control the ' valency ' ( preferred number of complementary neighbours ) of a particle . [SEP]
[CLS] this is interesting because conventional approaches to tune the valency of colloids are based on the use of different number of interacting ' patches ' . [SEP]
[CLS] however , the preparation of well - controlled patchy colloids is non - trivial . [SEP]
[CLS] in contrast , dnaccs with mobile B-property linkers can have a preferred valency without the need of physically creating real patches on the surface of the colloid . [SEP]
[CLS] even more complex effects should be expected for systems of mobile B-property strands where the colloid to which they are attached is deformable , as in the case of dna - functionalised vesicles , where the flexibility of the lipid B-material membrane I-material plays a nonnegligible role in determining the bond - mediated interaction . [SEP]
[CLS] for instance , hu and collaborators considered the interaction between two membranes that were coated with complementary proteins B-material ( rather than dna ) . [SEP]
[CLS] they found that the equilibrium constant for protein B-event - I-event protein I-event binding I-event ( i . e . dg bond in our nomenclature ) decreases by a factor that is controlled by the roughness of the membranes . [SEP]
[CLS] this effect , when translated to dna - mediated interaction , can be expected to have important consequences for the self - assembly . [SEP]
[CLS] modelling the interaction between nano - size dnaccs is more demanding than modelling their micron - sized counterparts . [SEP]
[CLS] the underlying reason is simple : the length - scale separation that justifies the factorisation between the different driving forces and facilitates modelling of micron - sized particles is less pronounced for nano - sized particles . [SEP]
[CLS] there are other differences as well : many of the earlier experiments on dnaccs ( although not the most recent ones ) used a bulky streptadivin - biotin construct to tether dna strands to the surface of micron - sized colloids . [SEP]
[CLS] in contrast , dna anchoring to gold B-material nano - particles is usually achieved by bonding the ( much smaller ) thiol B-material group to the gold B-material surface . [SEP]
[CLS] as a consequence , the density of the dna strands on nano - sized colloids is sufficiently high that the interaction between different dna - strands cannot be neglected for a quantitative modelling . [SEP]
[CLS] constructing a coarsegrained model of nano - sized dnaccs is therefore much more system - specific than for micron - sized colloids . [SEP]
[CLS] a case in point is the coarse - grained model developed by mladek et al . in an attempt to reproduce a typical experimental system . [SEP]
[CLS] construction of the model required three coarse - graining steps . [SEP]
[CLS] in the first step , simulations were performed on nano - colloids ( 12 nm diameter ) functionalised by 60 homogeneously charged freelyjointed chains representing the 665 base - pair constructs used in experiments . [SEP]
[CLS] at this level of description , no dna hybridisation was taken into account . [SEP]
[CLS] hence , the particles were simply treated as nano - colloids sterically stabilised with densely grafted chains . [SEP]
[CLS] the simulations probed the repulsive interaction between two such colloids . [SEP]
[CLS] subsequently , a multi - blob model was constructed to reproduce the computed repulsive interaction between the nano - colloids . [SEP]
[CLS] the ends of the coarse - grained chains were then functionalised with units that could hybridise . [SEP]
[CLS] however , for the nano - sized colloids , the spatial extent of the ' sticky ' end group could not be ignored , nor the fact that , upon hybridisation , two such groups combine to form a double helix that is more rigid than the unbound sticky ends . [SEP]
[CLS] hence , although in this model too the hybridisation free energy is calculated using the santalucia nearest neighbour rules , the conformational changes upon hybridisation are accounted for in the model ( see part 2b of fig . 5 ) . [SEP]
[CLS] using this fairly complex model , the authors computed the crystallization behaviour of the nano - sized dnaccs and they computed the structural properties of the crystalline solid . [SEP]
[CLS] encouragingly , the simulations predicted the correct stable crystal structure and gave very good estimates for the temperature at which the solid melts . [SEP]
[CLS] however , the estimated crystal lattice spacing was off by some 10 % ( presumably because the experimental estimates of the persistence length of ssdna show a considerable spread ) . [SEP]
[CLS] the other important finding was that three - body interactions play an important role and must be correctly included in order to predict the structure and melting of the crystalline phase . [SEP]
[CLS] as the above discussion shows , coarsegrained modelling of nano - sized dnaccs is not simple . [SEP]
[CLS] however , with the use of a coarse - grained polymer B-material model and the empirical santalucia nearest neighbour rules , it is still much more efficient than a brute force simulation of nano - size dnaccs . [SEP]
[CLS] indeed , both the system sizes ( hundreds to thousands of colloids ) and the timescale for hybridisation are such that atomistic simulations of the phase behaviour of dnaccs is out of the question for the foreseeable future , whilst calculation of the pair interaction between nano - sized dnaccs is , at lest at present , not an attractive proposition . [SEP]
[CLS] there are , however , good reasons to use models for dna that , whilst not atomistic , do reproduce the structure and dynamics at the nucleotide level . [SEP]
[CLS] in fact , much effort has been devoted to the development of coarse - grained models that are capable of reproducing the hybridisation free energy , the binding kinetics and the mechanical properties of dna complexes . [SEP]
[CLS] as the interactions that drive dna hybridisation are primarily the hydrophobic B-property forces between neighbouring bases , such models require a nucleotide - level description of the biopolymer B-material . [SEP]
[CLS] although there have been extensive and very successful simulations of systems with few dna strands , the models used are still not an attractive proposition to simulate the collective behaviour of dnaccs , as such simulations would require the study of systems of hundreds ( or thousands ) of colloids each coated with dozens of strands . [SEP]
[CLS] moreover , computing the thermodynamic properties from equilibrium simulations requires sampling times that are much longer than the time for a typical hybridisation / de - hybridisation events . [SEP]
[CLS] hence , at present , this is not yet possible with the models of ref . 62 - 64 . [SEP]
[CLS] for this reason , even more coarse - grained - but still nucleotidebased - descriptions have been developed starting with the work of starr and sciortino . [SEP]
[CLS] in the approach of ref . 59 and 60 , the nucleotides are described as ( repulsive ) spheres functionalised with a binding site . [SEP]
[CLS] neighbouring monomers B-material are bound together by a so - called fene potential . [SEP]
[CLS] a bending rigidity term between each three consecutive monomers B-material is also used . [SEP]
[CLS] a binding site , specifying the type of base , is connected to each monomer B-material via a fene potential . [SEP]
[CLS] these binding sites selectively interact with complementary sites via lennard jones potentials . [SEP]
[CLS] this model has been used to study the assembly of dendrimer - like structures functionalised by four dna arms , self - assembly mediated by linkers and crystallisation of nanoparticles B-nanoparticle . [SEP]
[CLS] a model that is closely related to that of starr and sciortino 59 was deployed by knorowski and collaborators ( see part 1b of fig . 5 ) . [SEP]
[CLS] as in ref . 59 and 60 , dna constructs were modelled by mean of beads carrying selective binding sites . [SEP]
[CLS] however , two flanking repulsive sites were added to every bead belonging to the sticky end part of the construct . [SEP]
[CLS] the function of the flanking beads is to provide directionality to the interaction between complementary bases and to prevent ( unphysical ) multi - base interaction . [SEP]
[CLS] this model has been used to probe the stability of different crystals and to investigate the dynamics of crystallisation . [SEP]
[CLS] recently the model of starr and sciortino 59 has been used by theodorakis and collaborators to study the pair interactions between nanoparticles B-nanoparticle functionalised by up to eight constructs . [SEP]
[CLS] this study highlighted the need for a potential further refinement of the model , as the simulations showed the presence of spurious bound states in which the ssdna strands that form a duplex were oriented parallel , rather than anti - parallel . [SEP]
[CLS] parallel hybridisation is not found in real dna , as the strongly polarity - dependent watson - crick pairing between complementary nucleotides uniquely favours anti - parallel hybridised sequences . [SEP]
[CLS] the model of knorowski has been further developed by li et al . in particular the size of the beads in the sticky part and in the spacer B-material part of the construct have been differentiated to qualitatively account for the different mechanical properties of the ssdna and of the dsdna spacers B-material . [SEP]
[CLS] this model is parametrised in such a way that each bead corresponds to e2 - 3 bases . [SEP]
[CLS] this relatively detailed model was subsequently used to parametrise an effective pair - potential that is composed of a short range repulsion due to the electrostatics of the system , and an attractive this journal is © the owner societies 2016 interaction provided by the hybridisation of the sticky - ends . [SEP]
[CLS] using the effective model the authors rationalised experimental finding on the dynamics of crystallisation , while the detailed model has been used to probe the stability of the crystals and to investigate the microscopic structure of the facets of dnaccsmade crystal . [SEP]
[CLS] detailed molecular models have also been used to investigate interaction between dna constructs and the nanoparticle B-nanoparticle substrate ( e . g . ref . 100 and 101 ) , and to study the changes in the structure of dsdna when used to assemble crystalline structures . [SEP]
[CLS] however , these studies go beyond the scope of the present review , where we focus on the physical properties of dnaccs that are related to the reversible biding of multiple dna complementary pairs , rather than the formation of a single pair formation . [SEP]
[CLS] there are , in principle , two ways to construct a proper coarsegrained model for dnaccs . [SEP]
[CLS] one ( the top - down ) approach is to derive a coarse - grained model from a fully atomistic model for dna , and use it to simulate dnaccs . [SEP]
[CLS] this type of approach thus starts with a system at the top of the ' ' complexity scale ' ' , and uses systematic coarse - graining techniques to move towards the bottom of the same scale . [SEP]
[CLS] such an approach still retains a molecular based description , and should be able to describe the hybridisation thermodynamics of dna . [SEP]
[CLS] the second approach is instead to start with the choice of the ingredients of the coarse - grained model ( e . g . ideal chains , rods , monomers B-material , blobs , hybridisation sites etc . ) and then to use comparison with the available experimental information to tune its parameters . [SEP]
[CLS] at present , the top - down approach is not yet as extensively used as that comparing coarse - grained models directly with experiment . [SEP]
[CLS] an encouraging example in this direction is the work of lequieu et al . and ding et al . , who recently reported studies of pair potentials between nanoparticles B-nanoparticle functionalised by few constructs of reactive dna , using the 3spn model previously used to study the dimerisation of dna in solution ( ref . 64 and 104 ) . [SEP]
[CLS] the oxdna model of ouldridge and coworkers is another valuable cg model that has been tested in multiple dna nanotechnology systems . [SEP]
[CLS] to our knowledge , oxdna has not yet been used to improve the modelling of dnaccs . [SEP]
[CLS] there are other aspects of the modelling that need to be improved . [SEP]
[CLS] one is related to the naive use of nearest - neighbour rules to compute dna B-event binding I-event free energies in constructs . [SEP]
[CLS] recent experimental / theoretical investigation by di michele et al . showed that the santalucia nearest - neighbour rules overestimate the hybridisation free energy dg 0 in the case of dna oligomers that are extended to include strings of noncomplementary bases . [SEP]
[CLS] considering for instance two complementary sequences of ssdna decorated by two inert strings at their 5 0 terminals ( snapshot of fig . 6a ) , ref . 103 reported that dg 0 increases with the length of the inert tails until reaching a plateau for a number of inert bases equal to e5 - 7 ( plot in fig . 6a ) . [SEP]
[CLS] in particular the presence of the inert tails destabilises double helix formation and hence decreases the melting temperature of the oligomers . [SEP]
[CLS] such an effect is due to the electrostatic interactions between the charged backbones of the dna that is not included in the nearest neighbour rules . [SEP]
[CLS] quite surprisingly ref . 103 showed that the shift in the hybridisation free energy due to the tails ( at salt B-material concentration that are typical of dna coated colloids experiments ) can be as large as the stacking contribution of the nearest - neighbour rules due to the first dangling ( i . e . non - hybridised ) base in the nucleotide sequence , a standard term entering the nearest - neighbour rules ( see fig . 6b ) . [SEP]
[CLS] the relevance of the inert tails to the calculation of the hybridisation free energy dg 0 seems to limit the degree of ' responsible ' coarse graining of dna mediated interactions ( also in other dna nanotechnology systems ) . [SEP]
[CLS] in particular if we consider models with explicit hybridisation thermodynamics , the importance of the electrostatic interactions between the inert tails and the dna duplex requires an explicit description of the charged backbones in the tails and in the duplex at the atomistic level . [SEP]
[CLS] interestingly the latest version of oxdna introduced debye - hu ¨ckel interactions between the backbone sites [SEP]
[CLS] the problem is that the binding free energy computed for a typical configurations of the dangling segments of the free oligomers , may not be transferable . this is the case , for instance , if the tails are more stretched when bridging two colloids than when they are free in solution . hence , estimates of dg 0 obtained from experiments for free strands might not be reliable once the same strands have been grafted . these ' minor ' effects are not so minor when one realises that a bias of one k b t in dg 0 results in a shift in the melting temperatures of about eae3 . 5 k ( see fig . 6b ) . hence , when analysing accurate experiments , neglect of the dangling tail effects can affect the interpretation of the results , once the experimental resolution gets better than a few kelvin . [SEP]
[CLS] there is also the ' no - free - lunch ' theorem of coarse graining . [SEP]
[CLS] as was pointed out in a different context many years ago by louis , the design of an optimal model that carefully reproduces the structural properties of a system may result in a model that is not representative of the thermodynamics of that system ( and vice versa ) . [SEP]
[CLS] finally we stress something that has been stressed many times : structures may be mechanically stable , but thermodynamically unstable . [SEP]
[CLS] demonstrating that one structure is more stable than another typically requires the calculation of the free energy ( or , more precisely , the chemical potential ) of both systems . [SEP]
[CLS] using mc or molecular dynamics to see what kind of structure forms by annealing from a disordered state is interesting , but it does not demonstrate that the structure that forms is stable ( in fact , ostwald ' s rule suggests the opposite ) . [SEP]
[CLS] the problem is that the first stages of the self - assembly process are those with a smaller barrier B-property from the starting disordered state . [SEP]
[CLS] these states can simply represent mechanically stable metastable states , which would transform to the true thermodynamically stable state at much longer times ( if at all ) . [SEP]
[CLS] these longer times may not be accessible with experiments , and are certainly many orders of magnitude larger than the time - scales accessible by computer simulations . [SEP]
[CLS] we also notice that , in order to correctly reproduce the stable phases observed in experiments , studies using this simulated annealing approach typically start with simulations boxes compatible with the shape and number of particle experimentally observed , which must thus be known a priori . [SEP]
[CLS] since maximal packing appears to be , at least for conventional binary dnaccs system , one of the major driving force behind formation of specific lattices , starting with experimentally available conditions necessarily biases the simulation toward the expected structure . [SEP]
[CLS] in short : constructing a phase diagram requires the calculation of the free energy of each possible phase , unless a direct , hysteresis - free transition between the two phases can be observed . [SEP]
[CLS] one of the main challenges in the simulation of the phase diagram of dnaccs was precisely the need to carry out free - energy calculations that properly account for the hybridisation between the binders B-property . [SEP]
[CLS] fortunately , efficient mc algorithms have been developed to calculate the density of states of an aggregate as a function of the number of bridges between particles , whose integration can then be used to calculate the free energy of the crystal . [SEP]
[CLS] these algorithms have been successfully combined with various techniques to select among the many possible candidate phases . [SEP]
[CLS] for example , in view of the rich polymorphism offered by dnaccs , genetic algorithms have been used to identify possible crystal structures , whose relative thermodynamic stability was then probed by free - energy calculations , providing phase diagrams in quantitative agreement with experiments without an a priori knowledge of crystals parameters . [SEP]
[CLS] theoretical and computational modelling have proven to be powerful tools to rationalise the observed behaviour of dnaccs , and , in some cases , to predict novel phenomena , with some theoretical predictions for unconventional yet experimentally accessible systems still waiting to be tested . understanding dnaccs behaviour means understanding their mutual interactions as a function of the system ' s parameters . [SEP]
[CLS] in this regard , we have shown how the presently available theoretical framework can be used to design interactions of almost arbitrary complexity : how these could lead to interesting effects ( see for instance ref . 114 and 115 for some applications ) is yet to be systematically investigated . [SEP]
[CLS] an important factor to highlight in this regard is that the analytical framework we described assumes thermodynamic equilibrium for bond formation , i . e . it implicitly considers the bonding network to be equilibrated on the timescale of colloidal diffusion . [SEP]
[CLS] hence , in order to connect the effective inter - particle potential predicted by theory with experimental observations ( that are of finite duration ) one requires both the bond - formation and bond - breaking rates to be fast compared to the timescale for colloidal diffusion . [SEP]
[CLS] until now , this was a strong assumption . [SEP]
[CLS] however , although this was not true for general bonding schemes previously typically employed , rogers and manoharan have recently shown a very powerful and general scheme exploiting toe - holding mechanisms that can make such assumption essentially correct . [SEP]
[CLS] proof of this point is already observable in their experimental results , whose trends could be rationalised using a local chemical equilibrium approach that implicitly makes such an assumption . [SEP]
[CLS] hence , we would expect such scheme to lead to a more reliable comparison between theory and experiments , opening new and truly exciting perspectives for this field . [SEP]
[CLS] this journal is © the owner societies 2016 [SEP]
[CLS] most of the focus in the dnaccs has probably been on their selfassembly property , and much remains to be done to exploit this system to its full potential , in particular in the design of functional materials for applications [SEP]
[CLS] in our view , however , other dnaccs based technologies will benefit from interpreting experiments in view of the presented framework , selective drug delivery and biosensing being two likely candidates . [SEP]
[CLS] it should be stressed that , besides describing dnaccs - systems that have important nanomedicine applications in their own regard - the theoretical and computational methods outlined here apply more generally to all systems whose interactions are dominated by the formation of non - covalent , multiple and reversible ligand - receptor B-material bonds , i . e . supramolecular , multivalent based systems . a wide variety of such systems are already under intense investigation for nanomedical applications , and we envisage important cross - fertilization of ideas between these fields and dnacc research . [SEP]
[CLS] in particular , understanding how to embed binding selectivity in multivalent - based systems , and not just increase their binding strength , seems to be a crucial point where theoretical work might prove useful to rationalise observed effects , and speed up the rational design of applications . [SEP]
[CLS] finally , we stress that there is much need for further theoretical and computational developments . [SEP]
[CLS] for applications that will require quantitative predictions , more accurate experiments are needed to inform the parametrisation of existing , or the development of , novel models . [SEP]
[CLS] an illustration is the tail effect discussed in section iii . [SEP]
[CLS] understanding the role of non - specific attractive interactions , and also of strand - strand interactions , are further challenges that have still not been properly investigated . [SEP]
[CLS] the need for such improved modelling has become urgent with the arrival of recent experiments of the pine group , who was able to reach coating B-material densities that are much higher than what was previously achievable . [SEP]
[CLS] interestingly these experiments reported enhanced crystallisation rates ; it would be interesting to understand these results using properly extended models . [SEP]
[CLS] finally , other challenges clearly arise when considering systems where the colloid itself can be deformed upon binding ( hence effectively coupling dg cnf with the shape of the colloidal core B-material ) , such as the case of functionalised vesicles . [SEP]
[CLS] after the long gestation period that followed the early work by mirkin and alivisatos , the field is moving rapidly . [SEP]
[CLS] exciting times lie ahead of the dnaccs field . [SEP]
[CLS] we hope that this review will have clarified some of the key unifying concepts that need to be understood in the move towards the rational design of dnacc - based structures . [SEP]
[CLS] in this regard , we make freely available a python - based set of routines that we developed to calculate ligand - receptor mediated interaction free energies using our selfconsistent equations at the following address https : / / github . com / sangiole / dnacc . [SEP]
[CLS] to illustrate the breakdown of lce and mean - field like approaches in the limit of small numbers of binding strands , we assume that the solutions are ideal . [SEP]
[CLS] then the partition function of species x ( x = a , b or ab ) is [SEP]
[CLS] where q int ( x ) is the contribution to the partition of x due to all internal degrees of freedom . [SEP]
[CLS] the equilibrium condition a1 can be written as [SEP]
[CLS] where c x is the number density of species x . [SEP]
[CLS] to compare with experiments , it is conventional to express the equilibrium condition such that the concentrations can be expressed in mol per liter . [SEP]
[CLS] in order to convert to number densities , we note that [ x ] = c x / r 0 where r 0 is the ' ' standard ' ' number density ( 1 molar = 6 . 022 a 10 26 molecules per m 3 ) . [SEP]
[CLS] then small compared to the effective length of the strands . on the other hand fig . 3 shows that the self - consistent approach performs very well also at low grafting density . in this case dg cnf can be measured comparing the time spent by the system in the free and in the bound state . [SEP]
[CLS] ) where c a ( b ) ( r ) are the equilibrium local concentrations , of free , unbound active sites of type a ( b ) ( note that in this formalism all this journal is © the owner societies 2016 phys . chem . chem . phys . , 2016 , 18 , 6373 - - 6393 | 6379 [SEP]
[CLS] 5 modelling dna hybridisation in dnacc systems . [SEP]
[CLS] ( 1 ) models that feature explicit dna thermodynamics attempt to recover dna hybridisation with an explicit modelling of the dimerisation process . [SEP]
[CLS] ( a ) ref . 59 and 60 used a bead - and - spring ssdna representation decorated by sites that selectively attract . [SEP]
[CLS] ( b ) this model was ameliorated by ref . 61 that introduced flanking beads to avoid binding between more than two bases . [SEP]
[CLS] although the molecular mechanism for binding is retained in these models , none of them tries to match the real thermodynamics of dna hybridisation but simply use an effective nucleotide - nucleotide interaction which should be considered as a tunable model parameter . [SEP]
[CLS] ( c ) nucleotide level models , such as snp3 or oxdna , have been parametrised using thermodynamic experiments . [SEP]
[CLS] ( 2 ) models that feature an implicit dna hybridisation rely on empirical estimates of the hybridisation free energy of the sticky ends ( dg 0 ij ) . [SEP]
[CLS] ( a ) chains , interacting by non selective hamiltonians ( h ns ) , hybridise by coalescing the two reactive end - elements according to dg 0 ij and to a configurational cost ( dg cnf ij ) , that depends on the algorithm that generates the hybridised chain . [SEP]
[CLS] notice that the non selective hamiltonians of the free and bound states are not the same . [SEP]
[CLS] ( b ) a similar scheme was previously proposed by mladek et al . that also considered the case in which more reactive sites bind resulting in a rigid dsdna segment . [SEP]
[CLS] ( c ) for long l - dna ref . 74 - 76 used a blob representation in which reactive blobs are chained according to an hybridisation free energy dg bnd . [SEP]
[CLS] ( 3 ) for ideal spacers B-material a local chemical equilibrium balance can be used to calculate the fraction of hybridised strands ( ab ) . [SEP]
[CLS] the concentrations of the sticky ends around the colloids c ( r ) are calculated by the end - to - end distribution functions of the tethered spacers B-material , while the equilibrium constant between sticky ends is dg 0 ij . [SEP]
[CLS] 6 ( a ) shift in the hybridisation free energy as due to the electrostatic interactions between the inert tails ( blue beads in the top rendering of the two oligomers ) and the paired bases as a function of the number of inert bases . [SEP]
[CLS] electrostatic interactions are modelled using the linearised debye - hu ¨ckel theory at three different salt B-material concentrations . [SEP]
[CLS] further details of the simulation method are reported in ref . 103 . [SEP]
[CLS] ( b ) santalucia hybridisation free energy of two oligomers ( 3 0 - aaccgacag - 5 0 and complementary strand ) with and without two ( g ) dangling bases attached to the 5 0 terminals of the reactive sequences for 100 mm nacl concentration . [SEP]
[CLS] the stacking contribution of the santalucia rules ( blue arrow in the figure ) is comparable with the tail contribution ( see part a ) . [SEP]
[CLS] notice that an error of ae1k b t in the nearest - neighbour rules results in a bigger incertitude of the melting temperature ( black arrows ) . [SEP]
[CLS] first consider the normal dimerisation reaction between two ' ' molecules ' ' a and b . [SEP]
[CLS] a + b " ab . [SEP]
[CLS] the condition for chemical equilibrium is m a + m b = m ab ( a1 ) [SEP]
[CLS] next consider the case where we still have an ideal gas mixture , but now with only very few molecules in the ' reaction volume ' ( in the case of tethers , this translates into : ' a tether of a given type ( say a ) can only bind with a small number of nearby complementary tethers . [SEP]
[CLS] in that case , lce breaks down . [SEP]
[CLS] to see this , consider the extreme ( but not unrealistic ) case of a volume v containing a single molecule of type a and a single copy of b . [SEP]
[CLS] as before , a and b can react to form ab . [SEP]
[CLS] let us denote by p the probability that molecule a ( or b ) is part of a dimer , then p / ( 1 a p ) is simply given by the ratio of the corresponding single - particle partition functions : now compare this with the lce expression . [SEP]
[CLS] the number density of a ( b ) equals p / v and the number density of ab is ( 1 a p ) / v . [SEP]
[CLS] 7inset bottom : the algorithm used in ref . 23 and 24 generates chains with rosenbluth weights that are distributed according to a distribution function ( p 0 ) which is different from the distribution obtained using equilibrium runs ( p 1 ) . [SEP]
[CLS] the average of the rosenbluth weight using p 0 ( right y - axis ) , together with the corresponding result for free constructs , is used in eqn ( 5 ) to calculate dg cnf . [SEP]
[CLS] using the overlapping between p 1 and p 0 and the results of ref . 121 we can calculate , in an independent way , aloghwi p 0 ( left y - axis and symbols ) . [SEP]
[CLS] the agreement between symbols and the straight line is perfect . [SEP]
[CLS] the algorithm of ref . 72 samples between free and bound states in a way that the rosenbluth weights are distributed with p 1 . [SEP]
[CLS] in this case dg cnf can be measured comparing the time spent by the system in the free and in the bound state . [SEP]
[CLS] assumptions used in the theoretical work main approximations implicit in analytical theories 1 . no strand - strand interaction besides those contained within dg bond ij 2 . no interactions besides excluded volume between colloids and strand features included in analytical theories 1 . effects induced by changes in binding strength in solution via dg 0 ij 2 . [SEP]
[CLS] effects of spacer B-material configurational entropy , i . e . spacer B-material structure 3 . presence of multiple bond formation 4 . competition for bonds between different strands - but only in general treatment of eqn ( 8 ) and ( 9 ) and equivalent lce , i . e . effects due to competing binding configurations [SEP]
[CLS] which is clearly not correct . [SEP]
[CLS] hence , we should not expect lce and in general mean field theories not accounting for the exact position of the tethering points ) to work if only small numbers of monomers B-material can interact . [SEP]
[CLS] however , this is precisely the situation if the grafting distance between strands is not [SEP]
[CLS] nanostructures have attracted huge interest as a rapidly growing class of materials for many applications . [SEP]
[CLS] several techniques have been used to characterize the size , crystal structure , elemental composition and a variety of other physical properties of nanoparticles B-nanoparticle . [SEP]
[CLS] in several cases , there are physical properties that can be evaluated by more than one technique . [SEP]
[CLS] different strengths and limitations of each technique complicate the choice of the most suitable method , while often a combinatorial characterization approach is needed . [SEP]
[CLS] in addition , given that the significance of nanoparticles B-nanoparticle in basic research and applications is constantly increasing , it is necessary that researchers from separate fields overcome the challenges in the reproducible and reliable characterization of nanomaterials B-material , after their synthesis and further process ( e . g . annealing ) stages . [SEP]
[CLS] the principal objective of this review is to summarize the present knowledge on the use , advances , advantages and weaknesses of a large number of experimental techniques that are available for the characterization B-technique of I-technique nanoparticles I-technique . [SEP]
[CLS] different characterization techniques are classified according to the concept / group of the technique used , the information they can provide , or the materials that they are destined for . [SEP]
[CLS] we describe the main characteristics of the techniques and their operation principles and we give various examples of their use , presenting them in a comparative mode , when possible , in relation to the property studied in each case . [SEP]
[CLS] nanoscale materials often present properties different from their bulk counterparts , as their high surface - to - volume ratio [SEP]
[CLS] dr stefanos mourdikoudis is a chemical engineer who obtained his phd degree from the department of physics , aristotle university of thessaloniki in greece in 2009 . [SEP]
[CLS] apart from his native country , he also worked in post - doctoral projects in france and spain . [SEP]
[CLS] currently he is working at the university college london ( ucl ) as a research associate . [SEP]
[CLS] his current research activity involves mostly work on the synthesis and characterization of magnetic B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] the variety of nanostructures he prepares are either directed for specific applications or simply inspired from curiosity to explore new protocols and characterize the resulting products . [SEP]
[CLS] stefanos mourdikoudisresults in an exponential increase of the reactivity at the molecular level . [SEP]
[CLS] such properties include electronic , optical and chemical properties , while the mechanical characteristics of the nanoparticles B-nanoparticle ( nps B-nanoparticle ) may also differ extensively . [SEP]
[CLS] this enables them to be an object of intensive studies due to their academic interest and the prospective technological applications in various fields . [SEP]
[CLS] such nanostructures may be synthesized by a wide number of methods , which involve mechanical , chemical and other pathways . [SEP]
[CLS] nowadays , many more types of nanomaterials B-material are synthesized than only a decade ago , and in higher amounts than before , requiring the development of more precise and credible protocols for their characterization . [SEP]
[CLS] however , such characterization is sometimes incomplete . [SEP]
[CLS] this is because of the inherent difficulties of nanoscale materials to be properly analysed , compared to the bulk materials ( e . g . too small size and low quantity in some cases following laboratory - scale production ) . [SEP]
[CLS] in addition , the multidisciplinary aspects of nanoscience and nanotechnology do not permit every research team to have easy access to a broad range of characterization facilities . [SEP]
[CLS] in fact , quite often a wider characterization of nps B-nanoparticle is necessary , requiring a comprehensive approach , by combining techniques in a complementary way . [SEP]
[CLS] in this context , it is desirable to know the limitations and strengths of the different techniques , in order to know if in some cases the use of only one or two of them is enough to provide reliable information when studying a specific parameter ( e . g . particle size ) . [SEP]
[CLS] nanoscience and nanotechnology are still undergoing constant growth , and the scientific community is rather aware that there may be certain differences between the way analytical characterization methods operate for nanomaterials B-material , in comparison with their more ' traditional ' modes of use for more ' conventional ' ( macroscopic ) materials . [SEP]
[CLS] herein we describe extensively the use of different methods for the characterization of nps B-nanoparticle . [SEP]
[CLS] these techniques are sometimes exclusive for the study of a particular property , while in other cases they are combined . [SEP]
[CLS] we discuss all these techniques in a comparative way , considering factors such as their availability , cost , selectivity , precision , non - destructive nature , simplicity and affinity to certain compositions or materials . [SEP]
[CLS] the techniques are analysed in depth , despite their big number presented herein . [SEP]
[CLS] there are microscopy B-technique - based techniques ( e . g . tem , hrtem , and afmthe full names of the techniques are provided later in the text , when presenting each one of them ) , which provide information on the size , morphology and crystal structure of the nanomaterials B-material . [SEP]
[CLS] other techniques are specialized for certain groups of materials , such as the magnetic B-property techniques . [SEP]
[CLS] examples of these techniques are squid , vsm , fmr , and xmcd . [SEP]
[CLS] many other techniques provide further information on the structure , elemental composition , optical properties and other common and more specific physical properties of the nanoparticle B-nanoparticle samples . [SEP]
[CLS] examples of these techniques include x - ray , spectroscopy B-technique and scattering techniques . [SEP]
[CLS] this review is organized in different sections , which will present numerous distinct characterization techniques for nps B-nanoparticle in relation to the properties studied ( see tables 1 and 2 ) . [SEP]
[CLS] the sections are categorized according to the different technique groups , as described above . [SEP]
[CLS] two of the main parameters studied in the characterization of nps B-nanoparticle are size and shape . [SEP]
[CLS] we can also measure size distribution , degree of aggregation , surface charge and surface area , and to some extent evaluate the surface chemistry . [SEP]
[CLS] size , size distribution and organic ligands present on the surface of the particles may affect other properties and possible applications of the nps B-nanoparticle . [SEP]
[CLS] in addition , the crystal structure of the nps B-nanoparticle and their chemical composition are thoroughly investigated as a first step after nanoparticle B-nanoparticle synthesis . [SEP]
[CLS] until now , there were no standardized protocols for this aim . [SEP]
[CLS] credible and robust measurement methods for nps B-nanoparticle will greatly affect the uptake of these materials in commercial applications and allow the industry to comply with regulation . [SEP]
[CLS] nevertheless , there are important challenges in the analysis of nanomaterials B-material because of the interdisciplinary nature of the field , the absence of suitable reference materials for the calibration of analytical tools , the difficulties linked to the sample preparation for analysis and the interpretation of the data . [SEP]
[CLS] in addition , there are unmet challenges in the characterization of nps B-nanoparticle such as the measurement of their concentration in situ and on - line , especially in a scaled - up production , as well as their analysis in complex matrices . [SEP]
[CLS] waste and effluent from mass production will also need to be monitored . [SEP]
[CLS] with the scale - up of nanoparticle B-nanoparticle manufacture , more reliable quantification techniques nguyen t . k . thanh [SEP]
[CLS] professor nguye [UNK] thi kim thanh , frsc , minstp ( http : / / www . ntk - thanh . co . uk ) , held a prestigious royal society university research fellowship ( 2005 - 2014 ) . [SEP]
[CLS] she was appointed a full professor in nanomaterials B-material in 2013 at the biophysics group , department of physics and astronomy , university college london , uk . [SEP]
[CLS] she leads a very dynamic group conducting cutting edge interdisciplinary and innovative research on the design and synthesis of nanomaterials B-material for biomedical applications from diagnostics to treatment of diseases such as cancer . [SEP]
[CLS] she has published nearly 100 peer reviewed journal articles and book chapters with over 5000 citations so far . [SEP]
[CLS] she has been a visiting professor at various universities in france , japan , china and singapore . [SEP]
[CLS] she has been an invited speaker at over 200 institutes and scientific meetings . will be required . [SEP]
[CLS] for this reason , it is crucial to characterize the nanomaterials B-material prepared in several ways to the maximum extent . [SEP]
[CLS] we do not only focus on the characterization of the nanoparticle B-nanoparticle core B-material , but also on the surface ligands that influence the physical properties . [SEP]
[CLS] in addition , we do not present only techniques that one might classify as ' common ' , but we also show examples of modern in situ operando techniques that are used to monitor the kinetics of nanoparticle B-nanoparticle formation and study through some recent advances in the topic the controlled defects that affect nanoparticle B-nanoparticle properties in a crucial manner . [SEP]
[CLS] x - ray diffraction ( xrd ) is one of the most extensively used techniques for the characterization of nps B-nanoparticle . [SEP]
[CLS] typically , xrd provides information regarding the crystalline structure , nature of the phase , lattice parameters and crystalline grain size . [SEP]
[CLS] the latter parameter is estimated by using the scherrer equation using the broadening of the most intense peak of an xrd measurement for a specific sample . [SEP]
[CLS] an advantage of the xrd techniques , commonly performed in samples of powder form , usually after drying their corresponding colloidal solutions , is that it results in statistically representative , volume - averaged values . [SEP]
[CLS] the composition of the particles can be determined by comparing the position and intensity of the peaks with the reference patterns available from the international centre for diffraction data ( icdd , previously known as joint committee on powder diffraction standards , jcpds ) database . [SEP]
[CLS] however , it is not suitable for amorphous materials and the xrd peaks are too broad for particles with a size below 3 nm . [SEP]
[CLS] upadhyay et al . determined the average crystallite size of magnetite nps B-nanoparticle using x - ray line broadening , and it was found to be in the range of 9 - 53 nm . [SEP]
[CLS] the broadening of xrd peaks was mainly caused by particle / crystallite size and lattice strains other than instrumental broadening . [SEP]
[CLS] the xrd - derived size is usually bigger than the so - called magnetic B-property size , due to the fact that smaller domains are present in a particle where all moments are aligned in the same direction , even if the particle is single domain . [SEP]
[CLS] on the contrary , the tem - deduced size was higher than that calculated using xrd , for samples with very large particles ; in fact , when the particle size is bigger than 50 nm , there are more than one crystal boundary on their surface . [SEP]
[CLS] xrd cannot distinguish between the two boundaries ; therefore the actual ( tem ) size of certain samples can be in reality bigger than the 50 - 55 nm calculated by the scherrer formula . [SEP]
[CLS] dai and co - workers prepared ultra - small au nps B-nanoparticle which were very likely to be more developed along the [UNK] 111 [UNK] direction ( rather than the [UNK] 220 [UNK] one ) as the peak corresponding to the former direction was much more intense in their xrd measurement . [SEP]
[CLS] similarly , li and colleagues noticed that after preparing copper B-material telluride nanostructures with different shapes ( i . e . cubes , plates , and rods ) , the relative intensities between the different xrd peaks varied in relation to the particle shape . [SEP]
[CLS] x - ray absorption B-technique spectroscopy I-technique ( xas ) includes both extended x - ray absorption fine structure ( exafs ) and x - ray absorption near edge structure ( xanes , also known as nexafs ) . [SEP]
[CLS] xas measures the x - ray absorption coefficient of a material B-material as a function of energy . [SEP]
[CLS] each element has a set of characteristic absorption edges corresponding to the different binding energies of its electrons , giving xas element selectivity . [SEP]
[CLS] as a highly sensitive technique , exafs is a convenient way to identify the chemical state of species which may occur even in very low concentrations . [SEP]
[CLS] synchrotrons are usually needed to acquire xas spectra ; therefore it is not a routine or readily available technique . [SEP]
[CLS] xanes probes the density of states of empty / partially filled electronic states by considering the excitation of an inner shell B-material electron to those states that are permitted by dipole selection rules . [SEP]
[CLS] pugsley et al . used in situ xas to examine the kinetics and mechanism of formation of germanium nps B-nanoparticle upon the reaction of mg 2 ge and gecl 4 . [SEP]
[CLS] actually , the exafs experiments and tem results indicated the formation of geo 2 nps B-nanoparticle along with the ge nps B-nanoparticle . [SEP]
[CLS] the analysis of exafs yielded a first - neighbour ge - ge distance of 2 . 45 a in good agreement with xrd . [SEP]
[CLS] moreover , chen et al . applied in situ exafs for the inspection of structural changes around germanium B-material atoms B-material in geo 2 nps B-nanoparticle . [SEP]
[CLS] surprisingly , they noticed that at high temperature ges 2 was formed as a product of the complete transformation of germanium dioxide , in the presence of a sulfur B-material source . [SEP]
[CLS] requejo and co - workers investigated the effects of sulfur B-material - palladium B-material interaction on the structural and electronic properties of alkyl thiol - capped pd nps B-nanoparticle . [SEP]
[CLS] the xanes and exafs analyses of the atomic structure and electronic properties of these nps B-nanoparticle showed that the sulfidation of pd clusters caused by the capping thiol B-material molecules took place not only on the surface but also in the bulk . [SEP]
[CLS] energy dispersive exafs helps to determine both structural and kinetic parameters in supported metal B-material catalysts B-property for reactions occurring on a timescale of a few seconds . [SEP]
[CLS] such a fast operation enables the aforementioned technique to be used at temperatures higher than 200 °c , which would hinder the use of surface enhanced raman B-technique spectroscopy I-technique ( sers ) , as the latter technique is not that fast under such conditions . [SEP]
[CLS] even on a timescale of tens of milliseconds , energy dispersive exafs can be used as a quantitatively suitable in situ probe of the dynamics of quick phase change in supported nanoparticulate metal B-material catalysts B-property . [SEP]
[CLS] bugaev and colleagues determined with exafs parameters the atomic structure of ptcu nps B-nanoparticle in ptcu / c catalysts B-property . [SEP]
[CLS] exafs is one of the most convenient techniques for the structural analysis of nps B-nanoparticle with sizes lower than 10 nm . [SEP]
[CLS] it possesses a high spatial resolution and provides information on the nearest environment of an atom B-material in a compound in the absence of long - range order . [SEP]
[CLS] the parameters derived in that study were partial coordination numbers , interatomic distances and debye - waller factors . [SEP]
[CLS] moreover , klasovsky and co - workers performed a physicochemical B-technique characterization I-technique of a new electron - conducting polymer B-material ( pani ) supported pto 2 catalyst B-property by electron paramagnetic B-property resonance ( epr ) , diffuse reflectance ftir spectroscopy B-technique ( drifts ) and exafs . [SEP]
[CLS] the importance of in situ / operando techniques was highlighted toward a better comprehension of the working oxidation catalyst B-property . [SEP]
[CLS] in another study , zhang and colleagues coated γ - fe 2 o 3 nps B-nanoparticle with sodium B-material dodecylbenzene sulphonate ( dbs ) , stearic acid and hexadecyltrimethylammonium bromide B-material ( ctab ) surfactants B-property by the microemulsion method . [SEP]
[CLS] the role of the surfactants B-property was investigated through exafs analysis and it was found that all samples had a tendency to extend the fe - o bond length . [SEP]
[CLS] all these molecules possess large spatial resistance , with the ctab molecule having the largest one . [SEP]
[CLS] the lattice distortion and disorder at the interfaces could play a significant role in hindering the fast nanoparticle B-nanoparticle growth . [SEP]
[CLS] cufe 2 o 4 and cufe 2 o 4 - mo 2 ( m = sn , ge ) nps B-nanoparticle were investigated by bertagnolli and colleagues by means of exafs and xanes . [SEP]
[CLS] the authors state the importance of exafs for the acquisition of information concerning the coordination number , the nature of the scattering atoms B-material surrounding the absorbing atom B-material , the interatomic distance between absorbing and backscattering atoms B-material , as well as the debye - waller factor , which is related to a disorder because of static displacements and thermal vibrations . [SEP]
[CLS] the fourier transform ( ft ) of the exafs signal as a function of wavenumber is related to the radial distribution of backscattering atoms B-material in real space x ( r ) . [SEP]
[CLS] the possible phase shifts during the exafs process and interference effects from different scattering channels cause the modification of the position of the peaks in the ft , which become no longer identical to the geometric distance between the backscattering atoms B-material and the absorbing atom . [SEP]
[CLS] as an alternative method aiming to tackle the drawbacks of the ft approach , the wavelet transform ( wt ) has been proposed , as reported by c . schmitz antoniak . [SEP]
[CLS] the principal concept behind the wt is to replace the infinitely expanded periodic oscillations in a ft with located wavelets as a kernel for the integral transformation . [SEP]
[CLS] more details of that approach can be found in ref . 18 . [SEP]
[CLS] exafs can also be used to study copper B-material cation I-material inversion in cufe 2 o 4 as a function of saturation magnetization B-property . [SEP]
[CLS] xanes is more helpful to determine the oxidation states , vacant orbitals , electronic configuration and site symmetry of the absorbing atom B-material . [SEP]
[CLS] xanes measurements were in agreement with exafs , both suggesting that iron B-material ( fe ) ions B-material occupied more tetrahedral sites than octahedral sites . [SEP]
[CLS] overall , these researchers showed that the aforementioned investigation on their copper B-material ferrite nps B-nanoparticle illustrated that these nanostructures had a structure analogous to that of the corresponding bulk material B-material . [SEP]
[CLS] the incorporation of the tetravalent metal B-material ions B-material in the spinel structures did not modify the local environment around cu and fe ions B-material . [SEP]
[CLS] moroz reviewed the x - ray diffraction structure diagnostics of nanomaterials B-material and stated that a remarkable advantage of exafs over redd ( radial electron density distribution ) is its selectivity , whereas redd is better in providing accurate values of the interatomic distances ; in that case , exafs provides interatomic distances corrected for the phase shift . [SEP]
[CLS] ideally , redd should be combined with exafs , ftir and microscopy B-technique techniques to acquire knowledge on the relation between the structure and physicochemical properties of nanomaterials B-material . [SEP]
[CLS] in another work , gomes et al . combined xrd and exafs to determine the cation B-material distribution and other structural parameters , comparing the np B-nanoparticle - based sample spectrum with the standard bulk material B-material spectrum of the cu ferrite . [SEP]
[CLS] differences were found among the cation B-material redistribution at the nanoparticle B-nanoparticle samples with regard to the ideal copper B-material ferrite . [SEP]
[CLS] ceo 2 nps B-nanoparticle were characterized with exafs by zhu and coworkers . [SEP]
[CLS] the authors emphasized on the suitability of the technique under discussion for their materials due to its element selectivity and nondependence on the long - range order of materials . [SEP]
[CLS] from the acquired debye - waller factors and the ce - o bond lengths , it was deduced that the surface or interface of the nps B-nanoparticle coated with sodium B-material bis ( 2 - ethylhexyl ) sulfosuccinate ( aot ) surfactants B-property was quite ordered ; however the bond lengths were elongated . [SEP]
[CLS] swatsitang and colleagues analysed with exafs the impact of cation B-material distribution on the magnetic B-property properties of co 1−x ni x fe 2 o 4 nps B-nanoparticle prepared by a hydrothermal B-technique method I-technique . [SEP]
[CLS] the results implied that co and ni ions B-material could occupy both the tetrahedral and octahedral sites with the preference to occupy the octahedral site more than the tetrahedral site , which is different from the bulk sample where all cations B-material occupy only the octahedral site in an inverse spinel ferrite model structure . [SEP]
[CLS] furthermore , zhang et al . synthesized co @ sio 2 core - shell nps B-nanoparticle with the sol - gel approach . [SEP]
[CLS] in situ xrd was used along with exafs to monitor the oxidation process of the co cores after thermal treatment at 800 °c either in air or under an inert atmosphere . [SEP]
[CLS] interestingly , it was noticed that co was oxidized in three steps no matter if air or n 2 gas was employed during the annealing . [SEP]
[CLS] ni - p was another material B-material in nanoscale form studied by exafs . [SEP]
[CLS] in particular , exafs proved to be very robust for the screening of the initial crystallization behaviour of such amorphous nps B-nanoparticle by probing the atomic - level structural change . [SEP]
[CLS] its combination with xrd , hrtem and vsm helped to investigate in detail the structural changes of ni - p nps B-nanoparticle in both shortrange and long - range order during heating at high temperatures . [SEP]
[CLS] more specifically , xrd illustrated the crystalline phases and phase changes . [SEP]
[CLS] hrtem provided information on size , size distribution and shape . [SEP]
[CLS] exafs provided insights regarding the changes of a local atomic structure and the chemical valence , especially for xrd - amorphous samples . [SEP]
[CLS] vsm enabled the study of magnetic B-property properties corresponding to different crystallization stages . [SEP]
[CLS] metal B-material chalcogenides have also been analysed by exafs , as in the case of cds nps B-nanoparticle prepared by rockenberger et al . [SEP]
[CLS] they found exafs to be suitable for their samples since it does not rely on any long - range order , in contrast to xrd . [SEP]
[CLS] exafs can also be used for liquid samples or even in cluster beams in the gaseous phase , permitting the identification of intercluster interactions by comparison with solid state measurements . [SEP]
[CLS] it revealed that the stabilization of cds nps B-nanoparticle with 1 . 3 - 12 nm diameter affected the mean cd - s distance . [SEP]
[CLS] unlike xrd , exafs is only sensitive to the local geometrical arrangement of neighboring atoms B-material that surround the absorbing atom B-material . [SEP]
[CLS] o ' brien and colleagues investigated the local environment of phosphorus B-material in the capping agent on the surface of cdse quantum B-nanoparticle dots I-nanoparticle . [SEP]
[CLS] the binding mode of the capping agents onto the surface was analysed , depending on the use of two distinct synthetic routes followed for its preparation ( ligands : trioctylphosphine oxide B-material and / or trioctylphosphine selenide ) . [SEP]
[CLS] furthermore , lloyd and co - workers used various techniques , including exafs , to monitor the reduction of se ( iv ) to se ( ii ) by a microbial wholecell catalyst B-property ( veillonella atypica ) . [SEP]
[CLS] the reduction was found to proceed via an insoluble red amorphous se ( 0 ) phase and the formation of metal B-material selenide was shown by exafs analysis from both ex situ and in situ ways . [SEP]
[CLS] noble metal B-material nanostructures , either monometallic or bimetallic , have also been studied by exafs . [SEP]
[CLS] for example , the structural features of silver B-material nps B-nanoparticle embedded in silicate glass were investigated combining hrtem and exafs techniques . [SEP]
[CLS] bugaev ' s group employed hrtem , xrd , optical absorption and exafs to identify correlations between the plasmonic properties and the atomic structure of ag nps B-nanoparticle and their aggregates . [SEP]
[CLS] the processing of the ag k - edge exafs spectra resulted in the acquisition of values for the parameters of the atomic structure in ag - ag and ag - o bonds averaged over the ionic and neutral states of ag . [SEP]
[CLS] the determination of the atomic structure of metallic ag nps B-nanoparticle , such as the type of point symmetry in the interior ( core B-material ) region of small nps B-nanoparticle , the nearestneighbor ag - ag distances and the structure of the near - surface region , is a challenging problem for this system . [SEP]
[CLS] the same group analysed by exafs the changes in the atomic structure of ag nps B-nanoparticle in soda - lime glass after annealing at 550 °c for 8 h . [SEP]
[CLS] brunsch and co - workers found that exafs showed a higher accuracy than hrtem in the determination of lattice parameters for ag nps B-nanoparticle embedded in silicate glasses . [SEP]
[CLS] the exafs results were averaged parameters of atom - atom correlations summed up for all detected particles , different from the data derived by hrtem for single particles . [SEP]
[CLS] combining both techniques would be beneficial for precise structural characterization . [SEP]
[CLS] the same researchers achieved the determination of the thermal expansion coefficient of similar glassembedded silver B-material nps B-nanoparticle , in a wide range of temperatures . [SEP]
[CLS] exafs allowed the precise demonstration of the changes of bond lengths and the stress state of their materials on the basis of a thermal expansion mismatch . [SEP]
[CLS] the evaluation of the exafs data of small nps B-nanoparticle typically provides a decreased coordination number , a dilatation or a contraction of the lattice structure and an increased debye - waller factor , together with an increased static disorder . [SEP]
[CLS] in addition , exafs does not require operation under vacuum , in contrast to xps . [SEP]
[CLS] it was used by the parkin group to investigate the phase change in silver B-material speciation , during the photo - assisted growth of ag from agno 3 . [SEP]
[CLS] the most plausible transition from metallic silver B-material to ag 2 o was illustrated . [SEP]
[CLS] exafs has also been applied for the characterization of alloys such as pd x pt y nps B-nanoparticle since common analytical techniques , such as xrd , have a limited capacity to distinguish between the various compositions of the aforementioned alloy , i . e . these metals B-material are perfectly miscible in any relative proportion and they both possess an fcc structure with similar lattice constants . [SEP]
[CLS] furthermore , structural information can also be obtained from exafs measurements , even though sometimes with lower precision and difficulty in extraction in comparison with the use of xrd . [SEP]
[CLS] ingham has written a comprehensive review describing what x - ray scattering techniques such as exafs , in situ xrd and small - angle x - ray scattering ( saxs ) can offer in nanoparticle B-technique characterization I-technique . [SEP]
[CLS] beale and weckhuysen have reported how the ratio between coordination numbers varied as a function of shape , through the exafs data . [SEP]
[CLS] their study concerned a series of nanoscale structures with several shapes and fcc , hcp , or bcc structures , with a maximum isotropic diameter of 3 nm . [SEP]
[CLS] in the case of pt - ru nanoclusters , size could also be obtained from exafs analysis , due to the fact that the coordination number of nearest neighbors in nps B-nanoparticle is a non - linear function of the particle diameter if the latter parameter lies below the range of 3 - 5 nm . [SEP]
[CLS] sokolov and co - workers characterized their pd npssynthesized by two separate routesby x - ray reflectivity , exafs and electron B-technique microscopy I-technique . [SEP]
[CLS] the exafs - deduced size was lower than the tem one if a cuboctahedral fcc structural model of pd nps B-nanoparticle surrounded by thiol B-material was assumed . [SEP]
[CLS] compared to bulk pd , lattice expansion was noticed in all types of nps B-nanoparticle , by both hrtem and exafs . [SEP]
[CLS] regarding cobalt B-material nps B-nanoparticle , a report by cheng et al . showed that exafs was able to differentiate ε - co from the fcc and hcp crystal structures . [SEP]
[CLS] the combination of xanes and exafs for the characterization of cuo , cu 2 o / cuo and cuo / tio 2 nps B-nanoparticle was published by sharma et al . [SEP]
[CLS] these techniques probed the local electronic / atomic structure of these samples and the existence of the different oxide B-material phases was evidenced . [SEP]
[CLS] iron B-material oxide I-material nps B-nanoparticle were also studied by xanes : this technique provided information on the oxidation state and local structure of iron B-material atoms I-material , while saxs analysis was useful for the size determination of the particles . [SEP]
[CLS] the combination of time - resolved in situ saxs and xanes measurements enabled the study of the formation of maghemite nps B-nanoparticle in water B-material , on a structural and chemical level . [SEP]
[CLS] on the basis of the acquired data , a complex four - stage formation mechanism was proposed , using ferrous and ferric chlorides B-material as well as triethanolamine . [SEP]
[CLS] the acquired knowledge would facilitate the control of the formation of nps B-nanoparticle in solution and tailor the properties of the final product . [SEP]
[CLS] in another publication , leveneur et al . investigated the nucleation and growth of fe nps B-nanoparticle in sio 2 by tem , xps and xanes . [SEP]
[CLS] it was demonstrated that ion B-material implantation initially resulted in the formation of dilute cationic B-material fe 2 + species , while at higher dissolved iron B-material concentrations , the formation of small metallic nuclei was noticed , which seed the nanocluster growth during prolonged implantation or annealing . [SEP]
[CLS] xanes is a technique far more sensitive to coordination and bonding environment than xps since it probes the unoccupied electronic states of atoms B-material and therefore can provide information about the crystal field ( octahedral , square pyramidal or tetrahedral ) that the iron B-material cations I-material occupy . [SEP]
[CLS] the complementary use of both xps and xanes was considered to be handy for such types of nanostructures with complex compositions and various possible states for the valence of iron B-material . [SEP]
[CLS] similar to the xrd method presented above , the saxs technique allows elastic scattering processes into a given solid angle to be run ; however the detector in saxs covers only small scattering angles ( normally lower than 1° ) . [SEP]
[CLS] a scheme that illustrates an in situ setup which manages to record realtime saxs / waxs / uv - vis measurements during the formation of au nps B-nanoparticle is displayed in fig . 1 . [SEP]
[CLS] the pictured device conducts saxs and waxs and records the uv - vis spectra at the same time in a given sample volume . [SEP]
[CLS] waxs ( wide - angle x - ray scattering ) is similar to saxs , but the distance between the sample and the detector is smaller and therefore diffraction maxima at larger angles are observed . [SEP]
[CLS] the authors investigated the nucleation and growth kinetics of gold B-material nps B-nanoparticle as a function of parameters such as concentration , temperature , ligand ratio and solvent type . [SEP]
[CLS] stuhn and co - workers characterized exten - sively polystyrene - grafted sio 2 nps B-nanoparticle , using techniques such as saxs , sans , dls , tga and tem . [SEP]
[CLS] small angle neutron scattering ( sans ) provided direct access to the static structure of the polymer B-material layer . [SEP]
[CLS] both saxs and sans can be used to measure the particle size ; in that report , saxs gave a 25 . 6 nm value , while a 23 . 3 nm np B-nanoparticle size was derived by sans . [SEP]
[CLS] although sans and saxs are very similar in various aspects ( e . g . sans uses elastic neutron scattering ) , the advantages of sans over saxs include its sensitivity to light elements , the possibility of isotope B-material labelling and the strong scattering by magnetic B-property moments . [SEP]
[CLS] the ligand shells B-material on small zno nps B-nanoparticle were characterized by a combined sans / saxs approach . [SEP]
[CLS] standard in situ methods such as uv - vis and saxs are sensitive only to the zno core B-material ; however sans probes the organic stabilizer in dispersion thanks to the high sensitivity of neutrons to h 2 . [SEP]
[CLS] in the work under discussion , both techniques allowed the determination of the size distribution of the cores B-material of the nps B-nanoparticle and the distribution of the stabilizer molecules ( acetate shell B-material ) simultaneously in the native solution . [SEP]
[CLS] typically , saxs is used to determine the particle size , size distribution , and shape . [SEP]
[CLS] regarding size values , saxs results are more statistically average than tem imaging . [SEP]
[CLS] wang et al . employed saxs to investigate the structural change of pt nps B-nanoparticle with temperature . [SEP]
[CLS] for certain temperatures , the size obtained by xrd was different from the corresponding saxs value . [SEP]
[CLS] this is because saxs is sensitive to the size of the fluctuation region of electronic density , but xrd is sensitive to the size of the long - range order region . [SEP]
[CLS] saxs provides the actual particle size , while xrd yields the crystallite size . [SEP]
[CLS] it is important to note that the different size values of saxs and xrd are related to the growth mode of nps B-nanoparticle during thermal treatment . [SEP]
[CLS] the particle size acquired with saxs was found to be a little bigger than that obtained from tem . [SEP]
[CLS] the reason is that pt nps B-nanoparticle were coated with pvp , and the scattering intensity due to the pvp coating B-material cannot be easily removed . [SEP]
[CLS] it has to be noted that saxs is a low resolution technique and in certain cases further studies by xrd and / or electron diffraction techniques are indispensable for the characterization of nps B-nanoparticle . [SEP]
[CLS] in fact , ti et al . have written a lengthy review article dedicated to the role of saxs for nanoparticle B-nanoparticle research . [SEP]
[CLS] in the case of pva - stabilized ag nps B-nanoparticle , saxs enables a more quantitative understanding of the correlations between the localised surface plasmon resonance ( lspr ) behaviour and the aggregation phenomena . [SEP]
[CLS] structure - property correlations between the lspr behaviour and the saxs spectra are feasible as both are scattering phenomena , which happen in the sol state . [SEP]
[CLS] moreover , the size range of the nps B-nanoparticle which show lspr is similar to that measured by saxs . [SEP]
[CLS] bulavin and colleagues reported a combined approach with saxs , uv - vis and qels ( quasi - elastic light scattering ) to characterize silver B-material sols in polymer B-material matrices . [SEP]
[CLS] saxs analysis showed a monomodal scatterer size distribution , whereas qels and uv - vis yielded a multimodal particle size distribution . [SEP]
[CLS] this discrepancy might come from the ability of the latter techniques to register large particles or aggregates within the range of 30 - 60 nm . [SEP]
[CLS] these large particles and aggregates are not within the detection limits of saxs . [SEP]
[CLS] however , for relatively small particles , all the aforementioned methods were in good agreement for the evaluation of the particle size and polydispersity . [SEP]
[CLS] in the case of ag - cu alloy nps B-nanoparticle synthesized using cu - and ag - nitrates in water B-material with hydrazine as the reductant and starch as the sol - stabilizing B-property agent I-property , saxs demonstrated the formation of mass fractal aggregates . [SEP]
[CLS] a bimodal size distribution was noticed , with the smaller aggregates having ag - rich compositions , while larger aggregates with low mass fractal dimensions were cu - rich . [SEP]
[CLS] this bimodal composition mode was also evident in the lspr spectra . [SEP]
[CLS] it is important to note that in view of the length scale of the np B-nanoparticle aggregates , which is related to lspr changes , saxs is the most suitable non - invasive technique for these studies . [SEP]
[CLS] in typical invasive techniques , such as tem and sem , the substrate - particle interactions and the solvent drying kinetics may affect the nanostructures formed . [SEP]
[CLS] these techniques cannot thus be helpful to analyse sol structures and explain adequately their lspr behaviour . [SEP]
[CLS] in another work , hashimoto and co - workers focused on the kinetics of the reduction - reaction - induced self - assembling process of ( pd ) n in the polystyrene - block - polyisoprene ( ps - b - pi ) matrix by time - resolved saxs . [SEP]
[CLS] pd ( acac ) 2 was used as precursor , and it was found that its reduction to pd ( 0 ) obeyed the first - order kinetics in both the disordered and ordered ps - b - pi matrix . [SEP]
[CLS] takenaka and co - workers performed a ' screening ' of the photoreduction synthesis of rhodium B-material and palladium B-material nps B-nanoparticle in aqueous ethanol / pvp solution using in situ and time - resolved saxs . [SEP]
[CLS] the nucleation , growth and particle coalescence of metal B-material atoms B-material for the production of metal B-material nps B-nanoparticle were monitored successfully . [SEP]
[CLS] in general , saxs helps to quantify the mass or concentration of nps B-nanoparticle as well as their size simultaneously , as a function of time . [SEP]
[CLS] the evolution of size , size distribution , amount and total volume of these rh and pd nps B-nanoparticle was quantitatively determined by saxs . [SEP]
[CLS] moreover , the formation process of mesostructured ptru nps B-nanoparticle electrochemically reduced on a microemulsion lyotropic liquid - crystallic template was studied by in situ xrd , saxs and xanes . [SEP]
[CLS] these techniques , together with complementary measurements by sem , fe - tem and eds , facilitated the understanding of the structural evolution , starting from the metallic precursors to the subsequent atom B-material reduction , np B-nanoparticle formation and aggregation , and finally mesostructure formation . [SEP]
[CLS] several insights were acquired , for instance , the degree of alloying between both metals B-material was studied , and any composition distribution ( e . g . pt - rich core B-material and ru - rich shell B-material ) was attributed to the different reduction rates of the pt and ru precursors . [SEP]
[CLS] in another work , lagrow et al . used in situ synchrotron small - angle x - ray scattering to monitor the growth and interparticle interaction of ni nps B-nanoparticle in solution as a function of time , and for different trioctylphosphine / ni precursor proportions in order to understand the influence of the top amount on the growth kinetics . [SEP]
[CLS] it was found that the top / ni ratio affected radically the final ni np B-nanoparticle size because of the action of top as a nucleating agent , together with the fact that top hindered the capacity of the nickel B-material precursor to reach the np B-nanoparticle surface . [SEP]
[CLS] metal B-material oxide I-material nps B-nanoparticle have also been investigated extensively by saxs . [SEP]
[CLS] in particular , grazing - incident saxs ( gisaxs ) and afm were employed to study self - assembled iron B-material oxide I-material nps B-nanoparticle prepared by a high - temperature solution phase reaction , as well as silicon B-nanoparticle dots I-nanoparticle produced by ion bombardment . [SEP]
[CLS] gisaxs is a unique non - destructive technique , which detects the diffusely scattered x - ray intensity from nanoscale objects ( through a large illumination area ) , providing information on properties such as np B-nanoparticle size , shape and arrangement . [SEP]
[CLS] afm can fit well with gisaxs by delivering a localized morphological image of the surface of the material B-material . [SEP]
[CLS] tobler and colleagues tried to shed light on the nucleation and growth steps of sio 2 nps B-nanoparticle , through saxs and dls measurements . [SEP]
[CLS] dls is much more sensitive to the presence of aggregates in comparison with saxs ; thus it is more suitable to monitor the starting step of the aggregation process . [SEP]
[CLS] the saxs and dls results confirmed that the rate of silica polymerization and nanoparticle B-nanoparticle formation was enhanced by increasing the ionic strength and silica concentration . [SEP]
[CLS] sem and tem verified the results obtained by saxs and dls regarding the particle size and shape although under certain conditions ( sample dehydration , exposure to high vacuum ) , the microscopy B-technique techniques yielded smaller np B-nanoparticle size compared to saxs and dls . [SEP]
[CLS] titania B-material nps B-nanoparticle prepared by the reaction of ticl 4 with hcl were studied by a combined approach using saxs , dls and tem . [SEP]
[CLS] while dls provides information only on the average hydrodynamic diameter of the particles and not on their internal features , saxs and tem were employed to unravel such details . [SEP]
[CLS] the authors stated the importance of saxs for the investigation of condensed and solid matter as well as for processes in colloidal systems . [SEP]
[CLS] in another work , tio 2 nps B-nanoparticle were studied by saxs , dsc and waxs . [SEP]
[CLS] the capacity of saxs to determine the structure of a nanocomposite polymer B-material electrolyte was noted ; in the work under discussion , ( peo ) 8 zncl 2 polymer B-material electrolytes and nanocomposites were doped with 10 % of tio 2 nanograins and γ - irradiated . [SEP]
[CLS] the effect of the inserted nanoscale titania B-material grains on the electrical , elastic and morphological properties of the nanocomposites , and the influence of γ - radiation from a co - 60 source were investigated with the aforementioned tech - niques . [SEP]
[CLS] in contrast to waxs , which showed lines and nanocrystallites only in low temperature crystalline phases , saxs was able to demonstrate the existence of nanograins in both the low and high temperature phases . [SEP]
[CLS] while waxs evidences only nanocrystallites , saxs records the presence of both crystalline and amorphous structures . [SEP]
[CLS] all techniques together showed that this complex material B-material showed a transition from a crystalline - amorphous phase to a highly conductive superionic one . [SEP]
[CLS] in another work , sio 2 / tio 2 hollow nps B-nanoparticle in the size range of 25 - 100 nm were studied by a combination of saxs , gisaxs , sans , tem , dls and other techniques . [SEP]
[CLS] experimental broadening of the scattering was negligible in the case of saxs and gisaxs measurements , but it was not negligible in sans . [SEP]
[CLS] in general , the results for the np B-nanoparticle size derived by all techniques were in reasonable agreement . [SEP]
[CLS] saxs / gisaxs provided accurate information on the inner diameter , outer diameter and size distribution . [SEP]
[CLS] tem sometimes overestimated the shell B-material thickness , unless hrtem was used . [SEP]
[CLS] dls was considered to be able to provide fast and cheap analysis , but saxs was more reliable in determining polydispersity . [SEP]
[CLS] zno nps B-nanoparticle were also investigated by saxs in various cases . [SEP]
[CLS] more specifically , the structural evolution of zinc B-material species toward zno nps B-nanoparticle prepared with a sol - gel synthesis route using zinc B-material oxy - acetate as the zn source was studied by saxs , uv - vis and quick - xafs . [SEP]
[CLS] the precursor led to the formation of nps B-nanoparticle through a hydrolysis - condensation pathway in ethanol solution , induced by the addition of sodium B-material hydroxide B-material . [SEP]
[CLS] zno / sio 2 nanocomposite thin films were investigated by gisaxs / waxs / ellipsometry experiments . [SEP]
[CLS] these measurements helped to evaluate the film structure ( thickness , porosity , and density ) as an evolution of temperature , since annealing was needed to acquire the final structure , starting with teos and zncl 2 precursors . [SEP]
[CLS] post - synthetic thermal treatment improved the crystallinity of zno and helped the creation of oxygen - related defects from the grain boundaries , which could have radical influence on the photoluminescent B-property behaviour . [SEP]
[CLS] the above - mentioned measurements were also useful for the comprehension of the formation kinetics . [SEP]
[CLS] finally , zinc B-material oxide I-material nps B-nanoparticle encapsulated into zeolite - y were analysed through an in situ combined xrd , xafs and saxs approach . [SEP]
[CLS] naoh was employed to assist the encapsulation of zinc B-material into the zeolite using aqueous zinc B-material acetate as the zn source . [SEP]
[CLS] the particle sizes estimated by exafs and saxs were in agreement with the cavity size of zeolite - y . [SEP]
[CLS] in another report , the clustering and dispersion behaviour of carboxylic B-material acid I-material - modified zro 2 nps B-nanoparticle in several solvents was evaluated by saxs / sans measurements . [SEP]
[CLS] such experiments helped to identify with precision the structural details of surface - modified nps B-nanoparticle , including the sizes of the inorganic B-material core I-material and the organic B-material shell I-material , as well as their secondary clusters . [SEP]
[CLS] concerning the size measurement , the authors note that tem provides solely the structural details in the dry form , which can differ from what occurs in the liquid state . [SEP]
[CLS] on the other hand , dls refers to the dispersed state , but a correct assignment of the refractive B-property indexes I-property for the nps B-nanoparticle and the solvent is necessary to obtain reliable measurements . [SEP]
[CLS] for these reasons , the authors highlighted the relatively better precision that saxs / sans can offer for the np B-nanoparticle size determination . [SEP]
[CLS] tin B-material oxide B-material ( sno 2 ) nps B-nanoparticle were also investigated by saxs : the effect of an acetylacetonate ( acac ) complexing ligand on the formation and growth of such particles , generated by the thermohydrolysis of sncl 4−n ( acac ) n at 70 °c , was analysed . [SEP]
[CLS] saxs and exafs were also employed to study the formation process of sno 2 nps B-nanoparticle produced after dissolving tetrachloride pentahydrate in acid ethanol solution and subsequent heating at 70 °c . [SEP]
[CLS] a five - step formation mechanism was suggested . [SEP]
[CLS] time - resolved saxs experiments helped to monitor the evolution of the number and size of some intermediate B-property species , known as nanoscopic polynuclear tin - oxo clusters . [SEP]
[CLS] x - ray photoelectron spectroscopy B-technique ( xps ) is the most widely used analytical technique for surface chemical analysis , also employed for the characterization of nanoscale materials . [SEP]
[CLS] its underlying physical principle is the photoelectric effect . [SEP]
[CLS] xps is a powerful quantitative technique , useful to elucidate the electronic structure , elemental composition and oxidation states of elements in a material B-material . [SEP]
[CLS] it can also analyse the ligand exchange interactions and surface functionalization of nps B-nanoparticle as well as core / shell structures , and it operates under ultra - high vacuum conditions . [SEP]
[CLS] nag and co - workers have published a review paper describing the role of xps as an interesting means to study the internal heterostructures of nps B-nanoparticle . [SEP]
[CLS] for example , it has been used to investigate the environmentdependent crystal structure tuning of metal B-material chalcogenide nps B-nanoparticle of various sizes . [SEP]
[CLS] it can also distinguish between core / shell and homogeneous alloy structures , and identify the bonding mode of ligands such as trioctylphosphine oxide B-material ( topo ) onto the surface of metal B-material chalcogenide nps B-nanoparticle . [SEP]
[CLS] for example , if topo bonds preferentially to the surface metal B-material element , then the uncapped surface chalcogenide element may oxidize more easily upon exposure to air . [SEP]
[CLS] in comparison with microscopy B-technique techniques , like tem and tem / eels , which use lateral spatial resolution to identify elements in a direction vertical to the probing electron beam , xps probes the composition of the material B-material along the direction of the electron beam . [SEP]
[CLS] regarding core - shell nps B-nanoparticle , shard has published an article that reports a straightforward method to interpret the xps data for such types of particles . [SEP]
[CLS] it involves a direct and accurate empirical method to convert the xps intensities into overlayer thicknesses , mostly suitable for spherical nps B-nanoparticle . [SEP]
[CLS] as further advantages of xps the author mentions that it provides the depth information , similar to the size of nps B-nanoparticle ( up to 10 nm depth from the surface ) and it does not significantly damage the samples . [SEP]
[CLS] two drawbacks of xps analysis are the preparation of samples ( i . e . dry solid form is required without contamination ) and the interpretation of data . [SEP]
[CLS] in another study , the interaction of l - cysteine with naked au nps B-nanoparticle has been studied with xps : that report aimed to provide experimental spectroscopic support to the kinetic models of catalyst B-property deactivation , studying the role of low - coordinated au atoms B-material belonging to np B-nanoparticle edges and corners . [SEP]
[CLS] furthermore , minelli and co - workers wrote an article on the analysis of protein B-material coatings B-material on au nps B-nanoparticle by xps and liquid - based particle sizing techniques . [SEP]
[CLS] xps is robust and useful to study proteins B-material quantitatively , as well as peptides B-material adsorbed at au interfaces . [SEP]
[CLS] it can also characterize the molecular interface of au nps B-nanoparticle . [SEP]
[CLS] the chemical information from the np B-nanoparticle surface analysed by xps can be used to assess the thickness of np B-nanoparticle coatings B-material . [SEP]
[CLS] smirnov et al . used the so - called davis ' method to determine the size of au nps B-nanoparticle in the planar model au / c systems based on the data of xps . [SEP]
[CLS] the np B-nanoparticle size values derived by xps agreed well with those from the scanning tunneling miscroscope ( stm ) data , with the degree of similarity being related to the particle shape ( e . g . sphere , hemisphere and truncated hemisphere ) . [SEP]
[CLS] tunc et al . presented a simple method by applying an external voltage stress during the xps analysis of au @ sio 2 nps B-nanoparticle ; their method facilitated the detection , location and identification of charges developed on surface structures in a completely non - contact mode . [SEP]
[CLS] therefore , xps provided information not only on the chemical identity but also on the dielectric properties of nanomaterials B-material , by recording their charging / discharging behaviour . [SEP]
[CLS] in another work , polzonetti and colleagues used synchrotron xps and nexafs to study the interaction at the molecule - metal interface and ligand arrangement in the molecular shell B-material of au nps B-nanoparticle capped by aromatic thiols B-material . [SEP]
[CLS] the experimental results of both techniques were supported by density functional theory ( dft ) calculations , illustrating the presence of a hybrid system in which the metallic B-material au I-material core I-material was surrounded by a shell B-material of aromatic thiol B-material molecules , whose thickness could be assessed by xps . [SEP]
[CLS] castner and coworkers quantified the impact of nanoparticle B-nanoparticle coatings B-material and nonuniformities on xps analysis , for the case of au @ ag core - shell nps B-nanoparticle . [SEP]
[CLS] they analysed the benefits of a complementary approach using xps , stem and simulated electron spectra for surface analysis ( sessa ) simulations to characterize the structure and composition of nps B-nanoparticle with nonideal geometries . [SEP]
[CLS] for instance , stem provided information concerning the metallic B-material cores I-material and shells B-material , while xps provided information regarding organic species and contaminants that were difficult to identify by stem . [SEP]
[CLS] in another report , ag nps B-nanoparticle capped with taurine B-material were investigated by sers and xps . [SEP]
[CLS] the latter method together with dft calculations showed that the gauche tautomer of taurine was the main component of the ag np B-nanoparticle surface . [SEP]
[CLS] xps confirmed the binding of taurine B-material through the oxygen B-material atoms I-material of the sulfonate group , denoting the existence of 71 % ag - o in taurine - functionalized ag nps B-nanoparticle . [SEP]
[CLS] this protocol provided a quantitative understanding of the interaction of the above molecule with ag nps B-nanoparticle . [SEP]
[CLS] furthermore , ramstedt and franklyn produced ag nps B-nanoparticle inside a poly ( 3 - sulfopropyl methacrylate ) brush and studied their formation process with tem , uv - vis and xps . [SEP]
[CLS] the apparent oxidation state of ag in the different forms ( nps B-nanoparticle , films , and clusters ) was investigated using xps and a chemical state plot . [SEP]
[CLS] in the case of bimetallic ag / pd colloids , prepared by galvanic replacement onto ag colloids pre - synthesized by laser ablation , the final product was investigated by uv - vis , xps , sers and ζ - potential . [SEP]
[CLS] these measurements showed that the nanostructures were mainly coated with metallic pd . [SEP]
[CLS] nevertheless , it was not easy to distinguish between ag ( 0 ) and ag ( i ) through xps analysis . [SEP]
[CLS] the galvanic replacement process using pd ( ii ) nitrate was monitored by the ζ - potential measurements on the basis of the fact that the charged species on the ag surface had a progressively modified adsorption due to the oxidative action of air diluted in an aqueous medium . [SEP]
[CLS] cds @ ag 2 s core / shell nps B-nanoparticle prepared with the aot / n - heptane / water B-material microemulsion technique were characterized by xps and sem - edx ( edx stands for energy - dispersive x - ray spectroscopy B-technique ) . [SEP]
[CLS] the authors emphasize on the benefit of the high sensitivity of xps , since every element has a particular set of peaks in the photoelectron spectrum at kinetic energies determined by the photon energy and the respective binding energies . [SEP]
[CLS] the intensity of the peaks is a function of the concentration of the respective element . [SEP]
[CLS] the xps and sem - edx results supported the core - shell formation . [SEP]
[CLS] in another work , ag , ni , and agni nps B-nanoparticle synthesized by the derived seed - mediated growth method on a transparent conductive indium B-material tin B-material oxide B-material substrate were studied by xps , xrd and optical spectroscopy B-technique . [SEP]
[CLS] xps determined the oxidation states of ag and ni at the outer layers of the nps B-nanoparticle . [SEP]
[CLS] it was shown that the surface of ag nps B-nanoparticle was not oxidized , while ni nps B-nanoparticle were oxidized to nickel B-material oxide B-material and hydroxide B-material . [SEP]
[CLS] it is interesting to note that no peaks of nio or ni ( oh ) 2 were detected in the xrd measurements , highlighting the utility of xps to identify amorphous species . [SEP]
[CLS] in the case of bimetallic agni nps B-nanoparticle , fcc - ag , silver B-material in ag ( ii ) state and oxidized ni atoms B-material were detected . [SEP]
[CLS] this indicated the presence of a ag core @ nio - ni ( oh ) 2 shell B-material structure . [SEP]
[CLS] kalinkin et al . investigated the particle size influence in the oxidation of small pt nps B-nanoparticle on graphite with nio 2 , using xps and stm . [SEP]
[CLS] the combination of these methods provided the most complete information regarding the composition , state and structure of the surface of the nps B-nanoparticle and could facilitate the understanding of the origin of the size effect . [SEP]
[CLS] in fact , only pt nps B-nanoparticle with a size smaller than 2 . 5 nm were found to oxidize to a mixture of pto and pto 2 under the experimental conditions of that study . [SEP]
[CLS] chakroune et al . studied acetate - and thiol - coated ru nps B-nanoparticle with xps , xas and hrtem . [SEP]
[CLS] for nps B-nanoparticle stored in polyol / acetate , surface oxidation limited to one monolayer and a surface coating B-material with mainly acetate ions B-material were evidenced by xps measurements . [SEP]
[CLS] for particles capped with thiol B-material after being prepared in polyol , the formation of a ru - s bond was shown for very small ( 2 nm ) ruthenium B-material particles . xanes and xps were in agreement with charge transfer from ru to s atoms B-material . [SEP]
[CLS] rhodium B-material nps B-nanoparticle , prepared by reducing rhcl 3 • 3h 2 o in a water / ethanol / pvp mixture , were characterized by several techniques , including xps and nexafs . [SEP]
[CLS] these techniques investigated the chemical states and indicated that the chlorine B-material moiety derived from the precursor remained at the obtained nps B-nanoparticle at both surface and bulk volume but heating may have caused its removal . [SEP]
[CLS] in another work , ir nps B-nanoparticle were produced by decomposing [ ir ( cod ) cl ] 2 in dichloromethane in the presence of an ionic liquid under a hydrogen B-material atmosphere . [SEP]
[CLS] xps helped to identify the interaction of ir ( 0 ) np B-nanoparticle surface with ionic species of the imidazolium ionic liquid 1 - ethyl - 3 - methylimidazolium ethylsulfate ( emi • etso 4 ) . [SEP]
[CLS] zerovalent fe nps B-nanoparticle were applied by li and zhang for the removal of water B-material contaminants , such as cd ( ii ) , pb ( ii ) , etc . [SEP]
[CLS] hr - xps confirmed that these fe nps B-nanoparticle had a core - shell structure , which resulted in remarkable properties for concurrent sorption and reductive precipitation of metal B-material ions B-material . [SEP]
[CLS] such measurements facilitated the identification of the type of element present at the np B-nanoparticle surface , the chemical and valence states of these elements and the ratio between the different chemical states of each element . [SEP]
[CLS] these core / shell fe / fe oxide B-material nps B-nanoparticle were highly efficient in metal B-material removal . [SEP]
[CLS] moreover , sheng et al . used zerovalent fe nps B-nanoparticle immobilized onto diatomite for the sequestration of uranyl ( u ( vi ) ) in water B-material . [SEP]
[CLS] the xps experiments implied that the diatomite - supported fe nps B-nanoparticle helped to reduce the highly toxic B-property and mobile B-property uo 2 to less toxic B-property and mobile B-property uo 2 . [SEP]
[CLS] complementary characterization with exafs illustrated that diatomite could act as a scavenger for insoluble products like uo 2 , therefore enabling more reactive sites to be used for u ( vi ) reduction . [SEP]
[CLS] the utility of xps to acquire information of ligand binding on nps B-nanoparticle coated with several types of ligands was demonstrated by lee and co - workers . [SEP]
[CLS] the particles studied were cdse / zns quantum B-nanoparticle dots I-nanoparticle capped with topo , 3 - mercaptopropionic acid ( mpa ) or 1h , 1h , 2h , 2h - perfluorooctanethiol ( pfot ) . [SEP]
[CLS] their analysis with xps imaging had low sensitivity and limited lateral resolution ; however , it provided a statistical , non - destructive method to characterize the ligand - qd binding mode . [SEP]
[CLS] in addition , near ambient pressure ( nap ) - xps was employed to record the changes in the oxidation state of palladium B-material in pdo nps B-nanoparticle supported on tio 2 in a temperature range of 30 - 120 °c . [SEP]
[CLS] pdo was used to catalyse the oxidation of 2 - propanol . [SEP]
[CLS] lab - based nap - xps instead of synchrotron facilities showed distinct advantages : the instrument is available upon need and it can be integrated permanently with other devices for optimal sample analysis , but there are also some disadvantages : a synchrotron source results in photoelectron peaks with higher intensity , thus obtaining measurements at a higher resolution . [SEP]
[CLS] however , care needs to be taken because a high - intensity radiation source can destroy certain types of samples . [SEP]
[CLS] overall , nap - xps is an effective technique to study in situ the steady - state conditions at the solid - gas interface , which are significant in the domains of catalysis , electrochemistry , corrosion and environmental science . [SEP]
[CLS] the authors mention that additional screening with techniques such as mass spectrometry was needed for a more complete picture of the catalytic process ( oxidation of 2 - propanol by pdo nps B-nanoparticle in this case ) in such reactions . [SEP]
[CLS] there are also several other techniques that help in the determination of the structure , composition , size and other basic features of the nps B-nanoparticle . [SEP]
[CLS] fourier transform infrared B-technique spectroscopy I-technique ( ftir ) is a technique based on the measurement of the absorption of electromagnetic radiation with wavelengths within the mid - infrared region ( 4000 - 400 cm −1 ) . [SEP]
[CLS] if a molecule absorbs ir radiation , the dipole moment is somehow modified and the molecule becomes ir active . [SEP]
[CLS] a recorded spectrum gives the position of bands related to the strength and nature of bonds , and specific functional groups , providing thus infor - mation concerning molecular structures and interactions . [SEP]
[CLS] feliu and co - workers studied how pt nanostructures performed on ethanol oxidation , using a combined approach with in situ atr - ftir and differential electrochemical mass spectroscopy B-technique ( dems ) . [SEP]
[CLS] these techniques helped to probe adsorbates B-property electrochemically and detect volatile reaction products . [SEP]
[CLS] their results were in agreement with previous findings , showing that the preferred decomposition products were related to surface structures , with co ads formation on ( 100 ) domains and acetaldehyde / acetic acid formation on ( 111 ) domains . [SEP]
[CLS] in another report , carbon - supported platinum B-material nps B-nanoparticle ( 3 - 8 nm size ) were used for the co oxidation reaction and this catalytic process was monitored using drifts and quadrupole mass spectrometry ( qms ) . [SEP]
[CLS] the ftir measurements of adsorbed co confirmed the variations of co ad and o ad in different steps of the experiment , in accordance with the results from qms , while modifications in the co distributing over various types of pt surface sites were also noticed . [SEP]
[CLS] overall , drifts was regarded as an important tool for the probation of the surface structure of pt nps B-nanoparticle under in situ conditions . [SEP]
[CLS] 90 shukla et al . published a paper devoted to the ftir investigation of the surfactant B-property bonding to fept nps B-nanoparticle that were synthesized in the presence of oleic acid and oleylamine . [SEP]
[CLS] the former ligand was found to bond to fept nps B-nanoparticle in both monodentate and bidentate forms , while oleylamine bonded to fept molecularly with the nh 2 group intact . [SEP]
[CLS] furthermore , au / ag bimetallic nps B-nanoparticle stabilized with dodecanethiol and soluble B-property in nonpolar solvents were produced through a two - phase synthetic route in water B-material / toluene mixtures . [SEP]
[CLS] the most important insight derived from xps and ftir measurements was that ag atoms B-material were enriched at the outer part of the alloy clusters in comparison with the au atoms B-material . [SEP]
[CLS] in another work , the influence of the ag np B-nanoparticle content on the photocatalytic degradation of oxalic acid adsorbed on tio 2 nps B-nanoparticle was evaluated by atr - ftir . [SEP]
[CLS] various ag np B-nanoparticle amounts were tested , and it was demonstrated that the incorporation of only a small quantity ( 2 % ) boosted the photocatalytic performance of tio 2 nps B-nanoparticle substantially . [SEP]
[CLS] afm and xps were used to characterize the topography and chemical structure / composition of the composite np B-nanoparticle films . [SEP]
[CLS] tzitzios et al . synthesized ni nps B-nanoparticle with a hexagonal crystal structure in the size range of 13 - 25 nm via the reduction of nickel B-material stearate in the presence of peg , oleic acid and oleylamine . [SEP]
[CLS] ftir spectra showed the presence of the characteristic groups B-nanoparticle at I-nanoparticle the I-nanoparticle surface I-nanoparticle of the nps B-nanoparticle , such as the - hcvcharrangement in oac and oam , while the binding modes of the ligands onto the np B-nanoparticle surface were also examined . [SEP]
[CLS] copper zinc tin B-material sulpho - selenide ( czts x se 1−x ) nanocrystals were prepared by haram and colleagues with a hot - injection process . [SEP]
[CLS] the precursors were dissolved in oam and heated at t > 200 °c for the synthesis . [SEP]
[CLS] ftir measurements showed the adsorption of oam onto the surface of the particles . [SEP]
[CLS] characteristic bands arising from the moieties existing at the molecule of oam and indicating its successful coordination with the nps B-nanoparticle were spotted . [SEP]
[CLS] superparamagnetic B-property ferrite nps B-nanoparticle ( mfe 2 o 4 , m = ni , co , zn , mn ) with high crystallinity and size below 10 nm were synthesized by sabale et al . with a simple ' polyol ' method . [SEP]
[CLS] the observation of tetrahedral ( v1 ) frequency at the ftir spectrum verified the presence of the spinel ferrite structure . [SEP]
[CLS] bands assigned to the - oh and c - o groups denoted the presence of diethylene glycol , thus revealing its successful coating B-material around the ferrite nps B-nanoparticle , endowing them a high solubility B-property in I-property water I-property . [SEP]
[CLS] in another report , the presence of fe - o - p bonds was shown by ftir measurements for hydrophobic B-property iron B-nanoparticle nanoparticles I-nanoparticle which were treated with alkyl phosphonic acid - based ligands in order to turn them into water - soluble B-property iron - iron oxide core - shell nps B-nanoparticle . [SEP]
[CLS] ftir spectroscopy B-technique was also employed to characterize multifunctional fe 3 o 4 @ c @ ag hybrid nps B-nanoparticle , which were prepared with a facile route based on the direct adsorption and spontaneous reduction of ag ions B-material onto the surface shell B-material of c - coated magnetic B-property nps B-nanoparticle . [SEP]
[CLS] the presence of carboxyl B-material and hydroxyl B-material groups I-material on the np B-nanoparticle surface was shown by ftir . [SEP]
[CLS] certain bands attributed to carboxyl B-material vibration implied that carbonyl and other reductive groups were oxidized by ag ions B-material , which was an indirect sign for the presence of ag in the products . [SEP]
[CLS] these hybrid nanostructures displayed a remarkable photocatalytic activity for the photodegradation of neutral red dye under visible light irradiation . [SEP]
[CLS] duong et al . have shown that ftir can be successfully used to assess the affinity of polymers B-material bearing phosphate groups as surface ligands for nayf 4 : yb / er upconversion nanoparticles B-nanoparticle . [SEP]
[CLS] trioctylphosphine - stabilized cds nanorods B-nanoparticle synthesized by chen et al . were also characterized by ftir , revealing c - p stretching peaks related to the aforementioned molecule . [SEP]
[CLS] 100 table 3 presents the ir vibrational assignments of several characteristic functional groups which are involved in nanoparticle B-nanoparticle synthesis . [SEP]
[CLS] nuclear magnetic B-property resonance ( nmr ) spectroscopy B-technique is another important analytical technique in the quantitative and structural determination of nanoscale materials . [SEP]
[CLS] it is based on the nmr phenomenon exhibited by nuclei that possess non - zero spin when placed in a strong magnetic B-property field , which causes a small energy difference between the ' spin - up ' and ' spin - down ' states . [SEP]
[CLS] transitions between these states can be probed by electromagnetic radiation in the radio wave range . [SEP]
[CLS] nmr is typically used to study the interactions or coordination between the ligand and the surface of diamagnetic B-property or antiferromagnetic nps B-nanoparticle . [SEP]
[CLS] it is , however , not suitable to characterize ferri - or ferromagnetic materials , as the large saturation magnetization B-property of such materials causes variations in a local magnetic B-property field , which lead to shifts of the signal frequency and dramatic decreases in relaxation times . [SEP]
[CLS] as a result , significant broadening of the signal peaks occurs , making the measurements practically inutile and unable to be interpreted . [SEP]
[CLS] marbella and millstone have written a comprehensive review article on the nmr techniques for noble metal B-material nps B-nanoparticle . [SEP]
[CLS] nmr spectroscopy B-technique can help toward the routine , straightforward , molecular - scale investigation of np B-nanoparticle formation and morphology in situ , in both solution and solid phase . [SEP]
[CLS] it is particularly useful for analyzing both the formation and final architecture of noble metal B-material nps B-nanoparticle . [SEP]
[CLS] the capping ligands are also typically studied by nmr , and such measurements can yield information on the properties of the particle core B-material ( e . g . electronic structure , atomic composition , or compositional architecture ) . [SEP]
[CLS] insights into ligand density , arrangement and dynamics can also be derived . [SEP]
[CLS] besides facilitating the monitoring of the chemical evolution of ligand precursors and their role in particle growth , nmr is also employed to probe the role of capping ligands for the determination of particle shape . [SEP]
[CLS] overall , nmr can screen the chemical conversion of np B-nanoparticle precursors in both the solution and solid phase , with high spatial and chemical resolution , under distinct reaction conditions , and for diverse metal B-material identities ; this helps in the better comprehension of the reaction mechanisms for np B-nanoparticle synthesis . [SEP]
[CLS] moreover , nmr is useful for the monitoring of the process and final products of ligand exchange , when the initial capping ligands need to be replaced . [SEP]
[CLS] the 1 h nmr chemical shift behaviour is sensitive to the surrounding electronic environment ; this includes the electronic structures and bonding environment of the nucleus . [SEP]
[CLS] consequently , any changes in the handedness of a molecule can be ' felt ' by neighboring spin positions and observed as changes in chemical shift . [SEP]
[CLS] this renders nmr significant to assess the chirality or absence of chirality of small , moleculelike nanoclusters . [SEP]
[CLS] nmr can also be applied for the direct monitoring of the diffusion of adsorbed gases onto the surface of metal B-material nps B-nanoparticle . [SEP]
[CLS] finally , nmr is utile for the measurement of the hydrodynamic radius of metal B-material nps B-nanoparticle and thus constitutes an important complement to more standard np B-nanoparticle sizing techniques , such as tem and dls . [SEP]
[CLS] similar to dls , nmr spectra are used to define the np B-nanoparticle size via the analysis of particle diffusion . [SEP]
[CLS] in particular , nmr helps to extract the diffusion coefficient of well - dispersed species in solution diffusing according to brownian motion only . [SEP]
[CLS] then the hydrodynamic size can be calculated through a rearrangement of the stokes - einstein equation . [SEP]
[CLS] finally , a phenomenon known as ' knight shift ' , which is induced by some metals B-material and can be present upon nmr measurements , is also described in ref . 102 . [SEP]
[CLS] it has to be noted that the particle size , which can be safely analysed by nmr , can exceed by far the 100 nm in the case of polymerhybrid particles , 103 , 104 whereas metallic nps B-nanoparticle have to be at around the size range of 1 - 5 nm in order to acquire meaningful nmr measurements . [SEP]
[CLS] h solution nmr has been reviewed by hens and martins as a tool for the investigation of the surface chemistry of colloidal nps B-nanoparticle . [SEP]
[CLS] diffusion - ordered nmr ( dosy - nmr ) offers the possibility to distinguish in situ free ligands from bound ligands , while the distribution of these species can also be quantified . [SEP]
[CLS] solution nmr can be employed to identify tightly bound ligands and quantify their surface density of sterically stabilized colloidal nps B-nanoparticle . [SEP]
[CLS] jicsinszky and co - workers studied hydrophilic B-property heptakis ( 6 - deoxy - 6 - thio ) cyclomaltoheptose capped au nps B-nanoparticle with dosy - nmr . [SEP]
[CLS] this technique proved to be an effective , reliable and rapid way to investigate the role of the total concentration of gold B-material in solvated metal B-material atom I-material ( sma ) solutions as well as of the au / capping ligand molar ratio on np B-nanoparticle sizes . [SEP]
[CLS] nmr measurements also helped to acquire some basic information on the drug transport and release capabilities of au nps B-nanoparticle . [SEP]
[CLS] this was achieved through the analysis of the nature of supramolecular aggregation processes and the ability of ( au ) n / β - cdsh nanoaggregates to act as hosts for deoxycytidine ( dc ) . [SEP]
[CLS] 106 dosy - nmr has also been employed to determine the nanoparticle B-nanoparticle size , e . g . in the case of au nps B-nanoparticle prepared by canzi et al . [SEP]
[CLS] this was achieved by analysing the 1 h spectrum of the protecting ligands using 2d dosy nmr , a method that could be facilely adapted also for other metal B-material and semiconductor nanocrystals . [SEP]
[CLS] size estimates were acquired by using diffusion coefficient ratios derived from the proton signals from the alkyl thiolate groups bound to au nps B-nanoparticle and a ferrocene internal standard . [SEP]
[CLS] the authors stated that dosy nmr was a reliable alternative method to calculate the np B-nanoparticle size , being quicker and more cost - effective than tem . [SEP]
[CLS] coelho and colleagues used nmr spectroscopy B-technique to determine particular intermolecular B-property interactions I-property and mechanisms of drug immobilization and location into surface peg - modified au nps B-nanoparticle . [SEP]
[CLS] the authors highlighted the advantages of nmr as a non - destructive , highly reproducible method , sensitive to the structural details of molecules and molecular conjugates , which could be employed for both qualitative and quantitative characterization . [SEP]
[CLS] information of size , shape , dynamics , chemical structure , intermolecular B-property interactions I-property , and binding and exchange processes in complex nano - systems could be obtained . [SEP]
[CLS] the combined use of nmr with ftir , uv - vis , dls and tem could yield significant insights regarding important physicochemical properties of drug delivery systems , which influence their therapeutic efficacy . [SEP]
[CLS] in another report , deuterium ( 2 h ) nmr was employed to study the intramolecular ligand dynamics in d 15 - ( pph 3 ) - capped au nps B-nanoparticle . [SEP]
[CLS] the authors made use of the ability of nmr to probe ligand structures and surface binding properties on nps B-nanoparticle by the in situ analysis of chemical shifts and resonance lines in the solid and liquid states . [SEP]
[CLS] a specific feature of 2 h nmr is its simplicity and the capacity to distinguish the type of dynamics in amorphous and crystalline domains , for organic compounds that are isotopically labelled with deuterons . [SEP]
[CLS] smith et al . [SEP]
[CLS] used nmr to investigate the extent of ligand exchange between distinct kinds of thiolated molecules on the surface of au nps B-nanoparticle . [SEP]
[CLS] in particular , they determined ligand density values for single - moiety ligand shells B-material and then used these data to describe the ligand exchange behaviour with a second , thiolated molecule . [SEP]
[CLS] triphenylphosphine - capped 1 . 8 nm au nps B-nanoparticle have been characterized by multinuclear nmr to investigate their surface structure and ligand binding environment . [SEP]
[CLS] in solution , the ligand exchange kinetic reactions were screened by h , 2 h and 31 p nmr to analyse the exchange process . [SEP]
[CLS] doyen et al . used uv - vis and nmr to study the formation of au nps B-nanoparticle by the citrate reduction method . [SEP]
[CLS] 1d - 1 h and dosy - nmr measurements showed that citrate aggregates with au ( i ) and au ( 0 ) were formed . [SEP]
[CLS] that work suggested that citrate , apart from being the reductant and the stabilizing B-property agent I-property for au nps B-nanoparticle , might act as a ' molecular linker ' , which could help in the particle formation . [SEP]
[CLS] the coordination of amine B-material ligands on ag nps B-nanoparticle was evaluated by nmr , sers and dft . [SEP]
[CLS] it was found by sers that the amidine moiety , coming from the silver B-material amidinate precursor , remained bound to the metal B-material surface , whereas the hexadecylamine ligand was in a fast exchange between a surface - bound state and free floating in solution , as revealed by nmr . solution nmr spectroscopy B-technique was a powerful tool for the analysis of short timescale effects . long - residence - time molecules at the np B-nanoparticle surface could not be monitored by this technique due to their very slow tumbling . the sers analysis of the nps B-nanoparticle combined with dft modelling demonstrated that unexpected organic groups were observed by this latter technique , in contrast with what was shown by solution nmr . [SEP]
[CLS] sers is efficient if molecules are in a close contact with the ag surface , whereas nmr spectroscopy B-technique examines molecules in the first and second coordination sphere of the nps B-nanoparticle . [SEP]
[CLS] despite this , the complementarity of sers with nmr is beneficial to reveal the molecular environment of the prepared nps B-nanoparticle . [SEP]
[CLS] amidine hindered the np B-nanoparticle aggregation , while hexadecylamine ( hda ) helped toward a narrow size distribution of stabilized ag 0 nps B-nanoparticle . [SEP]
[CLS] ag np B-nanoparticle / π - conjugated polyelectrolyte systems were investigated by nmr , ftir and sers and increased regularity of the high - cis polymers B-material was documented . [SEP]
[CLS] the ir spectra supported the conclusions drawn from the 1 h nmr measurement of the polymer B-material ; both techniques consistently illustrated the cis - rich configuration of polymers B-material formed by the solution polymerization in acetonitrile and the cis / trans configuration of the polymers B-material formed by the bulk polymerization . [SEP]
[CLS] velders and coworkers focused on the use of 1 h nmr spectroscopy B-technique to determine the np B-nanoparticle size in the case of dendrimer - encapsulated pd nps B-nanoparticle . [SEP]
[CLS] the advantage of nmr in comparison with tem consisted of its capacity to probe the total population of the nps B-nanoparticle , providing more representative information regarding the average np B-nanoparticle size . [SEP]
[CLS] in addition , in situ operation was possible with nmr , and this enabled the monitoring of the changes in the size and the capping ligand environment of the nps B-nanoparticle during catalytic reactions . [SEP]
[CLS] solution nmr spectroscopy B-technique has been extensively used also for the characterization of oxide B-material nanoparticle systems . [SEP]
[CLS] kahn and co - workers characterized zno nps B-nanoparticle by h and dosy - nmr . [SEP]
[CLS] they emphasized on the ability of the latter technique to sort species according to their size , as the diffusion coefficient is inversely proportional to the hydrodynamic radius . [SEP]
[CLS] their study , performed on zno nps B-nanoparticle stabilized by amine B-material molecules , showed that a fast exchange between free and coordinated amine B-material molecules was deduced within the nmr measurement timescale . [SEP]
[CLS] overall , the nmr spectra showed that the seemingly simple stabilization of zno nps B-nanoparticle by amine B-material molecules appeared to be much more complicated than considered beforehand . [SEP]
[CLS] the same group published a study dedicated to the use of nmr techniques for the investigation of the role of amine B-material ligands together with oleic acid on the formation of zno np B-nanoparticle superlattices in c 7 d 8 . [SEP]
[CLS] their experiments demonstrated the dependence of the type of ligand adsorbed on the np B-nanoparticle surface on the concentration of the colloidal np B-nanoparticle solutions . [SEP]
[CLS] it was suggested that the driving force of the superlattice formation was the presence of ion - paired ammonium carboxylate B-material shells B-material around each particle . [SEP]
[CLS] [UNK] and colleagues investigated phosphonic acid - capped sno 2 nps B-nanoparticle with sizes lower than 5 nm , using multinuclear solution and solid - state magic angle spinning ( mas ) nmr . [SEP]
[CLS] the latter technique indicated the absence of acidic protons of the phosphonic acid groups , strongly supporting the formation of p - o - sn linkages B-property . [SEP]
[CLS] insights into the ligand structure and the extent of phosphonic acid protonation upon binding the np B-nanoparticle surface were obtained . [SEP]
[CLS] in the case of ca 2 sno 4 nps B-nanoparticle prepared by the mechanochemical synthetic route , sn mas - nmr and sn mossbauer were employed to probe the local environment of sn nuclei , so as to acquire important insights into the local structural disorder of these nps B-nanoparticle . nmr spectroscopy B-technique provided information on the magnetic B-property and chemical interactions , while mossbauer measurements revealed the quadrupolar interactions experienced by the nuclei of sn . [SEP]
[CLS] magnetite - silica nps B-nanoparticle prepared by a two - stage procedure by bogachev et al . were characterized by nmr relaxometry , afm and uv - vis spectroscopy B-technique . [SEP]
[CLS] the aggregation process in the colloidal solutions of fe 3 o 4 - sio 2 nps B-nanoparticle was investigated . [SEP]
[CLS] dextran - coated γ - fe 2 o 3 nps B-nanoparticle were studied by papavassiliou and colleagues with fe nmr , mossbauer , tem and magnetization B-property measurements . the low temperature mechanism of collective magnetic B-property excitation in magnetic B-property nps B-nanoparticle , which originated from the fluctuations of the magnetization B-property direction around an energy minimum corresponding to an easy direction of magnetization B-property , was investigated . [SEP]
[CLS] 10 nm nanosized samples at low t displayed similar nmr spectra , and thus similar hyperfine fields to the bulk material B-material , implying that the samples had the same magnetic B-property structure . [SEP]
[CLS] gossuin et al . characterized gadolinium B-material hydroxide B-material and dysprosium oxide B-material nps B-nanoparticle using xrd , magnetometry and nmr relaxometry . [SEP]
[CLS] nuclear magnetic B-property relaxation dispersion profile represented the evolution of the longitudinal relaxation rate with respect to the magnetic B-property field and provided interesting information about the longitudinal relaxation mechanism . [SEP]
[CLS] finally , hfo 2 and zro 2 nps B-nanoparticle synthesized using the karlsruhe microwave - plasma process were characterized by several techniques such as 1 h mas nmr , xps , xrd and electron diffraction . [SEP]
[CLS] among these techniques , nmr and xps helped to identify the chemical composition of the as - prepared nps B-nanoparticle . [SEP]
[CLS] a hydrate surface layer with a hydrogen B-material content of 5 - 10 wt % , composed of chemisorbed hydroxyl B-material groups I-material and organic precursor fragments , was detected by 1 h - mas nmr . [SEP]
[CLS] solid - state nmr ( ss nmr ) spectroscopy B-technique is an important characterization tool to investigate the behaviour of solid catalysts B-property and chemical processes occurring at their surface . [SEP]
[CLS] such technique may help to resolve not only interactions at the ligand - solvent interface but also result in the acquisition of significant insight into ligand - particle bonding at the hardsoft matter interface . [SEP]
[CLS] for example , p is a very sensitive nmr nucleus with 100 % natural abundance and high gyromagnetic ratio and it is quite easy to measure the 31 p nmr spectra with a good signal to noise ratio even in systems with low ligand concentrations [SEP]
[CLS] j - resolved 31 p solid - state nmr spectroscopy B-technique combined with dft calculations can provide important information about the structure of heterogenized species and also provide insights into the immobilization of homogeneous metal phosphine catalysts B-property . [SEP]
[CLS] gutmann and co - workers have highlighted the crucial role of liquid and partly solidstate nmr techniques for the detection of surface molecules and the discrimination between different binding sites on nanoscale catalysts B-property . [SEP]
[CLS] in particular , 2 h mas nmr has been employed to study chemical reactions such as the hydrogenation B-event of olefins , being capable of detecting reactive intermediates B-property . [SEP]
[CLS] the authors denoted a weakness of the nmr measurements , which was related to their sensitivity . [SEP]
[CLS] solid state p nmr was used to characterize phosphinine - stabilised au nps B-nanoparticle and a phosphinine - au complex , as reported by mallissery and gudat . [SEP]
[CLS] nmr spectra showed that in addition to metalbound intact phosphinine units , several surface - bound species generated by the chemical transformation of the initially supplied ligands were also detected . [SEP]
[CLS] in another work , two different tripeptides attached on au nps B-nanoparticle were analysed by ss nmr and dft calculations . [SEP]
[CLS] substantial structural differences between cysalaala and alaalacys on au nps B-nanoparticle were evidenced through the aforementioned techniques . [SEP]
[CLS] in particular , the location of the carboxylate B-material moiety relative to the s atom B-material that served to anchor the peptide B-material to the surface played a significant role in determining these structures . [SEP]
[CLS] novio et al . have used ss nmr and ftir to characterize the location and dynamics of carbon B-material monoxide coordination on ru nps B-nanoparticle . [SEP]
[CLS] two different sets of 2 nm ru nps B-nanoparticle were tested , prepared under a h 2 atmosphere , stabilized by either pvp or a bidentate phosphine ligand ( dppb ) . [SEP]
[CLS] it was demonstrated that co groups were mobile B-property on the np B-nanoparticle surface , while the bulky ancillary ligand dppb slowed down the fluxionality of co and prevented the exchange at certain positions . [SEP]
[CLS] ss nmr was also employed to characterize 1 - 2 nm ru nps B-nanoparticle capped by either 1 , 3 , 5 - triaza - 7 - phosphaadamantane or pph 3 ligands and exposed to a co gas atmosphere . [SEP]
[CLS] that paper presented a new way to analyse interactions and calculate approximate distances between phosphine ligands and co probe molecules on the surface of ru nps B-nanoparticle employing 31 p - 13 c redor nmr . [SEP]
[CLS] and [ pt ( ch 3 ) 2 ( cod ) ] [ dimethyl ( 1 , 5 - cyclooctadiene ) platinum ( ii ) ] organometallic complexes to produce small core - shell rupt nps B-nanoparticle in the presence of pvp at room temperature . [SEP]
[CLS] several characterization techniques were combined for determining the structural composition of the particles , and [SEP]
[CLS] co was used for adsorption as a probe molecule . [SEP]
[CLS] ftir and ss nmr results were in agreement with the coordination of co to pt and in this way the presence of a segregated ru core / pt shell B-material structure was indicated . [SEP]
[CLS] measurements by waxs , hrtem , exafs and other techniques corroborated these findings . [SEP]
[CLS] electrically conductive al - doped zno nps B-nanoparticle prepared by avadhut et al . were characterized by ss nmr spectroscopy B-technique : a core - shell structure model was proposed for these nps B-nanoparticle , which were synthesized with a microwave - assisted polyol method . [SEP]
[CLS] a combination of different 1d al , 1 h , c and 2d 27 al { 1 h } ss nmr techniques helped to gain insight into the particle structure and explain the macroscopically observed conductivities as a function of the np B-nanoparticle composition . [SEP]
[CLS] nanoscale fluorinedoped sno 2 nps B-nanoparticle , prepared with a microwave assisted polyol approach were studied by several techniques , including ss nmr . [SEP]
[CLS] sn ( ii ) could be distinguished from sn ( iv ) using nmr , similar to what mossbauer spectroscopy B-technique can do . [SEP]
[CLS] heteronuclear nmr experiments helped to characterize intraparticle interfaces in polycrystalline nps B-nanoparticle . [SEP]
[CLS] the fluorine doped particles showed an increased conductivity , after annealing , in comparison with undoped sno 2 nps B-nanoparticle . [SEP]
[CLS] davidowski and holland employed ss nmr to characterize mixed phosphonic acid ligand binding and organization on sio 2 nps B-nanoparticle . [SEP]
[CLS] multinuclear ( 1 h , si , 31 p ) and multidimensional solid - state nmr techniques were used , while the phosphonic capping ligands were methylphosphonic acid and phenylphosphonic acid . [SEP]
[CLS] for instance , p nmr spectra showed that phosphonic acid functionalized silica nps B-nanoparticle displayed three different ligand environments , attributed to physisorbed , monodentate and bi / tridentate . [SEP]
[CLS] the combination of multinuclear ss nmr and dft calculations has been employed to investigate the structure of nayf upconverting nps B-nanoparticle . [SEP]
[CLS] a detailed analysis of the crystal lattice and ionic distribution was achieved by these techniques . [SEP]
[CLS] in particular , 89 y nmr was employed to probe the chemical environment of y 3 + ions B-material in the nayf 4 structure . [SEP]
[CLS] the presence of a solid solution type cubic structure in which cation B-material sites were randomly occupied was observed . [SEP]
[CLS] finally , for the characterization of surface species and substratesurface interactions on metal B-material nps B-nanoparticle , the groups of pruski and emsley have shown that dynamic nuclear polarization surface enhanced nmr can be a very useful tool for the further increase of the sensitivity of ss nmr . [SEP]
[CLS] the brunauer - emmett - teller ( bet ) technique is also used for the characterisation of nanoscale materials . [SEP]
[CLS] it is based on the principle of physical adsorption of a gas on a solid surface , and it was named by the initials of the surnames of its developers , brunauer , emmett and teller . [SEP]
[CLS] it is widely used for the determination of the surface area of nanostructures , being a relatively accurate , rapid and simple method for this purpose . [SEP]
[CLS] sahoo and co - workers prepared biocompatible B-property ferrofluid containing dye - functionalized fe 3 o 4 nps B-nanoparticle , which can serve as fluorescent B-property markers . [SEP]
[CLS] several techniques were used for the characterisation including bet , ftir and others . [SEP]
[CLS] the surface area measured by bet was smaller than the estimate obtained from the size distribution and density values of the studied material B-material ; this deviation might be caused by the agglomeration of smaller nps B-nanoparticle resulting in larger ones , thereby effectively reducing the collective surface area . [SEP]
[CLS] such agglomeration risk is probably aggravated considering that the np B-nanoparticle samples need to be dried for such measurements : strong hydrogen B-material bonding might occur among the np B-nanoparticle surfaces , thus , inducing a certain error . [SEP]
[CLS] in another work , mesoporous polymer B-material microspheres with au nps B-nanoparticle inside their pores were produced , to observe the adsorption behaviour of these nps B-nanoparticle , considering their surface functionality and porosity . [SEP]
[CLS] bet experiments of au / poly ( ethylene glycol dimethacrylate - co - acrylonitrile ) composite microspheres , used to measure the microsphere porosity , revealed that the adsorption of au nps B-nanoparticle into the pores kept the pore structure intact and turned it more porous . [SEP]
[CLS] ma et al . synthesized fe 3 o 4 nps B-nanoparticle by the co - precipitation method of ferrous and ferric species , resulting in a product with high specific surface area ( 286 . 9 m 2 g −1 ) . [SEP]
[CLS] this value was much higher than those already reported in the literature for such particles . [SEP]
[CLS] thermal gravimetric analysis ( tga ) . [SEP]
[CLS] while ftir offers information about the np B-nanoparticle - stabiliser interaction and confirmation of the stabiliser type , it does not provide insights into the extent of surface coverage or the mass to mass ratio of np B-nanoparticle to stabiliser , which is important to normalize the values of saturation magnetization B-property to purely metallic content , for instance . [SEP]
[CLS] tga provides information concerning the mass and composition of the stabilisers . [SEP]
[CLS] with this technique , a nanomaterial B-material sample is heated and components with different degradation temperatures decompose and vaporise , and a change of mass is recorded . [SEP]
[CLS] the temperature and the loss of mass are recorded by the tga device and , taking into account the starting sample mass , the type and quantity of np B-nanoparticle organic ligands are determined . [SEP]
[CLS] a method known as microthermogravimetric analysis ( μ - tga ) uses the same thermal decomposition principle as tga , but the mass of the sample investigated is in the order of 1 μg , with mass changes lower than 1 nanogram being able to be detected . [SEP]
[CLS] in this way , the detection limits of conventional tga can be improved to a significant extent . [SEP]
[CLS] mansfield et al . used μ - tga to identify the presence and quantity of surfacebound ligand coverage on au nps B-nanoparticle and verify the existence of peg coating B-material on silica nps B-nanoparticle . [SEP]
[CLS] their results demonstrated that the aforementioned technique is a valid one to determine quantitatively the nps B-nanoparticle coatings B-material , while information on the purity and compositional data of the nps B-nanoparticle can also be acquired sometimes . [SEP]
[CLS] the authors highlighted the advantages of tga , which is a simple and direct technique without any special need for sample preparation , apart from having the sample in dry state . [SEP]
[CLS] a drawback of conventional tga is the need to have a few milligrams of the nanomaterial B-material sample , which may raise the cost or lab - scale production feasibility issues . [SEP]
[CLS] these researchers used a variety of np B-nanoparticle systems to illustrate the utility and limitations of μ - tga and its comparison with convention - al tga . [SEP]
[CLS] for example , similar results of both techniques were obtained concerning the oxidation temperature and the residual mass measurements in carbon B-nanoparticle nanotubes I-nanoparticle . [SEP]
[CLS] in addition , the ability to identify layer - by - layer coatings B-material on a au np B-nanoparticle core B-material was evidenced by both techniques . [SEP]
[CLS] the same research group analysed the surface density of peg on au nps B-nanoparticle by using μ - tga . [SEP]
[CLS] the speed and reliability of tga to determine the fractions of thermally stable and unstable masses of a sample were exploited . [SEP]
[CLS] usually , the surface coverage for inorganic particles with combustible ligands can be calculated if particle size and ligand molecular weights are well known . [SEP]
[CLS] the authors measured the peg surface densities on au nps B-nanoparticle using both μ - tga and fluorescence B-technique spectroscopy I-technique . [SEP]
[CLS] the lower values for surface densities determined from the latter technique might be attributed to incomplete displacement of the ligands from the au surfaces . [SEP]
[CLS] in another report , thiol - terminated pegcoated au nps B-nanoparticle in aqueous solution were studied by tga and other techniques , aiming to elucidate their structure and hydration . [SEP]
[CLS] combining mass density , sans , saxs and tga resulted in the acquisition of precise information on the au core B-material size and on the capping polymer B-material chains . [SEP]
[CLS] sans fits reached their optimal minimizations with a three shell model : the inner one related to the au core B-material , while the other two are characterized by different polymer B-material - water B-material mixtures with distinct scattering densities . [SEP]
[CLS] on the other hand , saxs was principally sensitive to the dimension of the au core B-material , considering that the contrast in the electron densities between the polymer B-material and the solvent is low . [SEP]
[CLS] the results of the structural data of the scattering experiments and the volumetric data derived from mass density and tga measurements were consistent , revealing the complementarity and correctness of this overall characterization approach . [SEP]
[CLS] jia et al . prepared au and pd nps B-nanoparticle via a surfactant - free single phase solution route . [SEP]
[CLS] high - temperature tga coupled with mass spectroscopy B-technique ( ms ) was used to find the relative amounts of ionic contaminants , since protecting thiolate groups and inorganic contaminants were removed in separate weight loss events . [SEP]
[CLS] tga - ms helped to achieve a more accurate determination of the thiolate to au ratios , revealing a complex composition of the nps B-nanoparticle presented therein . [SEP]
[CLS] tga - ms could also distinguish between the evaporation of the original thiolate ligands and their oxidized species . [SEP]
[CLS] the limitations of the above technique include the fact that non - volatile compounds such as li 2 o cannot be detected ; however , xps , ftir and xrd can help toward such detection . [SEP]
[CLS] in addition , the quantification of the content of certain groups and compounds based on tga is only precise if their weight losses take place at distinct temperatures . [SEP]
[CLS] events that happen at similar temperatures can be separated by optimizing the heating program . [SEP]
[CLS] however , overlapping events may be identified by ms , but the quantification of the intensities recorded in the ms data is not simple . [SEP]
[CLS] magnetite nps B-nanoparticle with fatty acid ( ricinoleic ) adsorbed on their surface were investigated with a tga device coupled with ftir . [SEP]
[CLS] the decomposition of ricinoleic acid was studied by tga under an inert atmosphere , while gas phase ftir helped to gain information on the decomposition gases released . [SEP]
[CLS] the impact of the autoxidation of the fatty acids was presented , while an extended reduction of magnetite from carbonaceous residues was also noticed . [SEP]
[CLS] slight discrepancies between the results from the tga and xrd experiments on the exact composition of the iron B-material oxides I-material might originate from the formation of oxidized residues in these two different measurements . [SEP]
[CLS] in another work , nava - etzana and co - workers reported the synthesis of bifeo 3 nanostructures by a combustion reaction , in the presence of tartaric acid or glycine B-material as the promoter . [SEP]
[CLS] the origin of a high purity bifeo 3 nanomaterial B-material together with the formation of certain by - products was described on the basis of metal - ligand interactions . [SEP]
[CLS] such high product purity demonstrated by xrd analysis was corroborated with the results from tga . [SEP]
[CLS] furthermore , tga / ftir and a combination of tg - gas B-technique chromatography I-technique - mass spectrometry ( tg / gc - ms ) were employed to characterize the effect of different types of dopants ( e . g . sio 2 nps B-nanoparticle , multi B-nanoparticle - I-nanoparticle walled I-nanoparticle carbon I-nanoparticle nanotubes I-nanoparticle , and montmorillonite ) on the thermal decomposition of polypropylene sebacate ( ppseb ) . [SEP]
[CLS] it was evidenced through the mass detection analysis of the generated decomposition compounds ( aldehydes B-material , alcohols B-material , acids , etc . ) that the ppseb degradation involved mainly β - hydrogen B-material bond scission and also α - hydrogen scission . [SEP]
[CLS] the insertion of nps B-nanoparticle led to the increase of the thermal stability of the polymer B-material . [SEP]
[CLS] low - energy ion B-material scattering ( leis ) is a modern analytical method that permits the rapid thickness characterization of self - assembled monolayers ( sam ) , for example , in the case of au nps B-nanoparticle . [SEP]
[CLS] in this technique , a sample is exposed to low - energy gas ions B-material , and the scattering and subsequent loss of energy of these ions B-material can be related to the elemental composition of the outer layer surface . [SEP]
[CLS] high sensitivity leis ( hs - leis ) offers better sensitivity for the investigation of distinct atomic layers with an extensive reduction in surface damage . [SEP]
[CLS] hs - leis illustrated that a complete sam was formed in the case of c 16 cooh - functionalized 14 nm au nps B-nanoparticle . [SEP]
[CLS] the estimated sam thickness was in good agreement with previous results from simulated electron spectra for the surface analysis of the xps data . [SEP]
[CLS] the leis thickness values were consistent with the values obtained by afm , x - ray reflection and sputter depth profiling . [SEP]
[CLS] the high sensitivity of hs - leis concerns the top [UNK] nm of the surface atomic layers . [SEP]
[CLS] this method is fast and rather direct , whereas sessa simulations require a lengthy analysis of the results for the thickness , but can yield more information on chemical composition . [SEP]
[CLS] kauling et al . used hs - leis to analyse the outer layer of both functionalized and non - functionalized imidazolium ionic liquids on au nps B-nanoparticle . [SEP]
[CLS] the description of its operation principle is described therein , together with its capacity to analyse the atomic composition and thickness of the surface of ionic liquids . [SEP]
[CLS] finally the formation of ruthenium B-material - gold B-material core - shell nps B-nanoparticle prepared by the physical B-technique vapor I-technique deposition I-technique method on the tio 2 surface was studied by stm and leis , in an article published by ovari et al . [SEP]
[CLS] the chemical composition of the nps B-nanoparticle was studied by leis , and it was found that when rh was deposited on tio 2 previously covered by au , rh atoms B-material impinged to au clusters moved to subsurface sites ; as a consequence , the outermost atomic layer of these clusters remained almost pure au . [SEP]
[CLS] stm and leis results showed that very limited mixing between au and rh in the bimetallic nps B-nanoparticle took place ( if any ) . [SEP]
[CLS] uv - vis spectroscopy B-technique ( uv - vis ) is another relatively facile and low - cost characterization method that is often used for the study of nanoscale materials . [SEP]
[CLS] it measures the intensity of light reflected from a sample and compares it to the intensity of light reflected from a reference material B-material . [SEP]
[CLS] nps B-nanoparticle have optical properties that are sensitive to size , shape , concentration , agglomeration state and refractive B-property index I-property near the np B-nanoparticle surface , which makes uv - vis spectroscopy B-technique an important tool to identify , characterize and investigate these materials , and evaluate the stability of np B-nanoparticle colloidal solutions . [SEP]
[CLS] gold B-material , silver B-material and copper B-material nanostructure sols exhibit characteristic uv - vis extinction spectra due to the existence of a lspr signal in the visible part of the spectrum . [SEP]
[CLS] in certain cases ( e . g . metal B-material chalcogenide nps B-nanoparticle and anisotropic gold B-material or silver B-material nanostructures ) , lspr bands at the near - infrared ( nir ) wavelength region can also appear . [SEP]
[CLS] besides characterizing the np B-nanoparticle optical properties , the size and molar concentration of zerovalent au , for example , can also be obtained from the uv - vis measurements . [SEP]
[CLS] for this calculation , which can also be performed in situ under certain conditions , the position of the lspr and the extinction at this wavelength , as well as the ratio of extinctions at the wavelength of the lspr and at 450 nm ( a lspr / a 450 ) , are needed . [SEP]
[CLS] the absorbance at 350 - 400 nm wavelength can also be used to measure the gold B-material colloid concentration , however with an uncertainty up to 20 - 30 % due to a rather slight influence of parameters such as np B-nanoparticle size , surface modification and oxidation state . [SEP]
[CLS] if these factors are taken into account upon calculation , the uncertainty in determining the au np B-nanoparticle concentration can be decreased extensively . [SEP]
[CLS] in fact , the maximum absorbance at the uv - vis spectra has also been successfully used for the calculation of the concentration of citrate - coated silver B-material nps B-nanoparticle . [SEP]
[CLS] haiss et al . have published a very high profile study on the utility of uv - vis spectra to determine the size and concentration of au nps B-nanoparticle . [SEP]
[CLS] the colloidal stability of au nps B-nanoparticle can be quantitatively characterized by uv - vis absorbance spectroscopy B-technique , as shown by pennathur and colleagues . [SEP]
[CLS] particle instability parameter ( pip ) is a universal technique to quantitatively characterize the stability of plasmonic nanomaterials B-material based on uv - vis absorbance spectroscopy B-technique that does not depend on the colloid system and can fully record the evolution of a given studied system over time . [SEP]
[CLS] it is a robust and generalizable approach , not only for au nps B-nanoparticle , but also for plasmonic nps B-nanoparticle as a whole . [SEP]
[CLS] another use of uv - vis spectroscopy B-technique involves the ability to detect molecules such as thiamine , by mixing a solution of thiamine in water B-material with a au np B-nanoparticle solution . [SEP]
[CLS] the presence of thiamine could be detected visually with a color change in the np B-nanoparticle solution from red to greenish - grey . [SEP]
[CLS] au nps B-nanoparticle tested for this application were in the range of 20 - 30 nm , whereas the limit of detection of thiamine was between 0 . 5 and 1 μm . [SEP]
[CLS] in another report , au and pt nps B-nanoparticle prepared by photoreduction synthesis in an aqueous medium containing dodecyltrimethylammonium chloride B-material ( dtac ) and peg were studied by uv - vis , exafs and other techniques ( tem , saxs ) . [SEP]
[CLS] exafs confirmed the metallic character of the nps B-nanoparticle while saxs implied that the structure of dtac and peg could be fitted with the hard - sphere model having the interaction radius ( r hs ) and the spherically shaped core - shell structure . [SEP]
[CLS] the time evolution of the saxs profiles was consistent with the uv - vis spectral change during the first 30 min of photoirradiation . [SEP]
[CLS] behzadi et al . reported the development of a colorimetric sensor array to define the physicochemical properties of nps B-nanoparticle dissolved in water B-material with ultra - low concentrations . [SEP]
[CLS] the effects of several dyes on different types of nps B-nanoparticle were probed using variations in the visible spectrum of the dyes . [SEP]
[CLS] the system should produce unique composite responses to each np B-nanoparticle , similar to the well - established colorimetric array that is used to identify toxic B-property chemical vapors . [SEP]
[CLS] the authors prepared four different types of gold B-material nanostructures and they employed their uv - vis approach to detect and discriminate these particles . [SEP]
[CLS] overall , this method can be considered low - cost , non - destructive and quick for the recognition of np B-nanoparticle systems and types . [SEP]
[CLS] ag nanostructures have also been extensively studied by uv - vis spectroscopy B-technique . [SEP]
[CLS] jha and co - workers investigated the influence of maturing time and concentration of nabh 4 on size with uv - vis . [SEP]
[CLS] their method , under the framework of the mie theory , was employed to determine the particle size and size distribution . [SEP]
[CLS] in fact , the lspr of nps B-nanoparticle is affected by size , shape , interparticle interactions , free electron density and surrounding medium , and this helps to obtain a screening of the electron injection and aggregation of nps B-nanoparticle . [SEP]
[CLS] in this way , it was possible to characterize the ag np B-nanoparticle formation kinetics and the final colloidal stability . [SEP]
[CLS] in another work , ag nps B-nanoparticle were prepared via a green synthesis involving the flowers of the moringa oleifera ( mo ) plant . [SEP]
[CLS] this plant acted as a reducing and stabilizing B-property agent I-property , and the resulting particles were studied by ftir , uv - vis and other techniques . [SEP]
[CLS] ftir experiments demonstrated that proteins B-material in the mo flower extract were adsorbed on ag nps B-nanoparticle , acting as capping agents . [SEP]
[CLS] it also indicated that retinoic acid , a component of the mo flower extract , acted as a reductant . [SEP]
[CLS] uv - vis analysis verified the existence of lspr in the produced particles and as the concentration of the mo flower extract increased , the absorption spectra showed a blue shift with decreasing np B-nanoparticle size . [SEP]
[CLS] photoluminescence B-property ( pl ) spectroscopy B-technique is another technique used to study nanoscale materials ; it monitors the light emitted from atoms B-material or molecules that have absorbed photons . pl is typically useful as the characterization technique for fluorescent B-nanoparticle nanoparticles I-nanoparticle , such as quantum B-nanoparticle dots I-nanoparticle , as well as metal B-material nanoclusters . [SEP]
[CLS] recently , the inherent pl of metallic nps B-nanoparticle received remarkable interest . [SEP]
[CLS] despite the fact that the quantum efficiency of the emission process is low , this inefficiency can be compensated by the large excitation cross sections at the plasmon resonances . [SEP]
[CLS] in addition , the pl of metal B-material nps B-nanoparticle is free of photobleaching and photoblinking . [SEP]
[CLS] thus , pl can be regarded as a better alternative than fluorescent B-property molecules for optical labeling applications . [SEP]
[CLS] single - photon and multi - photon excitation pl has been acquired using plasmonic nanostructures of several shapes . [SEP]
[CLS] gong and co - workers studied the pl behaviour of a single au nanoflower , a highly branched plasmonic nanostructure . [SEP]
[CLS] it was demonstrated that the pl measurements of such single au nanoflower revealed some rather more complex features in comparison with simple nanostructures . [SEP]
[CLS] such pl properties of the au nanoflower were strongly dependent on the excitation wavelength and polarization , and they were further studied in situ . [SEP]
[CLS] the pl experiments and emission measurements comprised a complementary approach to the optical scattering method , and they are targeted to benefit potential applications in domains such as optical B-technique imaging I-technique and sensing . [SEP]
[CLS] andersen et al . illustrated the pl wavelength and polarization engineering by exploiting arrayed au nps B-nanoparticle atop a subwavelength - thin dielectric spacer B-material and optically thick au film , a configuration that supports gapsurface plasmon resonances . [SEP]
[CLS] on the other hand , quantum B-nanoparticle dots I-nanoparticle such as metal B-material chalcogenide nps B-nanoparticle have widely been studied by pl . [SEP]
[CLS] for instance , the extinction and photoluminescence B-property of cu 2−x s , cu 2−x se and cu 2−x te nps B-nanoparticle have been investigated by feldmann and co - workers and the tunability and control over those properties have been discussed through the active manipulation over their copper B-material deficiency under oxidative / reductive conditions . [SEP]
[CLS] it was demonstrated that the presence of nir lsp resonances in these nps B-nanoparticle had a crucial effect on the exciton recombination . [SEP]
[CLS] for example , the pl of cu 2 s nanoclusters was quenched during their gradual transformation to non - stoichiometric nanoclusters ( x > 0 ) under an oxidative environment . [SEP]
[CLS] metal B-material oxides I-material such as zno nps B-nanoparticle are also photoluminescent B-property . [SEP]
[CLS] saliba et al . synthesized zinc B-material oxide I-material nps B-nanoparticle in the presence of branched thermotropic liquid crystals . [SEP]
[CLS] three emissions were observed for their particles , depending on the excitation wavelength . [SEP]
[CLS] the origin of such emissions was attributed to several factors , such as surface defects ( e . g . oxygen B-material vacancies ) . [SEP]
[CLS] another example of nanoscale materials with photoluminescent B-property properties is cesium lead halide perovskites . [SEP]
[CLS] protesescu et al . synthesized cesium lead halide nanocrystals using inexpensive commercial precursors and they studied their photoluminescence B-property properties . [SEP]
[CLS] the colloidal cspbx 3 ( x = cl , br , i and mixed cl / br , br / i ) nps B-nanoparticle were bright ( quantum yield = 50 - 90 % ) , stable , and spectrally narrow and had tunable bandgap energies . [SEP]
[CLS] dynamic B-technique light I-technique scattering I-technique ( dls ) is a widely employed technique to find the size of nps B-nanoparticle in colloidal suspensions in the nano - and submicrometer ranges . [SEP]
[CLS] the nps B-nanoparticle dispersed in a colloidal solution are in continuous brownian motion . [SEP]
[CLS] dls measures light scattering as a function of time , which combined with the stokes - einstein assumption are used to determine the np B-nanoparticle hydrodynamic diameter ( i . e . diameter of the np B-nanoparticle and the solvent molecules that diffuse at the same rate as the colloid ) in solution . [SEP]
[CLS] in dls , a relatively low np B-nanoparticle concentration is needed so that a multiple scattering effect is avoided . [SEP]
[CLS] lim et al . have reviewed the characterization of nps B-nanoparticle by dls ( fig . 2 ) focusing in the case of magnetic B-property particles . [SEP]
[CLS] they present how various factors such as suspension concentration , particle shape , colloidal stability and surface coating B-material of mnps influence the size value obtained by dls measurements . [SEP]
[CLS] a comparison between the results derived from dls and other techniques , such as tem and afm , is performed and the origins for any discrepancies in the sizing , for either small or larger particles , are discussed , while the working size range for each technique is also given . [SEP]
[CLS] for example , for small - sized nps B-nanoparticle , the radius of curvature effect is the principal contributing factor for the large difference observed for the diameter measured by tem and dls . [SEP]
[CLS] middle - sized fe 3 o 4 nps B-nanoparticle capped with oleic acid and oleylamine seem to have size values that show the best match among dls and tem measurements . [SEP]
[CLS] the authors highlight the use of dls also for the measurement of the colloidal stability of mnps . [SEP]
[CLS] moreover , dls has been proven useful to monitor the transient behaviours of β - feooh nanorods B-nanoparticle : these structures self - assemble in a side - by - side fashion to form highly oriented 2 - d nanorod B-nanoparticle arrays , eventually leading to the formation of 3 - d layered architectures . [SEP]
[CLS] overall , the realtime screening of nps B-nanoparticle by dls provides important insights into their aggregation process , since it measures quantitatively the size of the particle clusters formed . [SEP]
[CLS] the sensitivity of dls to large particles is crucial for its excellent diagnostic capability to detect aggregation . [SEP]
[CLS] nevertheless , the authors denote that careful analysis is required for the best possible interpretation of the dls results as they are affected by the factors previously mentioned ( shape , coating B-material agents , etc . ) . [SEP]
[CLS] the advantages of dls include its quick , easy and precise operation for monomodal suspensions and the fact that it is an ensemble measurement method , yielding a good statistical representation of each np B-nanoparticle sample . [SEP]
[CLS] it is highly sensitive and reproducible for monodisperse , homogeneous samples . [SEP]
[CLS] a limitation of dls is the necessary conditions for the particles to be in suspension and undergoing brownian motion . [SEP]
[CLS] large particles scatter much more light and even a small number of large particles can obscure the contribution from smaller particles . [SEP]
[CLS] therefore , its resolution for polydisperse , heterogeneous samples is rather low . [SEP]
[CLS] dls requires transformative calculations with assumptions that must be taken into account when interpreting the dataparticularly with polydisperse samples . [SEP]
[CLS] although dls can sometimes measure anisotropic nanostructures , it generally assumes spherical shaped particles . [SEP]
[CLS] overall , dls measures the hydrodynamic radius accurately but lacks the resolution to detect small aggregates . [SEP]
[CLS] however , when coupled with differential centrifugal sedimen - tation ( dcs ) , for example , it can result in valuable information for core - shell nps B-nanoparticle , as in the case of those prepared by minelli and co - workers : when dcs confirms that the samples are not aggregated , the measurements by dls can be safely considered as accurate . [SEP]
[CLS] coleman et al . have compared several methods used to obtain information on particle size distributions . [SEP]
[CLS] for instance , if [UNK] % of larger particles exist in a sample , in comparison with the majority of the particles ( e . g . two - fold or three - fold larger than the average size of 99 % of the particles ) , dls is significantly affected , giving higher values than tem ( e . g . 42 nm for a given silica reference sample compared to 25 nm by tem ) . [SEP]
[CLS] moreover , dcs , apart from its above - mentioned ability to detect agglomerate clusters , is able to characterize samples with broad size distributions . [SEP]
[CLS] driskell and co - workers employed dls to elaborate a fast one - step screening method for the characterization of the specificity of antibody - antigen I-event binding using antibody - conjugated au nps B-nanoparticle . [SEP]
[CLS] the advantages of dls detection over the more classic colorimetric technique include better detection limits and higher sensitivity . [SEP]
[CLS] dls was used to measure the formation of aggregates produced from virus - antibody B-material binding . [SEP]
[CLS] the extent of aggregation was employed to assess the interaction between the antibody B-material and the virus . [SEP]
[CLS] their novel approach offers an important improvement regarding screening time in comparison with elisa assays , while giving similarly precise results as the conventional method . [SEP]
[CLS] dls has also been combined with dosy - and noesy - nmr techniques to explore the partitioning behaviour of secondary surfactants B-property added to suspensions of reverse micelles B-material containing either au or ag nps B-nanoparticle . [SEP]
[CLS] the critical role of nps B-nanoparticle and the surfactant B-property amount on the efficiency of surfactant - assisted np B-nanoparticle extraction was investigated . [SEP]
[CLS] examples of the surfactants B-property tested were oleylamine , oleic acid and dodecanethiol . [SEP]
[CLS] the average particle diameters acquired by tem imaging were lower than those measured by dls , since the dls values reflect the outer diameter of the np B-nanoparticle - containing aot reverse micelles B-material together with any related solvent molecules . [SEP]
[CLS] dls helped in the monitoring of the irreversible penetration of reverse micelles B-material by specific secondary surfactants B-property . [SEP]
[CLS] fissan et al . used an aerosol technique , named scanning mobility B-property particle sizer ( smps ) , to characterize au - pvp and ag - pvp nps B-nanoparticle and they compared these results with the ones obtained from techniques such as sem and dls . [SEP]
[CLS] for samples with binary dispersion , dls failed to provide a correct feedback on the particle size , whereas sem , smps and analytical disk centrifugation ( adc ) managed to identify the two different particle size populations . [SEP]
[CLS] in particular , adc has a high resolution and can distinguish mixtures if the components cover different size ranges or have distinct densities . [SEP]
[CLS] adc is though time - consuming in some cases and it can somewhat underestimate the np B-nanoparticle size . [SEP]
[CLS] combining smps with a nebulizer may result in a method with a higher resolution than adc . [SEP]
[CLS] grobelny and co - workers investigated the size and size distribution of polydisperse silver B-material np B-nanoparticle colloids using dls and uv - vis . [SEP]
[CLS] although dls is more sensitive than uv - vis , its usual drawback has to do with the difficulty in detecting the pres - ence of smaller nps B-nanoparticle ; in addition , the uv - vis spectra did not contain any separate peaks for nps B-nanoparticle of different sizes . [SEP]
[CLS] therefore , the authors concluded that uv - vis should not be used for size determination in the case of polydisperse samples . [SEP]
[CLS] uv - vis and dls are low - cost and fast methods , but care is needed when interpreting their results , especially for the aforementioned types of samples , which do not contain a single np B-nanoparticle population . [SEP]
[CLS] complementary measurements with afm and tem / sem will be certainly needed for polydisperse samples . [SEP]
[CLS] kestens et al . used numerous techniques ( dls , cls , sem , tem , afm , and pta ) to measure the size of a ' standard ' sio 2 nanomaterial B-material sample . [SEP]
[CLS] measurements from several researchers working in distinct laboratories were studied . [SEP]
[CLS] the authors presented the nanomaterial B-material tested as a new reference material B-material with certified values and uncertainties that can be used for assessing the reliability of several particle size analysis methods . [SEP]
[CLS] murdock et al . characterized a broad range of nanomaterials B-material in solution using dls and tem , before assessing their in vitro toxicity B-property . [SEP]
[CLS] metal B-material and metal oxide I-material nps B-nanoparticle , such as al , al 2 o 3 , sio 2 and cu nps B-nanoparticle , as well as carbon - based materials such as carbon B-nanoparticle nanotubes I-nanoparticle , were tested . [SEP]
[CLS] dls measurements showed that depending on the material B-material examined , when the nps B-nanoparticle are in solution they do not necessarily retain their nanoscale size . [SEP]
[CLS] nanoparticle B-nanoparticle tracking analysis ( nta ) is a relatively new , but quickly adopted , technique that can measure np B-nanoparticle size , and having a lower concentration detection limit compared to dls . [SEP]
[CLS] it utilises the properties of both light scattering and brownian movement so as to acquire a np B-nanoparticle size distribution of samples in liquid dispersion . [SEP]
[CLS] the details of its operation principle ( fig . 3 ) and further technical information are provided by hole et al . that paper examined the reproducibility of results acquired by nta by investigating a wide range of nanoparticle B-nanoparticle systems and size ranges , in different media . [SEP]
[CLS] the measurements were performed in 12 distinct laboratories , aiming to obtain a wide database . [SEP]
[CLS] examples of the types of nanomaterials B-material tested were au , sio 2 and polystyrene nps B-nanoparticle , dispersed in water B-material or in biological media . [SEP]
[CLS] an important advantage that nta offers in comparison with other size measurement techniques is that it is not biased toward larger nps B-nanoparticle or aggregates . [SEP]
[CLS] furthermore , its confirmed accuracy and reproducibility verified the suitability of nta to determine the size populations of bimodal samples . [SEP]
[CLS] the comparison between nta and dls was also examined by jiskoot and colleagues , investigating standard polystyrene beads in the size range of 60 - 1000 nm . [SEP]
[CLS] physical mixtures of samples with different np B-nanoparticle sizes were also evaluated . [SEP]
[CLS] it was shown that nta yielded precise values for the size distribution of both monodisperse and polydisperse samples . [SEP]
[CLS] the average size values recorded by nta were slightly smaller and more exact to the nominal ones than those obtained by dls . [SEP]
[CLS] nevertheless , nta is slower and has a somewhat more difficult operation mode compared to dls . [SEP]
[CLS] that study corroborated the above - mentioned findings of other researchers which mention that dls results are not easily interpreted in the case of polydisperse samples , whereas nta is able to identify two different sample populations in the same sample . [SEP]
[CLS] overall , nta tracks single particles , while dls studies an ensemble of particles and it is strongly biased to the biggest particles , which are present in the sample . [SEP]
[CLS] nta was also studied by hassellov and co - workers for its capacity to determine the size distributions and concentrations of nps B-nanoparticle in liquid samples . [SEP]
[CLS] apart from the differences among dls and nta , the authors concluded that nta allows the measurement of large amounts of particles , compared to tem . [SEP]
[CLS] therefore , the statistical confidence is increased and the absence of any particle changes because of the preparation mode of the specimen tested is ensured . [SEP]
[CLS] additionally , nta can potentially use the intensity of light scattered by individual particles to discriminate particles composed of distinct materials within a given size range . [SEP]
[CLS] it is important to note that the sensitivity of nta is related to the size and composition of the nanomaterials B-material studied . in another report , ryu et al . prepared cawo 4 and camoo 4 nps B-nanoparticle via the pulsed laser ablation method , and they used several techniques to characterize them , including nta . [SEP]
[CLS] the latter technique can dynamically analyse the paths the nps B-nanoparticle take under brownian motion over a suitable time range ( e . g . 10 - 20 s ) and visualize deeply sub - micron particles in real time and in a liquid medium . [SEP]
[CLS] nta combined with image analysis determined the particle size distribution function of the aforementioned samples . [SEP]
[CLS] the results for the mean np B-nanoparticle size were in accordance with the values derived by tem and xrd . [SEP]
[CLS] nta has also been employed to analyse the capping efficiencies of several biomass - derived stabilizers of colloidal ag suspensions in water B-material . [SEP]
[CLS] the nta software identifies and tracks single nps B-nanoparticle that undergo brownian motion and correlates the velocity of the movement with the np B-nanoparticle size . [SEP]
[CLS] for instance , bigger nps B-nanoparticle and heavy aggregates move with a slow speed , in comparison with smaller nps B-nanoparticle , which have less weight and move faster . [SEP]
[CLS] it was found that a biorefinery - derived residual syrup acted as an efficient stabilizing B-property agent I-property for silver B-material nps B-nanoparticle in solution . [SEP]
[CLS] another use of nta , presented by van leeuwen and co - workers , is the determination of the refractive B-property index I-property which dictates the interaction between light and nps B-nanoparticle . [SEP]
[CLS] heterogeneous nps B-nanoparticle were tested , with sizes < 500 nm in suspension , and nta was capable of discriminating between sio 2 and polystyrene beads on the basis of their different refractive B-property indexes I-property . [SEP]
[CLS] the authors noted that nta can overestimate the mean diameter of the beads in comparison with tem . [SEP]
[CLS] this was attributed to the uncertainty in the measured diffusion coefficient and to the difference between the hydrodynamic diameter measured by nta and the physical diameter measured by tem . [SEP]
[CLS] dcs measures particle size on the basis of their sedimentation rate , which depends upon their size and density . [SEP]
[CLS] while dls is a lower resolution analysis method , dcs can be used to detect and resolve peaks down to 2 nm , and differing in size by as little as 2 % . [SEP]
[CLS] minelli and co - workers determined the thickness of immunoglobulin B-material g I-material ( igg ) protein B-material on 105 nm polystyrene particles by dcs , dls and saxs . [SEP]
[CLS] while dls provides precise results for the hydrodynamic size of the particles , comprising their polymeric B-material core I-material and the surrounding protein B-material shell B-material , dcs results are dependent on the density of the particle core B-material and that of the protein shell B-material . [SEP]
[CLS] on the other hand , as mentioned before , saxs enables traceable particle size measurements for sufficiently monodisperse particles , and it is a robust tool to identify their size distribution in terms of size and polydispersity , although it relies upon correct modelling for core / shell B-material particles . [SEP]
[CLS] dcs yielded somewhat larger size than the other two methods . [SEP]
[CLS] nevertheless , all techniques showed an increase of the igg shell B-material thickness with increasing protein B-material concentration during incubation B-technique with the nps B-nanoparticle , but model refinement was required for their full consistency . [SEP]
[CLS] the same group also published a comparative study of several emerging and established techniques for the characterization of the size of submicron particles , evaluating their sizing accuracy and relative resolution . [SEP]
[CLS] they also demonstrated the variety of the physical principles upon which they are based , aiming to develop a framework in which they can be compared . [SEP]
[CLS] the particles tested were stober silica ones , and it was found that dcs measurements could provide additional information concerning particle porosity that was not accessible to the other techniques . [SEP]
[CLS] on the other hand , dcs , nta and sios ( scanning ion B-material occlusion sensing ) were considered to be compact , easy to use and cost - effective . [SEP]
[CLS] dcs offered a high resolution , which is important for particles with complex structures such as coreshell ones . [SEP]
[CLS] smps had large dynamic range , good resolution and precision . [SEP]
[CLS] dls displayed the second highest precision . [SEP]
[CLS] shape information could not be provided by sios , dcs or nta , although complementary characterization with tem could help in this direction . [SEP]
[CLS] mass spectrometry ( ms ) has drawn interest as a strong tool for the analytical characterisation of nps B-nanoparticle in a reliable way . [SEP]
[CLS] ms offers invaluable elemental and molecular information on the composition , structure and chemical state of nps B-nanoparticle , and their bioconjugation to target biomolecules . [SEP]
[CLS] furthermore , it can be used for bioconjugation quantification , as explained by montoro bustos et al . in ref . 186 . [SEP]
[CLS] ms is compatible with any type of sample , apart from being a highly sensitive technique . [SEP]
[CLS] in addition , it is easily coupled with separation B-technique techniques I-technique to obtain real - time information . [SEP]
[CLS] in this way , varied and novel insights into the nature of nps B-nanoparticle and their final uses and applications can be potentially acquired . [SEP]
[CLS] inductively coupled plasma - ms ( icp - ms ) is used for the elemental analysis of nps B-nanoparticle . [SEP]
[CLS] it is characterized by robustness , high sensitivity and wide dynamic range , as well as high selectivity and virtual matrix independence . [SEP]
[CLS] in addition , it is straightforward , usually requiring simple calibration protocols . [SEP]
[CLS] it allows the reliable quantification and elemental composition characterisation of metallic nps B-nanoparticle , and it can determine metallic impurities B-property in nonmetallic nps B-nanoparticle . [SEP]
[CLS] molecular ms techniques , e . g . with electrospray ionisation B-property ( esi ) and matrix - assisted laser desorption / ionisation B-property ( maldi ) , can provide information on the protecting ligands that surround the nps B-nanoparticle and also correlate the entire clusters with their chemical composition . [SEP]
[CLS] moreover , coupling size - exclusion chromatography B-technique with icp - ms helps to gain information on the size distribution of au nps B-nanoparticle and their elemental characterization . [SEP]
[CLS] certain characterization techniques , including capillary B-technique electrophoresis I-technique , hydrodynamic chromatography B-technique , ion B-material mobility B-property spectrometry and field flow fractionation ( fff ) , also offer useful information about the size and size distribution of nps B-nanoparticle . [SEP]
[CLS] they can be coupled with icp - ms , for example , fff - icp - ms can study the multi - elemental composition and size distribution of natural colloids . [SEP]
[CLS] the use of groundbreaking ' single particle operation mode ' icp - ms ( spicp - ms ) has helped to identify the concentration and size distribution of nps B-nanoparticle . [SEP]
[CLS] in that case , highly diluted sample np B-nanoparticle suspensions should be used for their characterisation . [SEP]
[CLS] mclean and colleagues have written a review article on the characterization of thiolate - capped au nps B-nanoparticle by mass spectrometry . [SEP]
[CLS] they reported that apart from characterizing the stabilising ligands and the elemental composition of the nps B-nanoparticle , they can also measure the core B-material size and molecular stoichiometry . ms is a formidable tool for elucidating the size distribution of small clusters . [SEP]
[CLS] it can also observe ligand mixtures with discrete stoichiometry . [SEP]
[CLS] other techniques , such as nmr spectroscopy B-technique , can give population averages , providing only the percentage coverage of different thiolate ligands on an average nanoparticle B-nanoparticle . [SEP]
[CLS] for instance , regarding au nps B-nanoparticle , icp - ms considers the gold B-material core B-material to be of constant mass . [SEP]
[CLS] this allows the study of the variations in the stoichiometry of distinct ligands on the basis of mass in the following manner : if one characterizes gold B-material nps B-nanoparticle containing mixed ligands with icp - ms , he / she compares ligands of distinct masses and each population of ligands will correspond to a unique mass . [SEP]
[CLS] this allows the differentiation between the distinct ligands in the cases of nps B-nanoparticle capped with more than one ligand . [SEP]
[CLS] icp - ms can also determine the size distribution and number concentration of nps B-nanoparticle in a single , fast analysis . [SEP]
[CLS] it strongly depends on the matrix of the sample solution . [SEP]
[CLS] a scheme of the processes involved in the icp - ms analysis of au nps B-nanoparticle with ( a ) and without ( b ) previous au dissolution is depicted in fig . 4 . [SEP]
[CLS] regarding its capacity for the size characterisation of au nps B-nanoparticle , helfrich et al . have published a relevant article . [SEP]
[CLS] they presented an on - line coupling of liquid B-technique chromatography I-technique or gel B-technique electrophoresis I-technique with icp - ms for the size determination and compared the results with other techniques . [SEP]
[CLS] in particular , they mentioned that dls is generally expected to give higher values than other techniques because the measured parameter is the hydrodynamic radius of the nanoparticle B-nanoparticle , but the results obtained by tem provide information about the diameter of the au core B-material . [SEP]
[CLS] their results illustrated that the performance of on - line ge - icp - ms is strongly related to the chemical structure of the np B-nanoparticle surface composition . [SEP]
[CLS] good agreement was found between the different methods used for the size determination of their au nps B-nanoparticle . [SEP]
[CLS] as mentioned before , the possibility to measure the size of au nps B-nanoparticle was also demonstrated using spicp - ms . [SEP]
[CLS] it has to be noted that for this determination , the chemical composition , density and shape of the nps B-nanoparticle are needed to be known . [SEP]
[CLS] winchester and co - workers illustrated that precise size measurements by spicp - ms in the range of 20 - 200 nm can be achieved by operating the icp - ms instrument in reduced sensitivity modes using a lower extraction voltage , collision cell / ked or higher mass resolution . [SEP]
[CLS] in addition , spicp - ms can detect and quantify the dissolved and nanoparticulate forms of au at the same time . [SEP]
[CLS] the detection of au nps B-nanoparticle by the method in discussion is straightforward , but accurate measurement requires careful experimental design and data interpretation . [SEP]
[CLS] the characterization of complex , polydisperse np B-nanoparticle suspension by spicp - ms will require careful experimental design and data interpretation . [SEP]
[CLS] pace et al . also used spicp - ms to count and size nps B-nanoparticle . [SEP]
[CLS] they mentioned the abovewritten advantages of the former method , but they also presented its drawbacks and future challenges . [SEP]
[CLS] a major hurdle with spicp - ms is the improvement of the size detection limit . [SEP]
[CLS] for multi - element particles and less ideal systems , spicp - ms may struggle to detect and size particles within the nanoscale range . [SEP]
[CLS] arsenic B-material was also determined using icp - ms by pereira et al . they detected as ( iii ) and as ( v ) in environmental and biological samples with the assistance of cyst - capped thoria ( tho 2 ) nps B-nanoparticle . [SEP]
[CLS] large amounts of the inorganic as species were successfully removed from polluted water B-material samples . [SEP]
[CLS] in another report , spicp - ms was employed to monitor the detection and characterization of nps B-nanoparticle in complex matrices , such as food and biological tissues . [SEP]
[CLS] np B-nanoparticle size , size distribution and particle concentration values were calculated . [SEP]
[CLS] the size detection limits for four types of nps B-nanoparticle studied were 20 nm for au and ag nps B-nanoparticle , 50 nm for titania B-material nps B-nanoparticle and 200 nm for silica nps B-nanoparticle . [SEP]
[CLS] the authors agree with previous reports for the need to combine icp - ms with separation B-technique techniques I-technique such as hydrodynamic chromatography B-technique and field flow fractionation in order to obtain a more reliable view on the np B-nanoparticle features . [SEP]
[CLS] olesik and gray have also discussed the use of icp - ms to calculate the number of particles per litre , for the case of either nanoscale or microscale particles . [SEP]
[CLS] the main advantages and drawbacks of the method under discussion were the same as mentioned by other researchers . [SEP]
[CLS] the minimum particle size that can be detected will depend on a number of variables including the sensitivity and the signal due to a dissolved analyte or other continuous signal sources . [SEP]
[CLS] the minimum quantity of particles per litre of the suspension that is required for detection depends on the equivalent volume of suspension liquid delivered to the icp in the total measurement time . [SEP]
[CLS] besides , thiol B-material ligand density was quantified at selfassembled monolayers on au nps B-nanoparticle by icp - ms . [SEP]
[CLS] gold B-material and sulfur B-material concentrations could be determined simultaneously by icp - ms , and were obtained as ensemble averages of the particle distributions , as shown by lammerhofer and colleagues . [SEP]
[CLS] the surface coverage of au nps B-nanoparticle was studied quantitatively based on the linear relationship of the gold B-material / sulfur B-material ( au / s ) ratio measured by icp - ms , and the au np B-nanoparticle size measured by tem . [SEP]
[CLS] their method proved to be a valuable tool for the quantification of ligand densities on the surface of au nps B-nanoparticle . [SEP]
[CLS] spicp - ms was also employed combined with tissue extraction for the quantification and characterization of pvp - capped au and ag nps B-nanoparticle in environmentally relevant biological tissues . [SEP]
[CLS] the authors described a size detection limit of 20 nm for these ag and au nps B-nanoparticle , but they noted that this value depends on instrument sensitivity and the ionic background for the metal B-material of interest . [SEP]
[CLS] spicp - ms was also employed to characterize tio 2 and au nps B-nanoparticle during water B-material purification , in addition to the ag nps B-nanoparticle . [SEP]
[CLS] parameters such as the np B-nanoparticle concentration , size , size distribution and dissolved metal B-material element concentration in surface water B-material as well as in purified water B-material were evaluated . [SEP]
[CLS] understanding the fate of ti , ag and au during real potable water B-material treatment processes is important since human exposure to these nps B-nanoparticle will eventually occur by drinking water B-material . [SEP]
[CLS] donovan and co - workers found that lime softening followed by alum coagulation in combination with powdered activated carbon B-material adsorption resulted in the complete removal of au and ag nps B-nanoparticle and almost complete removal of tio 2 nps B-nanoparticle . [SEP]
[CLS] the presence of titania B-material nps B-nanoparticle was also investigated in sunscreens , using spicp - ms . [SEP]
[CLS] the aforementioned parameters were studied ( size , size distribution and np B-nanoparticle concentration ) , and the developed method was considered of high throughput , reproducible , lowcost and sensitive . [SEP]
[CLS] the method under discussion has also been applied to detect lanthanide B-material metals B-material doped into the iron B-material cores B-material of superparamagnetic B-property iron B-material oxide I-material nps B-nanoparticle in tissue and blood samples . [SEP]
[CLS] with spicp - ms , more than 10 different np B-nanoparticle formulations with distinct physicochemical properties could be directly analysed at the same time . [SEP]
[CLS] as a proof of concept , their approach was used to study the influence of np B-nanoparticle size and surface charge on tumor B-material delivery , biodistribution and blood clearance in vivo . [SEP]
[CLS] secondary ion B-material mass spectrometry ( sims ) is a mass spectral technique which can be used to obtain molecular chemical information from nps B-nanoparticle . [SEP]
[CLS] it is a surface analysis technique where primary ions B-material , which can be atomic or polyatomic , are used to sputter positively and negatively charged secondary ions B-material . [SEP]
[CLS] the secondary ions B-material ( sis ) originate from the outmost nanometer of the sample . [SEP]
[CLS] sims is in particular suitable for the analysis of nps B-nanoparticle by virtue of detection sensitivity and lateral ( [UNK] nm ) and depth ( [UNK] nm ) resolution . [SEP]
[CLS] it is worth mentioning that the secondary ion B-material signature of nps B-nanoparticle may be distinct in comparison with the one of bulk materials having the same composition . [SEP]
[CLS] however , it is necessary to have a well - working methodology to deconvolute the analytical results . [SEP]
[CLS] blanc et al . used sims to analyse the composition of dielectric nps B-nanoparticle localized in a silica glass matrix in the core B-material of optical fibers . [SEP]
[CLS] they performed sims imaging at high spatial resolution ( nanosims 50l ) and their goal was to gain more understanding on the spectroscopic properties of the luminescent B-property ions B-material in these fibers . [SEP]
[CLS] the authors mentioned that in sims the depth resolution is much better than the lateral resolution , which is related to the size of the probe . [SEP]
[CLS] the partitioning of p , mg and er into phase - separated zones was demonstrated , and this indicated that the particle composition was related to the mg concentration . [SEP]
[CLS] schweikert and co - workers noted that nano B-nanoparticle - I-nanoparticle objects I-nanoparticle of ' subcritical assay dimension ' have a sims signature that is specific to their physical and chemical features and their environment . [SEP]
[CLS] a question that arises is how the sims response would be influenced in the case of a single layer of nps B-nanoparticle with varied composition . [SEP]
[CLS] the researchers presented an investigation of a single layer of a mixture of ag and au nps B-nanoparticle . [SEP]
[CLS] cluster sims was employed to study individual nps B-nanoparticle . [SEP]
[CLS] time of flight secondary ion B-material mass spectrometry ( tof - sims ) is a material B-material characterisation technique that possesses high chemical sensitivity , high surface sensitivity ( upper 2 - 3 nm probed ) and molecular specificity . [SEP]
[CLS] this method can analyse the nanoparticle B-material drug I-material delivery formulations I-material . [SEP]
[CLS] in fact , tof - sims is extensively used to characterize the nano - zones of larger components , such as electronic devices and thin to ultrathin films of either organic or inorganic nature . [SEP]
[CLS] the technique under discussion is also utile for the investigation of the surface coating B-material or functional groups of nps B-nanoparticle , for example , to analyse peptides B-material coupled to au nps B-nanoparticle and multilayer plasmadeposited organic coatings B-material on al 2 o 3 nps B-nanoparticle . [SEP]
[CLS] laus and colleagues noted that sims can be destructive while conducting the analysis . [SEP]
[CLS] even though the ion B-material dose maximum limit can be adjusted to tackle the molecule destruction issue , the nps B-nanoparticle tested may still undergo melting . [SEP]
[CLS] these authors used sims for the depth profiling of certain types of nps B-nanoparticle ( au - sio 2 and ag - sio 2 configurations ) and they investigated the depth profiles for melting issues , combining their sims study with additional characterization by sem imaging . [SEP]
[CLS] in all cases , the interpretation of the sims depth profiles illustrated that melting took place , although it is possible that with ultralow energy cs + this effect was limited to its minimum . [SEP]
[CLS] 5 shows a scheme which explains how tof - sims is used to probe nps B-nanoparticle . [SEP]
[CLS] nps B-nanoparticle are adsorbed on a surface ; the bombardment of the primary ions B-material results in the desorption of molecules ( nps B-nanoparticle or np B-nanoparticle conjugates ) , which then results in the emission of secondary ions B-material from the outermost 1 - 1 . 5 nm molecular layers . [SEP]
[CLS] the secondary ions B-material are fragments of adsorbed molecules : metallic nps B-nanoparticle have high secondary ion B-material yields , whereas organic nps B-nanoparticle yield chemical - specific fragments that help to determine the surface ligands . [SEP]
[CLS] kim et al . mention that when tof - sims is combined with several np B-nanoparticle - based signal enhancing strategies , it can probe the functionalization of nps B-nanoparticle as well as their locations and interactions in biological systems . [SEP]
[CLS] np B-nanoparticle - based sims is important for label - free drug screening because signal - enhancing nps B-nanoparticle can be designed to directly measure the enzyme B-property activity I-property . [SEP]
[CLS] it can also be employed to monitor ligand - exchange processes . [SEP]
[CLS] the benefit of tof - sims , compared to maldi - ms ( matrix - assisted laser desorption / ionization B-property ) , is the straightforward analysis of targets without any matrix use . [SEP]
[CLS] therefore , tof - sims provides molecular information about functional groups , molecular orientation and conformation as well as denatured species from chemicals and / or from biomolecules . [SEP]
[CLS] it can also be used to gain information on the core B-material composition of nps B-nanoparticle , apart from their surface . [SEP]
[CLS] the types of nps B-nanoparticle usually probed by tof - sims are popular in domains such as biosensing and bio - imaging . [SEP]
[CLS] nevertheless , the spatial resolution of tof - sims is limited to only hundreds of nanometers and tof - sims is not particularly sensitive to high mass fragments . [SEP]
[CLS] for a higher sensitivity and higher spatial resolution for the ability to detect metals B-material in organic matrices , tof - sims can be coupled with laser secondary neutral mass spectrometry ( laser - snms ) . [SEP]
[CLS] high - resolution nanosims can provide monoatomic and diatomic secondary ions B-material with a better sensitivity and spatial resolution than tof - sims . [SEP]
[CLS] rafati et al . used tof - sims to investigate polymer B-material microspheres for the controlled release of a therapeutic protein B-material from an implantable scaffold . [SEP]
[CLS] the ability of tof - sims imaging to spatially image the polyvinyl alcohol B-material ( pva ) surfactant B-property and protein B-material adsorbed onto the surface of the microspheres was shown for the first time . [SEP]
[CLS] the surfactant B-property layer had a thickness of about 4 nm and it could be readily removed under sputtering with c 60 , as also confirmed by afm measurements . [SEP]
[CLS] indeed , afm can act as a complementary technique to tof - sims providing nanometer spatial resolution of the surface topography . [SEP]
[CLS] both techniques were able to chemically and physically visualize correspondingly the integrity and pattern of the surfactant B-property across the surface of the nps B-nanoparticle . [SEP]
[CLS] their work is a good example of what tof - sims imaging can offer , such as the spatial location of the protein B-material , the surfactant B-property and the polymer B-material substrate . [SEP]
[CLS] confocal raman B-technique spectroscopy I-technique can also be combined with tof - sims to study the bulk distribution of the protein B-material within the microparticles . [SEP]
[CLS] wiesmann and coworkers also employed tof - sims to detect protein B-material coatings B-material on np B-nanoparticle surfaces by tof - sims and advanced electron B-technique microscopy I-technique techniques . [SEP]
[CLS] in addition to its other characteristics , this technique can detect all isotopes B-material and offers a simultaneous imaging of the surface distribution of detected molecules and elements . [SEP]
[CLS] the thicknesses of the different protein B-material coatings B-material of collagen ( two different collagen types ) were measured by tem . [SEP]
[CLS] tof - sims permitted one to distinguish and identify the masses of typical amino B-material acids I-material of the two protein B-material matrixes . [SEP]
[CLS] cowin and colleagues employed tof - sims and sem for an in situ study of 5 nm goat anti - mouse igg au nps B-nanoparticle in a novel portable vacuum compatible microfluidic device . [SEP]
[CLS] characteristic signals of the conjugated au nps B-nanoparticle were successfully spotted through the aperture by edx in sem and tof - sims . [SEP]
[CLS] in another report , tof - sims and xps were used together to study the aging of plasma - mediated coatings B-material with embedded ag nps B-nanoparticle on stainless steel . [SEP]
[CLS] the variation of film composition ( silver B-material release , matrix composition , and thickness ) with immersion time in saline solution was analysed . [SEP]
[CLS] coating B-material modifications , caused by immersion , were found to depend on the starting ag content . [SEP]
[CLS] lee et al . employed and validated an approach combining tof - sims and a confocal B-technique laser I-technique scanning I-technique microscopy I-technique ( clsm ) imaging method for the cytotoxicity B-property study of zno nps B-nanoparticle in hacat cells B-material . [SEP]
[CLS] several compositional and toxicological analysis methods were applied to evaluate the size , shape and other features of the zno nps B-nanoparticle . [SEP]
[CLS] furthermore , their dissolution behaviour and effect on hacat cell B-property viability I-property in the presence of various concentrations in water B-material was also studied . [SEP]
[CLS] comparative and correlative analyses of the above - mentioned results with tof - sims and clsm imaging demonstrated a reasonable and acceptable outcome and allowed the consideration of this approach as reliable , quick and sensitive . [SEP]
[CLS] niehuis and coworkers used tof - sims to study the effect of primary ion B-material parameters ( species and energy ) on a model system ( hfo 2 on si ) as well as on lumidot core - shell nps B-nanoparticle . [SEP]
[CLS] it was indicated that the energy values used in tof - sims caused the melting - up or evaporation of nps B-nanoparticle after direct or grazing impact of primary ions B-material . [SEP]
[CLS] therefore , although atomic layer - deposited films of hfo 2 on si were well suited for studies on the information depth of tof - sims , experiments on lumidot nps B-nanoparticle implied that the information gained using ald references cannot be easily transferred to nps B-nanoparticle . [SEP]
[CLS] moreover , with mass spectrometry techniques , the sample needs to undergo ionization B-property and subsequent sorting based on the mass to charge ratio in magnetic B-property and electric fields . [SEP]
[CLS] the desorption and ionization B-property process can be assisted by ablation with a high energy laser ( matrix assisted laser desorption / ionization B-property , maldi ) or a salvo of inert gases ( fast atom bombardment ) . [SEP]
[CLS] maldi - tof ms can characterize very small nps B-nanoparticle as it can quantify many particles at a time leading to an improved estimate of dispersity . [SEP]
[CLS] the size range of the particles that can be analysed is very large and highly sensitive . [SEP]
[CLS] maldi - tof was successfully employed by hyeon and co - workers to estimate the np B-nanoparticle size of spherical ag nps B-nanoparticle in 9 - nitroanthracene . [SEP]
[CLS] the size values matched well with the ones measured by tem . [SEP]
[CLS] it was shown that the method under discussion can be used as a generic methodology to estimate with high precision the size and size distribution of nps B-nanoparticle with several shapes and sizes . [SEP]
[CLS] maldi - tof was also employed to characterize colloidal pt nps B-nanoparticle prepared by navin et al . the particles analysed were in the 1 - 4 nm size range and they were stabilized by pvp . [SEP]
[CLS] particle sizes determined from mass spectra were found to be in good accordance with those derived from tem and xrd experiments . [SEP]
[CLS] zhang et al . used high - performance liquid B-technique chromatography I-technique coupled with mass spectrometry for the analysis of ultrasmall pd nps B-nanoparticle . [SEP]
[CLS] reverse - phase hplc is expected to offer more accurate determinations of the catalytic , electronic , optical and toxicological properties of metal B-material nps B-nanoparticle . [SEP]
[CLS] among several separation B-technique techniques I-technique , hplc can be considered as an effective approach to isolate different metal B-material np B-nanoparticle species . [SEP]
[CLS] the authors employed rp - hplc to separate and analyse for the first time water - soluble dmf - pd nps B-nanoparticle . [SEP]
[CLS] the measurements by maldi - tof ms were in agreement with the chemical compositions of the fractions . [SEP]
[CLS] the aforementioned technique is the most popular ms technique in determining the number of metal B-material atoms B-material of np B-nanoparticle fractions . [SEP]
[CLS] it is further anticipated that rp - hplc combined with ms can be applied to investigate the growth mechanism of pd nps B-nanoparticle . [SEP]
[CLS] resonant mass measurement microelectro - mechanical system ( rmm - mems ) is a technique used to detect and count subvisible and sub - micron particles in a material B-material , and to measure their size and mass and the distributions of these properties . [SEP]
[CLS] a micro electro - mechanical system ( mems ) sensor , containing a resonating cantilever with a microfluidic channel embedded in its surface , is employed . [SEP]
[CLS] when a particle with a size between 50 nm - 5 μm flows through the fluidic channel , it alters the resonating frequency of the cantilever , which indicates the buoyant mass , and also the dry mass and size of the particle . [SEP]
[CLS] the information on sample concentration , viscosity B-property , density and volume can also be obtained by the sensor . [SEP]
[CLS] voevodin and colleagues synthesized au / pd bimetallic nps B-nanoparticle with a biotemplated approach and deposited them on au mems switch contacts as a np B-nanoparticle - based lubricant . [SEP]
[CLS] the authors of that study noted that since the melting point of nps B-nanoparticle is generally lower than that of the bulk materials , np B-nanoparticle size and size distribution are important factors for using nps B-nanoparticle as mems switch lubricants . [SEP]
[CLS] the bimetallic nps B-nanoparticle synthesized by these authors were found to be excellent candidates as surface B-property modifiers I-property / lubricants for mems switch lubricants . [SEP]
[CLS] zeta B-property potential I-property ( ζ - potential ) . [SEP]
[CLS] the ζ - potential of a sample is a key indicator B-property of the stability of colloidal dispersions . [SEP]
[CLS] highly positively or negatively charged particles tend to repel each other , thus forming stable colloidal solutions which show only minor trends to agglomerate . [SEP]
[CLS] such highly charged particles are related to ph values which are far from the so - called ' isoelectric point ' of a solution which refers to the ph value at which the zeta B-property potential I-property is zero . [SEP]
[CLS] on the other hand , a low value for the ζ - potential of a colloidal np B-nanoparticle dispersion causes the flocculation of the colloids and it corresponds to values closer to the isoelectric point of the system . [SEP]
[CLS] in general , colloids with values for the ζ - potential in the range of ±20 - 30 mv or higher are considered stable . [SEP]
[CLS] this property can be tuned through the modification of the surface chemistry , so the stabilisation of the colloidal suspension is obtained via electrostatic repulsion . [SEP]
[CLS] the ζ - potential is influenced by the concentration of the suspension and composition of the solvent and other additives . [SEP]
[CLS] since dls can also provide indications on the aggregation tendency of a sol , it can be combined with ζ - potential measurements for a more complete characterization . [SEP]
[CLS] branda et al . employed dls and ζ - potential studies ( which in fact can be carried out in the same device with modern instruments ) to analyse the influence of the exposure to growth media on the size and surface charge of silica - based stober nps B-nanoparticle . [SEP]
[CLS] these techniques appeared to be valuable tools to investigate the fate of nps B-nanoparticle in biological environments . [SEP]
[CLS] compared to tem and sem , the above - mentioned techniques offer the benefit that the nps B-nanoparticle are not exposed to the risk of clustering during sample preparation because of solvent evaporation . [SEP]
[CLS] dobson and colleagues synthesized and characterized ultra - small superparamagnetic B-property iron B-material oxide I-material nps B-nanoparticle thinly coated with sio 2 . [SEP]
[CLS] the authors noted that characterizing the np B-nanoparticle surface properties was important for the understanding of properties under physiological conditions and optimizing the conjugation chemistry . [SEP]
[CLS] surface charge was characterized by ζ - potential analysis . [SEP]
[CLS] acid washes using hno 3 reversed the ζ - potential of the fe 3 o 4 colloid and removed any remaining ammonium ions B-material , but also caused the material B-material to release fe 2 + , converting magnetite to maghemite , with no reduction in particle size . [SEP]
[CLS] ph . [SEP]
[CLS] the ph is another property frequently measured in colloidal np B-nanoparticle solutions . [SEP]
[CLS] aroca and co - workers tailored the size and shape of au nps B-nanoparticle in fulvic acid colloidal solution by modifying the ph and concentration of the acid . [SEP]
[CLS] the reasoning behind the ability to vary the acquired morphology came from the fact that a different ph affected the reaction kinetics . [SEP]
[CLS] the reversible aggregation of au nps B-nanoparticle was induced by ph - dependent modifications in a self - assembled monolayer of disulfide modified poly ( l - glutamic acid ) . [SEP]
[CLS] the change in the aggregation behaviour with ph took place within minutes and in a narrow range of ph from 4 . 5 to 5 . 5 . [SEP]
[CLS] in another report , cappellari et al . synthesized ultrasmall cysteine - coated au nps B-nanoparticle by ph switching of the au ( i ) - cysteine B-material polymer B-material . [SEP]
[CLS] by characterizing their products with several techniques such as xanes and exafs , the authors concluded that the ph affected not only the charge state of the polymer B-material , but it also caused a modification in the oxidation state of the metallic centers . [SEP]
[CLS] the size of the nps B-nanoparticle was controlled by the ph value and ultrasmall sizes ( [UNK] . 6 nm ) appeared for a 4 - 9 ph range . [SEP]
[CLS] qin and co - workers synthesized au nps B-nanoparticle by a biosynthetic approach : the products had a tunable shape by simply changing the ph of the reaction solution at room temperature . [SEP]
[CLS] the structural configuration of moss protein B-material could be induced by ph solutions . [SEP]
[CLS] hamlett et al . published a study on the ph - dependent adsorption of au nps B-nanoparticle on chemically modified si 3 n 4 mems devices . [SEP]
[CLS] the maximum adsorption of citrate - passivated au nps B-nanoparticle took place at ph = 5 , in agreement with afm and xps experiments . [SEP]
[CLS] the mass adsorption experiments were performed using aminofunctionalised si 3 n 4 ' flap ' resonators . [SEP]
[CLS] the ph values can also affect the toxicity B-property of nanomaterials B-material , as in the case of ag nps B-nanoparticle reported by oukarroum et al . [SEP]
[CLS] the size distribution of their particles depended on the ph of the culture medium . [SEP]
[CLS] the ag np B-nanoparticle toxicity B-property on the green alga chlamydomonas acidophila was ph - dependent as shown by the cytotoxicity B-property mediated through the induction of oxidative stress . [SEP]
[CLS] pavlopoulou et al . monitored the synthesis of pt nps B-nanoparticle using ph - responsive microgel particles . [SEP]
[CLS] saxs was employed to study the structure of ph - responsive microgels before and after metal B-material incorporation . [SEP]
[CLS] the decrease in the microgel radius together with an increase of the fractal dimension f when increasing the solution ph confirmed the ph - responsive character of the microgels . [SEP]
[CLS] these tertiary amine I-material - based microgels were used as nonreactors for the preparation of pt nps B-nanoparticle . [SEP]
[CLS] bradu and colleagues published an article on the influence of ph on the catalytic activity and selectivity of pd - cu nps B-nanoparticle supported on titania B-material in the nitrate reduction reaction . [SEP]
[CLS] the presence of titania B-material endowed an increased catalytic activity of the nanomaterials B-material studied . [SEP]
[CLS] gwak et al . studied the physicochemical changes of zno nps B-nanoparticle with different sizes and surface chemistries under physiological ph conditions . [SEP]
[CLS] the zno nps B-nanoparticle were found to enhance the ph under the physiological ph conditions to a neutral ( in the case of the gastric conditions ) or basic range ( in the case of the intestinal and plasma conditions ) , showing a dependency on the size and surface chemistry . [SEP]
[CLS] in another report , samarium oxide B-material nps B-nanoparticle were synthesized by yousefi and co - workers through a cathodic electrodeposition approach . [SEP]
[CLS] the effect of the ph on the morphology of the nps B-nanoparticle was studied . [SEP]
[CLS] with the increase of ph , parameters such as the weight , density and adhesion of the deposit on the electrode were decreased remarkably . [SEP]
[CLS] engelbrekt et al . synthesized selectively cu 2 ( oh ) 3 cl and tenorite cuo nps B-nanoparticle with a one - pot protocol and the obtained product was tuned according to the solution ph . [SEP]
[CLS] in particular , acidic ph values prohibited the formation of nps B-nanoparticle , and neutral ph resulted in cu 2 ( oh ) 3 cl , whereas cuo nps B-nanoparticle were generated in a basic ph environment . [SEP]
[CLS] the np B-nanoparticle morphology was also tuned by controlling the ph . [SEP]
[CLS] finally , the influence of ph and calcination temperature on the structural and optical properties of al 2 o 3 nps B-nanoparticle was studied by amirsalari and shayesteh . [SEP]
[CLS] it was evidenced that the alumina particles had an optical direct bandgap and the energy gap decreased with increasing calcination temperature and ph of the reaction . [SEP]
[CLS] the crystalline size of nps B-nanoparticle increased according to the ph of the solution . [SEP]
[CLS] electrophoretic B-property mobility I-property ( epm ) is measured to evaluate the surface charge of nanomaterials B-material . [SEP]
[CLS] the aggregation and disaggregation of iron B-material oxide I-material nps B-nanoparticle in relation to np B-nanoparticle concentration , ph and natural organic matter were reported . [SEP]
[CLS] low epm values were associated with the formation of large aggregates , whereas very high epm values were observed in the case of very stable nps B-nanoparticle for a prolonged time . [SEP]
[CLS] in another report , dls and electrophoretic B-property mobility I-property measurements were used to monitor the evolution of silica colloid to silica colloid - polyelectrolyte - iron B-material oxide I-material composites . [SEP]
[CLS] au nps B-nanoparticle , prepared by merga and co - workers upon the reduction of au 2 o 3 by h 2 , were characterized by several techniques , including epm . [SEP]
[CLS] conductivity measurements showed that most of the unreduced au ions B-material are in solution , but a small fraction resides on the particle . [SEP]
[CLS] epm measurements help to obtain the ζ - potential values . [SEP]
[CLS] in fact , minelli and co - workers compared several techniques in a systematic way for the determination of the ζ - potential of silica nps B-nanoparticle in a biological medium . [SEP]
[CLS] the ζ - potential is directly related to the electrophoretic B-property mobility I-property through the henry equation and the smoluchowski or huckel models . [SEP]
[CLS] the authors used one ensemble and two particle - by - particle techniques : electrophoretic light scattering ( els ) , tunable resistive pulse sensing ( trps ) and zeta particle tracking analysis ( z - pta ) . [SEP]
[CLS] despite differences between the basic measurement principles of the three methods , the results were overall in good agreement [SEP]
[CLS] luminescent B-property au nps B-nanoparticle decorated with bifunctional ligands possessing thiol B-material and carboxylic B-material acid I-material functional groups I-material were characterized by electrophoresis B-technique , which revealed a monodisperse distribution of nps B-nanoparticle . [SEP]
[CLS] it was suggested that the mercaptoalkanoic acid ligand used to form a au - s charge transfer complex behaves as a ph - responsive collapsible molecular brush at the surface of the au nps B-nanoparticle . [SEP]
[CLS] gel permeation chromatography B-technique ( gpc ) , also known as size exclusion chromatography B-technique , is a highly valuable tool that separates molecules based on their hydrodynamic volume or size . [SEP]
[CLS] with advanced detection systems coupled to gpc , information about polymers B-material , such as molecular weight ( m w ) distribution , average molecular mass , and degree of branching , can be acquired . [SEP]
[CLS] tadros and colleagues characterized the adsorption of poly ( hydroxystearic acid ) to tio 2 nps B-nanoparticle using gpc . [SEP]
[CLS] the latter technique was able to resolve and quantify the nonadsorbed molecules by size . [SEP]
[CLS] in another work , gpc was used , together with ftir and nmr , to characterize a series of succinate linearly linked plga - peg - sa - peg - plga multiblock copolymers which were conjugated with au nps B-nanoparticle . [SEP]
[CLS] gpc helped to determine the average m w and m w distribution of the copolymer samples . [SEP]
[CLS] differential scanning calorimetry ( dsc ) is a thermoanalytical technique in which the difference in the amount of heat required to increase the temperature of a sample and a reference is measured . [SEP]
[CLS] badia et al . used dsc to detect the phase transitions of c 18 sh - derivatized au nps B-nanoparticle . [SEP]
[CLS] these phase transitions could be associated with the reversible disordering of the alkyl chains . [SEP]
[CLS] actually , ss nmr measurements show that the chain melting arose from an increased frequency of gauche bonds in the au - tethered alkanethiol chains . [SEP]
[CLS] ftir spectroscopy B-technique established that the chain melting starts at the chain terminus and propagates toward the middle of the chain with increasing temperature . [SEP]
[CLS] the melting behaviour of pb and sn 3 . 5 ag nps B-nanoparticle has also been investigated by dsc studies . [SEP]
[CLS] the latter technique has also been used to measure the specific heat capacity and thermal conductivity of peek / ag nps B-nanoparticle . [SEP]
[CLS] inductively coupled plasma optical emission spectrometry ( icp - oes ) is a highly sensitive technique that can characterize the core B-material nps B-nanoparticle and also their coating B-material ligands . [SEP]
[CLS] it can reach tracelevel concentrations , small changes in concentration can be identified , and multiple elements can be detected at the same time . [SEP]
[CLS] therefore , it can provide information on surface species conjugated on au nps B-nanoparticle and quantify the ligand packing density . [SEP]
[CLS] in addition , icp - oes offers a wide dynamic linear range and it is well reproducible . [SEP]
[CLS] magnetic B-property solid phase extraction ( mspe ) combined with icp - oes has been used to identify chromium B-material ions B-material in environmental water B-material samples . [SEP]
[CLS] in addition , trace amounts of cr , cu and pb can also be spotted by the combination of the aforementioned techniques . [SEP]
[CLS] electrospray differential mobility B-property analysis ( es - dma ) is a rapid technique ( analysis timescales on the order of 1 - 100 min ) with sub - nanometer resolution . [SEP]
[CLS] it can determine the np B-nanoparticle concentration , and it is a quick , low - cost technique , with statistically significant results ; however it does not offer the atomic - scale resolution of other techniques such as sans or x - ray crystallography . [SEP]
[CLS] the size values derived by es - dma can match the ones derived from electron B-technique microscopy I-technique and light scattering techniques . [SEP]
[CLS] a technique belonging to the latter type is elliptically polarized light scattering ( epls ) , which is accurate , fast , and non - intrusive and allows in situ function . [SEP]
[CLS] it can provide information on the size , size distribution , shape and structure of agglomerates . [SEP]
[CLS] moreover , the thermal lens spectrometry ( tls ) technique can be employed to measure the thermal diffusivity of np B-nanoparticle solutions , e . g . in the case of 15 nm au nps B-nanoparticle at different ph values at constant np B-nanoparticle size and concentration . [SEP]
[CLS] it provides a reliable alternative to evaluate , with high sensitivity , the thermal diffusivities of semitransparent materials as well as low thermal diffusivities . [SEP]
[CLS] quartz crystal microbalance ( qcm ) . [SEP]
[CLS] compared to icp and micro - computerized tomography B-technique , qcm can be used for the mass measurement of nps B-nanoparticle , and it offers the advantages of real - time monitoring , greater sensitivity and lower cost . [SEP]
[CLS] burg et al . described the use of suspended microchannel resonators as a means to weigh single nps B-nanoparticle , single bacterial cells B-material and sub - monolayers of adsorbed proteins B-material in water B-material with subfemtogram resolution ( 1 hz bandwidth ) . [SEP]
[CLS] in another work , link and co - workers have shown in a review article the utility of single particle spectroscopy B-technique for the characterization of plasmonic nps B-nanoparticle with arbitrary size and shape , especially when combined with correlated electron imaging and detailed electromagnetic calculations . [SEP]
[CLS] they present single nanoparticle B-nanoparticle spectroscopy B-technique performed with several scattering , absorption and extinction methods . [SEP]
[CLS] magnetic B-property nps B-nanoparticle find applications in a broad range of domains , such as magnetic B-property resonance contrast media and as therapeutic agents in cancer treatment . [SEP]
[CLS] akbarzadeh et al . have written a review paper on the preparation and physical properties of magnetic B-property nps B-nanoparticle as well as their applications , with emphasis on the biomedical ones . [SEP]
[CLS] in this section we focus on the characterization techniques that are employed to evaluate the magnetic B-property properties of such nps B-nanoparticle . [SEP]
[CLS] superconducting quantum interference device magnetometry ( squid ) is a tool for measuring the magnetic B-property properties of nanoscale materials . [SEP]
[CLS] nanomaterials B-material in particular exhibit different properties to those in the bulk state due to their small size and sensitivity to local conditions . [SEP]
[CLS] as a material B-material decreases in size , it progresses from multi - domain , to single domain and finally to superparamagnetic B-property status . [SEP]
[CLS] typical squid measurements yield properties such as the magnetization B-property saturation ( m s ) , magnetization B-property remanence ( m r ) and blocking temperature ( t b ) . [SEP]
[CLS] apart from nps B-nanoparticle , the magnetic B-property response of individual molecules can also be measured by squid . [SEP]
[CLS] in fact , a scanning magnetic B-property microscope including a nanosquid has also been developed recently , fabricated on the apex of a sharp quartz . [SEP]
[CLS] nanosquid is considered as a highly promising probe for nanoscale magnetic B-property imaging and spectroscopy B-technique . [SEP]
[CLS] a nanosquid sensor requires deep sub - micron josephson junctions , which are provided by two dayem nanobridges ( nano - constriction of a superconducting film ) , fabricated by electron beam lithography or focused ion B-material beam ( fib ) with a length and width comparable to the coherence length . [SEP]
[CLS] the main requirement for a squid designed for the detection of magnetic B-property nps B-nanoparticle is a very small squid area . [SEP]
[CLS] ideally , to gain the best coupling factor , the loop size should be comparable to those of the nps B-nanoparticle directly coupled to it . [SEP]
[CLS] regarding the magnetic B-property resonance force microscopy B-technique or magneto - optic spin detection , nanosquids offer the advantage of direct measurement of magnetization B-property changes in small spin systems . [SEP]
[CLS] the dayem nanobridges of a nanosquid , apart from their easy fabrication by a single nanopatterning step , are also resilient to the magnetic B-property field applied in the plane of the squid loop . [SEP]
[CLS] the experimental setup of a nanosquid is shown in fig . 6 . [SEP]
[CLS] fiorani and co - workers demonstrated that the latter type of squid device is a useful and reliable tool to investigate the magnetic B-property properties of iron B-material oxide I-material nps B-nanoparticle . [SEP]
[CLS] gamarra et al . used squid magnetometry B-property and ferromagnetic resonance ( fmr ) to carry out static and dynamic measurements of a biocompatible B-property ferrofluid based on fe 3 o 4 nps B-nanoparticle . [SEP]
[CLS] such measurements were performed as a function of field , temperature and driving fre - quency . [SEP]
[CLS] their magnetization B-property results as a function of the external field showed that for temperatures above t b the hysteresis cycle did not exhibit coercivity , indicating the superparamagnetic B-property behaviour of the material B-material . [SEP]
[CLS] in another report , nickel B-material ferrite nps B-nanoparticle synthesized by malik et al . were investigated by squid and mossbauer . [SEP]
[CLS] the effect of size on the coercivity and saturation magnetization B-property as derived from the hysteresis loop and the hyperfine parameters obtained from the mossbauer spectra were reported . [SEP]
[CLS] the bimodal size distribution was reflected only in the zero - field - cooled - field - cooled ( zfc - fc ) measurement done at a very low field , which is also borne out by numerical calculations . [SEP]
[CLS] vibrating sample magnetometry ( vsm ) is another method that can be used to record the m - h loops for magnetic B-property nanomaterials B-material and obtain parameters such as m s and m r . [SEP]
[CLS] the magnetic B-property properties of nps B-nanoparticle are studied as a function of magnetic B-property field , temperature and time . [SEP]
[CLS] the feco nps B-nanoparticle examined the magnetic B-property properties of superparamagnetic B-property feco @ sno 2 nps B-nanoparticle on graphene - polyaniline . [SEP]
[CLS] their enhanced electromagnetic wave absorption properties were investigated . [SEP]
[CLS] a series of feco , feco @ sno 2 and feco @ sno 2 @ graphene @ pani composites were characterized by vsm . [SEP]
[CLS] the feco nps B-nanoparticle display strong magnetic B-property dipolar interactions and if an external magnetic B-property field is applied , their magnetic B-property moments would be aligned in the same direction with the field . [SEP]
[CLS] fabris and colleagues prepared size - controlled magnetite nps B-nanoparticle through a direct reduction - precipitation method in the presence of tetramethylammonium hydroxide B-material . [SEP]
[CLS] all studied samples were found to be superparamagnetic B-property , as evidenced by both zero coercivity and zero remanence on the magnetization B-property loop . [SEP]
[CLS] the saturation magnetization B-property was a linear function of the np B-nanoparticle size . [SEP]
[CLS] kumari et al . described the concept of first order reversal curves , which is an assembly of partial hysteresis loops originating from the major loop . [SEP]
[CLS] first order reversal curves ( forc ) are efficient for the identification of domain size , composition and interaction in a magnetic B-property system . [SEP]
[CLS] it is a significant method to obtain a semi - quantitative measure of the effective magnetic B-property particle size . [SEP]
[CLS] under certain conditions , forc may facilitate the revealing of the presence of secondary / minor magnetic B-property contributions , thus helping in a more precise characterization of the magnetic B-property properties . [SEP]
[CLS] vsm magnetometry was used to obtain such forc measurements . [SEP]
[CLS] in another work , feco nps B-nanoparticle with anisotropic long chain structure were prepared by a sputter based gas - condensation method and their magnetic B-property properties were analysed by vsm . [SEP]
[CLS] a strong exchange coupling interaction between nps B-nanoparticle was evidenced in a chain - like sample , while well - dispersed samples showed a distinct magnetic B-property performance . [SEP]
[CLS] zfc / fc curves and time dependent remanent magnetic B-property moment measurements helped to gain information on the thermal stability of the studied nps B-nanoparticle . [SEP]
[CLS] apart from the full hysteresis loop properties of the magnetic B-property media , there has been increased interest in the measurement of remanence curves . [SEP]
[CLS] the measurement of remanence determines only the irreversible component of magnetization B-property and therefore enables the phenomena of switching to be deconvoluted from the hysteresis measurement , which in general includes a reversible component . [SEP]
[CLS] two main remanence curves exist : the isothermal remanence and the dc demagnetization curve . [SEP]
[CLS] the former is measured after the application and removal of a field with the sample initially demagnetized . [SEP]
[CLS] the dc demagnetization curve is measured after the saturated state by the application of increasing demagnetizing fields . [SEP]
[CLS] these remanence curves can be obtained by vsm measurements and they can provide the true switching field distribution of the materials . [SEP]
[CLS] mossbauer spectroscopy B-technique is a valuable analytical tool that is based on the recoil - free resonance fluorescence B-property of γ - photons in matter with mossbauer - active elements , such as fe . [SEP]
[CLS] mossbauer can be used to evaluate the oxidation state , the symmetry and spin state as well as the magnetic B-property ordering of the fe atoms B-material in a np B-nanoparticle sample and thus identify the magnetic B-property phases in a sample . [SEP]
[CLS] furthermore , for magnetically B-property ordered materials , mossbauer spectra recorded as a function of temperature can be used to estimate the magnetic B-property anisotropy energy and quantify the thermal unblocking ( superparamagnetism B-property ) . [SEP]
[CLS] the mossbauer spectroscopy B-technique isomer shift is an important parameter that arises from the nuclear - energy shift that is caused by the coulombic interaction between the nucleus and the electron density at the site of the nucleus . [SEP]
[CLS] the isomer shift values of fe 2 + and fe 3 + are significantly distinct from each other and mossbauer spectroscopy B-technique has been generally accepted as the method of choice to determine the oxidation number . [SEP]
[CLS] in the case of doped fe - zno nps B-nanoparticle , whilst the mossbauer isomer shift is related to the charge on the ion B-material in the structure , there is not necessarily a correlation with its oxidation state . [SEP]
[CLS] the mossbauer isomer shift cannot act as a necessary determinant of the oxidation number of dopant atoms B-material . [SEP]
[CLS] in fact , mossbauer spectroscopy B-technique directly probes the charge on the nucleus site . [SEP]
[CLS] evidently , this charge is sensitive to the local environment of the atom B-material , both structurally and chemically . [SEP]
[CLS] a schematic diagram of a transmission mossbauer spectrometer system is depicted in fig . 7 . [SEP]
[CLS] oh et al . investigated the magnetic B-property properties of feco nps B-nanoparticle synthesized by the chemical vapor condensation process and it was found by mossbauer that the nps B-nanoparticle had α - feooh , γ - feooh and fe 3 o 4 at their surface . [SEP]
[CLS] with the complete fit of mossbauer spectra , the fraction of each phase was quantitatively determined . [SEP]
[CLS] lange and co - workers mentioned that mossbauer spectroscopy B-technique utilizes hyperfine interactions between nuclei and their surrounding environment . [SEP]
[CLS] thus , this method is very sensitive to the surroundings of a given isotope B-material used as a probe . [SEP]
[CLS] the fe mossbauer effect can yield information concerning the local chemical and structural environments around the fe nuclei , permitting the determination of fe - containing phases as well as the quantitative analysis of their relative proportions . [SEP]
[CLS] fitting of the mossbauer spectra can help to identify parameters such as the magnetic B-property hyperfine field ( h hf ) and isomer shift for six - line spectral components , electric quadrupole splitting and isomer shift for quadrupole doublets , and isomer shift for single lines . [SEP]
[CLS] tiano et al . employed mossbauer spectroscopy B-technique to probe the nature of metal B-material cation I-material occupancies in mfe 2 o 4 systems ( m = mg , fe , co , ni , cu or zn ) . [SEP]
[CLS] squid and mossbauer measurements helped to systematically probe their ferrite nps B-nanoparticle in an attempt to correlate the magnetic B-property properties with the np B-nanoparticle size and composition . [SEP]
[CLS] superparamagnetism B-property was found in particles with sizes smaller than 4 nm , whereas the presence of spin canting , uncompensated surface spins and magnetic B-property anisotropy was observed for the majority of the samples . [SEP]
[CLS] mossbauer analysis supported the squid data , showing that the occupancies of the tetrahedral fe ( a ) and octahedral fe [ b ] sites were significantly modified , thereby emphasizing the importance of the synthetic method , size and chemical composition . [SEP]
[CLS] in another work , pankhurst and co - workers used fe mossbauer spectroscopy B-technique to find the composition of magnetite / maghemite mixtures and the stoichiometry of magnetite / maghemite solid solutions . [SEP]
[CLS] they presented the data on high - purity magnetite and maghemite powders and mixtures thereof , as well as the comparison literature data from nanoparticulate mixtures and solid solutions to demonstrate that there is a linear correlation between the ' centre of gravity ' parameter δ rt ( also known as area weighted mean isomer shift at room temperature ) and the numerical proportion of iron B-material atoms I-material in the magnetite environment . [SEP]
[CLS] it has to be noted that xrd cannot distinguish between fe 3 o 4 and γ - fe 2 o 3 , as their reflections coincide , rendering mossbauer a successful alternative in this case . [SEP]
[CLS] still , it has been reported that the mossbauer spectra of nps B-nanoparticle are much more complex compared to the ones of the bulk state . [SEP]
[CLS] sharma and colleagues synthesized iron B-material oxide I-material nps B-nanoparticle by the thermal decomposition of fe - precursors in ar and vacuum environments with controlled size distribution and phase composition . [SEP]
[CLS] detailed xrd , xanes and mossbauer experiments demonstrated that the prevailing chemical phase was γ - fe 2 o 3 in both environments . [SEP]
[CLS] fe mossbauer spectroscopy B-technique is a powerful tool to characterize iron B-material oxide I-material nps B-nanoparticle undergoing superparamagnetic B-property relaxation . [SEP]
[CLS] rumenapp et al . monitored the aging of magnetite nps B-nanoparticle using mossbauer spectroscopy B-technique . [SEP]
[CLS] the measurements were performed at 4 . 2 k in order to identify the oxidation state of the iron B-material in the core B-material of the nps B-nanoparticle . [SEP]
[CLS] in mossbauer spectra , fe ( ii ) and fe ( iii ) can be easily distinguished by their different isomer shifts . [SEP]
[CLS] the authors noticed that the magnetite content of naked magnetic B-property nps B-nanoparticle with sizes below about 10 nm decreased rather rapidly after synthesis and use of hydrous solutions or drying in air . [SEP]
[CLS] however , diethylene glycol provided a resistance to the oxidation of magnetite to maghemite . [SEP]
[CLS] sundar and co - workers investigated the local structure and magnetic B-property properties of cubic iron B-material oxide I-material nps B-nanoparticle formed in zeolite , with the use of mossbauer spectroscopy B-technique . [SEP]
[CLS] this method was employed to distinguish between the isolated superparamagnetic B-property nps B-nanoparticle of iron B-material oxides I-material . [SEP]
[CLS] the mossbauer study revealed a strong binding of fe 3 o 4 nps B-nanoparticle in zeolite . [SEP]
[CLS] in another report , mossbauer measurements helped to study the disordered surface spins in core / shell ferrite nps B-nanoparticle . [SEP]
[CLS] the nps B-nanoparticle tested had a nickel B-material ferrite core B-material and a maghemite shell B-material . [SEP]
[CLS] their experiments showed that the magnetization B-property temperature dependence of gas - like diluted dispersions of independent nps B-nanoparticle is well described by a monodomain ordered core B-material and a surface layer of disordered spins . [SEP]
[CLS] domracheva et al . performed magnetic B-property resonance and mossbauer studies on superparamagnetic B-property γ - fe 2 o 3 nps B-nanoparticle encapsulated into liquid - crystalline poly ( propylene imine B-material ) dendrimers B-nanoparticle . [SEP]
[CLS] mossbauer measurements showed that these nps B-nanoparticle were composed of an α - fe core B-material and a γ - fe 2 o 3 shell B-material . [SEP]
[CLS] in another report , siddique et al . investigated the particle size effect on mossbauer parameters in maghemite nps B-nanoparticle . [SEP]
[CLS] these particles were synthesized by a chemical co - precipitation approach . [SEP]
[CLS] the presence of a quadrupole doublet indicated the existence of single domain particles . [SEP]
[CLS] it was evidenced that the internal magnetic B-property field increased with the increase of np B-nanoparticle size and the superparamagnetic B-property component remained almost stable . [SEP]
[CLS] the authors noted that mossbauer spectroscopy B-technique is a very effective and sensitive method to identify the np B-nanoparticle size effect and the spin structure in order to analyse the supertransferred hyperfine interactions in nanostructured B-material materials I-material . [SEP]
[CLS] γ - fe 2 o 3 nps B-nanoparticle were also the topic of the study of the hyeon group and their co - workers : the 57 fe mossbauer spectra were recorded and a muon spin relaxation study of the magnetodynamics of these monodisperse oleic acid - capped nps B-nanoparticle was also carried out . [SEP]
[CLS] mossbauer and magnetic B-property susceptibility measurements helped to estimate the magnetic B-property anisotropy constant values . [SEP]
[CLS] in fact , the relaxation frequencies obtained by mossbauer spectroscopy B-technique and μ - spin relaxation were found to be different and not directly comparable . [SEP]
[CLS] mossbauer spectroscopy B-technique yielded larger relaxation frequencies than those measured by muonspin relaxation , a difference which is in agreement with the characteristic times of the two techniques . [SEP]
[CLS] the mossbauer spectra are not fully sensitive to the monodisperse nature of the nps B-nanoparticle due to substantial np B-nanoparticle interactions , which can appear despite the np B-nanoparticle coating B-material by oleic acid . [SEP]
[CLS] cofe 2 o 4 nps B-nanoparticle prepared by a hydrothermal B-technique method I-technique were also studied by mossbauer , so as to evaluate their magnetic B-property properties and the cation B-material distribution . [SEP]
[CLS] concerning ferrite nps B-nanoparticle , in addition to the cation B-material distribution , mossbauer can provide information on the magnetic B-property domain structure , spin polarization and s - electron density around the mossbauer probe nuclei . [SEP]
[CLS] the results of the mossbauer spectra indicated that these cobalt B-material ferrite nps B-nanoparticle had a complete magnetic B-property order . [SEP]
[CLS] complementary vsm measurements facilitated a better understanding of the magnetic B-property properties of these materials through the modification of the main magnetic B-property properties ( m s , h c ) with the reaction time and the np B-nanoparticle size . [SEP]
[CLS] fe mossbauer spectroscopy B-technique was used by tirado and colleagues to investigate iron B-material nps B-nanoparticle obtained in situ in conversion ferrite electrodes . [SEP]
[CLS] important information was derived concerning parameters such as oxidation state , local environment and magnetic B-property ordering of cofe 2 o 4 in electrodes cycled vs . lithium B-material . [SEP]
[CLS] moreover , the thermal reduction of hematite into magnetite was monitored using mossbauer spectroscopy B-technique by lyubutin and coworkers : the data from zfc - fc magnetization B-property curves were combined with those from mossbauer and it was found that the nps B-nanoparticle , prepared by the thermal treatment of α - fe 2 o 3 under inert conditions in octadecene solvent , were strongly coupled by magnetic B-property interactions up to 300 k . [SEP]
[CLS] mossbauer spectra illustrated that 95 % of the iron B-material was in the magnetite phase while the rest 5 % was still in the hematite one . [SEP]
[CLS] joos et al . studied by mossbauer iron B-material oxide I-material nps B-nanoparticle prepared in diethyleneglycol . [SEP]
[CLS] they described a protocol to distinguish between maghemite and magnetite using a magnetic B-property field of 0 . 7 t , at room temperature . [SEP]
[CLS] this was a remarkable achievement , considering that normally nps B-nanoparticle smaller than 15 nm are affected by superparamagnetic B-property relaxation , which hinders their characterization by mossbauer spectroscopy B-technique . [SEP]
[CLS] in another work , β - feooh nps B-nanoparticle were prepared in a microemulsion system with the use of a non - ionic surfactant B-property . [SEP]
[CLS] several characterization techniques were employed to study the properties of the product , and fe mossbauer spectra showed that the magnetic B-property structure transformed below 150 k , and two kinds of fe - o octahedra were present in the lattice of the modified β - feooh nps B-nanoparticle . [SEP]
[CLS] an approximate neel temperature ( t n ) in a range of 10 degreescan also be derived from the mossbauer measurements . [SEP]
[CLS] another significant feature of mossbauer spectroscopy B-technique is that it does not require the periodic lattice of a crystal , unlike xrd , also knowing that the mossbauer effect is limited to only a few elements in the periodic table . [SEP]
[CLS] mossbauer is powerful in selecting the resonant isotope B-material ( e . g . fe ) in the presence of other atoms B-material in a sample . [SEP]
[CLS] giersig and co - workers performed the mossbauer studies of core - shell nps B-nanoparticle , and they found out that the magnetic B-property splitting increased with the concentration of maghemite and decreased for magnetite . [SEP]
[CLS] the mossbauer spectra of pure γ - fe 2 o 3 , pure fe 3 o 4 and fe 3 o 4 @ γ - fe 2 o 3 as well as γ - fe 2 o 3 @ fe 3 o 4 core - shell nps B-nanoparticle were very different from each other . [SEP]
[CLS] the technique under discussion was also employed to study the biodegradation B-property of magnetic B-property nps B-nanoparticle in rat brain , three months after their injection . [SEP]
[CLS] the presence in the injected ferrofluid of both magnetite nps B-nanoparticle and an additional chemical compound containing ferric ion B-material in the high - spin state was evidenced . [SEP]
[CLS] mazeika et al . studied the effect of the interactions to the properties of ultrasmall cofe 2 o 4 nps B-nanoparticle using mossbauer spectroscopy B-technique : these nps B-nanoparticle were prepared by the co - precipitation method and their size was in the range of 1 - 3 nm . [SEP]
[CLS] the blocking temperature of the nps B-nanoparticle can be determined by several methods , including mossbauer . [SEP]
[CLS] in addition , the experimental evidence of the dependence of the curie temperature on the size of nps B-nanoparticle is another interesting task where the application of mossbauer spectroscopy B-technique is valuable . [SEP]
[CLS] moreover , gupta and colleagues employed mossbauer , raman and xrd to study superparamagnetic B-property [UNK] nm nife 2 o 4 nps B-nanoparticle prepared by a sol - gel auto - combustion method . [SEP]
[CLS] mossbauer measurements recorded at 5 k and under 5 t applied magnetic B-property field demonstrated a mixed spinel structure and canted spin order for the nps B-nanoparticle , while a collinear spin order with an inverse spinel structure was observed for larger particles . [SEP]
[CLS] a prominent central doublet was present at room temperature mossbauer spectra , showing the superparamagnetic B-property character of the sample at ambient temperature . [SEP]
[CLS] the measurements from the different techniques concluded that these nickel B-material ferrite nps B-nanoparticle consist of a single phase , which is not common with this method of preparation . [SEP]
[CLS] besides , ni - substituted mn 0 . 5 zn 0 . 5 fe 2 o 4 nps B-nanoparticle were prepared by thota et al . with a citrate method . [SEP]
[CLS] these researchers studied the cation B-material distribution of these nps B-nanoparticle and the techniques used were raman , mossbauer , xrd and electron spectroscopy B-technique . [SEP]
[CLS] mossbauer spectroscopy B-technique showed the trivalent iron B-material ion B-material distribution between tetrahedral and octahedral sites for all samples with nearly 70 - 75 % of fe 3 + ions B-material sitting on the octahedral sites . [SEP]
[CLS] nio nps B-nanoparticle were also studied with mossbauer spectroscopy B-technique by bahl and co - workers . [SEP]
[CLS] these particles were prepared by a combination of chemical precipitation and heating stages . [SEP]
[CLS] mossbauer measurements indicated that the nano - material B-material was composed of a mixture of ferromagnetic and antiferromagnetic phases . [SEP]
[CLS] magnetization B-property measurements yielded larger magnetic B-property moments in comparison with those obtained from the mossbauer data . [SEP]
[CLS] this can be explained by interparticle interactions in the samples as well as a difference in the sensitivity of magnetization B-property measurements and mossbauer spectroscopy B-technique to a particle size distribution . [SEP]
[CLS] in addition , fe 3 + - doped ceo 2 nps B-nanoparticle were analysed by xrd , hrtem and mossbauer . [SEP]
[CLS] these particles were prepared by a sol - gel method using ferric nitrate and cerium B-material nitrate as precursors in an alcohol B-material solution . [SEP]
[CLS] mossbauer measurements implied the existence of exchange interaction and a sextet pattern observed was assigned to hematite . [SEP]
[CLS] magnetic B-property susceptibility also showed the presence of α - fe 2 o 3 , and this was an interesting finding considering that xrd could not confirm the presence of hematite , unlike both of the aforementioned techniques . [SEP]
[CLS] iron - doped sno 2 nps B-nanoparticle were prepared with a hydrothermal route I-technique by diamandescu and colleagues . [SEP]
[CLS] these particles were characterized by electron magnetic B-property resonance ( emr ) and mossbauer spectroscopies B-technique . [SEP]
[CLS] the emr data had features attributed to fe ions B-material in low symmetry crystalline fields and could be related to paramagnetic B-property ions B-material in distorted crystalline positions . [SEP]
[CLS] both emr and mossbauer studies demonstrated the disordered distribution of iron B-material ions B-material in the bulk and on the surface of sno 2 nps B-nanoparticle . [SEP]
[CLS] in another work , kovalenko and coworkers unraveled the core - shell structure of ligand - capped sn / sno x nps B-nanoparticle by surface - enhanced ss nmr , mossbauer and xas . [SEP]
[CLS] oleate or inorganic ligands were employed for the coating B-material of the nps B-nanoparticle . [SEP]
[CLS] xas and sn mossbauer spectroscopies B-technique were able to identify and quantify amorphous sno and sno 2 nps B-nanoparticle but could not provide insight into the arrangement of these phases within the surface oxide B-material shell B-material . [SEP]
[CLS] surface - enhanced ss nmr demonstrated that the outer shell B-material of the nps B-nanoparticle was composed exclusively of amorphous sno 2 . [SEP]
[CLS] xrd and tem showed a crystalline β - sn core B-material , whereas xas and mossbauer measurements detected an interlayer of amorphous sno and the atomic fraction of each of the three phases . [SEP]
[CLS] sn nmr signals were not observed due to the low sensitivity of nmr spectroscopy B-technique . the combined use of all these techniques resulted in a core / shell 1 / shell 2 model of sn / sno / sno 2 nps B-nanoparticle coated with organic and inorganic ligands , where the only crystalline component was a metallic β - sn core I-material . [SEP]
[CLS] in particular , the sn mossbauer spectroscopy B-technique was considered as a highly sensitive tool to determine the oxidation state and chemical environment of tin B-material atoms B-material for several materials , including nps B-nanoparticle . [SEP]
[CLS] fesb 2 nps B-nanoparticle were prepared by tremel and co - workers with a wet - chemical approach , and they were analysed by mossbauer spectroscopy B-technique . [SEP]
[CLS] fe mossbauer measurements elucidated the remaining iron B-material - containing species during the formation process and determined the purity of the final fesb 2 nps B-nanoparticle . [SEP]
[CLS] any discrepancies between the xrd and fe mossbauer data are not surprising due to the fact that these two methods are sensitive to different characteristics , for example , the xrd cannot detect amorphous phases . [SEP]
[CLS] mossbauer measurements not only contributed to the comprehension of the formation of the fesb 2 nps B-nanoparticle but also pro - vided further proof of the quality of the prepared nanomaterials B-material . [SEP]
[CLS] the surface oxidation of co nps B-nanoparticle prepared by linderoth and colleagues was analysed by mossbauer spectroscopy B-technique . [SEP]
[CLS] the structure of coo formed onto the surface of cobalt B-material particles was considerably well ordered in comparison with the surface oxide B-material formed on iron B-material particles . [SEP]
[CLS] concas et al . synthesized a cobalt B-material - iron B-material alloy with varying iron B-material content by a sol - gel method followed by thermal treatment under a hydrogen B-material atmosphere . [SEP]
[CLS] these nps B-nanoparticle were embedded in a silica matrix . [SEP]
[CLS] mossbauer spectra showed the formation of an ordered component with isomer shift and hyperfine fields characteristic of a fe - co alloy only when fe - and co - acetate salts B-material were used as precursors , unlike the case of nitrate salts B-material . [SEP]
[CLS] the atomic arrangement in magnetic B-property fept nps B-nanoparticle was analysed by sakuma and coworkers . [SEP]
[CLS] xrd and mossbauer techniques were employed for the analysis of these particles . [SEP]
[CLS] the order parameter q was introduced and discussed by the authors , and its value was deduced from mossbauer measurements . [SEP]
[CLS] q denotes the probability of the appearance of the l1 0 - type atomic arrangement . [SEP]
[CLS] q is a short - range order parameter , while another parameter named ' s ' is a long - range order one . [SEP]
[CLS] the coercivity of the fept nps B-nanoparticle was found to be more dependent on q than s . [SEP]
[CLS] another bimetallic np B-nanoparticle system is the fe / au , and such nanomaterials B-material were studied by xrd , magnetic B-property and mossbauer experiments by kauzlarich and colleagues . [SEP]
[CLS] the authors noted that the xrd pattern that they obtained had a notable simplicity , which was strikingly different from the complexity of the mossbauer spectra . [SEP]
[CLS] the latter technique indicated that both uncoated and au - coated fe nps B-nanoparticle prepared by reduction had three major ironcontaining components in their composition . [SEP]
[CLS] these components were α - fe , fe 1−x b x alloy and several poorly crystallized iron B-material oxides I-material species . [SEP]
[CLS] fecu nps B-nanoparticle were prepared using an aerosol process by molins and co - workers , and they were analysed by xrd and mossbauer . the latter technique played a great role in understanding the processes of formation and decomposition of metastable fecu alloys . [SEP]
[CLS] in another work , europium B-material sulfide nps B-nanoparticle were synthesized with a colloidal approach and they were characterized by mossbauer spectroscopy B-technique . [SEP]
[CLS] this technique allows a close monitoring of the oxidation state of eu . [SEP]
[CLS] the blocking temperature of 20 nm nps B-nanoparticle , derived from magnetic B-property measurements , was above 15 k , which is close to the value deduced from the mossbauer experiments . [SEP]
[CLS] ferromagnetic resonance ( fmr ) is a spectroscopic technique that probes the magnetization B-property of ferromagnetic materials , including nanoscale ones . [SEP]
[CLS] it has similarities with epr and nmr : for instance fmr probes the sample magnetization B-property that results from the magnetic B-property moments of dipolar - coupled but unpaired electrons , whereas nmr probes the magnetic B-property moments of atomic nuclei that are screened by the atomic or molecular orbitals surrounding such nuclei of non - zero nuclear spin . [SEP]
[CLS] fmr spectra can provide important information on the average shape and size of catalyst B-property particles , which are composed of ferromagnetic elements ( fe , ni , co ) , and are used for the production of carbon B-nanoparticle nanotubes I-nanoparticle . [SEP]
[CLS] the fmr line width of metal B-material magnetic B-property films is related to the film thickness and depends on the surface anisotropy , defect density and other reasons . [SEP]
[CLS] the treatment of si / sio 2 / co substrates in h 2 plasma at 350 - 400 °c resulted in an isotropic fmr spectrum that suggested either the disordered arrangement of catalyst B-property particles or their spherical form on the average . [SEP]
[CLS] increasing temperature induced the strong angular dependence of the resonant magnetic B-property field of fmr due to the flattening of the non - spherical and ordered catalyst B-property nps B-nanoparticle . [SEP]
[CLS] in fact , the fmr of magnetic B-property nps B-nanoparticle differs from the resonance behaviour in bulk materials since the skin depth generally exceeds the particle size , and the multi - magnetic B-property domain structure is excluded from line shape . [SEP]
[CLS] increasing the np B-nanoparticle size or decreasing the temperature is followed by a shift in the resonance field , an increasingly asymmetric line shape , and an enhanced broadening of the fmr . [SEP]
[CLS] surface effects in nmr were revealed at lower temperatures by murray and co - workers when they studied superparamagnetic B-property cobalt B-material nps B-nanoparticle with different crystalline structures and sizes in the range of 4 - 9 nm by fmr . [SEP]
[CLS] the comparison of fmr from crystalline magnetic B-property nps B-nanoparticle to magnetic B-property nps B-nanoparticle with an imperfect structure made it clear that the coherence of the lattice is equally important in describing the anisotropy and hence inhomogeneity of the magnetic B-property properties of the nps B-nanoparticle . [SEP]
[CLS] in total , these authors consider fmr as a sensitive probe of crystallographic imperfection , particle shape and surface composition . [SEP]
[CLS] morgunov et al . employed fmr spectroscopy B-technique to study the magnetic B-property properties of spherical ( 5 - 9 nm ) co nps B-nanoparticle in a polymer B-material shell B-material . [SEP]
[CLS] the fmr spectra recorded for cobalt B-material particles did not show any hysteresis , suggesting the existence of the internal field and the presence of remanent magnetization B-property in the nps B-nanoparticle . [SEP]
[CLS] it was found that the saturation magnetization B-property of these nps B-nanoparticle was higher than that of the bulk state . [SEP]
[CLS] in addition , the blocking temperature of the particles was much larger than ambient temperature . [SEP]
[CLS] the high blocking temperature indicated strong anisotropy , which can be associated with the surface effects in the nps B-nanoparticle . [SEP]
[CLS] complementary characterization with epr spectroscopy B-technique suggested that the polymer B-material shell B-material interacts with the embedded nps B-nanoparticle . [SEP]
[CLS] stepanov and colleagues investigated co and ni nps B-nanoparticle implanted in the sio 2 matrix by fmr and tem methods . [SEP]
[CLS] fmr signals acquired at room temperature from ensembles of co and ni nps B-nanoparticle implanted in sio 2 exhibited an out - of - plane uniaxial magnetic B-property anisotropy , typical for thin magnetic B-property films . [SEP]
[CLS] fmr is in general a suitable method for the evaluation of the magnetic B-property properties of nanogranular media and thin - film systems as it allows the identification of the magnetization B-property value , magnetic B-property anisotropy constants and demagnetization field of a given sample . [SEP]
[CLS] hochepied and pileni published a study on the study of the fmr behaviour of nonstoichiometric zinc B-material ferrite nps B-nanoparticle doped with co 2 + ions B-material or undoped . [SEP]
[CLS] fmr measurements on texturated samples ( particles subjected to a magnetic B-property field during sample preparation ) provided reliable information on the relative thermal variation of the anisotropy constant , and therefore the latter parameter could be evaluated approximately for 3 . 7 nm zinc B-material ferrite nps B-nanoparticle . [SEP]
[CLS] the fmr spectra of these materials were characterized by an invariant point at a given field , h 0 . [SEP]
[CLS] the anisotropy constant varied linearly with temperature and vanished at about ambient temperature . [SEP]
[CLS] the role of dipolar interactions in magnetic B-property nps B-nanoparticle was studied by lezama and co - workers : the fmr measurements of discontinuous multilayers composed of co 80 fe 20 / al 2 o 3 were recorded as a function of the angle of the applied magnetic B-property field with respect to the sample at ambient temperature . [SEP]
[CLS] angular dependent measurements demonstrate how fmr can be employed to assess interparticle interactions . [SEP]
[CLS] overall , fmr can provide significant information not only on ' bulk ' magnetic B-property properties , but is also useful in evaluating surface magnetic B-property properties and interactions . [SEP]
[CLS] the g - factor of nps B-nanoparticle is one of the parameters that frequency - dependent fmr is able to evaluate . [SEP]
[CLS] many of the previously reported fmr studies of nps B-nanoparticle had focused on the temperature dependence of the resonance field . [SEP]
[CLS] dunlop and co - workers have published a study to discuss the 2nd order fmr in nps B-nanoparticle ; two principal processes in fmr are : the first order absorption of a photon and the creation of a single magnon , which means that the magnon wave - vector should have zero value . [SEP]
[CLS] consequently , only the uniform precession magnon ( or magnetostatic modes ) at the center of the zone can be excited . [SEP]
[CLS] the second order involves the absorption of a photon , which causes the creation of two magnons of equal and opposite wave - vector . [SEP]
[CLS] the applications of the 2nd order photon decay of the magnons in fmr include the remagnetization of dilute assemblies of magnetic B-property nps B-nanoparticle with high power microwave fields , and the isolation and measurement of magnetic B-property overprints . [SEP]
[CLS] in another report , the magnetic B-property states and fmr in geometrically frustrated arrays of multilayer ferromagnetic nps B-nanoparticle ordered on triangular lattices were presented . [SEP]
[CLS] it was shown that the interlayer coupling resulted in the remarkable splitting of the fmr spectrum . [SEP]
[CLS] in addition , any magnetizing B-property and remagnetizing of the multilayer systems caused transitions between different ferro - , antiferro - or mixed f / af interlayer ordering , which were accompanied by dramatic changes in the fmr spectra . [SEP]
[CLS] lue and colleagues investigated the change from paramagnetic B-property to ferromagnetic resonance for iron B-material nps B-nanoparticle produced by the sol - gel method . [SEP]
[CLS] in esr ( electron spin resonance ) the ions B-material are diluted and non - interacting , whereas in fmr the ferromagnetic ions B-material are clustered and interact with each other by the exchange force . [SEP]
[CLS] actually both fmr and broadened spr are relevant to the long - range exchange interaction within the nps B-nanoparticle . [SEP]
[CLS] gamarra et al . used fmr to quantify the amount of superparamagnetic B-property iron B-material oxide I-material nps B-nanoparticle in biological materials under both in vitro and in vivo conditions . [SEP]
[CLS] moreover , fmr was employed to study a phase transition in magnetic B-property field - aligned hematite nps B-nanoparticle . [SEP]
[CLS] measurements of the temperature dependence of the fmr signal in oriented 9 nm α - fe 2 o 3 showed anomalies in the intensity , line width and field position in the vicinity of 200 k , implying the occurrence of a phase transition . [SEP]
[CLS] this transition corresponds to a previously observed morin transition but having a lower transition temperature than the bulk material B-material . [SEP]
[CLS] the experiments indicate a transition from a weak ferromagnet to a stronger one at high temperature , whereas in bulk state such transition is from an af form to a ferromagnetic one . [SEP]
[CLS] another interesting use of fmr is the determination of the size distribution of nps B-nanoparticle . [SEP]
[CLS] such possibility was demonstrated by de biasi and gondim for the case of γ - fe 2 o 3 nps B-nanoparticle produced by a sol - gel method . [SEP]
[CLS] by measuring at the temperature range of 10 - 300 k the relative intensity of the spectrum due to superparamagnetic B-property particles , and the anisotropy field of the spectrum due to ferromagnetic nps B-nanoparticle , the size distribution of the particles was obtained . [SEP]
[CLS] the overall shape of the fmr spectrum of randomly oriented nps B-nanoparticle reflected the magnetic B-property anisotropy of the particles . [SEP]
[CLS] their work showed that fmr can be used to acquire the size distribution not only in ferrimagnetic precipitates , but also for randomly oriented particles , since the standard deviation of the particle size distribution is nearly the same as the one derived by tem . [SEP]
[CLS] the size value measured by tem is around 40 % larger than the corresponding value measured by fmr , because of the presence of a disordered layer in the surface of the particles that makes the ' magnetic B-property size ' of the nps B-nanoparticle to be smaller than the physical size . [SEP]
[CLS] depending on the application , the ' magnetic B-property size ' of the nps B-nanoparticle may be more important than the physical size . [SEP]
[CLS] there are other ways to obtain the size distribution of small magnetic B-property nps B-nanoparticle from magnetic B-property measurements , such as from hysteresis loops and from zfc / fc curves . [SEP]
[CLS] the fmr method is most suitable when the magnetocrystalline anisotropy is relatively small and the particles are approximately spherical . [SEP]
[CLS] in that case , a quick and quite precise estimation of the size distribution of the magnetic B-property nps B-nanoparticle can be achieved . [SEP]
[CLS] in another work , [UNK] nm maghemite nps B-nanoparticle in a pmma polymer B-material matrix were studied by fmr and dsc . [SEP]
[CLS] iron B-material hydroxide B-material gel was used as a precursor for the np B-nanoparticle synthesis , and the fmr experiments exposed the temperature range of a superparamagnetic B-property regime ( 60 - 290 k ) and the blocking temperature , t b [UNK] 60 k . [SEP]
[CLS] the significance of the dipole B-property - I-property dipole I-property interaction I-property for a high concentration of maghemite and temperatures above 220 k was demonstrated . [SEP]
[CLS] owens studied the ferromagnetic resonance of magnetic field oriented magnetite nps B-nanoparticle in frozen ferrofluids : it was shown that by freezing magnetic B-property nps B-nanoparticle suspended in a fluid in a magnetic B-property field it is possible to determine the orientational dependence of the fmr spectrum and abstract parameters such as the g value and magnetic B-property anisotropy constant , k . [SEP]
[CLS] comparing the data with the fmr measurements in the bulk material B-material indicated that the magnetic B-property phase transition at 136 k does not happen in the nps B-nanoparticle until a lower temperature in the range of 25 k . [SEP]
[CLS] in another report , the ferromagnetic resonance of magnetostatically coupled shifted chains of nps B-nanoparticle in an oblique magnetic B-property field was published . [SEP]
[CLS] the resonance field is what routinely measured in fmr measurements with a rotating applied magnetic B-property field , and it permits the characterization of the system with regard to its physical parameters . [SEP]
[CLS] in that study , this could be useful to characterize , inter alia , the magnetostatic interaction between the chains and to investigate the critical shift as a function of the applied field ( restricted to a dimer ) . [SEP]
[CLS] fept - au nps B-nanoparticle were also the subject of a study with fmr . [SEP]
[CLS] the author of that study notes that a relatively small amount of material B-material is capable of providing a good signal to noise ratio . [SEP]
[CLS] it was shown that the experimental spectra noticed in partially ordered fept - au films arise mainly in the low anisotropy disordered phase . [SEP]
[CLS] vargas et al . characterized by fmr the order - disorder transformation in fept nps B-nanoparticle . [SEP]
[CLS] these particles were studied in both as - made and annealed forms . [SEP]
[CLS] the as - prepared particles were synthesized in phenyl ether B-material , they crystallized in the low magnetic B-property anisotropy fcc phase and their diameter was in the range of 2 - 4 nm . [SEP]
[CLS] the aim of that study was to evaluate , by means of fmr , the dynamical response of as - made and thermally treated fept nps B-nanoparticle . [SEP]
[CLS] fmr helped to estimate the magnetic B-property anisotropy in a collection of fept nps B-nanoparticle annealed at various temperatures . [SEP]
[CLS] fmr spectroscopy B-technique was also employed to investigate magnetic B-property nickel B-material nps B-nanoparticle that are generated through the thermal decomposition of the layered lithium - aluminum double hydroxide B-material with intercalated nickel - edta complexes . [SEP]
[CLS] the curie temperature ( t c ) of the resulting nps B-nanoparticle measured using fmr spectroscopy B-technique was close to the corresponding one for bulk nickel B-material . [SEP]
[CLS] a numerical simulation of the fmr spectra of these systems was carried out , and the information on the size and shape of ni nps B-nanoparticle was acquired , being consistent with the data obtained through other methods . [SEP]
[CLS] in addition , insights into the early generation stages of a ferromagnetic phase were gained . [SEP]
[CLS] in another work , romero and colleagues analysed the surface and frustration evidence in co - ni - b nps B-nanoparticle through fmr experiments . [SEP]
[CLS] these particles were amorphous and the measurements were performed as a function of temperature . [SEP]
[CLS] the fmr measurements provided microscopic information on the internal magnetic B-property order of the particles , which may be hidden by interparticle interactions in magnetization B-property measurements . [SEP]
[CLS] furthermore , the ferromagnetic resonance in ni - zn ferrite nps B-nanoparticle in different aggregation states was studied by ammar and co - workers . [SEP]
[CLS] these particles were prepared through force hydrolysis in polyol using acetate salts B-material of the corresponding metals B-material as precursors . [SEP]
[CLS] the products ranged from isolated particles with a size around 5 nm to 20 nm clusters . [SEP]
[CLS] in fmr experiments , where the absorption is measured by the microwave field , the time window is smaller than in squid experiments and thus it shows an ordered magnetic B-property structure for considerably higher temperatures . [SEP]
[CLS] any inconsistency in the results derived by the aforementioned techniques is attributed to their different timescales . [SEP]
[CLS] for instance , at certain temperatures a given sample can appear to be ferromagnetic with one technique , whereas the other technique could characterize it as superparamagnetic B-property . [SEP]
[CLS] x - ray magnetic B-property circular dichroism ( xmcd ) is a technique which is utilized as a local probe for the study of the site symmetry and the magnetic B-property moments of transition metal B-material ions B-material in ferro - and ferrimagnetic materials . [SEP]
[CLS] xmcd uses the differential absorption of left and right circularly polarized light in a magnetic B-property field . [SEP]
[CLS] the external magnetic B-property field is applied along the x - ray propagation vector and the measurement is recorded at the l 2 , 3 edges of the transition elements . [SEP]
[CLS] for example , xmcd and xas were employed to study the effects of the size of γ - fe 2 o 3 nps B-nanoparticle on their chemical and magnetic B-property structures . [SEP]
[CLS] xmcd allows the separation and quantification of the magnetic B-property contributions of fe a 3 + and fe b 3 + ions B-material to the magnetization B-property . [SEP]
[CLS] in the case of a phosphate - modified surface ( for particles coated with phosphoric acid ) , xmcd experiment results implied that the surface disordered spins could be mainly fe b 3 + spins . [SEP]
[CLS] xmcd signals recorded for 2 . 7 and 8 nm particles and acquired by decreasing values of the external applied field helped to detect a greater disorder of fe b 3 + spins with respect to the field direction than for fe a 3 + spins . [SEP]
[CLS] the existence of a preferential spin canting of fe b 3 + spins at the surface was evidenced , and overall these results were in agreement with a core - shell model of the magnetic B-property structure previously proposed for the particles . [SEP]
[CLS] the same group published another work on maghemite nps B-nanoparticle measured by xmcd : the experiments were carried out at the l 2 , 3 edges of iron B-material to analyse the site - specific magnetic B-property contribution of ions B-material in the nps B-nanoparticle of γ - fe 2 o 3 . [SEP]
[CLS] the site - specificity of xmcd renders it a robust tool to analyse the magnetic B-property contributions of the different atoms B-material in the nps B-nanoparticle of spinel oxides B-material . [SEP]
[CLS] in that study , xmcd experiments helped to investigate the magnetic B-property order on tetrahedral and octahedral sites in those nps B-nanoparticle at liquid he temperature as a function of the external magnetic B-property field . [SEP]
[CLS] from such measurements on a single size of particles , it was not possible to conclude whether this magnetic B-property disorder was a surface effect or a core B-material effect . [SEP]
[CLS] cai et al . analysed the orbital and spin moments of fe 3 o 4 nps B-nanoparticle with size in the range of 5 to 11 nm using the xmcd method . [SEP]
[CLS] unlike magnetometry , xmcd is element - specific . [SEP]
[CLS] their results implied that while the magnetic B-property moment in the larger nps B-nanoparticle appears somewhat to the corresponding one in fe 3 o 4 single crystal , it may be reduced by a number of factors associated with the nanostructuration : preparation method , particle ligand environment , and np B-nanoparticle shape and size . [SEP]
[CLS] manna and co - workers published a study on the structural and magnetic B-property deconvolution of fept / feo x - nps B-nanoparticle using xmcd . [SEP]
[CLS] xas fit parameters represent the ' real ' chemical material B-material contribution , whereas the xmcd fit parameters represent the magnetic B-property contribution of each component of the material B-material . [SEP]
[CLS] the potential of xas / xmcd techniques for an accurate structural and magnetic B-property characterization with a high spatial resolution at the nanoscale was shown in this work . [SEP]
[CLS] a core - shell - like structure seemed to be a suitable term to describe this type of structureand not a ' dimer - like ' one . [SEP]
[CLS] takahashi et al . employed xmcd to study the orbital magnetic B-property moment and coercivity of sio 2 - coated fept nps B-nanoparticle . [SEP]
[CLS] in xmcd , one can eliminate the extrinsic magnetic B-property signals , such as those from oxidized fe and those from the diamagnetic B-property sio 2 coating B-material . [SEP]
[CLS] ferh nps B-nanoparticle were also studied with xmcd , by chaudret et al . [SEP]
[CLS] xmcd constitutes a valuable tool to unravel the role of each element in the overall magnetic B-property behaviour of bimetallic nps B-nanoparticle . [SEP]
[CLS] powerful sum rules permit the direct identification from the experimental spectra not only the value of spin and orbital contributions to the total magnetic B-property moment , but also its orientation . [SEP]
[CLS] it was observed that the spin and orbital moments induced on rh could be strongly influenced by the chemical composition of nps B-nanoparticle and by their synthesis process . [SEP]
[CLS] moreover , xmcd spectra were recorded at the l 2 , 3 edges of co , cu , ag and au and at the k edges of co and cu for a series of multilayer systems of partially selfassembled co nps B-nanoparticle , both coated with al 2 o 3 and with different metals B-material ( cu , ag and au ) . [SEP]
[CLS] because of its element selectivity and high sensitivity , xmcd proved useful to provide information regarding the orbital and spin moment components of the co and the capping metals B-material independently . [SEP]
[CLS] direct evidence of the hybridization of the interatomic 3d - nd and the co intraatomic 3d - 4p bands was acquired through the xmcd measurements . [SEP]
[CLS] these experiments resulted in the acquisition of the values for the spin and orbital moments averaged over the core B-material and surface of the particle , and the number of holes n h in the empty exchange split nd subbands . [SEP]
[CLS] in another work , prado et al . probed using xmcd the magnetic B-property anisotropy of cyanide - bridged core B-material and core - shell coordination nps B-nanoparticle . [SEP]
[CLS] xmcd allows the determination of the relative orientation of the magnetic B-property moments throughout the coreshell nps B-nanoparticle . [SEP]
[CLS] this method is particularly useful for core - shell nanostructures , in which three different magnetic B-property ions B-material are present . [SEP]
[CLS] in comparison with squid measurements for cocontaining nps B-nanoparticle , the xmcd magnetization B-property curve reaches the maximum magnetization B-property more gradually . [SEP]
[CLS] this difference might be due to the sensitivity of the xmcd technique to the surface . [SEP]
[CLS] hochepied et al . used xas and xmcd to measure at the fe and co l 2 , 3 edges of mixed cobalt B-material - zinc B-material ferrite nps B-nanoparticle . [SEP]
[CLS] such measurements allowed the identification of their magnetic B-property structure and cationic B-material distribution . [SEP]
[CLS] the advantage of xmcd compared to neutron diffraction is that the former method can also be used for particles that are not well crystallized , and for particles with relatively small size . [SEP]
[CLS] elements can be easily separated by the values of their l 2 , 3 edges . [SEP]
[CLS] furthermore , xmcd is sensitive to the site symmetry of the absorbing ions B-material , and to the orientation and amplitude of the local magnetic B-property moments . [SEP]
[CLS] isotropic spectra are sensitive to the ratio between octahedral and tetrahedral site occupancy , whereas xmcd signals are sensitive to the ratio of magnetic B-property moments of the two sites . [SEP]
[CLS] the complementarity of the information extracted from isotropic and from xmcd spectra was confirmed . [SEP]
[CLS] any discrepancies between the xmcd results and magnetization B-property curves could be assigned to the sample preparation , since for xmcd measurements a powder of particles is inserted into layers , resulting in strong interactions between the nps B-nanoparticle and radical shape effects , while in squid experiments , the particles were dispersed in a polymer B-material matrix . [SEP]
[CLS] in another study , pd nps B-nanoparticle prepared under a high purity atmosphere showed ferromagnetic properties and were characterized by xmcd . [SEP]
[CLS] this technique proved useful for the evaluation of the electronic and magnetic B-property states of pd nps B-nanoparticle . [SEP]
[CLS] the researchers who authored that study claim that this was the first observation of the inherent ferromagnetic moment in pd nps B-nanoparticle achieved by performing xmcd measurements . [SEP]
[CLS] besides , yamamoto et al . published an xmcd study of polymer - protected au nps B-nanoparticle . [SEP]
[CLS] this was considered as a direct observation of the spontaneous spin polarization of au nps B-nanoparticle using the technique under discussion . [SEP]
[CLS] the magnetization B-property assessed by xmcd was in accordance with the values derived by dc magnetization B-property . [SEP]
[CLS] the origin of spin polarization observed was assigned to an interaction between the protecting polymer B-material and the nps B-nanoparticle , although this assumption was not that clear . [SEP]
[CLS] xmcd has also provided evidence of the zno np B-nanoparticle ferromagnetic behaviour . [SEP]
[CLS] the size of the particles was 20 nm , and three different surfactants B-property were used : trioctylphosphine , dodecylamine and dodecanethiol . [SEP]
[CLS] the occurrence of ferromagnetic - like ( fml ) property up to room temperature was shown . [SEP]
[CLS] the zn k - edge xmcd measurements revealed the coexistence of two distinct magnetic B-property contributions : a paramagnetic B-property response from the core B-material of the np B-nanoparticle , and a ferromagnetic - like contribution stemming from the interface formed between the zno core B-material of the np B-nanoparticle and the organic molecule . [SEP]
[CLS] in another work , kataoka et al . used xmcd to investigate the origin of room temperature ferromagnetism in fe - doped zno nps B-nanoparticle . [SEP]
[CLS] these particles were prepared by a chemical pyrophoric reaction approach . [SEP]
[CLS] the xmcd spectral line shape of the zn 0 . 9 fe 0 . 1 o nps B-nanoparticle was different from that of fe metal B-material , implying that the magnetism B-property in this sample was not due to metallic fe clusters , but assigned to the ionic fe atoms B-material with localized 3d electrons . [SEP]
[CLS] the xas results indicated that iron B-material ions B-material were mainly in the trivalent state , together with a small amount of fe 2 + . [SEP]
[CLS] room - temperature ferromagnetism for these nps B-nanoparticle was primarily attributed to the antiferromagnetic coupling between unequal amounts of fe 3 + ions B-material occupying two sets of non - equivalent positions in the region of the xmcd probing depth of 2 - 3 nm . [SEP]
[CLS] magnetic B-property susceptibility . [SEP]
[CLS] measuring the magnetic B-property susceptibility of a nanomaterial B-material is another way to measure its magnetic B-property properties . [SEP]
[CLS] the susceptibility indicates whether a material B-material is attracted into or repelled out of a magnetic B-property field , which has implications for practical applications . [SEP]
[CLS] it is expressed as the ratio of the magnetization B-property to the applied magnetizing B-property field intensity . [SEP]
[CLS] herrera et al . reported that poly ( n - isopropylacrylamide ) [ pnipam ] - coated magnetic B-property nps B-nanoparticle showed aggregation through ac susceptibility measurements , which was not evident from dls experiments . [SEP]
[CLS] sans measurements supported the above information derived from ac susceptibility . [SEP]
[CLS] usually , magnetic B-property susceptibility measurements are performed over a range of temperatures , rather than frequencies , because of the limited available frequencies of most susceptometers . [SEP]
[CLS] broadband alternating current magnetic B-property susceptibility measurements were employed to characterize magnetic B-property nps B-nanoparticle in natural materials . [SEP]
[CLS] rinaldi and co - workers carried out ac susceptibility measurements of cobalt B-material ferrite nps B-nanoparticle to determine the viscosity B-property of mineral oil . [SEP]
[CLS] oleic acid was used a capping ligand for the suspended nps B-nanoparticle in the oil and an excellent agreement was found between the nanoscale and macroscale viscosities B-property . [SEP]
[CLS] lima and colleagues were able to evaluate the size and size dispersity of magnetic B-property nps B-nanoparticle in polymeric templates through susceptibility measurements . [SEP]
[CLS] they also managed to evaluate the magnetocrystalline anisotropy values of magnetite nps B-nanoparticle considering the field dependence of the susceptibility peak . [SEP]
[CLS] np B-nanoparticle size parameters acquired from the analysis by the dynamic susceptibility data were in accordance with the values obtained from the fitting of the tem data . [SEP]
[CLS] in another work , enpuku and co - workers used ac susceptibility measurements to detect magnetic B-property fe 3 o 4 nps B-nanoparticle with a weight down to 7 ng . [SEP]
[CLS] to achieve this , an excitation field was applied to the particles , and the resulting signal field from the particles was detected with a pickup coil . [SEP]
[CLS] an advantage of the susceptibility measurement is that the magnetic B-property signal is determined only by the total weight of the particles and is nearly independent of the size of each particle . [SEP]
[CLS] a disadvantage of the susceptibility measurement is that the magnetic B-property signal must be measured in the presence of an excitation field , while the signal can be measured in the absence of the excitation field , in the case of relaxation , and remanence measurements for the detection of nps B-nanoparticle . [SEP]
[CLS] magnetic B-property susceptibility measurements were also employed to quantify pva - coated fe 3 o 4 nps B-nanoparticle in granular sludge . [SEP]
[CLS] the authors of that study mentioned that compared to other analytical methods , magnetic B-property susceptibility did not require any sample preparation and enabled the straightforward quantification of engineered magnetic B-property nps B-nanoparticle in both water B-material phase and granular sludge . [SEP]
[CLS] their approach allowed the development of a calibration and correlation of the measured magnetic B-property susceptibility with the iron B-material concentration of the nps B-nanoparticle . [SEP]
[CLS] the fe concentration for the calibration was identified by icp - oes . [SEP]
[CLS] in fact , measuring the magnetic B-property susceptibility with magnetic B-property susceptibility balance ( mbs ) offers a simple , quick and high accuracy method to determine the concentration of added magnetic B-property nps B-nanoparticle without special sample preparation in complex matrices . [SEP]
[CLS] it was observed in another work that if ni 0 . 6 zn 0 . 4 fe 2−x cr x o 4 ( x = 0 - 0 . 5 ) ferrite nps B-nanoparticle were randomly orientated , the overall susceptibility was decreased by decreasing temperature . the temperature dependence of the real and imaginary parts of the effective magnetic B-property susceptibility was measured . fitting the experimental data of susceptibility with a neel - brown model yields unphysical high values for relaxation time and implies the presence of strong interactions between ferrite nps B-nanoparticle . [SEP]
[CLS] in another report , the temperature variation of the low - field magnetic B-property susceptibility for antiferromagnetic nps B-nanoparticle of ferritin and ferrihydrite in the superparamagnetic B-property regime was studied . [SEP]
[CLS] the authors of that study managed to show why the temperature variation of the lowfield susceptibility in antiferromagnetic nps B-nanoparticle , such as the above mentioned ones , deviates from the curie law variation even without invoking the interparticle interaction . [SEP]
[CLS] in addition , fept nps B-nanoparticle produced in the presence of polyol and pvp were found to possess a high magnetic B-property susceptibility to alternate ac fields at around ambient temperature for biomedical applications , such as magnetic B-property sensing devices for diagnostics and magnetic B-property hyperthermia . [SEP]
[CLS] the ac magnetic B-property susceptibility reached its maximum value at a temperature near the blocking temperature , and the blocking temperature of the fept nps B-nanoparticle was required to be adjusted at approximately room temperature to ameliorate biomedical performances . [SEP]
[CLS] crystallite size and blocking temperature were increased with higher synthesis reaction temperature , resulting in the enhancement of magnetic B-property susceptibility in the range of 300 - 350 k . [SEP]
[CLS] magnetophoretic mobility B-property arises from the motion of an electrically neutral body in a viscous medium when exposed to an inhomogeneous magnetic B-property field . [SEP]
[CLS] it is defined as the ratio of a particle - field interaction parameter to the particle friction coefficient . [SEP]
[CLS] the particle mobility B-property is a significant factor in predicting the separation when a mixture of particles of different mobilities B-property is exposed to an external field . [SEP]
[CLS] superparamagnetic B-property iron B-material oxide I-material nps B-nanoparticle ( spions ) were used by lee et al . in an approach based on the concept that such particles can act as a magnetophoretic mobility B-property switch . [SEP]
[CLS] more specifically , these particles undergo aggregation only in the presence of target analytes . [SEP]
[CLS] these authors developed a new lspr detection technique based on the programmed assembly of spions and the respective change in mobility B-property under external magnetic B-property fields . [SEP]
[CLS] they noticed a substantial improvement in lspr response , permitting a selective detection of target molecules without the need to immobilize receptors on the sensor surface . [SEP]
[CLS] the sensing performance could be tuned by modifying the concentrations of the reactants and the np B-nanoparticle sizes . [SEP]
[CLS] yang and co - workers published a study on the magnetophoretic mobility B-property and superparamagnetism B-property of core - shell iron B-material oxide I-material nps B-nanoparticle with dual targeting and imaging functionality . [SEP]
[CLS] the efficiency of magnetic B-property targeting depends mainly on the magnetophoretic mobility B-property , a parameter that can be increased only by increasing the size of the magnetic B-property nps B-nanoparticle . [SEP]
[CLS] preliminary in vivo investigation confirmed the suitability of utilizing these nps B-nanoparticle in yielding distinctive magnetic B-property resonance imaging of the brain tumor B-material in a rat model . [SEP]
[CLS] the calculated magnetophoretic mobility B-property of a range of magnetic B-property compounds has identified feco to be an alternative for magnetite in vitro biological cell B-material applications . [SEP]
[CLS] in a simple model , the magnetophoretic mobility B-property of a magnetic B-property np B-nanoparticle is deduced for a spherical magnetic B-property carrier , which moves slowly in a liquid medium under the effect of an applied inhomogeneous magnetic B-property field . [SEP]
[CLS] the nps B-nanoparticle tested were capped by oleic acid , their size was in the range of 1 - 11 nm and the stoichiometric ( fe 50 co 50 ) alloy was the best one from the magnetophoretic mobility B-property point of view . [SEP]
[CLS] bharti et al . published a report on the magnetophoretic assembly of flexible nps B-nanoparticle / lipid B-material microfilaments . [SEP]
[CLS] in the presence of a uniform magnetic B-property field , the magnetophoretic attraction of the particles combined with interparticle dipole - dipole attraction drives the microfilament assembly . [SEP]
[CLS] the magnetophoretic assembly is guided by the distribution of the external magnetic B-property field . [SEP]
[CLS] in this way , the aggregation of lipid - coated sticky iron B-material oxide I-material nps B-nanoparticle into unusually thick and flexible microfilaments takes place . [SEP]
[CLS] in another work , bakuzis and co - workers reported a mass magnetophoretic experiment applied for the separation of biocompatible B-property magnetic B-property nps B-nanoparticle with the potential to magnetohyperthermia . [SEP]
[CLS] these researchers performed a mass magnetophoretic experiment to segregate nps B-nanoparticle according to their diameter and size dispersion . [SEP]
[CLS] the analysis of hrtem images proved that with a few hours of exposure to the gradient field , the mean diameter and size dispersion of the nps B-nanoparticle near the surface of the fluid showed a significant change . [SEP]
[CLS] magnetophoretic separation is one of the most promising approaches for harvesting microalgae since the utilization of iron B-material oxide I-material nps B-nanoparticle are both technically and economically competent to remove the suspended cells B-material from the surrounding media . [SEP]
[CLS] toh et al . investigated the real - time kinetic behaviour of the magnetophoretic separation of chlorella sp . and the biointeraction between the chlorella sp . and surface - functionalized iron B-material oxide I-material nps B-nanoparticle under low gradient magnetic B-property separation . [SEP]
[CLS] the reliability of magnetophoretic separation for microalgal biomass collection was demonstrated , and this method could be employed as an effective downstream process for biofuel production . [SEP]
[CLS] faraudo and colleagues published an article on the simulation of magnetophoretic separation processes in dispersions of superparamagnetic B-property nps B-nanoparticle in the non - cooperative regime . [SEP]
[CLS] the magnetophoretic separation process of a mixture containing nps B-nanoparticle with different sizes and magnetic B-property responses was studied . [SEP]
[CLS] it was demonstrated that the homogeneous magnetophoretic conditions created by a closed type separator ( high magnetic B-property field over almost the whole sample and constant magnetic B-property gradient ) enhance the separation process , resulting in a better control over the process , and decreasing the expected separation time when compared to the open - type version of the separator . [SEP]
[CLS] in addition , non - magnetic B-property particles were also affected by the application of a magnetic B-property field gradient in magnetic B-property media . [SEP]
[CLS] the so - called negative magnetophoresis was used to separate such non - magnetic B-property nps B-nanoparticle based on their size . [SEP]
[CLS] superparamagnetic B-property relaxometry ( spmr ) is a technique that combined the use of sensitive magnetic B-property sensors and the superparamagnetic B-property properties of fe 3 o 4 nps B-nanoparticle . [SEP]
[CLS] it is an emerging technology with applications in various fields , including cancer research where the functionalization of nps B-nanoparticle with biomarkers B-property permits the specific binding to cancer cells B-material . [SEP]
[CLS] in magnetorelaxometry , the magnetic B-property moments of the nps B-nanoparticle are aligned by a magnetizing B-property field pulse of amplitude of a few mt and length of some seconds , and after abruptly switching off the field , the decay of the net magnetic B-property moment of the sample is recorded . [SEP]
[CLS] the magnetic B-property flux density from the sample ' s net magnetic B-property moment is obtained using high - sensitivity magnetic B-property field sensors , such as squids ( fig . 8 ) and fluxgates . [SEP]
[CLS] to ac susceptibility , magnetorelaxometry provides information on the relaxation times ( magnetization B-property dynamics ) for mnps in a carrier liquid or for immobilized magnetic B-property nps B-nanoparticle . [SEP]
[CLS] flynn and colleagues reported that the sprm technology can be employed to specifically determine different types of ab and cancer cell B-material lines through incubation B-technique measurements . [SEP]
[CLS] superparamagnetic B-property nps B-nanoparticle were conjugated to biomarkers B-property and they could be detected through spmr measurements , ensuring high contrast in vivo . [SEP]
[CLS] unbound nps B-nanoparticle did not give any spmr signal , falling in the measurement time window . [SEP]
[CLS] overall , their experiments demonstrated that spmr is an ideal approach for cancer detection and treatment monitoring . [SEP]
[CLS] ludwig et al . published a comparative study on the characterisation of magnetic B-property core - shell nps B-nanoparticle by fluxgate magnetorelaxometry , ac susceptibility , tem and photocorrelation spectroscopy B-technique ( pcs ) . [SEP]
[CLS] the samples tested were commercial fe 3 o 4 nps B-nanoparticle with polyacrylic acid shells B-material . [SEP]
[CLS] there was good agreement between the hydrodynamic size determined from the magnetorelaxometry and ac susceptibility measurements and that obtained from pcs . [SEP]
[CLS] this suggests that , although clustering occurred , magnetic B-property interactions were negligible and that the models were applicable . [SEP]
[CLS] in comparison with other methods , magnetorelaxometry is very rapid , it can be performed in opaque media and its signal is less dominated by bigger particles than in ac susceptibility or pcs . [SEP]
[CLS] it can also be used to characterize both the core B-material properties and the hydrodynamic size distribution of magnetic B-property nps B-nanoparticle and clusters , respectively . [SEP]
[CLS] in magnetorelaxometry , the magnetic B-property moments of the nps B-nanoparticle are aligned by an external magnetic B-property field of the order of 1 - 2 mt for typically 1 - 2 s , and the decay of the net magnetic B-property moment of the sample is recorded after abruptly switching off the magnetizing B-property field . [SEP]
[CLS] compared to magnetorelaxometry utilizing sensitive squid sensors , the differential fluxgate magnetorelaxometry setup has the advantages that the measurements can be carried out without magnetic B-property shielding and that the whole magnetization - relaxation cycle can be recorded . [SEP]
[CLS] in a magnetorelaxometry experiment , the relaxation of the superparamagnetic B-property nps B-nanoparticle can happen via the brownian and neel mechanisms . [SEP]
[CLS] the magnetorelaxometry - derived size obtained by these authors was found to be slightly larger than that determined from tem imaging . [SEP]
[CLS] magnetite nps B-nanoparticle were characterized by adolphi et al . by squid - relaxometry and magnetic B-property needle biopsy . [SEP]
[CLS] they found that the magnetization B-property detected by squid - relaxometry was 0 . 33 % of that detected by susceptometry , implying that the sensitivity of squid - relaxometry could be significantly improved through better control of the np B-nanoparticle size . [SEP]
[CLS] these researchers developed squid relaxometry as a highly sensitive platform for detecting and localizing superparamagnetic B-property magnetite nps B-nanoparticle specifically bound to cell B-material - surface antigens ( or other disease targets ) in vivo . [SEP]
[CLS] both relaxometry and susceptometry can be used together in a complementary way , in order to quantitatively analyse nanoparticle - cell binding experiments and to evaluate the results obtained by the moment superposition model analysis . [SEP]
[CLS] in fact , both magnetic B-property relaxometry and mri can be used to detect and locate targeted magnetic B-property nps B-nanoparticle , noninvasively and without ionizing B-property radiation . [SEP]
[CLS] magnetic B-property relaxometry has specificity ( only nps B-nanoparticle are detected ) and linear dependence of the relaxometry signal on the number of nps B-nanoparticle present . [SEP]
[CLS] relaxometry is well suited for therapeutic monitoring applications where the quick and precise measurement of a high concentration of magnetic B-property nps B-nanoparticle is needed . [SEP]
[CLS] squid - detected magnetorelaxometry has been reported to offer accurate quantification over a wider range of np B-nanoparticle concentrations compared to mri . [SEP]
[CLS] in addition , the former technique can be more rapid and cheaper than the mri . [SEP]
[CLS] urbano - bojorge et al . evaluated and compared alternating gradient field magnetometry and relaxometry as effective tools to assess the biodistribution of the magnetic B-property nps B-nanoparticle and to detect them on ex vivo tissue . [SEP]
[CLS] to do this , a standard dose of the fe - oxide B-material core B-material and dextran coated magnetic B-property nps B-nanoparticle were injected in the retro - orbital sinus on mice . [SEP]
[CLS] the relaxometry time and magnetometry data were consistent with the distribution of magnetic B-property nps B-nanoparticle and specific uptake in the reticuloendothelial system . [SEP]
[CLS] another comparison between fluxgate and squid magnetorelaxometry techniques for the characterization of magnetic B-property core - shell nps B-nanoparticle was reported by schilling and colleagues . [SEP]
[CLS] they mention that the advantages of using fluxgate magnetometers are that they are easier to operate since they do not need cryogenic cooling and since they are less susceptible to magnetic B-property disturbances . [SEP]
[CLS] in addition , the fluxgate method measures the absolute value of the corresponding vector component of the magnetic B-property field , and not just flux / field changes as squid magnetometers ; therefore the complete magnetization B-property - relaxation process can be recorded and zero signal is defined . [SEP]
[CLS] whereas fluxgate approach acts as a compact , user - friendly and affordable tool for the standard magnetic B-property characterization , squid relaxometry shows a high sensitivity performance . [SEP]
[CLS] peng et al . published a study on engineered water - soluble B-property twodimensional magnetic B-property nanocomposites , aiming for high magnetorelaxometry properties . [SEP]
[CLS] in terms of mri activity , the relaxometric properties of nanoparticulate contrast B-technique agents I-technique were structure - related and highly dependent on the interaction between water B-material protons and the core B-material magnetic B-property nps B-nanoparticle within the magnetic nanocomposites . [SEP]
[CLS] these researchers used hydrophobic B-property mn - doped ferrite nps B-nanoparticle and they turned them into hydrophilic B-property colloids through a one - step direct solvent evaporation method , involving aqueous graphene oxide B-material solution as a phase transfer agent . [SEP]
[CLS] the resultant unique two - dimensional magnetic B-property nanocomposite construct showed improved water B-material accessibility and water B-material retention in between the aggregated hydrophobic B-property mn - doped ferrite samples . [SEP]
[CLS] thus , it resulted in enhanced relaxometric properties . [SEP]
[CLS] transmission B-technique electron I-technique microscopy I-technique ( tem ) is a microscopy B-technique technique that exploits the interaction between a uniform current density electron beam ( i . e . the energies are usually within a range of 60 to 150 kev ) and a thin sample . [SEP]
[CLS] when the electron beam reaches the sample , part of the electrons are transmitted , while the rest are elastically or inelastically scattered . [SEP]
[CLS] the magnitude of the interaction depends on several factors , such as size , sample density and elemental composition . [SEP]
[CLS] the final image is built with the information acquired from the transmitted electrons . [SEP]
[CLS] as it is clear from the previous sections , size and morphology define the unique set of physical properties , such as optical , 361 magnetic B-property , 362 electronic 363 and catalytic , 364 of nps B-nanoparticle , as well as their interaction with biological systems . [SEP]
[CLS] 365 , 366 tem is the most common technique to analyse nanoparticle B-nanoparticle size and shape , since it provides not only direct images of the sample but also the most accurate estimation of the nanoparticle B-nanoparticle homogeneity . [SEP]
[CLS] nevertheless , some limitations have to be considered when using this technique , such as the difficulty in quantifying a large number of particles or misleading images due to orientation effects . [SEP]
[CLS] when characterizing very homogeneous samples , other techniques that analyse larger amounts of nps B-nanoparticle can provide more reliable results , such as saxs for larger and spherical nps B-nanoparticle , or xrd by exploiting the bordering of the xrd reflections and the scherrer formula . [SEP]
[CLS] however , a previous analysis has to be performed to ensure sample homogeneity . [SEP]
[CLS] nanoparticle B-nanoparticle properties not only depend on their size and morphology but also other factors , like interparticle distance . [SEP]
[CLS] for instance , when two metal B-material nps B-nanoparticle are brought in close proximity , their plasmons couple , red - shifting their plasmon band and changing their colour . [SEP]
[CLS] therefore , tem has been used to characterize the nanoparticle B-nanoparticle aggregation for different biomedical applications , including ( 1 ) sensing and diagnostics , where the aggregation depends on the presence of a biomarker B-property or analyte ; [SEP]
[CLS] ( 2 ) therapy , where the aggregation causes an increase of the nanoparticle B-nanoparticle therapeutic effect ; 371 and ( 3 ) imaging , where the aggregation improves the response signal . [SEP]
[CLS] in order to obtain reliable results , extra care should be taken for sample preparation , since an inadequate protocol can result in sample alteration or artefact creation , 373 e . g . aggregation during the drying of the colloid suspension . [SEP]
[CLS] thus , tem is usually combined with other techniques that can measure larger numbers of particles , and require less sample preparation , such as uv - vis and dls . [SEP]
[CLS] in recent years strong control over the nanoparticle B-nanoparticle assembly has been achieved , and a controlled np B-nanoparticle self - assembly can lead to welldefined np B-nanoparticle superlattices . [SEP]
[CLS] the systematic assembly of different nanocrystals yields new multifunctional structures that combine the features of the individual building blocks , as well as the rise of new and exciting properties . [SEP]
[CLS] tem has been one of the techniques used to characterize the formation of different super - lattice nanocomposites , which can be isostructural to several atomic crystal systems ( fig . 9 ) . [SEP]
[CLS] these new three - dimensional arrays are made of different nps B-nanoparticle ( e . g . quantum B-nanoparticle dots I-nanoparticle , metals B-material and magnetic B-property nps B-nanoparticle ) , and their final structure and composition can be controlled by tailoring the colloid surface charge 377 or directional bonding with dna . [SEP]
[CLS] in the last few years , the scientific community has started to view nps B-nanoparticle as dynamic systems , where their structure and properties can evolve over time as they interact with their surroundings . [SEP]
[CLS] therefore , it is important to characterize their dynamic transformations in order to optimize their performance in many applications . [SEP]
[CLS] for instance , sunlight has been reported to aggregate ag nps B-nanoparticle and decrease their cytotoxicity B-property . [SEP]
[CLS] tem imaging showed that nanobridges were formed between the nps B-nanoparticle upon sunlight exposure . [SEP]
[CLS] these morphological changes combined with surface sulfidation affected the nanoparticle B-nanoparticle dissolution rate , which caused the toxicity B-property to decrease . [SEP]
[CLS] furthermore , tem and dls have been used to study the biodegradation B-property of the nanoparticle B-nanoparticle polymeric I-nanoparticle coating B-material by bacteria . [SEP]
[CLS] the loss of the particle coating B-material caused colloidal aggregation , which affected their mobility B-property and cytotoxicity B-property . [SEP]
[CLS] furthermore , traditional tem cannot be used to study the growth of nps B-nanoparticle in solution ( this topic is further discussed in the liquid - tem section of this review ) . [SEP]
[CLS] nevertheless , it can be used to characterize the formation of colloids from solid precursors . [SEP]
[CLS] for example , tem has been used to image the growth dynamics of copper B-material nps B-nanoparticle . [SEP]
[CLS] these were synthesized in a heating holder by reducing copper B-material phyllosilicate platelets with hydrogen B-material . [SEP]
[CLS] the in situ visualization allowed the characterization of the phase transformation of copper B-material as the reaction was pro - gressing . [SEP]
[CLS] another use of nps B-nanoparticle concerns the field of therapeutic carriers , since they can enhance the efficiency of drugs by improving their stability and cellular uptake . [SEP]
[CLS] two main techniques are used to study the interaction between nps B-nanoparticle and cells B-material : tem and clsm . [SEP]
[CLS] both techniques are complementary and frequently used together , since tem provides higher resolution than any other imaging B-technique technique I-technique while clsm allows the live cell imaging and fluorescent B-property labelling of different cell B-material components . [SEP]
[CLS] nps B-nanoparticle are internalized through endocytosis B-event after interacting with cell B-material membrane receptors , such as scavenger receptors . [SEP]
[CLS] however , in order to increase their therapeutic effect , nps B-nanoparticle need to escape from the vesicles and be released into the cytoplasm . [SEP]
[CLS] thus , tem has been used to assess the location of nps B-nanoparticle within a cell B-material . [SEP]
[CLS] for instance , it was used to study the au np B-nanoparticle shape and size requirements for higher cellular uptake and later vesicle escape ( fig . 10 ) . [SEP]
[CLS] as mentioned in an earlier section of this review , the aggregation of nps B-nanoparticle can change their physical properties . [SEP]
[CLS] therefore , tem has been applied to characterize the dispersion of nps B-nanoparticle after their internalization . [SEP]
[CLS] for example , au nps B-nanoparticle grafted with peg were well dispersed , and in low proportions within the intracellular vesicles of macrophages , while non - grafted au nps B-nanoparticle mostly accumulated as aggregates in the vesicles . [SEP]
[CLS] an additional advantage of tem is that it allows the assessment of the changes of subcellular structures caused by the nps B-nanoparticle . [SEP]
[CLS] for instance , apoptosis - related vacuoles were observed in melanoma cells B-material after magnetic B-property field hyperthermia treatment with iron B-material oxide I-material nps B-nanoparticle was applied . [SEP]
[CLS] this observation helped to understand the cell B-event death I-event pathways in response to magnetic B-property field hyperthermia . [SEP]
[CLS] finally , tem has also been employed to define the degree of penetration of nps B-nanoparticle through different tissues , such as tio 2 nps B-nanoparticle through the skin for sunscreen applications . [SEP]
[CLS] high - resolution tem ( hrtem ) is an imaging mode of transmission B-technique electron I-technique microscopy I-technique that uses phase - contrast imaging , where both transmitted and scattered electrons are combined to produce the image . [SEP]
[CLS] in comparison with traditional tem imaging , hrtem requires a larger objective aperture in order to employ the scattered electrons . [SEP]
[CLS] phase - contrast imaging is the technique with the highest resolution ever developed and allows the detection of the arrays of atoms B-material in crystalline structures . [SEP]
[CLS] hrtem provides important information on the nanoparticle B-nanoparticle structure ; in particular , while conventional electron B-technique microscopies I-technique can provide the statistical assessment of np B-nanoparticle morphology , they do not have enough resolution to image the single particle crystal structure . [SEP]
[CLS] thus , hrtem has become the most common technique to characterize the internal structure of nps B-nanoparticle . [SEP]
[CLS] for instance , hrtem has been used to study the effect of ligands in the final structure of pt nps B-nanoparticle grown by organometallic synthesis . [SEP]
[CLS] similarly , it has been employed to observe that the pd nanoclusters ( sizes between 1 and 1 . 5 nm ) synthesized by a different organometallic protocol are a mixture of four different structured crystals with comparable energy levels , see fig . 11 and 12 . [SEP]
[CLS] furthermore , hrtem can distinguish between single crystal and polycrystalline anisotropic au nps B-nanoparticle that present similar optical properties . [SEP]
[CLS] hrtem also allows the characterization of structural transitions , such as the thermal transition from disordered face - centred cubic to ordered l1 0 in iron B-material - platinum B-material nps B-nanoparticle . [SEP]
[CLS] this thermal - induced event yields nps B-nanoparticle with enhanced coercivity and larger magnetocrystalline anisotropy , which are necessary qualities to build permanent magnets . [SEP]
[CLS] the imaging of single crystals also offers the opportunity to identify structural defects that may explain the unusual properties . [SEP]
[CLS] for instance , it had been reported that the lattice constant of ceo 2 nps B-nanoparticle increased with decreasing particle size . [SEP]
[CLS] 396 nevertheless , no explanation had been found for such abnormal behaviour . [SEP]
[CLS] a later study using hrtem showed that these changes were not caused by either disclinations ( line defects ) or volume expansions in the high angle boundaries . [SEP]
[CLS] combining these results with the ones from raman B-technique spectroscopy I-technique , the authors concluded that the lattice expansion was the result of an increased number of point defects at smaller particle sizes . [SEP]
[CLS] even though hrtem is a powerful technique , it is worth mentioning that the characterization of nps B-nanoparticle is not always feasible by this technique . [SEP]
[CLS] due to the random orientation of the crystals relative to the electron source , there may be directions where the atoms B-material are not well aligned , resulting in complex images that cannot be directly used to define the structure . [SEP]
[CLS] insights regarding np B-nanoparticle growth and structure - related properties can also be gained through hrtem observations . [SEP]
[CLS] for instance , zhang et al . studied the formation of cuo nps B-nanoparticle by in situ hrtem . [SEP]
[CLS] they observed that the leading mechanism was coalescence , which was much faster than others , such as nanocrystal reshaping . [SEP]
[CLS] furthermore , they witnessed that if the colloids were aligned before merging , the resulting nps B-nanoparticle were single crystals . [SEP]
[CLS] furthermore , hrtem has been used to clarify the effect of substrates on the properties of metal B-material nps B-nanoparticle . [SEP]
[CLS] for instance , it was observed that cu nps B-nanoparticle deposited on graphite substrates presented a distinctive melting behaviour and selective superheating , in comparison with nps B-nanoparticle supported on cuo 2 . [SEP]
[CLS] based on the hrtem images and molecular dynamics , the authors of the study attributed the distinctive behaviour to the absorption of a thin layer of carbon B-material on the np B-nanoparticle surface , which improved their thermal stability . [SEP]
[CLS] liquid tem [SEP]
[CLS] as mentioned earlier a fundamental component of tem is the vacuum system , which prevents the damage of the filament and decreases the electron beam scattering . [SEP]
[CLS] traditional tem imaging has been solely used on solid and dried samples , since the evaporation of liquids could com - promise the vacuum . [SEP]
[CLS] thus , the characterization of solidliquid systems at the nanoscale has been neglected for many decades . [SEP]
[CLS] early attempts to characterize liquid samples date back to the 1930s , when l . marton imaged biological samples trapped between aluminum thin foils . [SEP]
[CLS] nevertheless , the technical challenge of preserving the vacuum and avoiding the liquid evaporation prevented any significant advancement until recent years , when the nanofabrication of sealed liquid cells B-material was developed . [SEP]
[CLS] in 2003 , frances m . ross and collaborators developed a tem liquid cell B-material using epoxy - sealed silicon B-material nitride ( sin ) membranes . [SEP]
[CLS] these membranes were electron transparent and confined the liquid sample , preserving the microscope vacuum . [SEP]
[CLS] ross et al . were able to image the growth of cu nanoclusters with 5 nm spatial resolution and a time resolution of 30 images per second . [SEP]
[CLS] since then , several modifications have been introduced to the liquid tem grid . [SEP]
[CLS] for instance , better cell B-material sealing was achieved by replacing the original sio 2 spacers B-material with softer indium B-material thin films . [SEP]
[CLS] sin window grids were fabricated by binding commercially available sin wafers with polymer B-material o - rings . [SEP]
[CLS] the polymers B-material greatly simplified the cell B-material fabrication , decreasing the assembly times down to 10 to 15 min , and allowing the re - use of the wet cells B-material . [SEP]
[CLS] sin tem grids are currently commercially available with electrochemistry and heating packages , making them the most popular option among the different tem liquid cells B-material . [SEP]
[CLS] however , these grids suffer from lower image resolution , due to the sin membrane and liquid layer thickness , which contribute to scatter the electron beam ( fig . 13 ) . [SEP]
[CLS] an alternative to sin membrane grids is imaging solid - liquid systems in low vapor pressure ionic liquids ( ils ) . [SEP]
[CLS] the solids are dispersed in the ils and imaged without sealing , since ils do not evaporate . [SEP]
[CLS] the absence of cell B-material membrane provides lower electron scattering and better image resolution . [SEP]
[CLS] nevertheless , working with ils is technically demanding and the number of systems that can be imaged is very limited , since ils react with a wide range of components . [SEP]
[CLS] recently , a new kind of liquid cell B-material has been developed by enclosing liquid samples between two thin graphene sheets . [SEP]
[CLS] in these , the thickness of both liquid layer and sealing membrane is highly minimized , decreasing the electron beam scattering and achieving images with atomic resolution . [SEP]
[CLS] furthermore , the van der waals forces between the graphene layers keep the system assembled and no further sealing step is required . [SEP]
[CLS] although graphene membrane cells B-material have become a hot topic , they still present some limitations that must be considered before using them . [SEP]
[CLS] for example , low operation voltages are required ( 80 kv ) in order to minimize the electron knock - off effects on the graphene atoms B-material . [SEP]
[CLS] most of the imaging B-technique techniques I-technique only provide information at a single time point , usually after the nanoparticle B-nanoparticle growth has finished . [SEP]
[CLS] therefore , they can characterize the final nanoparticle B-nanoparticle structure but not the growing mechanism . [SEP]
[CLS] liquid tem allows the tracking of the nanoparticle B-nanoparticle trajectory while this is growing , providing direct observation of the nanoparticle B-nanoparticle evolution . [SEP]
[CLS] for instance , liquid tem has been used to study the growth mechanism of platinum B-material nps B-nanoparticle , which can follow two different growing mechanisms , i . e . monomer B-material attachment or coalescence , and still yield the same final nanoparticle B-nanoparticle size . [SEP]
[CLS] interestingly , graphene liquid cell resolution is high enough to study the facet - dependence interaction between nps B-nanoparticle . [SEP]
[CLS] alivisatos ' group observed that the platinum B-material nanoparticle B-nanoparticle growth through the coalescence mechanism is facet - specific , where the attachment is favoured on the lowest energy surfaces . [SEP]
[CLS] other interesting mechanistic observations imaged through liquid tem include the oscillatory growth dynamics of bismuth nps B-nanoparticle , where both ostwald and anti - ostwald ripening occur , the kirkendall effect on the synthesis of hollow bismuth oxide B-material nps B-nanoparticle 410 and the galvanic replacement on the formation of hollow palladium B-material nps B-nanoparticle . [SEP]
[CLS] furthermore , in situ imaging can be used to calculate the redox reaction rates on the growth of heterocomplexes , such as core - shell gold B-material - palladium B-material nps B-nanoparticle . [SEP]
[CLS] nps B-nanoparticle within fluids are under constant movement . [SEP]
[CLS] in addition to brownian motion , several other parameters can contribute to their movement , such as chemical - induced changes of the environment or liquid flow . [SEP]
[CLS] liquid tem has been used to characterize some of them . [SEP]
[CLS] for instance , the groups of alivisatos and dahmen recorded the movement of inorganic nps B-nanoparticle during fluid evaporation . [SEP]
[CLS] nevertheless , the most exciting application of recording nanoparticle B-nanoparticle motion is the 3d reconstruction of the colloid . [SEP]
[CLS] park et al . imaged the free movement of platinum B-material nps B-nanoparticle in liquid in order to reconstruct their structure at the near - atomic scale . [SEP]
[CLS] nanoparticle B-nanoparticle assembly and superlattice formation are emerging as important fields of research within nanoscience because they can present different properties in comparison with the individual nps B-nanoparticle and bulk materials . [SEP]
[CLS] the fundamental understanding of their formation mechanisms requires characterizing not only the final structure but also the assembly process . [SEP]
[CLS] in this direction , liquid tem has been used for the direct observation of pt np B-nanoparticle superlattice formation , which includes an initial amorphous agglomerate condensation and a subsequent array crystallization . [SEP]
[CLS] cryo - electron microscopy B-technique ( cryo - tem ) is a subclass of tem that allows the visualization of the near - unaltered samples in their frozen - native environment by vitrifying them at cryogenic temperatures . [SEP]
[CLS] very recently , the 2017 nobel prize of chemistry was awarded to dubochet , frank and henderson for the development of cryo - electron B-technique microscopy B-technique for the high resolution structure determination of biomolecules in solution . [SEP]
[CLS] liquid nitrogen B-material is usually employed to freeze the samples . [SEP]
[CLS] this technique is commonly used in molecular biology and colloid chemistry due to the lack of factors ( i . e . staining and sample ' s preservation in non - physiological environments ) that can alter the conformation or assembly of the sample ' s molecules . [SEP]
[CLS] the liquid samples are usually vitrified by commercial automated plunge - freezers , which freeze water B-material solutions by decreasing their temperature extremely fast , so the water B-material molecules cannot reorganize in long - range ordered crystal lattices . [SEP]
[CLS] this results in an amorphous state that is similar to the native liquid . [SEP]
[CLS] plunge - freezers accomplish this amorphous state in four steps : ( 1 ) placing the liquid sample in the carbon - coated copper B-material grid , ( 2 ) removing the excess of liquid in order to produce a thin film , ( 3 ) plungefreezing the grid into the liquid n 2 and ( 4 ) storing the vitrified sample in a storage box that contains liquid n 2 . [SEP]
[CLS] before liquid tem became commercially available , cryo - tem was one of the two most common techniques used to visualize the nanoparticle B-nanoparticle growth ( the other one involved arresting the nps B-nanoparticle at intermediate B-property reaction stages , and performing normal tem characterization ) . [SEP]
[CLS] cryo - tem has been used to study complex growth mechanisms , such as the aggregative growth of zeolite crystals , where several amorphous aggregates are formed before they rearrange into the final crystals . [SEP]
[CLS] other studied systems include the formation of biphasic particles , 420 made of silica and polystyrene , and the " popcorn " like growth of gold B-material nanorods B-nanoparticle . [SEP]
[CLS] the latter is a good example to highlight the strengths and weaknesses of this technique . [SEP]
[CLS] on the one hand , it can provide direct images of the nps B-nanoparticle while growing in their native environment . [SEP]
[CLS] on the other hand , the concentration of the particles usually is too low ( in the nanomolar range ) to provide statistical information . [SEP]
[CLS] in addition to the np B-nanoparticle growth , cryo - tem has been used to visualize the molecular templates , such as block copolymers and ctab , that direct the growth of lanthanide - based nps B-nanoparticle 422 and au nanorods B-nanoparticle , 423 respectively . [SEP]
[CLS] the morphology and volume transitions of thermoresponsive core - shell nps B-nanoparticle have also been imaged by cryo - tem , where the morphology of the thermosensitive shell B-material is preserved after the plunge freezing , and clearly visible without staining . [SEP]
[CLS] lastly , it is worth mentioning that cryo - tem can achieve sub - nanomolar resolutions . [SEP]
[CLS] for instance , the au ( 200 ) planes of 15 nm au nps B-nanoparticle have been imaged with structural resolutions below 0 . 2 nm . [SEP]
[CLS] cryo - tem has been used to study complex nanoparticle B-nanoparticle aggregation mechanisms , such as the kinetic manipulation of block copolymer nanostructures ( fig . 14 ) or the assembly of binary np B-nanoparticle superlattices using protein B-material cages . [SEP]
[CLS] furthermore , cryo - tem imaging is usually required to confirm the unusual assembly behaviours , since the fast plunge freezing avoids the particle rearrangement during the sample preparation and visualization . [SEP]
[CLS] as an example , conventional tem showed that cellulose nps B-nanoparticle laterally self - assemble into flat objects . [SEP]
[CLS] nevertheless , cryo - tem imaging was required to confirm that these assemblies were not drying or staining artefacts . [SEP]
[CLS] in addition to qualitative characterization , cryo - tem can also be used to quantify the thermodynamic forces involved in the formation of assemblies . [SEP]
[CLS] even though several theoretical models have been developed to explain the contribution of these forces , 429 there is very limited amount of experimental data available . [SEP]
[CLS] the formation free energy of quantum B-nanoparticle dot I-nanoparticle nanostructures was calculated from cryo - tem images . [SEP]
[CLS] the free energy was later separated into the entropic and enthalpic contributions , exploiting the variation of the assemblies with temperature . [SEP]
[CLS] electron diffraction ( ed ) , also known as selected area electron diffraction ( saed ) , is another important microscopy B-technique tool for the study of the crystal structure of nps B-nanoparticle . [SEP]
[CLS] experiments are usually performed in a tem , or a scanning electron microscope ( sem ) as electron backscatter diffraction . [SEP]
[CLS] in these instruments , electrons are accelerated by an electrostatic potential in order to gain the desired energy and determine their wavelength before they interact with the sample to be studied . [SEP]
[CLS] the periodic structure of a crystalline solid acts as a diffraction grating , scattering the electrons in a predictable manner . [SEP]
[CLS] working back from the observed diffraction pattern , it may be possible to deduce the structure of the crystal producing the diffraction pattern . [SEP]
[CLS] buffat discussed the use of electron diffraction and hrtem to investigate multiply - twinned structures and dynamical events in metal B-material nps B-nanoparticle . [SEP]
[CLS] the author noted that measuring the shrinkage of the lattice space by xrd may be complicated , as instrumental parameters , reflection broadening due to a very small np B-nanoparticle size and matrix effects can lead to unclear xrd results . [SEP]
[CLS] with ed , particles are lying rather free on a substrate , lower material B-material quantity is needed for the measurement , and correlation with direct images of the crystals is possible . [SEP]
[CLS] however , the study of size effects in au and pt by ed requires a careful interpretation of its results due to the complex multiply - twinned or icosahedral - like structure that appears in nps B-nanoparticle to lower the total free energy . [SEP]
[CLS] the saed technique is limited by the fact that many nps B-nanoparticle contribute to the diffraction pattern because of the relatively large size of the illuminated area , making their individual study difficult . [SEP]
[CLS] in the more modern ' nanodiffraction ' technique , the area of the sample which contributes to the diffraction pattern is limited by the size of the electron probe , which in a field emission tem can be as small as 0 . 1 nm . [SEP]
[CLS] in a paper concerning decahedral au nps B-nanoparticle , the ' nanodiffraction ' approach was employed enabling the study of single nps B-nanoparticle , but it was demonstrated that the beam convergence produced a loss of symmetry from 10 - to 5 - fold in the diffraction pattern of the nps B-nanoparticle . [SEP]
[CLS] schamp and jesser used ed to calculate interplanar spacings and other lattice parameters of au nps B-nanoparticle . [SEP]
[CLS] an improved calculation of such parameters allows a more precise determination of the anisotropic strains in the gold B-material nps B-nanoparticle . [SEP]
[CLS] in another work , the origin of the ' forbidden ' reflections present in the [ 111 ] and [ 112 ] electron diffraction patterns of triangular - flatthin au nps B-nanoparticle was explained . [SEP]
[CLS] regarding another noble metal B-material , ag , smyslov and co - workers combined saxs , ed and microscopy B-technique experiments to determine the size and phase composition of ag nps B-nanoparticle in a gel film of bacterial cellulose . [SEP]
[CLS] in that report , saxs provided a reliable estimate of the size of the nps B-nanoparticle in the moisture - containing composite ; ed and electron B-technique microscopy I-technique permitted the performance of phase analysis , obtain images of nps B-nanoparticle and visualize their arrangement in the composite matrix . [SEP]
[CLS] bismuth nps B-nanoparticle have also been studied by electron diffraction : the authors of that study noted that the diffraction pattern is produced from the whole ensemble of the nps B-nanoparticle , and if there are populations of nps B-nanoparticle with two different sizes , the larger size nps B-nanoparticle will contribute more to the diffraction intensity than the smaller ones . [SEP]
[CLS] fe - based nps B-nanoparticle ( alloys and oxides B-material ) have often been investigated with electron diffraction . [SEP]
[CLS] sato and hirotsu used ed to study the order - disorder transformation in l1 0 - fepd nps B-nanoparticle . [SEP]
[CLS] the disappearance of the long - range atomic order in such 10 nm nps B-nanoparticle was examined by ed using a specimen heating stage attached to a tem , for an in situ annealing . [SEP]
[CLS] a particle size dependence of the order - disorder transformation temperature of 10 nm sized fepd isolated nps B-nanoparticle was evidenced . [SEP]
[CLS] compared to the bulk alloy , such temperature was lower by around 80 k for 13 . 5 nm fepd nps B-nanoparticle . [SEP]
[CLS] the same group employed ed to determine the long - range order ( lro ) parameters of two - dimensional epitaxially - grown dispersed mono - crystalline 10 nm l1 0 - fepd nps B-nanoparticle . [SEP]
[CLS] in that case , the very small volume of the 2d sample would hinder the applicability of the usual xrd measurements for the calculation of the lro parameters . [SEP]
[CLS] similarly , the lro parameters of l1 0 - fept nps B-nanoparticle were also determined through the use of ed . [SEP]
[CLS] it is reported that when using transmission electron diffraction with fast electrons , the scattering power of atoms B-material for electrons is about 10 4 times larger than that for x - rays . [SEP]
[CLS] thus , ed has a great advantage in acquiring superstructure reflections for these bimetallic nps B-nanoparticle with ordered structure and 2d dispersion . [SEP]
[CLS] nevertheless , the dynamical scattering effect complicates the analysis of ed intensity . [SEP]
[CLS] still , the lro parameters of such fept nps B-nanoparticle can be calculated with accuracy by ed taking into account the multiple scattering effect . [SEP]
[CLS] li et al . analysed the structure of cofe - fe 3 o 4 core - shell nps B-nanoparticle by electron imaging and diffraction . [SEP]
[CLS] these researchers employed both ed and hrtem to find out if the core B-material is composed of cofe 2 o 4 . [SEP]
[CLS] hrtem images can provide significant information on the real - space structure , but only the nps B-nanoparticle oriented along specific directions and the lattice planes that are large enough to be resolved by tem can give rise to lattice fringes in the image . [SEP]
[CLS] electron diffraction patterns recorded from a large number of nps B-nanoparticle have a unique advantage , i . e . all of the lattice planes are represented in the diffraction pattern . [SEP]
[CLS] hrtem , ed and eds microanalysis helped in the combination for determining the structure and composition of such coreshell nps B-nanoparticle . [SEP]
[CLS] langguth and co - workers combined ed and xrd for the structural characterization of iron B-material oxide I-material / hydroxide B-material nps B-nanoparticle in 9 different parenteral drugs for the treatment of iron deficiency anaemia . [SEP]
[CLS] while xrd permits a higher resolution of small d - distances because of the low wavelength of about λ = 0 . 154 nm , the combination of stem with diffraction analyses allows the selective investigation of crystalline areas in the sample . [SEP]
[CLS] finally , electron imaging and diffraction were used in a complementary way to determine the crystalline planes and directions of the surface facets and edges of hematite nps B-nanoparticle as well as to calculate their miller indices . [SEP]
[CLS] in scanning B-technique transmission I-technique electron I-technique microscopy I-technique ( stem ) , the electron beam is focused to a fine spot that is then scanned over the sample in a raster , unlike conventional tem . [SEP]
[CLS] the rastering of the beam across the sample makes stem appropriate for techniques such as z - contrast annular dark - field imaging ( explained below ) and spectroscopic mapping by energy dispersive x - ray ( edx ) spectroscopy B-technique or electron energy loss spectroscopy B-technique ( eels ) . [SEP]
[CLS] using edx or eels spectroscopy B-technique in the stem it is possible to obtain elemental maps that show features down to the atomic scale . [SEP]
[CLS] for the proper operation of stem , the experimental determination of the absolute cross section is very challenging , as electron donors of high dynamical range are required . [SEP]
[CLS] this has hampered the application of the stembased technique to a broad range of particle sizes , as one would wish . [SEP]
[CLS] nevertheless , mass information can be acquired through stem - based mass measurements if a known mass standard can be established . [SEP]
[CLS] for the characterization of the 3d morphology of nps B-nanoparticle , stem electron B-technique tomography I-technique ( analysed later in this review ) is a very powerful technique and has been suc - cessfully employed for embedded and stable nps B-nanoparticle . [SEP]
[CLS] the main restriction of the method is the time needed to take full tomographs and this might exclude many electron beam sensitive samples from analysis . [SEP]
[CLS] to tackle that difficulty , palmer and coworkers developed a ' single - shot ' approach to a three - dimensional measurement problem , using au nps B-nanoparticle as the model system . [SEP]
[CLS] haigh and co - workers published a paper on the investigation of the limitations and optimisation of edx tomography B-technique within a stem , focusing on the application of the technique to characterize the 3d elemental distribution of bimetallic agau nps B-nanoparticle . [SEP]
[CLS] a key question they worked on for edx tomography B-technique was whether the characteristic x - ray intensity generated in the stem meets the requirements for the constraints of a particular sample and detector geometry . [SEP]
[CLS] ag nps B-nanoparticle exposed to light and humic substances were investigated by a combination of high resolution stem , eels and uv - vis techniques . this multimethod approached facilitated the acquiring of information on np B-nanoparticle morphology , surface chemistry transformations and corona formation . [SEP]
[CLS] despite the signal loss , probably by dissolution , that was noticed , there was no direct evidence of oxidation from the stem - eels . [SEP]
[CLS] the palmer group has reported that not many applications of quantitative stem exist in nanomaterial B-material characterization . [SEP]
[CLS] therefore , they demonstrated a new approach to quantify the imaging contrast I-technique in stem using size - selected clusters . [SEP]
[CLS] the nanoclusters used consisted of pd ( z = 46 ) and au ( z = 79 ) . [SEP]
[CLS] finally , deiana et al . used stem - edx to investigate the core - shell structure of bimetallic pd - hg nps B-nanoparticle , which proved to be a crystalline coreshell structure , with a pd core B-material and a pd - hg ordered alloy shell B-material . [SEP]
[CLS] the ordered shell B-material was considered to be responsible for the high oxygen B-material reduction selectivity to h 2 o 2 . [SEP]
[CLS] high - angle annular dark - field imaging ( haadf - stem ) . [SEP]
[CLS] annular dark - field imaging is a method of mapping samples in a stem . [SEP]
[CLS] these images are formed by collecting scattered electrons with an annular dark - field detector . [SEP]
[CLS] an annular dark - field image formed only by very high angle , incoherently scattered electrons ( rutherford scattered from the nucleus of the atoms B-material ) as opposed to bragg scattered electronsis highly sensitive to variations in the atomic number of atoms B-material in the sample ( z - contrast images ) . [SEP]
[CLS] this technique is also known as high - angle annular dark - field imaging ( haadf ) . [SEP]
[CLS] haadf - stem is a valuable tool to observe local atomic structures and has been successfully applied to the imaging of various material B-material interfaces . [SEP]
[CLS] akita et al . used this technique to observe au nps B-nanoparticle supported on ceo 2 for the first time to investigate the mechanism of the cyclic structural change according to the switching on and off of the electron beam . [SEP]
[CLS] the sequential haadf - stem observations can directly detect the atomic process of the structural change as well as the detailed behaviour of au atoms B-material at the perimeter edge . [SEP]
[CLS] haadf - stem images can also represent the correct atomic column positions of au and ce as maximum intensity positions without any artifact under their observation conditions . [SEP]
[CLS] this is a difference from the usual hrtem images , where intensive image simulations are essential to decide atomic positions due to the significant occurrence of artifacts , especially at surfaces or interfaces . [SEP]
[CLS] haadf - stem has straightforward interpretability , although multislice simulation is often required in order to take into account the strong dynamical screening effect if quantitative structure information is needed . [SEP]
[CLS] the technique under discussion has applications in tomography B-technique ( discussed below ) , size mass / thickness measurement at the atomic scale , structure characterization and composition measurement . [SEP]
[CLS] haadf - stem uses a sharply focused beam to scan across the specimen , and the annular dark - field ( adf ) detector collects only the scattered electrons . [SEP]
[CLS] the observation of au nps B-nanoparticle on tio 2 was also achieved using haadf - stem . [SEP]
[CLS] this technique together with hrtem is indispensable for the direct observation of the atomic structure of heterointerfaces . [SEP]
[CLS] haadf - stem can resolve atomic configurations directly without image simulations considering the defocus value and the thickness of the samples , although it is hard to image the light atoms B-material , such as oxygen B-material . [SEP]
[CLS] in the study by haruta and co - workers , the distance between the au and ti layers at the interface is estimated from the haadf - stem image , which is essential to evaluate the status of oxygen B-material layers affecting the catalytic activity . [SEP]
[CLS] the oxygen B-material columns on the tio 2 surface and in the bulk tio 2 region were not detected in the haadf - stem image , because oxygen B-material atoms I-material are light compared with titanium B-material , and the signal - to - noise ratio was not high enough . [SEP]
[CLS] stem images are easily distorted during image acquisition by the sample drift or mechanical and electronic vibrations . [SEP]
[CLS] although the atomic columns are detected in the haadf - stem image , it is hard to detect the local displacement of each atom B-material . [SEP]
[CLS] the complementary combination of haadf - stem imaging and first - principles calculations should be a promising approach to elucidate the atomic and electronic structure at the interfaces . [SEP]
[CLS] li et al . mention that haadf - stem is appealing to probe the 3d - structure of nps B-nanoparticle because its intensity is strongly dependent not only on the atomic number z of the observed atoms B-material but also on the number of atoms in a column . [SEP]
[CLS] they combined quantitative haadf - stem analysis with molecular - dynamicsbased model structure search procedures , and realistic image contrast simulations in order to identify not only the size and shape but also the structure and orientation of soft - landed au nanoclusters . [SEP]
[CLS] badonneau et al . studied by haadf - stem the au and ag nps B-nanoparticle embedded in dielectric capping . [SEP]
[CLS] the authors illustrated that this method is a convenient tool for revealing the morphology of buried nps B-nanoparticle , and highlighting the influence of the np B-nanoparticle size and the dielectric - capping layer on the aspect ratio and optical response of the nps B-nanoparticle . [SEP]
[CLS] the information on the long - range order and the random ( or not ) orientation of the nps B-nanoparticle can be derived . [SEP]
[CLS] in comparison with cross - sectional brightfield tem , the haadf - stem data represent a statistical average over 10 3 nps B-nanoparticle . [SEP]
[CLS] the morphological parameters derived from a haadf - stem analysis can be used to simulate accurately the absorption spectra obtained in the visible range by spectroscopic ellipsometry measurements of the sandwiched ag nps B-nanoparticle , thus confirming the validity of the haadf - stem analysis . [SEP]
[CLS] the haadf - stem analysis helps to reveal the average shape ( in - plane diameter and height ) of the individual embedded nps B-nanoparticle , with no need for cross - section preparation or z - contrast tomography B-technique measurements , which require a big number of projections to be collected over a wide tilt range . [SEP]
[CLS] in another report , microscopy B-technique techniques , including haadf - stem , were employed to characterize bimetallic cu - au nps B-nanoparticle with size in the range of 1 - 7 nm . [SEP]
[CLS] the researchers noted that the haadf - stem imaging provides thickness contrast , which is linearly proportional to specimen thickness , and atomic number contrast , which is proportional to the atomic number z . [SEP]
[CLS] the compositional sensitivity of haadf images allows the investigation of heterogeneous materials with components of very different atomic numbers present . [SEP]
[CLS] in that paper , the haadf - stem imaging of a cubo - octahedral particle supported a mixed cu - au configuration . [SEP]
[CLS] calvino and colleagues focused on the characterization of au catalysts B-property supported on a ce - tb - zr mixed oxide B-material . [SEP]
[CLS] in general , haadf - stem operates well when metal B-material nps B-nanoparticle are dispersed within light support materials , such as zeolites or alumina , for which large differences between metal B-material and support element atomic numbers contribute to a high contrast in the images . [SEP]
[CLS] the quantitative 3d haadf - stem tomographic analysis of nanometer - sized noble metal B-material particles supported on oxides B-material of high atomic number ( ce , tb and zr ) was proved to be feasible . [SEP]
[CLS] quantitative haadf imaging at the atomic level can also be used to measure the number of atoms B-material contained in a np B-nanoparticle or a cluster . [SEP]
[CLS] for instance , bimetallic 8 nm fepd nps B-nanoparticle were studied by haadf - stem to determine their chemical composition . [SEP]
[CLS] particularly , haadf was used to identify the chemical variations of a population of nps B-nanoparticle , i . e . measure the statistical dispersion in chemical composition . [SEP]
[CLS] filippousi et al . studied with haadf - stem the polyhedral iron B-material oxide I-material core - shell nps B-nanoparticle in a biodegradable B-property polymeric matrix , and they found out that the nps B-nanoparticle consisted of well - defined polyhedral structures with multiple facets . [SEP]
[CLS] aberration - corrected electron B-technique microscopy I-technique . [SEP]
[CLS] the performance of electron microscopes may be limited by spherical aberration , a feature of all round lenses that causes image distortion and limits the resolution . [SEP]
[CLS] the relatively recent development of aberration correctors for the objective lens resulted in a radical improvement in the resolution limits of haadf - stem microscopes . [SEP]
[CLS] the palmer group used aberration - corrected electron B-technique microscopy I-technique and atomistic computer simulations to demonstrate the hierarchy of metastability in the deposited , sizeselected au nanoclusters . [SEP]
[CLS] they have also investigated the atomic structure of the au 55 ( pph 3 ) 12 cl 6 schmid cluster by using aberration - corrected stem combined with the multislice simulation of stem images . [SEP]
[CLS] the combination of size - fractionation by the stem mass balance method and atomic structure determination in the aberration - correction regime might be able to reveal the isomeric structures of other types of nps B-nanoparticle too . [SEP]
[CLS] the use of chromatic aberration correction is in general expected to allow a much larger fraction of the incident electrons to be used to record high spatial resolution images than by using energy filtering . [SEP]
[CLS] in another report , aberrationcorrected stem was used to probe , one cluster at a time , the atomic structure of a statistical ensemble of 79 au clusters as a function of irradiation time . [SEP]
[CLS] each cluster contained 923 ± 23 atoms B-material . [SEP]
[CLS] midgley and co - workers used high resolution aberration - corrected electron B-technique microscopy I-technique and 3d electron B-technique tomography I-technique to localize au nps B-nanoparticle supported on tio 2 . [SEP]
[CLS] the aberration correctors in the haadf - stem imaging helped to gain insights into the atomic level structure critical to understanding the reactivity properties of nanocatalysts . [SEP]
[CLS] rellinghaus and colleagues used aberration - corrected hrtem for the quantitative measurement of the surface self - diffusion on au nps B-nanoparticle . [SEP]
[CLS] in another report , aberration - corrected stem provided the direct atomic - resolution imaging of surface migration , coalescence and atomic rearrangement of au clusters on a y : zro 2 support . [SEP]
[CLS] bimetallic nps B-nanoparticle , such as au / pd nps B-nanoparticle , have also been investigated by aberration - corrected stem . [SEP]
[CLS] ferrer et al . used this technique to study the atomic structure of three - layer au / pd nps B-nanoparticle , in combination with theoretical simulations and single particle diffraction . [SEP]
[CLS] the authors note that the aberration corrector offers the possibility to study atomic structures at a resolution lower than 0 . 1 nm , enabling the acquiring of more detailed information . [SEP]
[CLS] esparza et al . also used the technique under discussion for au - pd core - shell nps B-nanoparticle and they observed the presence of au nps B-nanoparticle with preferential surfaces enriched with pd atoms B-material . [SEP]
[CLS] these nps B-nanoparticle were synthesized using au nps B-nanoparticle as core B-material seeds and the final au - pd particles reached an average size of 5 . 5 nm . [SEP]
[CLS] ricolleau and co - workers performed aberration - corrected electron B-technique microscopy I-technique measurements and revealed in an unambiguous way the existence of long - range chemical orders in au - pd nps B-nanoparticle . [SEP]
[CLS] these ordered au - pd nps B-nanoparticle may offer a new class of advanced nanocatalysts for various chemical reactions . [SEP]
[CLS] jose - yacaman and co - workers combined aberration - corrected stem with spectral and chemical analysis stem - eds and stem - eels to identify and better understand the interface structure of pd - au nps B-nanoparticle . [SEP]
[CLS] the atomistic structure and alloying of pd - au - pd tri - layer nps B-nanoparticle were investigated . [SEP]
[CLS] in another report , co / au and pd / au nps B-nanoparticle were deposited on grids aiming to study the coalescence of the different metals B-material . [SEP]
[CLS] the as - synthesized materials ( co / au ) were sintered by thermal treatment or by strong beam irradiation and the subsequent characterization was performed in situ in an aberration corrected stem . [SEP]
[CLS] jian and palmer investigated the variation of the core B-material atomic structure of thiolated ( au x ag 1−x ) 321±55 nanoclusters with composition using aberration - corrected haadf - stem . [SEP]
[CLS] 15 demonstrates a comprehensive set of high - resolution haadf - stem images of auag alloy clusters with their respective simulated images ( for bare au 309 clusters ) . [SEP]
[CLS] cu - au core / shell clusters synthesized through cluster - beam synthesis were also analysed by aberration - corrected stem . [SEP]
[CLS] insights were obtained into the growth kinetics of the bimetallic clusters leading to the controlled , selective and efficient production of different metastable but practical core / shell np B-nanoparticle morphologies . [SEP]
[CLS] furthermore , herzing et al . showed that aberration - corrected stem - edx can provide important high spatial resolution compositional information on ( i ) alloy homogeneity and phase segregation effects within individual nps B-nanoparticle , ( ii ) particle - size composition correlations , ( iii ) the detection of trace amounts of the alloying element and ( iv ) metal B-material component distribution in extremely highly dispersed catalyst B-property systems for the case of au - ag and au - pd bimetallic np B-nanoparticle systems . [SEP]
[CLS] the disclinations in those decahedral pd nanostructures with d 5h symmetry were studied by aberration - corrected hrtem . [SEP]
[CLS] these researchers mentioned that the advantage of the aforementioned technique is to minimize the possibility of image artefacts that might confuse the geometric phase analysis of the nps B-nanoparticle . [SEP]
[CLS] the coalescence and sintering of small ( < 3 nm ) pt nps B-nanoparticle under the influence of the electron beam was also studied by aberration - corrected haadf - stem in real time . [SEP]
[CLS] the authors of that study showed that this technique allows single atomic columns to be clearly identified within each nanoparticle B-nanoparticle . [SEP]
[CLS] such measurements are significant in order to understand how particle size influences mass transport in nanoscale materials . [SEP]
[CLS] hashimoto et al . used aberration - corrected scanning confocal electron B-technique microscopy I-technique for the 3d analysis of pt nps B-nanoparticle on carbon B-material nanohorn aggregate supports . [SEP]
[CLS] in comparison with haadf - stem , the confocal electron B-technique microscopy I-technique improves the depth resolution because in the former method such resolution is limited by the lateral size of the objects and the illumination angle . [SEP]
[CLS] it is expected that a continuous improvement in aberration correction will enable the use of larger convergence and collection angles , or image - processing techniques , such as the deconvolution method may result in depth resolution values close to those theoretically predicted . [SEP]
[CLS] in this way , such approach can become a routine one for structural np B-nanoparticle characterization . [SEP]
[CLS] the same researchers used the aberration - corrected tem for the in situ observation of pt nps B-nanoparticle on graphene layers . [SEP]
[CLS] the structural changes and motions at the pt colloids under high temperature were also characterized by the assistance of eels measurements . [SEP]
[CLS] the ability of single atom B-material detection even at high temperature by aberration - corrected tem facilitates the understanding of the interactions between catalytic nps B-nanoparticle or atoms B-material and graphene on an atomic scale , resulting in the development of more efficient catalyst B-property - graphene composites . [SEP]
[CLS] pt / γ - al 2 o 3 nps B-nanoparticle ( pt clusters on an alumina support ) were investigated by sinkler et al . through a combined approach using aberration - corrected tem ( ac - tem ) and in situ xafs . [SEP]
[CLS] in comparison with stem , aberration - corrected tem uses a broad coherent electron beam and thus can offer advantages relative to stem for the structure determination of fine clusters ; this is because of the reduced tendency of the structures to deteriorate under the electron beam upon using ac - tem . [SEP]
[CLS] the complementarity of ac - tem with the xafs measurements is assured because it provides an ensemble - averaged view of the structures . [SEP]
[CLS] ling and zhang used aberration - corrected stem ( ac - stem ) to map the reactions of cr ( vi ) in fe nps B-nanoparticle . [SEP]
[CLS] 16 provides stem - eds elemental mapping of fe ( kα ) , cr ( kα ) , o ( kα ) and corresponding color overlays of the spent iron B-material nps B-nanoparticle after 24 h of reactions with hexavalent chromium B-material . [SEP]
[CLS] ortalan et al . demonstrated the use of ac - stem for the study of supported rh - ir clusters , combined with dynamic multislice image simulations , so as to identify individual atoms B-material , map the full structure and determine changes in the positions of metal B-material atoms B-material in sequential images . [SEP]
[CLS] the outmost goal of their approach was to help the development of new and improved catalysts B-property and other functional nanostructures . [SEP]
[CLS] the combination of ac - stem with the simulations provided the critical experimental input required to determine the full 3dstructure of a nanocluster composed of rh and ir atoms B-material . [SEP]
[CLS] the resolution achieved went down to the atomic scale and the authors confirmed the capacity of ac - stem to provide information on the size , shape , number and types of atoms B-material in a nanocluster , as well as how they change with processing conditions and under the influence of reactants [SEP]
[CLS] lead chalcogenide nps B-nanoparticle were also investigated using aberration - corrected stem ; in that study , pbse and pbte nps B-nanoparticle were chemically synthesized and a combination of electron diffraction , edxs , eels and ac - stem was employed to acquire information related to their morphology , crystal structure and composition . [SEP]
[CLS] the results obtained implied the presence of a np B-nanoparticle surface rich in pb and poor in chalcogen , with no oxygen B-material , and a clear c signal that might be attributed either to the supporting film or to the presence of carbon B-material in the capping layer as well . [SEP]
[CLS] electron energy loss spectroscopy B-technique ( eels ) is a family of techniques that measure the change in kinetic energy of electrons after their interaction with a material B-material . [SEP]
[CLS] the sample tested is exposed to a beam of electrons with a known , narrow range of kinetic energies . [SEP]
[CLS] some of the electrons will undergo inelastic scattering , which means that they lose energy and have their paths slightly and randomly deflected . [SEP]
[CLS] the amount of energy loss can be measured via an electron spectrometer and interpreted in terms of what caused the energy loss . [SEP]
[CLS] eels is typically used to identify the atomic structure and chemical properties of a sample , including : the type and quantity of atoms B-material present , chemical state of atoms B-material and collective interactions of atoms with their neighbors . [SEP]
[CLS] schaffer et al . compared energy - filtering tem ( ef - tem ) and stem - eels for the plasmon mapping of au nps B-nanoparticle in a monochromated tem . [SEP]
[CLS] they found out that the stem eels approach provides higher energy resolution , and thus allows the accurate mapping of peak positions , whereas the eftem technique provides spatially highly resolved information over large fields of view in a comparably short acquisition time . [SEP]
[CLS] it is thus the ideal technique to monitor long distance effects as encountered in coupled systems . [SEP]
[CLS] mccomb and co - workers demonstrate the use of eels - stem as a powerful tool for the study of lspr in silver B-material nps B-nanoparticle . [SEP]
[CLS] plasmon modes were highly sensitive to changes in local geometry and could be affected by electron beam damage , although special care in specimen preparation techniques could minimize such damage . [SEP]
[CLS] experimental data were in good agreement with theoretical predictions . [SEP]
[CLS] collins et al . noted that it is possible to combine eels with electron B-technique tomography B-technique in order to image surface plasmon resonances qualitatively at the nanoscale in a 3d mode . [SEP]
[CLS] the eigenmode tomography B-technique enables eels to analyse not a particular electron - induced response , but the underlying geometric modes characteristic of particle surface plasmons . [SEP]
[CLS] the precise optical analysis of single particles , particle ensembles and plasmonic devices is possible . [SEP]
[CLS] 17 shows an eels analysis of a plan - view silver right bipyramid that highlights many key issues in 2d imaging . [SEP]
[CLS] in another report , wei et al . noted that the high spatial and energy resolution eels - stem approach could be used to study several coupling interactions of a plethora of metal - semiconductor nanocomposite systems . [SEP]
[CLS] in particular these researchers observed a strong exciton - plasmon coupling between zno nanowires B-nanoparticle and ag nps B-nanoparticle by the monochromated eels - stem technique . [SEP]
[CLS] ni nps B-nanoparticle encapsulated in carbon B-material , prepared by rojas and colleagues , were characterized by tem , eels and eftem . [SEP]
[CLS] the combined analysis indicated that the ni nanocrystallites were surrounded by amorphous c , which provided some protection to the metallic ni from oxidation . [SEP]
[CLS] the eels technique records core level absorption edges in an analogous way to xas but provides information at a microscopic level . [SEP]
[CLS] in another report , the location and role of al in al - modified titania B-material nps B-nanoparticle were determined using low - temperature heat capacity , eels and xrd . [SEP]
[CLS] eels measurements confirmed that al entered the tio 2 lattice but it also indicated that the short - ranged structure around the al atoms B-material shifted from a tio 2 - like environment toward an al 2 o 3 - like environment , as the dopant concentration increased . [SEP]
[CLS] xrd showed that the long - range order of the nps B-nanoparticle decreased as the dopant concentration increased but retained a basic tio 2 - like structure . [SEP]
[CLS] the heat capacity data showed that lattice vacancies increased significantly with the addition of the al dopant , suggesting that the al 3 + cations B-material entered the titania lattice and created vacancies due to the charge difference between al 3 + and ti 4 + . [SEP]
[CLS] crozier and colleagues used monochromated eels - stem to measure bandgap states in individual non - stoichiometric praseodymium - ceria nps B-nanoparticle . [SEP]
[CLS] the authors of that study reported that the combination of eels and ac - stem offers new opportunities for the local nanoscale probing of bandgap states , and correlation with structure and chemistry at the 0 . 1 nm level . [SEP]
[CLS] eels allows the width and energy position of the state to be determined with respect to the top of the valence band , while optical observations of chemicallyinduced color changes are employed to provide further information on the energy shift of the inter - band state when the pr oxidation state is changed . [SEP]
[CLS] in that paper , high spatial and energy resolution monochromated eels helped to detect a state within the bandgap of [UNK] nm nps B-nanoparticle composed of pr x ce 1−x o 2−δ . [SEP]
[CLS] that inter - band state was associated with pr 4 + 4f levels . [SEP]
[CLS] the ultra - high resolution stem - eels permits inter - band states to be probed with high spatial resolution and should be applicable to other systems where nanocharacterization is necessary , such as grain boundaries , dislocations and precipitates . [SEP]
[CLS] de la fuente and co - workers used spatially - resolved eels to analyse the antibody B-material distribution on biofunctionalized core - shell fe 3 o 4 nps B-nanoparticle . [SEP]
[CLS] spatially resolved eels - stem analysis was performed on such biofunctionalized nps B-nanoparticle on the basis of its suitability to gain insight not only into the morphology and chemical composition of the np B-nanoparticle surface , but also into the direct visualization and spatial localization of the organic biomolecules . [SEP]
[CLS] in that report , the authors showed that their functional moieties ( i . e . the antibodies B-material ) were located only in specific areas of the np B-nanoparticle surface , namely those in which n was detected . [SEP]
[CLS] both biochemistry techniques and tem studies provided complementary information for the evaluation and understanding of the validity of their functionalization protocol . [SEP]
[CLS] another interesting ability of quantitative eels measurements is to distinguish the core B-material from the shell B-material in nps B-nanoparticle with such configuration , for example in the case of the mno x / mno y and feo x / mno x core / shell B-material , in a study published by peiro and colleagues . [SEP]
[CLS] the eels data obtained from spectrum lines across several nps B-nanoparticle showed that the mn oxidation state was 3 + at the outer part of the nps B-nanoparticle ( where no fe signal was found ) and decreased moderately towards the centre of the nps B-nanoparticle . [SEP]
[CLS] importantly , it appears that the power of the quantitative eels technique to resolve core B-material / shell B-material structures is sufficient even in cases where hrtem or haadf cannot distinguish them . [SEP]
[CLS] tem and relevant techniques give a two - dimensional ( 2d ) projection of three - dimensional ( 3d ) objects . [SEP]
[CLS] to tackle this problem , 3d electron B-technique microscopy I-technique or so - called ' electron B-technique tomography I-technique ' ( et ) has been developed . [SEP]
[CLS] apart from 3d structural information , the chemical composition can be analysed in 3d by combining the concepts of tomography B-technique with analytical tem techniques . [SEP]
[CLS] in this way , electron B-technique tomography I-technique is now considered as an important tool for the comprehension of the relation between the properties and structure of nps B-nanoparticle . [SEP]
[CLS] nowadays , et can provide quantitative 3d information down to the atomic scale . [SEP]
[CLS] in addition to nps B-nanoparticle , et can also be employed for the study of np B-nanoparticle assemblies . [SEP]
[CLS] with et , typically a tilt of photos ( snapshots ) is acquired by tilting the sample in tem over a large tilt angle range . [SEP]
[CLS] using a mathematical algorithm , the tilt series is combined into a 3d reconstruction of the original object . [SEP]
[CLS] in this manner , several different 3d images of the np B-nanoparticle are obtained , together with a video that is a sum of all projections . [SEP]
[CLS] an example of such reconstruction is shown in fig . 18 . [SEP]
[CLS] in that image , a reconstruction of the structure of au nanorods B-nanoparticle is shown . [SEP]
[CLS] such experiments allow the study of the influence of the synthesis method on the final shape of au nanorods B-nanoparticle . [SEP]
[CLS] 19 shows a schematic summary of the function mode of the et . [SEP]
[CLS] in the paper containing that figure , meurig thomas et al . have described the concept of compressed sensing ( cs ) that can be allied to the et aiming to use the resultant cs - et approach , especially for particles of organic or biological materials , which are particularly prone to damage by the electron beam . [SEP]
[CLS] the aim of compressed sensing is to obtain a signal from fewer measurements than would normally be required . [SEP]
[CLS] van aken and co - workers used et to study the growth of 1d - cupcf 16 nanostructures onto au nps B-nanoparticle . [SEP]
[CLS] to understand this growth , it is necessary to know the shape of the 1d nanostructure and its geometrical relationship with the au particle . [SEP]
[CLS] the experimental results from the combination of tomography B-technique and hrtem provided a detailed 3d insight into the structural properties of the 1d self - organization of cupcf 16 molecules onto the au nps B-nanoparticle , which resulted in the proposed growth model of the 1d nanostructures . [SEP]
[CLS] the benefits of et for the characterization of the precise morphology of coreshell au @ ag nps B-nanoparticle and its implications on their plasmonic properties were also analysed by coronado and colleagues . [SEP]
[CLS] in their paper , it is noted that et provides more statistically significant information on core - shell systems compared to other commonly used techniques . [SEP]
[CLS] bright - field tem ( bf - tem ) imaging may easily lead to artifacts upon 3d reconstruction , whereas haadf - stem matches much better with the requirements needed for tomographic applications ( e . g . minimal diffraction or phase contrast ) . [SEP]
[CLS] other noble metals B-material such as pd have also been investigated using haadf - stem - electron tomography B-technique . [SEP]
[CLS] pd nps B-nanoparticle with complex shapes were the subject of the study of berhault and colleagues . [SEP]
[CLS] such 3d approach is expected to yield quantitative information , such as angle measurements and facets indexing , deduced from the acquired tomogram . [SEP]
[CLS] shapes such as pentagonal rods and bipyramids were among the pd nanostructures monitored in that study . [SEP]
[CLS] florea et al . used et to study the selective deposition of pd nps B-nanoparticle inside the bimodal porosity of β - sic . the spatial distribution and connection of the porous network of the medium surface area β - sic synthesized through the gas - solid reaction were investigated by et . [SEP]
[CLS] the obtained results illustrated the unique character of the et to shed light on the morphology , internal structure , and spatial distribution of a nanoscale material B-material . [SEP]
[CLS] such information is crucial for the field of catalysis , for instance . [SEP]
[CLS] in another report , blacher and co - workers monitored the localization of pd nps B-nanoparticle within their silica support , in two heterogeneous catalysts B-property prepared by the sol - gel method , with different metal B-material loadings . [SEP]
[CLS] by using et , it was found that the presence of artifacts was associated with an overestimation of the size of the pd nps B-nanoparticle . [SEP]
[CLS] the resolution of the tomograms could be roughly estimated as the ratio of the thickness of the sample to the number of projections used for the reconstruction . [SEP]
[CLS] the pd nps B-nanoparticle were located deep inside the silica skeleton . [SEP]
[CLS] it was found that the dispersion manner of the pd nps B-nanoparticle also partially depended on their loading amount . [SEP]
[CLS] et has also been employed for the characterization of pt nanostructures , such as nanodendrites and small nps B-nanoparticle entrapped in zeolite . [SEP]
[CLS] the quantitative and qualitative location of the latter particles was achieved through et . [SEP]
[CLS] h 2 ptcl 6 was used as the pt source for the impregnation of ultrastable ( usy ) zeolite support and 3 - 4 nm pt nps B-nanoparticle did not show any preferential location in mesopores or at the surface of the crystals . [SEP]
[CLS] the size distribution as well as the distances between pt nps B-nanoparticle were also identified . [SEP]
[CLS] a comparison of the size values with those obtained by exafs was also included in that work . [SEP]
[CLS] ricolleau and colleagues published a paper on the comparison of et and hrtem slicing methods as tools to measure the thickness of copt nps B-nanoparticle deposited on a substrate . [SEP]
[CLS] regarding thickness measurements , et presents several advantages over hrtem , although the precision of the experiments obtained by these two techniques is similar . [SEP]
[CLS] et is a more direct ( in what concerns the recording of the thickness value ) and readily statistical approach , since more particles can be analysed at once . [SEP]
[CLS] concerning imaging modes , the bright - field one is associated with the presence of artifacts , as mentioned before . [SEP]
[CLS] the haadf - stem mode avoids the occurrence of diffraction contrast problems , but bf - tem involves short exposure time for each image , less distortion due to a residual specimen drift , limited sample contamination and small image distortion due to electrical instability . [SEP]
[CLS] iron B-material oxide I-material nps B-nanoparticle have also been extensively studied by et : for example , midgley and colleagues applied the compressed sensing - et approach in order to reveal the morphology of iron B-material oxide I-material nps B-nanoparticle with reactive concave surfaces in great detail , and with fewer artifacts in comparison with the use of more ' classic ' reconstruction algorithms . [SEP]
[CLS] the reduction of missing wedge and star artifacts allows the simpler and more objective segmentation of tomograms , leading to a greater reliability of the 3d quantitative analysis of nanostructures . [SEP]
[CLS] only a few projections are enough for the reconstruction of decent tomograms using cs - et , thus showing the ability of this technique for rapid data acquisition . [SEP]
[CLS] in another report , magnetic B-property np B-nanoparticle composites with a fe 3 o 4 core B-material and a hydroxyapatite coating B-material were synthesized using the precipitation method followed by hydrothermal treatment . [SEP]
[CLS] the combination of energy - filtered tem ( ef - tem ) and 3d - reconstructured electron B-technique tomography I-technique demonstrated that the nanocomposites consisted only of needle - like hydroxyapatite nanocrystals coating B-material the magnetite spherical nps B-nanoparticle which had internal nanopores . [SEP]
[CLS] the capacity of the compressed sensing anisotropic total variation algebraic reconstruction technique ( csatv - et ) to improve the quality and accuracy of tomograms using fewer datasets when compared to more ' common ' reconstruction techniques ( e . g . sirt and bp ) was illustrated by monsegue et al . also for the case of hematite nps B-nanoparticle . [SEP]
[CLS] scanning electron B-technique microscopy I-technique ( sem ) is a widely used method for the high - resolution imaging of surfaces that can be employed to also characterize nanoscale materials . [SEP]
[CLS] sem uses electrons for imaging , much as a light microscope uses visible light . [SEP]
[CLS] mazzaglia et al . combined field - emission sem ( fe - sem ) and xps measurements to study supramolecular colloidal systems of au nps B-nanoparticle / amphiphilic B-property cyclodextrin . [SEP]
[CLS] these two techniques provided important information on the morphology and nature of the interaction of ( thiohexyl carbon B-material chain ) sc 6 nh 2 and ( thiohexadecyl carbon B-material chain ) sc 16 nh 2 with au nps B-nanoparticle onto the silicon B-material surface . [SEP]
[CLS] sinclair and co - workers have reported that sem and nanosims can be employed to locate au nps B-nanoparticle in cells B-material . [SEP]
[CLS] sem analysis illustrated its superiority compared to nanosims for the analysis of inorganic nps B-nanoparticle in complex biological systems . [SEP]
[CLS] nanosims has a lower spatial resolution of around 50 nm while sem is able to achieve resolutions down to 1 nm . [SEP]
[CLS] the particles tested were raman - active au - core B-material nps B-nanoparticle and nanosims resulted in somewhat blurred images in certain cases due to its limited resolution . [SEP]
[CLS] however , nanosims has the unique capability to differentiate between isotopes B-material , although this is not relevant for the case of au nps B-nanoparticle . [SEP]
[CLS] high - resolution sem ( hrsem ) was used by goldstein et al . for the imaging of au nps B-nanoparticle in cells B-material and tissues . [SEP]
[CLS] the straightforward visualization of metallic nps B-nanoparticle is assured with this technique , and the sample preparation is fast and easy . [SEP]
[CLS] however , in case of biological specimens , the need to decrease charging artefacts might make metal B-material coating B-material necessary , thus increasing the risks of radiation damage for the samples . [SEP]
[CLS] the advantage of hrsem , compared to other imaging B-technique techniques I-technique , is the capacity to scale down and study the arrangements of nanometric elements in their wider context . [SEP]
[CLS] it allows the study of the specific spatial arrangement of nps B-nanoparticle and thus the examination of possible interactions between them . [SEP]
[CLS] the results of that study indicated the potential of hrsem as a relatively simple tool to qualitatively screen the factors that enhance au nps B-nanoparticle penetration , through the skin barrier B-property . [SEP]
[CLS] it can be considered as a powerful and diverse tool for the study of the interactions between biological systems and metallic nanostructures . [SEP]
[CLS] in another report , sem and afm measurements were compared for the same set of nps B-nanoparticle , that is , sio 2 and au nps B-nanoparticle on mica or silicon B-material substrates . [SEP]
[CLS] for example , afm observations enabled the measurement of the height of a nano B-nanoparticle - I-nanoparticle object I-nanoparticle with sub - nanometric accuracy , but the lateral measurements ( along the x and y axes ) had large errors because of the tip / sample convolution . [SEP]
[CLS] in contrast to the afm , sem cannot provide any metrological information on the height of the nps B-nanoparticle ; however , modern sem can give decent measurements of their lateral dimensions . [SEP]
[CLS] in fact , the measurements of nearly spherical sio 2 nps B-nanoparticle by using both techniques gave similar results , showing the coherency and complementarity of both instruments . [SEP]
[CLS] sem can be operated in the transmission mode , i . e . through the technique called ' transmission in scanning electron microscope ' ( t - sem ) ( see fig . 20 ) . [SEP]
[CLS] in the transmission mode , advanced np B-nanoparticle analysis can be carried out by gaining in - depth information as well as analysis of ensembles of nps B-nanoparticle . [SEP]
[CLS] in a paper by rades et al . , the combination of complementary techniques as sem , t - sem , edx and scanning auger microscopy B-technique ( sam ) was proven to be a powerful strategy for comprehensive morphological and chemical evaluation of the properties of individual silica and titania B-material nps B-nanoparticle . [SEP]
[CLS] on the other hand , methods such as saxs , dls , xps , xrd and bet would be suitable to characterize only the ensembles of the nps B-nanoparticle , and not single particles . [SEP]
[CLS] t - sem allows a quick examination of the np B-nanoparticle shape , though its lateral resolution is limited to np B-nanoparticle sizes down to 5 - 10 nm . [SEP]
[CLS] tem provides images with better quality , but t - sem can be easily combined with edx for a fast check of the np B-nanoparticle size and elemental composition . [SEP]
[CLS] hodoroaba et al . proved that t - sem imaging provides a size distribution that is slightly broader than that obtained by tem . for small sio 2 nps B-nanoparticle , the precise delimitation of the particles in the t - sem mode is definitely constrained by the lower spatial resolution achieved compared to that of conventional tem . [SEP]
[CLS] in addition , with the t - sem , the surface layer of the particles might not be always easily detected . [SEP]
[CLS] the same author noted in another paper that the conventional sem imaging mode could not detect the nps B-nanoparticle on the back side of the support film that was required for the observations . [SEP]
[CLS] therefore , an explicit knowledge of the t - sem operator is needed for the measurements . [SEP]
[CLS] the authors observed that the obtained sio 2 np B-nanoparticle size distributions by sem and tsem in their work and for various conditions agreed well with each other , within the associated measurement uncertainties . [SEP]
[CLS] in another report , 3d reconstruction by focused ion B-material beam ( fib ) cutting and sem imaging were combined to comprehend the evolution of pore volume , pore shape and other parameters during the two - step sintering of zno nps B-nanoparticle . [SEP]
[CLS] in this way , the sintering process at the nanoscale for such particles can be better understood . [SEP]
[CLS] ni - and cu - co - doped zinc B-material oxide I-material nps B-nanoparticle prepared by the co - precipitation method were investigated by ashokkumar and muthukumaran by microstructure , optical and ftir measurements . [SEP]
[CLS] the depicted shape by sem was in good agreement with the mathematical determinations from xrd , whereas ftir provided important information on chemical bonding . [SEP]
[CLS] electron backscatter diffraction ( ebsd ) is a microstructuralcrystallographic characterisation technique for the study of crystalline or polycrystalline materials , including nanoscale ones . [SEP]
[CLS] this technique aims at the comprehension of the structure , crystal orientation , and phase of the materials in sem . [SEP]
[CLS] normally , ebsd is employed to examine microstructures , revealing texture , defects , grain morphology and deformation . [SEP]
[CLS] high resolution and non - destructive analysis of these parameters can be achieved . [SEP]
[CLS] in ebsd , an electron beam hits a sample that is tilted at an angle of typically 70°towards the detector . [SEP]
[CLS] the detector , usually a phosphor screen , which captures the inelastically backscattered electrons from the sample surface , is able to make a diffraction pattern . [SEP]
[CLS] ebsd can effectively improve the statistics of the analysis of nps B-nanoparticle compared to tem , thus giving a better overview of a larger ensemble of nps B-nanoparticle . [SEP]
[CLS] the heteroepitaxial relationship of au nps B-nanoparticle with an average size of 60 - 80 nm on ( 001 ) [UNK] 1 ba 2 cu 3 o 7−δ has been investigated by ebsd . [SEP]
[CLS] in that case the small size of au nps B-nanoparticle compared to the spatial resolution of the ebsd caused a certain challenge on the orientation analysis . [SEP]
[CLS] in another report , ebsd was used to directly measure the crystallographic orientation of embedded y 2 bacuo 5 and y 2 ba 4 cumo x nps B-nanoparticle in melt - textured ybco , with a spatial resolution of around 40 nm . [SEP]
[CLS] the researchers of that study aimed to explore how the behaviour of the superconducting matrix was modified upon embedding a certain quantity of nps B-nanoparticle . [SEP]
[CLS] the interactions between these nps B-nanoparticle and the surrounding ybco matrix were studied . [SEP]
[CLS] a novel finding of that work concerned the observation of twin boundaries within the melt - textured ybco samples through the use of ebsd . [SEP]
[CLS] the ebsd analysis showed that the addition of depleted uranium B-material oxide B-material had a remarkable effect on the resulting microstructure of the meltprocessed ybco samples . [SEP]
[CLS] the same group found out that a homogeneous ybco matrix can be formed , even though a large number of embedded particles are present . [SEP]
[CLS] small et al . reported that the primary cause of the reduced ebsd pattern quality from nps B-nanoparticle is an increase in the diffuse background contribution or noise resulting from electron penetration through the small particles into thick , amorphous mounting substrates and not the loss of the coherent scattering intensity . [SEP]
[CLS] it was suggested that designing an ebsd sample holder that accommodates particles mounted on thin film substrates would help to decrease radically the background produced as a result of electron interactions with the mounting substrate . [SEP]
[CLS] this would lead to an increase in pattern quality , extending the application of ebsd phase identification analysis to relatively low - z , low - ρ particles as small as 120 nm in size . [SEP]
[CLS] atomic B-technique force I-technique microscopy I-technique ( afm ) is a microscopy B-technique technique capable of creating three - dimensional images of surfaces at high magnification . [SEP]
[CLS] it was initially developed by gerard binning and heinrich rohrer at ibm in 1986 . [SEP]
[CLS] afm is based on measuring the interacting forces between a fine probe and the sample . the probe is a sharp tip and is coupled to the end of a cantilever , which is made of silicon B-material or silicon nitride . [SEP]
[CLS] when the afm scans the sample , the cantilever gets deflected as a result of the attractive or repulsive forces between the tip and the sample surface ( fig . 21a ) . [SEP]
[CLS] the bending is quantified by a laser beam that reflects on the cantilever back side . [SEP]
[CLS] the forces are finally calculated by combining the information from the laser variation and the known cantilever stiffness . [SEP]
[CLS] afm can scan under three different modes depending on the degree of proximity between the probe and the sample , i . e . contact , non - contact and tapping mode ( also known as intermediate B-property or oscillating mode ) . [SEP]
[CLS] the latter is the most common when characterizing nps B-nanoparticle . however , it is very sensitive to the free amplitude of the oscillating tip . [SEP]
[CLS] in addition , other parameters , such as tip curvature radius , and surface energy and elasticity of the nanoparticle B-nanoparticle , influence the final topological values . [SEP]
[CLS] nevertheless , these factors can be minimized by plotting the particle height against the free amplitude of the oscillating probe , providing more reliable results . [SEP]
[CLS] alternatively , non - contact is preferred when the sample is very sensitive and can be influenced by the tipsample forces . [SEP]
[CLS] afm has the advantage that it does not require any surface modification or coating B-material prior to imaging . [SEP]
[CLS] thus , the topological analysis of small nps B-nanoparticle ( ≤ 6 nm ) , such as ion - doped y 2 o 3 , has been performed by afm without any special treatment . [SEP]
[CLS] low density materials , which present poor contrast in electron B-technique microscopy I-technique , have also been characterized . [SEP]
[CLS] for instance , afm was used to understand the formation mechanism of uniform patchy and hollow rectangular nanoplatelets made of polymer B-material mixtures . [SEP]
[CLS] side - by - side comparison between afm and electron B-technique microscopies I-technique , i . e . sem and tem , showed that afm provided comparable results when analysing np B-nanoparticle sizes . [SEP]
[CLS] afm has the advantage that images the sample in three dimensions and allows the characterization of the nanoparticle B-nanoparticle height . [SEP]
[CLS] furthermore , it has similar resolution to sem and tem , while costing much less and occupying smaller laboratory space . [SEP]
[CLS] nevertheless , afm displays slower scanning times than any electron microscope . [SEP]
[CLS] alternatively , spectroscopic techniques , such as dls and photon correlation spectroscopy B-technique ( pcs ) , have also been used to characterize the nanoparticle B-nanoparticle size . [SEP]
[CLS] dls and afm provided similar results when the sample analysed was monodisperse and uniform . [SEP]
[CLS] 523 however , only afm could properly characterize nps B-nanoparticle with bimodal distribution sizes . [SEP]
[CLS] the study of alumina nanopowder formation in the solvent by ultrasonic treatment , and posterior sedimentation to a thin film , showed that correlated information could be obtained by pcs and afm , even though the former analysed liquid samples and the latter solid ones . [SEP]
[CLS] the combination of different technique strengths , such as the high magnification of hrtem and height measurements of afm , has helped to understand longstanding problems in nanoscience , like the role of the dendrimer B-nanoparticle template on the growth of pt nps B-nanoparticle . [SEP]
[CLS] 526 afm and xrd were jointly used to characterize ag np B-nanoparticle films , where both techniques provided complementary information . [SEP]
[CLS] in particular , afm allowed the characterization of the grain size and the nanoparticle B-nanoparticle coverage of the surface , while xrd identified the preferential growth direction of the particles . [SEP]
[CLS] interestingly , at higher np B-nanoparticle coverage , afm showed that the film was made of larger particle grains . [SEP]
[CLS] nevertheless , xrd indicated that the crystal size remained the same . [SEP]
[CLS] this apparent contradiction suggested that the larger particles were formed by coalescence of different crystals , yielding larger polycrystalline grains . [SEP]
[CLS] it is worth mentioning that a densely packed nanoparticle B-nanoparticle film can be challenging to characterize by afm , since part of the particles are hidden by their neighbours . [SEP]
[CLS] therefore , several algorithms have been developed to estimate the nanoparticle B-nanoparticle size from the visible part of the image . [SEP]
[CLS] these algorithms can be applied to densely packed spherical and non - spherical particles . [SEP]
[CLS] the catalytic activity of rh nps B-nanoparticle in the polymerization of phenylacetylene was characterized by afm and tem . [SEP]
[CLS] both techniques were able to track the formation of poly - phenylacetylene fibers around the nps B-nanoparticle . [SEP]
[CLS] however , only tem could solve the pitch of the polymer B-material helical structure . [SEP]
[CLS] afm has also been used to characterize different np B-nanoparticle - based metal B-material substrates for sers sensing ( fig . 22 ) . [SEP]
[CLS] different parameters , such as np B-nanoparticle composition , size , shape and surface properties , were correlated to the measured enhanced factors , and near single molecule detection limit was achieved for one of the substrates . [SEP]
[CLS] in addition , afm was further employed to study the sers phenomenon at the single - np B-nanoparticle level . [SEP]
[CLS] lastly , afm and kelvin probe force microscopy B-technique could be combined to generate three - dimensional maps of nanoparticle B-nanoparticle surface potential distributions . [SEP]
[CLS] these were obtained by monitoring the corrosion behaviour of individual iron B-material and stainless steel nps B-nanoparticle under a sulfuric B-material acid environment ( fig . 23 ) . [SEP]
[CLS] magnetic B-technique force I-technique microscopy I-technique ( mfm ) is a type of scanning B-technique probe I-technique microscopy I-technique where a magnetic B-property probe is used to raster - scan the sample surface , of which its magnetic B-property field interacts with the magnetic tip to offer insight into the magnetic B-property properties ( fig . 21b ) . [SEP]
[CLS] mfm has the ability to separate the magnetic B-property interactions from the other tip sample forces ( such as van der waals , and other forces recorded in afm ) . [SEP]
[CLS] the most common measurement method is called the ' two - pass technique ' in which the sample is scanned twice , once to produce an afm image , and a second time to produce an mfm one . [SEP]
[CLS] the mfm advantages include its non - destructive character , lack of surface preparation or np B-nanoparticle modification , and no use of labels or tags . [SEP]
[CLS] mfm allows the determination of the magnetic B-property moment of a single np B-nanoparticle and study of how this measurement changes with the np B-nanoparticle size as well as probe distance from the sample , something which bulk magnetic B-property analysis is not capable of . [SEP]
[CLS] the development of mfm operating in liquid brings excellent possibilities of the studies of magnetic B-property nps B-nanoparticle under biologically relevant conditions , such as in the interior of cells B-material . [SEP]
[CLS] in addition , mfm can operate under ambient conditions , at varying temperatures and in a ultra - high vacuum environment . [SEP]
[CLS] furthermore , it can provide a resolution down to less than 10 nm . [SEP]
[CLS] neves et al . published a paper on how mfm can be used to discriminate between magnetic B-property and nonmagnetic nps B-nanoparticle . [SEP]
[CLS] mfm can detect and localize the magnetic B-property fields arising from nanoscopic magnetic B-property domains , such as magnetic B-property nps B-nanoparticle . [SEP]
[CLS] unfortunately , there are cases where mfm can give a strong response even on non - magnetic B-property nps B-nanoparticle or under circumstances where no magnetic B-property interaction would be expected , potentially giving rise to misleading results . [SEP]
[CLS] the magnetic B-property field from nps B-nanoparticle and consequently the phase shift that is detected in mfm depend very strongly on the particle diameter . [SEP]
[CLS] mfm is sensitive to magnetic B-property fields coming from magnetic B-property nps B-nanoparticle with diameters around 40 - 60 nm . [SEP]
[CLS] on the other hand , the evaluation by mfm of small ferromagnetic or superparamagnetic B-property nps B-nanoparticle is particularly challenging : in this case , the formation of the mfm contrast takes place under conditions of strong interaction of the probe field and the particle magnetic B-property moment , which complicates the interpretation of the experimental results . [SEP]
[CLS] the application of an external magnetic B-property field can result in the redistribution of the magnetic B-property contrast from low - coercive co nps B-nanoparticle , as reported by mironov et al . , making it possible to distinguish between the contribution of the magnetic B-property and van der waals interactions to the generation of a phase contrast of the mfm images . [SEP]
[CLS] therefore , the observation of mfm contrast from superparamagnetic B-property co nps B-nanoparticle smaller than 10 nm is still possible under magnetic B-property moment stabilization in a strong external magnetic B-property field . [SEP]
[CLS] asenjo and co - workers employed mfm for the study of superparamagnetic B-property versus blocked states in aggregated of fe 3−x o 4 nps B-nanoparticle . [SEP]
[CLS] two distinct magnetic B-property behaviours were observed depending on the particle size . [SEP]
[CLS] aggregates of nps B-nanoparticle of about 11 and 49 nm in size were investigated . [SEP]
[CLS] for the former sample , a homogeneous attractive tip - sample interaction was observed , displayed as a uniform dark contrast on the mfm images , arising from the coherent rotation of the spins within the aggregate as they align along the tip stray - field . [SEP]
[CLS] this reflected the predominant superparamagnetic B-property character of these small nps B-nanoparticle within the characteristic acquisition time of the mfm technique and at zero applied field . [SEP]
[CLS] for the sample with the 49 nm np B-nanoparticle aggregates , dark / bright contrast associated with the existence of magnetic B-property domains and magnetization B-property - polarization prevailed in the mfm images all along the magnetic B-property cycle . [SEP]
[CLS] this happened due to the fact that the net magnetization B-property of these large particles remained blocked during the acquisition time of the mfm images , even at zero applied field . [SEP]
[CLS] athanassiou and colleagues have published a study on a quantitative , high spatially resolved mfm imaging of samples based on 11 nm diameter superparamagnetic B-property iron B-material oxide I-material nps B-nanoparticle in air at room temperature , characterizing magnetic B-property textures down to the single particle level . [SEP]
[CLS] energy loss imaging in the tapping mode can provide high compositional sensitivity and magnetic B-property features as small as a few tens of nanometers lying under the surface were pointed out by mfm , whereas topographical imaging alone would not be able to detect them . [SEP]
[CLS] in this section we provide some examples of the literature where different methods are used at given samples to characterize their size , as this property is one of the most basic ones for nps B-nanoparticle and it deserves special attention . [SEP]
[CLS] akbari et al . used tem , pcs , bet and xrd to evaluate the size and size distribution of alumina nps B-nanoparticle . [SEP]
[CLS] the np B-nanoparticle size was found to be in the range of 5 - 95 nm . [SEP]
[CLS] xrd and tem size values were in agreement for these particles . [SEP]
[CLS] the authors of that study mention that pcs is well suited for the measurement of narrow particle size distribution in the range of 1 - 500 nm , but for systems where agglomeration occurs , comparison with other methods is rec - [SEP]
[CLS] focus on np B-nanoparticle sizedistinct examples of characterization with different techniquesommended . [SEP]
[CLS] the size value deduced from bet was also in accordance with the ones derived by tem and xrd , as expected for particles with a spherical shape , but the recorded pcs value was higher . [SEP]
[CLS] gollwitzer et al . compared several techniques for the size measurement of silica nps B-nanoparticle dispersed in water B-material and in the cell B-material culture medium . [SEP]
[CLS] the techniques used were dls , cls ( centrifugal liquid sedimentation ) , saxs and pta ( particle tracking analysis ) . [SEP]
[CLS] pta is practically the same as nta , but pta is a more generic term which covers a larger range of particle sizes . [SEP]
[CLS] the dls results in the cell B-material culture medium differed to a significant extent from the other methods , due to the presence of agglomerates , which diminish the dls accuracy . [SEP]
[CLS] the particle agglomeration caused by the cell B-material culture medium resulted in a significant size increase in pta , whereas the np B-nanoparticle size value remained stable for saxs and cls measurements . [SEP]
[CLS] saxs offered highly precise values while cls yielded detailed size distributions from which further information on the agglomeration state can be derived . [SEP]
[CLS] minelli and co - workers used tunable resistive pulse sensing ( trps ) , dcs and dls to measure the size of silica nps B-nanoparticle in serum . [SEP]
[CLS] also in this case , dls precision was not sufficient because of the presence of agglomerates . [SEP]
[CLS] dcs and trps values were quite similar , though . [SEP]
[CLS] the agglomeration measured by dcs was more significant than that observed by trps . [SEP]
[CLS] in fact , in contrast to dls and dcs , trps performs particle - by - particle measurements , providing a statistical distribution of the data across a np B-nanoparticle sample rather than average results . [SEP]
[CLS] the researchers authoring that study confirmed that trps is a sensitive and high resolution technique in the characterization of nps B-nanoparticle in biological media . [SEP]
[CLS] in another report , a certified reference material B-material , erm - fd100 , composed of sio 2 nps B-nanoparticle with a nominal equivalent spherical diameter of 20 nm , was characterized by researchers from 34 laboratories using dls , cls , em ( tem / sem ) , saxs and els . [SEP]
[CLS] participants from both the industry and academic institutions showed that a good agreement for the results by different methods was confirmed . [SEP]
[CLS] the good comparability of results enabled the certification of the colloidal sio 2 materials for np B-nanoparticle size analysis . [SEP]
[CLS] the size measurement uncertainties of nearmonodisperse , near - spherical nps B-nanoparticle composed of reference gold B-material and polystyrene materials were compared in a paper by mast and colleagues . [SEP]
[CLS] pta proved to be a precise and non - biased method for the determination of the modal hydrodynamic diameter in the range of 30 - 200 nm . [SEP]
[CLS] tem was accurate and nonbiased for the measurement of the mean area - equivalent circular diameter in the size range between 8 and 200 nm of the investigated near - monomodal near - spherical materials . [SEP]
[CLS] therefore , pta was found to be a good alternative to tem for measuring the np B-nanoparticle size , with the exception of 8 . 9 nm au nps B-nanoparticle , because that sample had a size below the detection limit of the former technique . [SEP]
[CLS] carney et al . described a 2d analytical ultracentrifugation approach for the determination of the size , density and molecular weight distributions of gold - based nps B-nanoparticle . [SEP]
[CLS] the extracted values for the sedimentation and diffusion coefficients from the analytical ultracentrifugation helped to find the above - mentioned parameters . [SEP]
[CLS] shard and coworkers used pta to quantify the igg protein B-material adsorption to gold B-material nps B-nanoparticle . [SEP]
[CLS] in the low protein B-material coverage regime , the measured amount of protein B-material depended upon the technique : nta and dls gave similar values that correlated well with the plasmon frequency shift . [SEP]
[CLS] dcs analysis underestimated the protein B-material shell B-material thicknesses in that regime . [SEP]
[CLS] dls and nta measurements resulted in larger diameters for the citrate - capped au nps B-nanoparticle than those provided by the supplier , with the dcs method giving smaller diameters . [SEP]
[CLS] dls and nta assess np B-nanoparticle size from the analysis of brownian motion and should be expected to result in identical diameters for a monodisperse sample . [SEP]
[CLS] it was noted that dcs was more precise than either dls or nta and less prone to artifacts . [SEP]
[CLS] apart from aggregates , dls is also sensitive to impurities B-property and nta is statistically limited by the number of significant observations of the position that can be made on a single particle before it moves from the field of view . [SEP]
[CLS] in another report , the combination of techniques such as waxs and dls that are sensitive to different characteristics of colloidal particles ( au , ni ( oh ) 2 ) , such as crystalline core B-material and overall size , permitted the estimation of the thickness of polymeric stabilizing layers . [SEP]
[CLS] waxs was efficient for the determination of the size and shape of dispersed colloidal particles . [SEP]
[CLS] finally , in a study concerning ag nps B-nanoparticle , quasielastic light scattering was employed for size measurement , and it was reported that it achieved rapid measurements , with a slightly higher size in comparison with tem . [SEP]
[CLS] this review described the role of several different techniques for the characterization of nanoscale materials . [SEP]
[CLS] through this comprehensive summary of np B-nanoparticle characterization methods , we demonstrated the uses of each one of them , emphasizing on their advantages and limitations , as well as on explaining how they can be effectively combined and how they can complement each other . [SEP]
[CLS] the acquisition of a full picture of the variety of features that are associated with a nanomaterial B-material requires typically the use of numerous techniques , often needing to use more than one of them for evaluating well and completely even a single property . [SEP]
[CLS] by presenting the role of each technique in a comparative way , our review will act as a robust guide , helping the scientific community to understand better the discussed topic . [SEP]
[CLS] in this way , researchers will be helped for the choice of the most suitable techniques for their characterization , together with the ability to assess their use in a more precise manner . [SEP]
[CLS] of course , there are challenges in the scientific community for the further improvement of the accuracy and resolution of many techniques . [SEP]
[CLS] therefore , we finally hope that a careful reading of this review will help to identify which valuable techniques merit efforts for further technical improvements . [SEP]
[CLS] roger m . pallares roger m . pallares received his bsc and msc degrees in chemistry from the ramon llull university ( barcelona , spain ) in 2009 and 2011 , respectively . [SEP]
[CLS] after a year working on the growth of 2d nanomaterials B-material at ntt basic research laboratories ( atsugi , japan ) , he started a joint doctoral program between the university college london ( ucl , london , uk ) and the agency for science , technology and research ( singapore ) , obtaining a phd in materials science from ucl in 2016 . [SEP]
[CLS] he is currently a postdoctoral fellow at northwestern university ( evanston , il ) . [SEP]
[CLS] his research interests focus on the use of nanomaterials B-material for biomedical applications . [SEP]
[CLS] 1 schematic presentation of the in situ setup employed for realtime saxs / waxs / uv - vis measurements during the formation of au nps B-nanoparticle . [SEP]
[CLS] the setup measures saxs , waxs and the uv - vis spectra simultaneously in the same sample volume . [SEP]
[CLS] reprinted with permission from ref . 43 . [SEP]
[CLS] copyright 2015 american chemical society [SEP]
[CLS] / 1200 - 1100 organic siloxane or silicone B-material ( si - o - si ) 1095 - 1075 / 1055 - 1020 organic siloxane or silicone B-material ( si - o - c ) 1100 - 1080 thiols B-material ( s - h stretch ) 2600 - 2550 thiol B-material or thioether B-material , ch 2 - s - ( c - s stretch ) 710 - 685 aliphatic chloro - compounds , c - cl stretch 800 - 700 ammonium ion B-material 3300 - 3030 / 1430 - 1390 [SEP]
[CLS] lara et al . decomposed [ ru ( cod ) ( cot ) ] [ ( 1 , 5 - cyclooctadiene ) ( 1 , 3 , 5 - cyclooctatriene ) ruthenium B-material ] [SEP]
[CLS] 2 optical configuration of the typical experimental setup for dynamic light measurements of a nanoparticle B-nanoparticle suspension . [SEP]
[CLS] the setup can be operated at multiple angles . [SEP]
[CLS] reproduced with permission from ref . 166 . copyright 2013 springer . [SEP]
[CLS] 3 schematic of the optical configuration used in nta . [SEP]
[CLS] reprinted with permission from ref . 177 . copyright springer 2013 . [SEP]
[CLS] spicp - ms was also employed by yang and co - workers to analyse ag and au nps B-nanoparticle in environmental water B-material . [SEP]
[CLS] the size distribution of these ag and au dispersions was in accordance with the tem results . 192 [SEP]
[CLS] 4 scheme of the processes involved in the icp - ms analysis of au nps B-nanoparticle with ( a ) and without ( b ) previous gold B-material dissolution . [SEP]
[CLS] reprinted with permission from ref . 188 . copyright 2003 springer . [SEP]
[CLS] 5 scheme of probing nps B-nanoparticle ( nps B-nanoparticle ) by using tof - sims . [SEP]
[CLS] polyatomic or monoatomic bombardment on the surface generates different types of secondary ions B-material from metal B-material nps B-nanoparticle that can be encapsulated or conjugated with ligands or biomolecules . [SEP]
[CLS] reproduced with permission from ref . 207 . [SEP]
[CLS] copyright 2015 wiley - vch [SEP]
[CLS] 6 scheme of the experimental setup for the np B-nanoparticle magnetization B-property measurements . [SEP]
[CLS] the variation of the critical current is obtained by averaging the switching current events measured by using a time of flight technique . [SEP]
[CLS] the resolution of the critical current measurements is about 1 part in 10 4 . [SEP]
[CLS] the feedback circuit allows the increase of the linear dynamic range of the sensor . [SEP]
[CLS] the picture shows the holder including the sample and the multiturn feedback coil . [SEP]
[CLS] reprinted with permission from ref . 257 . [SEP]
[CLS] copyright 2013 springer [SEP]
[CLS] 7 schematic diagram of a transmission mossbauer spectrometer system . [SEP]
[CLS] reprinted with permission from ref . 267 . copyright 2004 elsevier . [SEP]
[CLS] similar [SEP]
[CLS] 8 mrx experimental setup . [SEP]
[CLS] ( 1 ) lihe dewar ; ( 2 ) optional superconducting quantum interference detector ( squid ) magnetometer channel ( squid sensor not shown ) ; ( 3 ) seven - channel second - order squid gradiometers ( squid sensors not shown ) ; ( 4 ) helmholtz coil ; and ( 5 ) manually controlled non - magnetic B-property 3d stage with optical readout . [SEP]
[CLS] reprinted with permission from ref . 352 . [SEP]
[CLS] copyright 2015 degruyter . [SEP]
[CLS] 9 the depicted superlattices are assembled from a , 13 . 4 nm γ - fe 2 o 3 and 5 . 0 nm au ; b , 7 . 6 nm pbse and 5 . 0 nm au ; c , 6 . 2 nm pbse and 3 . 0 nm pd ; d , 6 . 7 nm pbs and 3 . 0 nm pd ; e , 6 . 2 nm pbse and 3 . 0 nm pd ; f , 5 . 8 nm pbse and 3 . 0 nm pd ; g , 7 . 2 nm pbse and 4 . 2 nm ag ; h , 6 . 2 nm pbse and 3 . 0 nm pd ; i , 7 . 2 nm pbse and 5 . 0 nm au ; j , 5 . 8 nm pbse and 3 . 0 nm pd ; k , 7 . 2 nm pbse and 4 . 2 nm ag ; and l , 6 . 2 nm pbse and 3 . 0 nm pd nps B-nanoparticle . [SEP]
[CLS] scale bars : a - c , e , f , i - l , 20 nm ; d , g , h , 10 nm . [SEP]
[CLS] the lattice projection is labelled in each panel above the scale bar . [SEP]
[CLS] reprinted with permission from ref . 377 . [SEP]
[CLS] copyright 2006 nature publishing group [SEP]
[CLS] 10 representative tem images of u87 cells B-material after treatment with np B-nanoparticle - sirna constructs indicate that larger constructs can distribute in the cytoplasm . [SEP]
[CLS] u87 cells B-material were treated with 0 . 5 nm of ( a ) 13 nm spheres , ( b ) 50 nm spheres , and ( c ) 40 nm stars for 24 h . [SEP]
[CLS] the images in the boxes ( lower panel ) indicate zoomed - in views . [SEP]
[CLS] the yellow arrows indicate nps B-nanoparticle distributed outside vesicles ; the orange arrows indicate locally disrupted vesicle membranes . [SEP]
[CLS] reprinted with permission from ref . 387 . [SEP]
[CLS] copyright 2017 american chemical society [SEP]
[CLS] 11 hrem images of pd particles with fcc structure . [SEP]
[CLS] ( a ) and ( b ) are in a [UNK] 1 1 0 [UNK] orientation and ( c ) is in a [UNK] 1 0 0 [UNK] orientation , while ( d ) corresponds to a particle with a hexagonal profile , which corresponds to a distorted [UNK] 1 1 0 [UNK] orientation . [SEP]
[CLS] the corresponding fft is included in each case . [SEP]
[CLS] reprinted with permission from ref . 393 . [SEP]
[CLS] copyright 2001 elsevier [SEP]
[CLS] 12 sequence of hrtem images for decahedral pd particles showing different orientations with respect to the one five - fold axis parallel to the electron beam . [SEP]
[CLS] a model shows in each case the orientation the corresponding fft is included in the figure . [SEP]
[CLS] reprinted with permission from ref . 393 . [SEP]
[CLS] copyright 2001 elsevier . [SEP]
[CLS] 13 ( a ) o - ring sealed in situ wet cell B-material design . [SEP]
[CLS] ( b ) sealless in situ liquid tem setup utilizing low vapor ionic liquids . [SEP]
[CLS] ( c ) illustration of an in situ liquid cell B-material formed by atomic thin graphene membranes . [SEP]
[CLS] ( d ) atomic resolution images obtained with c , showing the pt nanocrystal growth procedure . [SEP]
[CLS] reprinted with permission from ref . 405 . [SEP]
[CLS] copyright 2015 royal society of chemistry . [SEP]
[CLS] 14 tem images of directed gold B-nanoparticle nanoparticle I-nanoparticle assembly in the charged polyacrylic acid region . [SEP]
[CLS] ( a and b ) bright - field images . [SEP]
[CLS] dark stripes are concentrated gold B-nanoparticle nanoparticle I-nanoparticle areas . [SEP]
[CLS] the insert shows the proposed structures . [SEP]
[CLS] yellow dots denote gold B-material nps B-nanoparticle . [SEP]
[CLS] ( c ) high - resolution tem ( hrtem ) imaging of the lattice structure of gold B-material single crystals . [SEP]
[CLS] ( d and e ) high - angle annular dark - field ( haadf ) imaging of periodic gold B-material stripes . [SEP]
[CLS] gold B-material particles appear as bright stripes . [SEP]
[CLS] ( f ) tem image of periodic gold B-material stripes when polyamine functionalized gold B-material particles are used as counterions . [SEP]
[CLS] reprinted with permission from ref . 426 . [SEP]
[CLS] copyright 2007 aaas [SEP]
[CLS] 15 typical haadf - stem images of thiolated ( au x ag 1−x ) 312±55 clusters . [SEP]
[CLS] ( a ) - ( c ) , ( g ) - ( i ) and ( m ) - ( o ) are clusters assigned to cubooctahedral , ino - decahedral or icosahedral structures , based on ( d ) - ( f ) , ( j ) - ( l ) and ( p ) - ( r ) , the corresponding simulated images ( for bare au 309 clusters , which is the closest full shell B-material size of cuboctahedral , icosahedral and inodecahedral ) . [SEP]
[CLS] reprinted with permission from ref . 469 . [SEP]
[CLS] copyright 2015 american chemical society [SEP]
[CLS] 16 aberration - corrected stem - eds elemental mapping of nzvi reactions with cr ( vi ) : ( a ) fe , ( b ) o , ( c ) cr and ( d ) fe + o + cr overlay . [SEP]
[CLS] reprinted with permission from ref . 477 . [SEP]
[CLS] copyright royal society of chemistry 2014 . [SEP]
[CLS] 17plan - view eels spectrum imaging of a silver B-material right bipyramid . [SEP]
[CLS] ( a ) non - negative matrix factorization of eels for a selected area ( blue square , inset ) . [SEP]
[CLS] the decomposition is shown for a 4 . 3 nm × 4 . 3 nm ( 9 pixel × 9 pixel ) region . [SEP]
[CLS] blue dots represent the summed raw spectra , the black line is the sum of all decomposition components , the gray line corresponds to the spectral signature of the zero loss peak , and each of the remaining components corresponds to a spatial map that exhibits a dominant contribution matching a surface plasmon mode of the bipyramid ( α - ε ) , the bulk plasmon ( ζ ) , or the moo 3 substrate band edge ( η ) . [SEP]
[CLS] ( b ) nmf component maps ( exp . ) and simulated energy loss probability maps ( sim . ) . [SEP]
[CLS] intensities are plotted on a normalized scale for each map . [SEP]
[CLS] subscripts on β denote fully resolved peaks in simulated spectra represented in the single experimental non - negative matrix factorization component β . [SEP]
[CLS] energies refer to peak maxima in the respective experimental and simulated spectra . [SEP]
[CLS] scale bars are 25 nm . [SEP]
[CLS] reprinted with permission from ref . 482 . [SEP]
[CLS] copyright 2015 american chemical society [SEP]
[CLS] 17plan - view eels spectrum imaging of a silver B-material right bipyramid . [SEP]
[CLS] ( a ) non - negative matrix factorization of eels for a selected area ( blue square , inset ) . [SEP]
[CLS] the decomposition is shown for a 4 . 3 nm × 4 . 3 nm ( 9 pixel × 9 pixel ) region . [SEP]
[CLS] blue dots represent the summed raw spectra , the black line is the sum of all decomposition components , the gray line corresponds to the spectral signature of the zero loss peak , and each of the remaining components corresponds to a spatial map that exhibits a dominant contribution matching a surface plasmon mode of the bipyramid ( α - ε ) , the bulk plasmon ( ζ ) , or the moo 3 substrate band edge ( η ) . [SEP]
[CLS] ( b ) nmf component maps ( exp . ) and simulated energy loss probability maps ( sim . ) . [SEP]
[CLS] intensities are plotted on a normalized scale for each map . [SEP]
[CLS] subscripts on β denote fully resolved peaks in simulated spectra represented in the single experimental non - negative matrix factorization component β . [SEP]
[CLS] energies refer to peak maxima in the respective experimental and simulated spectra . [SEP]
[CLS] scale bars are 25 nm . [SEP]
[CLS] reprinted with permission from ref . 482 . [SEP]
[CLS] copyright 2015 american chemical society [SEP]
[CLS] 18 atomic scale reconstruction of au nanorods B-nanoparticle . [SEP]
[CLS] ( a , b ) orthogonal slices through the atomic scale reconstruction of au nanorods B-nanoparticle prepared using different surfactants B-property . [SEP]
[CLS] the side facets of these rods can be clearly recognized . [SEP]
[CLS] ( c ) strain measurement along the major axis of the nanorod B-nanoparticle . [SEP]
[CLS] reprinted with permission from ref . 489 . [SEP]
[CLS] copyright wiley - vch 2014 . [SEP]
[CLS] 19 the essence of et : an angular series of 2d projection images is recorded by tilting the specimen in the ( scanning ) transmission electron microscope . [SEP]
[CLS] the ' tilt - series ' of images are then back - projected into space to obtain a 3d reconstruction . [SEP]
[CLS] a variety of signals may be recorded , including haadf signals . [SEP]
[CLS] the bright - field detector can be removed to allow the transmitted electrons to pass through to a spectrometer and form an energy - loss spectrum . [SEP]
[CLS] reproduced with permission from ref . 490 . [SEP]
[CLS] copyright 2013 elsevier [SEP]
[CLS] 20 scheme of a sem / eds system operating in the transmission mode with the zeiss single - unit transmission setup ( pe : primary electrons ; se1 : secondary electrons emitted at the point of impact of the pe on the sample ; te : transmitted electrons ; bf : bright field ; df : dark field ; e - t : everhart - thornley detector ) . [SEP]
[CLS] reprinted with permission from ref . 506 . [SEP]
[CLS] copyright royal society of chemistry 2014 . [SEP]
[CLS] 21 schematic of afm and mfm imaging B-technique techniques I-technique . [SEP]
[CLS] ( a ) ( 1 ) an afm tip scans the surface of a sample to produce a topographical trace , ( 2 ) the cantilever is raised to a user - defined height away from the sample surface and the retrace follows the original topographical pattern from the first step ; ( 3 ) during the retrace , the magnetic B-property signal is scanned and recorded for the sample . [SEP]
[CLS] in all cases , the signals are recorded via the reflection of a laser beam off the back of the cantilever and onto a photodiode , where changes in cantilever deflection are detected . [SEP]
[CLS] ( b ) in the case of using magnetic B-technique force microscopy I-technique to scan magnetic B-property nps B-nanoparticle on mica substrates , a magnetically B-property coated tip is used to scan the sample surface and an mfm signal is obtained as it interacts magnetically B-property with the sample and its magnetic B-property domains or nps B-nanoparticle . [SEP]
[CLS] reprinted with permission from ref . 536 . [SEP]
[CLS] copyright 2016 united scientific group [SEP]
[CLS] 22 afm images of a nickel B-material plate before ( upper ) and after ( lower ) the deposition of silver B-material colloidal nps B-nanoparticle . [SEP]
[CLS] reproduced with permission from ref . 531 . [SEP]
[CLS] copyright 2007 elsevier [SEP]
[CLS] 23 afm ( left ) and kfm ( right ) images of pure iron B-nanoparticle nanoparticles I-nanoparticle in different concentrations of h 2 so 4 . reprinted with permission from ref . 535 [SEP]
[CLS] copyright 2014 esg . [SEP]
[CLS] efficient intracellular delivery of nucleic B-material acids I-material to achieve sensitive detection and gene regulation is essential for chemistry and biology . [SEP]
[CLS] here we developed a novel protein scaffolded dna tetrad , a four - arm dna nanostructure constructed using streptavidin ( sa ) protein B-material and four biotinylated hairpin dna probes for efficient nucleic B-material acid I-material delivery and ultrasensitive mirna imaging through crosslinking hybridization chain reaction ( chcr ) . [SEP]
[CLS] dna tetrads were easy to prepare and allowed precise control of the structure of the probes . [SEP]
[CLS] dna tetrads showed rapid intracellular delivery of dna probes and high efficiency in lysosome escape by using confocal images for individual cells B-material and flow B-technique cytometry I-technique for a large population of cells B-material . [SEP]
[CLS] chcr allowed generating clumps of crosslinked hydrogel networks specifically to target mirna , affording high sensitivity and spatial resolution for imaging . [SEP]
[CLS] to our knowledge , this is the first time that hcr amplification has been realized in situ on nanostructures . [SEP]
[CLS] moreover , the fret based design of chcr conferred improved precision with the use of dual - emission ratiometric imaging to avoid false signals in biological systems . [SEP]
[CLS] intracellular imaging experiments further showed that dna tetrad based chcr could realize ultrasensitive and accurate mirna imaging in living cells B-material . [SEP]
[CLS] moreover , dna tetrad based chcr provided a potential tool for quantitative measurement of intracellular mirna . [SEP]
[CLS] the results suggested that this developed strategy provided a useful platform for nucleic B-material acid I-material delivery and low level biomarker B-property imaging . [SEP]
[CLS] nucleic B-material acids I-material play critical roles in many fundamental cellular B-event processes I-event involving genetic information carriers , intracellular regulatory molecules , and catalytic enzymes . [SEP]
[CLS] because of their ability to interact with various molecules via canonical basepairing or conformation - tted binding , nucleic B-material acids I-material have become a valuable regulatory and analytical tool with exquisite specicity for cell B-material biology and medicine . [SEP]
[CLS] a prime quest to use nucleic B-material acids I-material for biomedical applications is the transfection system that allows their efficient delivery into different cells B-material . [SEP]
[CLS] dna / rna nanostructures represent an attractive option for intracellular delivery of nucleic B-material acids I-material . [SEP]
[CLS] prominent examples are spherical nucleic B-material acids I-material ( snas ) , and self - assembled nucleic B-material acid I-material nanostructures . [SEP]
[CLS] these nucleic B-material acid I-material nanostructures have demonstrated their ability to enter different cells B-material without the aid of cationic B-material carriers , affording a powerful platform for developing diagnostic assays , therapeutic drugs , and gene regulation agents for live cell B-material and in vivo applications . [SEP]
[CLS] nevertheless , major challenges still exist in facile synthesis or purication of dna / rna nanostructures , high - efficiency delivery of nucleic B-material acid I-material reagents into cells B-material and limited sensitivity for intracellular detection . [SEP]
[CLS] proteins B-material provide an alternative promising scaffold for constructing dna / rna nanostructures . [SEP]
[CLS] in contrast to synthetic nanoparticles B-nanoparticle , proteins B-material are characterized by a surface with unevenly distributed reactive sites and a core B-material with well - dened conformation and diverse oligomeric structures . [SEP]
[CLS] motivated by this advantage , protein - based snas , which use proteins B-material as the core B-material and utilize their surface amine B-material and thiol B-material groups for dense functionalization of dna , have been engineered . [SEP]
[CLS] this particular type of snas is shown to not only enhance cellular uptake of functional proteins B-material , but also offer building blocks for precisely modulating the nanoparticle B-nanoparticle superlattice architecture . [SEP]
[CLS] however , protein - scaffolded dna nanostructures that are facile in synthesis and purication and efficient for delivery of nucleic B-material acid I-material reagents have rarely been explored . [SEP]
[CLS] to allow facile synthesis of the protein - scaffolded dna nanostructure , we turn to employing high - affinity non B-property - I-property covalent I-property interactions I-property for assembling dna on the protein B-material surface . [SEP]
[CLS] streptavidin ( sa ) is known for its exceptionally tight binding of biotin with the dissociation constant down to 10 a14 m , and fusion to spytag has generated assemblies of sa subunits capable of binding a varying number of biotin conjugates [SEP]
[CLS] hence , sa and its recombinant protein B-material fusion have the potential to provide a modular scaffold for assembling biotinylated dna into nanostructures with precise control of the number and position of dna . [SEP]
[CLS] motivated by this hypothesis , we choose sa as the model scaffold and develop a simple dna nanostructure , a dna tetrad , in which the surface of sa is provided with four biotinylated dna oligonucleotides via high - affinity sa - biotin interactions . [SEP]
[CLS] additionally , nanostructures reported so far had dense dna probes on their surface , which may generate high steric hindrance and inhibit the dna - assembly based amplication ; the tiny dna tetrad developed here with only four dna probes resulted in a relatively loose structure , offering the possibility of hcr in situ on nanostructures . [SEP]
[CLS] motivated by this assumption and based on previous work on dna circuits for signal amplication by our group and others , 24 - 29 we further demonstrated the utility of dna tetrads for ultrasensitive imaging of mirna using a novel crosslinking hybridization chain reaction ( chcr ) , as shown in scheme 1 . [SEP]
[CLS] as a proof - of - principle , we chose mir - 21 , a known biomarker B-property overexpressed in many cancers , as a case of study to demonstrate the design of dna tetrads for intracellular chcr imaging . [SEP]
[CLS] in chcr , two biotinylated hairpin probes h1 and h2 are designed , one labeled with a cy3 uorescence donor and the other with a cy5 uorescence acceptor . [SEP]
[CLS] then , dna tetrads with h1 and h2 probes separately displayed on sa are synthesized . [SEP]
[CLS] in the presence of target mirna , it opens the hairpin probe h1 , which in turn initializes hcr with a cascade of alternating hybridization between h1 and h2 and induces crosslinking of the tetrads and form 3d crosslinked hydrogel networks . [SEP]
[CLS] in the chcr products , cy3 uorescence donors and cy5 acceptors are drawn into close proximity , which activates a fret signal as an indicator B-property for the expression of target mirna . [SEP]
[CLS] in the absence of target mirna , chcr between h1 and h2 probes does not occur and no fret signal from cy3 to cy5 is observed . [SEP]
[CLS] it is worth noting that , compared to current dna circuit based imaging strategies , this dna tetrad based strategy shows remarkable advantages . [SEP]
[CLS] first , dna tetrads provide a more robust , convenient and modular platform because of their fast and easy preparation , and precise control of the structure and concentration of the probes . [SEP]
[CLS] it is also demonstrated that dna tetrads are able to enter live cells B-material quickly and efficiently without the aid of transfection carriers . [SEP]
[CLS] most importantly , dna tetrads showed desirable efficiency in lysosome escape . [SEP]
[CLS] second , because each tetrad has four reactive sites , hcr can induce crosslinking of the tetrads and form 3d crosslinked hydrogel networks , affording high sensitivity and spatial resolution for imaging . [SEP]
[CLS] to our knowledge , this is the rst time that hcr amplication has been realized in situ on nanostructures , providing the rst integrated system for dna delivery and signal amplied imaging in living cells B-material . [SEP]
[CLS] finally , the foster resonance energy transfer ( fret ) based design confers improved precision because it allows the use of dual - emission ratiometric imaging to avoid false signals in complex biological environments . [SEP]
[CLS] therefore , the dna tetrad enabled chcr strategy can offer a highly robust , accurate , and ultrasensitive approach for rna based diagnostics . [SEP]
[CLS] to study the cellular permeability of dna tetrads , we synthesized a dna tetrad by incubating B-technique sa with probe h3 . [SEP]
[CLS] as sa is a tetramer protein B-material with four biotin - binding sites , four types of sa - dna complexes can be obtained , corresponding to the 1 : 1 sa - dna complex to the 1 : 4 sa - dna complex . [SEP]
[CLS] these sa - dna complexes were readily characterized by gel B-technique electrophoresis I-technique ( fig . 1a ) . [SEP]
[CLS] as anticipated , at different concentration ratios of h3 to sa , the gel image showed one or two bands with varying mobility B-property in each lane ( 2 - 4 ) , and four bands with different molecular weights were obtained in these lanes . [SEP]
[CLS] this result gave clear evidence for the formation of four types of sa - dna complexes . [SEP]
[CLS] because of the well - separated bands for sacomplexes , the dna tetrads were readily puried by cutting the corresponding dna band followed by dna isolation from the gel . [SEP]
[CLS] the puried product was also conformed in the gel image ( fig . s1 † ) . [SEP]
[CLS] fluorescence B-property anisotropy analysis and zeta B-property potential I-property analysis were both used to verify the interaction between dna probes and sa ( fig . 1b and c ) . [SEP]
[CLS] the cell B-material permeability of the dna tetrads was investigated . [SEP]
[CLS] to visualize the dna tetrads , we labelled the dna tetrads on sa protein B-material with cy3 or on the h2 probe with cy5 . [SEP]
[CLS] aer 60 min of incubation B-technique with the dna tetrads , the cells B-material displayed bright uorescence with typical cytosolic localization ( fig . 2b and c ) . [SEP]
[CLS] the results suggested that dna tetrads were capable of entering the cells B-material without the aid of transfection reagents . [SEP]
[CLS] by contrast , in a control experiment in which cells B-material were incubated B-technique with free sa protein B-material labelled with cy3 , no appreciable uorescence was observed ( fig . 2a ) . [SEP]
[CLS] according to this control , it was clear that surface - immobilized dna probes were essential for the cell B-material permeability of dna tetrads . [SEP]
[CLS] a further interrogation of cells B-material incubated B-technique using dna tetrads with sa protein B-material labelled using cy3 and the h2 probe tagged using cy5 showed very intense and highly colocalized uorescence signals in the cells B-material at both cy3 and cy5 channels ( fig . 2d - f ) . [SEP]
[CLS] this nding evidenced that dna tetrads retained their structural integrity in cells B-material with no dissociation of the biotinylated dna probes from sa protein B-material . [SEP]
[CLS] further investigation of cellular uptake of the dna tetrads in hela cells B-material was performed ( fig . s2 † ) . [SEP]
[CLS] the confocal microscopy B-technique images showed a bright uorescence when cells B-material were incubated B-technique with dna tetrads at 37 c for 1 h . [SEP]
[CLS] however , for cells B-material pretreated at 4 c before adding dna tetrads , a dramatic reduction of cy5 uorescence intensity was observed , which suggested that the reaction with dna tetrads involved a temperature - dependent cellular B-event uptake I-event process I-event . [SEP]
[CLS] another experiment by pretreating cells B-material with nan B-technique 3 , an inhibitor of atpase which is involved in all energy dependent endocytic pathways , 29 also displayed signicantly reduced cellular uptake of dna tetrads . [SEP]
[CLS] the trafficking dynamics of dna tetrad uptake were monitored using both ow cytometry for a large population of cells B-material and confocal uorescence microscopy B-technique for individual cells B-material . [SEP]
[CLS] as shown in fig . 3a , aer incubating B-technique the cells B-material with dna tetrads , a substantial uorescence peak shi was observed within 15 min , which saturated at $ 45 min , demonstrating an efficient delivery approach using dna tetrads . [SEP]
[CLS] confocal microscopy B-technique imaging further veried these results ( fig . s3 † ) . [SEP]
[CLS] the dependency of uptake efficiency on the concentration of dna tetrads was also investigated . [SEP]
[CLS] with the concentration of dna tetrads increased from 0 to 50 nm , the uorescence intensity was signicantly increased ( fig . 3b ) . [SEP]
[CLS] to elucidate whether the internalization of dna tetrads is dependent upon the conformation of the dna probe , a control experiment using dna tetrads with single strand dna l1 was performed ( fig . s4 † ) . [SEP]
[CLS] a large peak red - shi was also observed for cells B-material incubated B-technique with dna tetrads carrying l1 , verifying that dna tetrads afford an efficient intracellular delivery approach for either linear or hairpin dna probes . [SEP]
[CLS] we next studied the subcellular localization of dna tetrads . [SEP]
[CLS] with the incubation B-technique time increased from 15 to 60 min , the cy5 uorescence gradually increased and spread out from the lysosomes into the cytosol with lysosome colocalization coefficients decreasing from 69 . 1 % to 47 . 2 % to 26 . 8 % ( fig . 3c , and s5 † ) . [SEP]
[CLS] additionally , a z - axis optical sectioning study further veried the efficiency of dna tetrads in lysosome escape ( fig . s6 and s7 † ) . [SEP]
[CLS] these results suggested that dna tetrads could be a useful platform for nucleic B-material acid I-material delivery with efficiency in lysosome escape . [SEP]
[CLS] to our knowledge , hcr has not been realized in situ on nanostructures . [SEP]
[CLS] presumably , existing nanostructures with dense dna probes may generate huge steric hindrance that prevents the growth of hcr polymers B-material . [SEP]
[CLS] because of the tiny size of tetrads and sparsely tethered dna probes , we reasoned that dna tetrads offer the possibility of hcr amplication in situ on the nanostructures . [SEP]
[CLS] as a result , we then investigated the feasibility of dna tetrads for hcr amplication . [SEP]
[CLS] as shown in fig . 4a , almost no fret occurred without target mir - 21 due to the stable intramolecular hybridization of h1 and h2 on the tetrads , but a strong fret signal was observed in the presence of target mir - 21 . [SEP]
[CLS] a negative control experiment using inactive mir - 21 merely displayed a low fret signal . [SEP]
[CLS] agarose B-technique gel I-technique electrophoresis I-technique analysis was also performed to verify the design ( fig . 4b ) . [SEP]
[CLS] the formation of crosslinked hydrogel networks was further demonstrated by atomic B-technique force I-technique microscopy I-technique . [SEP]
[CLS] in contrast to the monomers B-material of dna tetrads with a $ 5 nm diameter ( fig . 4c ) , clumps with a size in the micron order for the chcr products was observed ( fig . 4d ) ; this result clearly evidenced the formation of crosslinked hydrogels in chcr assay . [SEP]
[CLS] the fret signals showed dynamic responses to mir - 21 with concentrations ranging from 0 . 05 to 100 nm with an estimated detection limit of 6 pm ( fig . 4e , and s8 † ) . [SEP]
[CLS] this detection limit was much better than those of the existing mirna imaging methods , indicating the advantage of our strategy in sensitivity enhancement . [SEP]
[CLS] we also made a comparison between this chcr and conventional hcr using dimer probes of h1 and h2 ( fig . s9 † ) . [SEP]
[CLS] interestingly , chcr displayed a higher signal for cy5 emission and a lower peak for cy3 emission than those for conventional hcr . [SEP]
[CLS] we ascribed the improved fret signals of chcr to the proximity of probes in dna tetrads , which facilitates the reaction kinetics of hcr in the tetrads . [SEP]
[CLS] besides the improved sensitivity , our design also afforded excellent selectivity . [SEP]
[CLS] as shown in fig . 4f , target mir - 21 triggered an obvious fret signal . [SEP]
[CLS] control experiments using other mirnas , a blank sample of the reaction buffer , ripm 1640 cell B-material culture medium or b - actin protein B-material in place of target mirna all displayed very low fret signals , demonstrating the high selectivity of our method to detect mir - 21 . [SEP]
[CLS] furthermore , a lysate of negative l - 02 cells B-material with no mir - 21 expression also did not produce an enhanced fret signal , suggesting that our design had excellent resistance to false signals in complex biological environments . [SEP]
[CLS] in other words , these results indicated that the design could improve the accurate image of mirna in complex biological systems . [SEP]
[CLS] on the basis of preliminary results , we next applied dna tetrads for imaging mir - 21 in living cells B-material . [SEP]
[CLS] when hela cells B-material were incubated B-technique with two dna tetrads carrying h1 and h2 , bright uorescence was observed both in the green channel and red channel , suggesting that mir - 21 initiated the chcr reaction to generate the fret signal ( fig . 5a , and s10 † ) . [SEP]
[CLS] to further verify the specicity of the uorescence response , hela cells B-material pretreated with an mir - 21 inhibitor , synthetic single - stranded rnas designed to specically bind to and selectively decrease the active concentration of mir - 21 , showed dim red uorescence but very bright green uorescence ( fig . 5b ) , indicating the specic response to mir - 21 . [SEP]
[CLS] moreover , hela cells B-material pretreated with a small chemically modied mir - 21 mimic to imitate mirna - 21 high expression displayed higher red uorescence intensity but lower green uorescence intensity than that observed in untreated hela cells B-material ( fig . 5c ) . [SEP]
[CLS] these results revealed that uorescence signals were dynamically correlated to the concentration of mir - 21 . [SEP]
[CLS] the ratiometric images also showed similar results , giving direct evidence for chcr and distinguishing different levels of intracellular mirna while avoiding false signals . [SEP]
[CLS] furthermore , we transfected hela cells B-material with dimer probes of h1 and h2 using the lipofectamine 3000 reagent for conventional hcr assay . [SEP]
[CLS] as anticipated , cells B-material with conventional hcr assay showed much weaker fret signals than those in dna tetrad based chcr assay ( fig . s11 † ) , demonstrating that chcr afforded higher amplication efficiency and better imaging contrast than conventional hcr . [SEP]
[CLS] we ascribed the improved contrast of chcr to the high delivery efficiency of dna tetrads , facilitated hcr kinetics in dna tetrads , and highly crosslinked hcr polymers B-material with more localized signals . [SEP]
[CLS] next , the potential of the strategy for quantitative evaluation of the mir - 21 expression in different cell B-material lines was explored . [SEP]
[CLS] fluorescence B-property images of varying brightness and ratiometric images were obtained ( fig . 6 ) . [SEP]
[CLS] hepg2 cells B-material showed the highest mir - 21 expression whereas l - 02 displayed the lowest expression , which were consistent with the previous report for mirna - 21 expression levels in these cells B-material . [SEP]
[CLS] ratiometric images were dynamically correlated to the concentration of mir - 21 , as analyzed by rt - pcr ( fig . s12 † ) . these results veried the ability of the chcr - based strategy for quantitative measurement of mirna in living cells B-material . [SEP]
[CLS] in summary , we have developed a novel dna tetrad as a facile platform for efficient nucleic B-material acid I-material delivery and ultrasensitive mirna imaging through chcr . [SEP]
[CLS] the dna tetrads were demonstrated to be a highly robust delivery agent because of facile synthesis and purication , precise control of the structure and concentration of the probes , rapid nucleic B-material acid I-material delivery and efficiency in lysosome escape . [SEP]
[CLS] conclusionschcr amplication allows generating clumps of crosslinked hydrogel networks specically to target mirna , affording sensitive and spatially resolved mirna imaging in living cells B-material . [SEP]
[CLS] to our knowledge , this is the rst time that hcr amplication has been realized in situ on nanostructures . [SEP]
[CLS] moreover , the fret based signal activation mode improved the precision of detection , avoiding false signals in complex biological systems . [SEP]
[CLS] in virtue of these advantages , dna tetrads may provide an effective strategy for nucleic B-material acid I-material delivery , low level biomarker B-property imaging and related theranostics . [SEP]
[CLS] 1 ( a ) agarose gel ( 3 % ) electrophoresis B-technique image of the four types of sa - dna complexes . [SEP]
[CLS] ( b ) fluorescence B-property anisotropy responses of h2 and the dna tetrad . [SEP]
[CLS] ( c ) zeta B-property potential I-property analysis of free sa and the dna tetrad . [SEP]
[CLS] 2 ( a - c ) pseudocolored fluorescence B-property images of free sa - cy3 ( a ) , dna tetrads using h4 and sa - cy3 ( b ) and dna tetrads using h2 - cy5 and sa ( c ) , respectively . [SEP]
[CLS] ( d - f ) pseudocolored fluorescence B-property images of each channel of dna tetrads using sa - cy3 and h2 - cy5 for cy3 ( d ) , cy5 ( e ) and merge fluorescence B-property ( f ) . [SEP]
[CLS] 3 ( a ) flow cytometric assay of hela cells B-material with varying incubation B-technique time with dna tetrads carrying h2 . [SEP]
[CLS] ( b ) flow cytometric assay of hela cells B-material treated with varying concentrations of dna tetrads carrying h2 . [SEP]
[CLS] ( c ) fluorescence B-technique imaging I-technique of subcellular localization of dna tetrads carrying h2 with varying incubation B-technique time followed by lysotracker staining . [SEP]
[CLS] 4 ( a ) fluorescence B-property spectral responses obtained by incubating B-technique dna tetrads carrying h1 ( black ) , dna tetrads carrying h1 and h2 ( blue ) , dna tetrads carrying h1 and h2 with the target rna and inhibitor ( pink ) , dna tetrads carrying h1 and h2 with the target rna ( red ) , and dna tetrads carrying h2 ( green ) . [SEP]
[CLS] ( b ) gel B-technique electrophoresis I-technique image for chcr assay . [SEP]
[CLS] lane 1 , dna marker ; lane 2 , h1 and h2 ; lane 3 , rna target plus h1 and h2 probes ; lane 4 , dna tetrads containing h1 and h2 probes ; lane 5 , rna target plus dna tetrads containing h1 and h2 probes . [SEP]
[CLS] afm images of the ( c ) dna tetrads and ( d ) chcr products . [SEP]
[CLS] ( e ) fluorescence B-property spectral responses to the rna target of varying concentrations ( 0 nm , 0 . 05 nm , 0 . 1 nm , 0 . 25 nm , 1 nm , 2 . 5 nm , 10 nm , 25 nm , and 100 nm ) . [SEP]
[CLS] ( f ) selectivity of dna tetrad based chcr . [SEP]
[CLS] 5 fluorescence B-property images of hela cells B-material treated with dna tetrads ( a ) , 300 nm mir - 21 inhibitor ( b ) and 300 nm mir - 21 mimic ( c ) . [SEP]
[CLS] 6 fluorescence B-technique imaging I-technique of l - 02 , hela , and hepg2 cells B-material by dna tetrad based chcr . [SEP]
[CLS] genetically encoded light - up rna aptamers afford a valuable platform for developing rna sensors toward live cell imaging . [SEP]
[CLS] however , quantitative imaging of intracellular rnas remains a grand challenge . [SEP]
[CLS] here we reported a novel genetically encoded rna sensor strategy using a plasmid that expresses a splittable fusion of the rna sensor and the gfp mrna in an individual transcript B-event using a single promoter system . [SEP]
[CLS] this splittable fusion design enables synchronous co - expression of the rna sensor with gfp mrna while alleviates the interference with correct folding of rna aptamers due to intramolecular hybridization . [SEP]
[CLS] this single - promoter system is applied to ratiometric imaging of survivin mrna in tumor B-material cells B-material . [SEP]
[CLS] the results reveal that the ratiometric images dynamically correlated with survivin mrna concentrations and allow quantitative imaging of survivin mrna in different tumor B-material cells B-material . [SEP]
[CLS] the rna sensor strategy may provide a new paradigm for developing a robust imaging platform for quantitative mrna studies in living cells B-material . [SEP]
[CLS] " seeing " the abundance of rnas as well as their localization and underlying dynamics in living cells B-material is essential for understanding cellular biology , since the behaviours of rnas drive all sorts of fundamental processes of life . [SEP]
[CLS] rna - targeting imaging probes , including uorescent protein B-material ( gfp ) - tagged rna binding B-event proteins I-event and hybridization probes such as molecular beacons and spherical nucleic B-material acids I-material ( sna ) - based nano - ares , have signicantly contributed to this area . [SEP]
[CLS] nevertheless , quantitative rna imaging of living cells B-material is still largely unexplored because of the perturbations from different analyte - independent factors such as changes in focuses and laser intensity . [SEP]
[CLS] ratiometric imaging corrects these factors by providing built - in self - calibration and has the potential to improve the quantitative measurement of cells B-material . [SEP]
[CLS] therefore , development of ratiometric rna imaging approaches is in high demand for live cell B-material studies . [SEP]
[CLS] genetically encoded rna sensors , based on light - up rna aptamers , [SEP]
[CLS] have emerged as a valuable platform for uorescent imaging in living cells B-material . [SEP]
[CLS] light - up rna aptamers , represented by spinach , broccoli 17 and mango , are a class of engineered rna molecules displaying " turn - on " uorescence upon binding to nonuorescent small - molecule dyes . [SEP]
[CLS] when integrated with target - specic sequences as the responsive module , light - up rna aptamers have enabled detection of varying targets including metabolites , proteins B-material 20 and rnas in varying biological media and even in living cells B-material [SEP]
[CLS] we have recently reported a genetically encoded rna sensor for ratiometric imaging of microrna via engineering a plasmid with two promoters , which allows stable expression and robust detection of the rna sensor in living mammalian cells B-material . [SEP]
[CLS] the design of using two promoters separately expressing the rna sensor and gfp involves two asynchronous transcription B-event events recruiting rna polymerases ii or iii , respectively . [SEP]
[CLS] such asynchronism may result in different concentration ratios of the rna sensor and gfp mrna at varying times , leading to asynchronous signals for the gfp reference and the rna sensor . [SEP]
[CLS] therefore , this system only calibrates the variations due to transfection B-property efficiency I-property in different cells B-material , but the asynchronism for the rna sensor and gfp is not precluded , limiting the ability of the rna sensor for quantitative rna imaging . [SEP]
[CLS] development of a genetically encoded sensor enabling quantitative ratiometric imaging has not been reported . [SEP]
[CLS] because the gfp uorescence is largely dependent upon the abundance of gfp mrna , we envisioned that a fusion transcript B-event of the rna sensor with gfp mrna had the potential to minimize the asynchronism of gfp and the rna sensor . motivated by this hypothesis , we develop a novel genetically encoded rna sensor strategy using a plasmid that expresses a fusion of the rna sensor and the gfp mrna in an individual transcript B-event using a single promoter system , as illustrated in scheme 1a . [SEP]
[CLS] the sensor is designed to be fused to the 3 0 utr of gfp mrna [SEP]
[CLS] view journal | view issue using a two - domain spacer B-material , an mrna - stabilizing triplex in the upstream domain and a dsrna dicer substrate 27 in the downstream . [SEP]
[CLS] this fusion design enables a synchronous coexpression of the rna sensor and gfp mrna , allowing synchronism of the reference gfp uorescence with the rna sensor . [SEP]
[CLS] however , this fusion can interfere with the folding of the rna sensor because of the intracellular hybridization of gfp mrna with the rna sensor . [SEP]
[CLS] to avoid this interference , a dsrna dicer substrate is incorporated between the rna sensor and gfp mrna , which allows the transcript B-event to be cleaved at the dsrna site by dicer enzyme that is ubiquitously present in mammalian cells B-material [SEP]
[CLS] furthermore , the mrnastabilizing triplex is introduced at the 3 0 terminal of gfp mrna to avoid fast degradation of gfp mrna . [SEP]
[CLS] according to this design , when the plasmid is transfected into mammalian cells B-material , a splittable rna fusion can be transcribed from a single cmv promoter in the cells B-material , as illustrated in scheme 1b . [SEP]
[CLS] in the cytosol , the transcript B-event is cleaved at the dsrna site , producing two separate rnas with one for the stabilized gfp mrna and the other for the rna sensor . [SEP]
[CLS] the rna sensor is incorporated into a trna scaffold , which improves stability of the rna sensor in mammalian cells B-material . [SEP]
[CLS] on hybridizing with target mrna , the sensor undergoes a conformational change and recovers the conformation of a light - up aptamer , sulforhodamine b ( sr ) aptamer . [SEP]
[CLS] it is recalled that the sr aptamer is capable of lighting up the quenched uorescence of a conjugate of sr with dinitroaniline ( dn ) . [SEP]
[CLS] therefore , the activated uorescence from sr gives an indicator B-property for the expression level of target mrna . [SEP]
[CLS] on the other hand , the stabilized gfp mrna still retains its activity to be translated into gfp , which delivers a green uorescence signal acting as the internal reference for ratiometric imaging . [SEP]
[CLS] because the single promoter mediated co - expression of gfp mrna and the rna sensor minimizes the asynchronism of gfp uorescence and the signal of the rna sensor , the ratiometric imaging afforded the possibility for quantitative imaging of mrna expression . [SEP]
[CLS] to demonstrate its potential , this novel rna sensor has been applied to ratiometric imaging of survivin mrna , a known biomarker B-property over - expressed in different tumors B-material , in living cells B-material . [SEP]
[CLS] the results revealed that the obtained ratiometric images were dynamically correlated with survivin mrna concentrations in varying cell B-material lines . [SEP]
[CLS] moreover , the results were conrmed by quantication using quantitative reverse transcription B-event pcr ( qrt - pcr ) , implying the promise of the rna sensor as a robust imaging platform for quantitative mrna studies in living cells B-material . [SEP]
[CLS] to our knowledge , there has been no previous study reporting genetically encoded light - up rna sensors of mrna imaging in mammalian cells B-material . [SEP]
[CLS] hence , our work represents the rst time to engineer genetically encoded lightup rna sensors for quantitative ratiometric imaging of mrna in living mammalian cells B-material . [SEP]
[CLS] a co - expressing rna sensor with gfp using one plasmid enabled calibrating the variations due to transfection B-property efficiency I-property in different cells B-material . [SEP]
[CLS] nevertheless , the asynchronism in the transcription B-event efficiency of two different promoters could lead to varied concentration ratios for the rna sensor to the gfp reference in different cells B-material due to their heterogeneity . [SEP]
[CLS] as a matter of fact , we found that the uorescence of the sr aptamer and gfp exhibited obvious differences in their relative intensity in different hela cells B-material ( fig . 1a ) aer the cells B-material were transfected using a plasmid with two promoters for separate expression of gfp and the sr rna aptamer . [SEP]
[CLS] the difference revealed signicant variations of cells B-material in expression of the rna aptamer and gfp , evidencing the asynchronism in their relative expression levels and thereby in their uorescence signals from cell B-material to cell . [SEP]
[CLS] as the reference uorescence did not vary in correlation with the sr aptamer signal , there was a limitation for the two - promoter system in quantitative imaging of living cells B-material . [SEP]
[CLS] co - expressing the rna aptamer with gfp in the same transcription B-event event had the potential to minimize the asynchronism in the expression of the rna aptamer and gfp . [SEP]
[CLS] based on the hypothesis , we designed a plasmid with a single promoter that could generate a fusion of the sr aptamer and gfp mrna in an individual transcript B-event . [SEP]
[CLS] surprisingly , aer transfecting the plasmid into hela cells B-material for 24 h , we merely observed green uorescence of gfp with no detectable red uorescence for the sr aptamer even in the presence of sr - dn ( fig . 1b ) , implying misfolding of the sr rna aptamer . [SEP]
[CLS] considering the close proximity of the gfp mrna located in the upstream of the fusion transcript B-event to the downstream rna aptamer , we reasoned that the folding of the rna aptamer was distorted by the gfp mrna due to the facilitated intramolecular folding of the fusion transcript B-event . [SEP]
[CLS] we then envisioned that splitting of the rna aptamer from the fusion could alleviate the inference with the folding of the sr aptamer from gfp mrna . [SEP]
[CLS] hence , we re - engineered a new plasmid with a dicer - cleavage site in the fusion between the rna aptamer and gfp mrna ( scheme s1 † ) . [SEP]
[CLS] aer the fusion transcript B-event was expressed in mammalian cells B-material , dicer enzyme split the fusion at the dsrna site in the cytosol into the gfp mrna with a stabilizing triplex tail and the rna aptamer in a trna scaffold . [SEP]
[CLS] to conrm the success of this design , the total rna extracts of hela cells B-material aer transfection with the plasmid were detected using rt - pcr with gel B-technique electrophoresis I-technique using three pairs of primers specic to the gfp mrna , dicer substrate site and the trna - scaffolded rna aptamer , respectively . [SEP]
[CLS] the bright gel bands that appeared at 171 bp , 109 bp and 138 bp , respectively , were consistent with the design ( fig . s1 † ) . [SEP]
[CLS] to testify the cleavage of the rna transcript B-event , qrt - pcr analysis was performed separately for the plasmids with or without the dicer substrate site . [SEP]
[CLS] a much lower concentration was found for the rna transcript B-event with the dicer substrate site than for that without the site ( fig . s2 † ) . [SEP]
[CLS] this result conrmed the efficient cleavage of the rna transcript B-event with the dicer substrate site in cells B-material . [SEP]
[CLS] additionally , confocal microscopy B-technique images of cells B-material transfected with the plasmid with the dicer substrate site showed bright red uorescence for the sr aptamer in the presence of sr - dn and green uorescence for gfp ( fig . 1c ) . [SEP]
[CLS] the images indicated successful expression and correct folding of the sr rna aptamer and gfp reference in the cells B-material . [SEP]
[CLS] the appearance of red uorescence actually veried our previous hypothesis that the correct conformation of the sr aptamer was distorted by gfp mrna due to intramolecular folding , considering that cells B-material transfected with the plasmid without the dicer substrate site did not display the red uorescence of the sr aptamer . [SEP]
[CLS] moreover , we observed that the red uorescence signals in different cells B-material changed in a consistent manner with the corresponding green uorescence , indicating the synchronism of the red and the green uorescence signals . [SEP]
[CLS] taken together , the results demonstrated the feasibility of co - expressing the correctly folded rna aptamer with the gfp reference using a single promoter that afforded synchronous uorescence signals in living mammalian cells B-material . [SEP]
[CLS] to further testify the synchronism of uorescence signals for the rna aptamer and gfp , qrt - pcr analysis for the total rna extracts from cells B-material transfected with a single - promoter plasmid revealed that the relative concentrations of the rna aptamer were approximate to those of gfp mrna at different times ( fig . s3 † ) . [SEP]
[CLS] this quantication result supported our design that a single promoter plasmid allowed synchronous expression of the rna aptamer and gfp mrna . [SEP]
[CLS] in addition to the rna level , we then investigated the uorescence signals for gfp and the rna aptamer using confocal images . [SEP]
[CLS] it was observed that the uorescence signals of gfp appeared very bright aer 24 or 48 h transfection , and the intensity decreased slightly aer 60 h transfection ( fig . 2 ) . [SEP]
[CLS] similar time - dependent changes were also found for uorescence signals of the rna aptamer . [SEP]
[CLS] this nding suggested that a single promoter plasmid afforded synchronous uorescence intensities for the rna aptamer and gfp in cells B-material , implying its potential for developing quantitative ratiometric genetically encoded rna sensors for imaging B-technique of I-technique living I-technique tumor I-technique cells B-material . [SEP]
[CLS] next , we utilized a single promoter plasmid to develop an rna sensor for quantitative imaging of mrna in living cells B-material . [SEP]
[CLS] it is known that mrna is critical in regulating cellular polarity , migration and development , and aberrant expression of mrna is highly implicated in the progression of various diseases including cancer . [SEP]
[CLS] in this study we chose mrna survivin 31 as the model target and designed the sr aptamer into an rna sensor for its detection in living cells B-material ( scheme 1a ) . [SEP]
[CLS] the sensor was engineered by fusing a stem - loop motif in response to survivin mrna in the terminal stem of the sr aptamer in light of our previous design . [SEP]
[CLS] the presence of target mrna allowed refolding of the sr - binding loop , activating the uorescence signals that illuminated the expression of survivin . [SEP]
[CLS] the as - prepared rna sensor was rstly examined via in vitro assays using a synthetic dna template for the rna sensor with fig . 2 fluorescence B-property images for hela cells B-material transfected using a singlepromoter plasmid expressing splittable fusion of the rna aptamer and gfp at different times . [SEP]
[CLS] a t7 promoter for in vitro transcription B-event [SEP]
[CLS] the sensor was found to display remarkable uorescence activation in response to survivin with an enhancement ratio of 11 in peak intensities ( fig . s4 † ) . [SEP]
[CLS] in addition , we found that the trna - scaffolded sensor has comparable performance with its prototype rna sensor without the trna scaffold ( fig . s5 † ) . [SEP]
[CLS] the sensor also exhibited high selectivity to survivin in contrast to other interferents including other rna sequences , proteins B-material and a lysate of c166 cells B-material , a cell line with negative expression for survivin ( fig . s6 † ) . [SEP]
[CLS] gel B-technique electrophoresis I-technique analysis gave further evidence for stable hybridization and uorescence activation of the rna sensor in response to survivin in the presence of sr - dn ( fig . s7 † ) . [SEP]
[CLS] moreover , the rna sensor displayed enhanced uorescence responses to survivin in a dose dependent manner in a concentration range of 1 nm to 5 mm ( fig . s8 † ) . [SEP]
[CLS] the detection limit was estimated to be 0 . 4 nm according to the 3s rule , suggesting the high sensitivity of the sensor for survivin detection . [SEP]
[CLS] on the basis of the preliminary results , we then used the single - promoter plasmid for transfection of living cells B-material for ratiometric imaging of survivin in the cells B-material ( scheme s2 † ) . [SEP]
[CLS] aer transfecting hela cells B-material using the plasmid in the presence of sr - dn for 24 h , very bright green uorescence signals from gfp appeared relatively evenly in the nuclei and in the cytosol . [SEP]
[CLS] bright red uorescence was also observed in the cytosol of hela cells B-material , a cell line with overexpressed survivin , which indicated activated uorescence of the rna sensor in response to survivin ( fig . 3a ) . [SEP]
[CLS] it was noticed that the uorescence response of the rna sensor was appreciably weaker than the signals generated using the rna aptamer obtained in the preliminary experiment ( fig . 2 ) . [SEP]
[CLS] this result was ascribed to the low abundance of survivin in hela cells B-material , which did not restore the conformation of all rna aptamers expressed in the cells B-material . [SEP]
[CLS] additionally , we observed uorescence signals arising from the rna aptamer both in the nuclei and in the cytosol , indicating that the trna scaffolded rna aptamer were localized throughout the cells B-material . [SEP]
[CLS] in contrast , the uorescence of the rna sensor was only found in the cytosol , which is ascribed to the cytosol localization of target survivin mrna . [SEP]
[CLS] to validate the selectivity of the uorescence response , we also performed a control experiment using the survivin - negative cell B-material line c166 ( ref . 32 ) ( fig . 3b ) . [SEP]
[CLS] the dim red uorescence displayed in c166 cells B-material suggested that the uorescence of the rna sensor was specically activated in the presence of survivin . [SEP]
[CLS] in addition , we also did not observe an enhanced uorescence response in hela cells B-material transfected using a single - promoter plasmid with no dicer - substrate site inserted between the gfp mrna and the rna sensor ( fig . s9 † ) , con - rming the necessity of the dicer - substrate site for the singlepromoter system . [SEP]
[CLS] next , we explored the rna sensor for ratiometric imaging of survivin in different cell B-material lines , mda - mb - 435s , hela , mcf - 7 and mcf - 10a ( fig . 4a ) . [SEP]
[CLS] aer transfecting these cells B-material using the plasmid for 24 h , bright green uorescence signals of gfp were observed in different cells B-material , affording a robust reference for the rna sensor . [SEP]
[CLS] by contrast , we observed red uorescence images of varying brightness and ratiometric images of different colors in these cells B-material . [SEP]
[CLS] mda - mb - 435s cells B-material gave the highest expression level of survivin , whereas mcf - 10a cells B-material had the lowest expression , and hela cells B-material gave a little higher expression than mcf - 7 cells B-material . [SEP]
[CLS] these results were in good agreement with previous studies , implying that the uorescence responses of the rna sensor had the potential for quantitative detection of survivin expression . [SEP]
[CLS] to further demonstrate its ability for quantitative imaging , we performed quantitative determination of survivin expression in these cell B-material lines using qrt - pcr ( fig . 4b ) . [SEP]
[CLS] as anticipated , the average values of the red - to - green uorescence ratio in the regions of interest ( rois ) , ten rois in each image , dynamically correlated with the relative concentrations of survivin ( fig . s10 † ) . [SEP]
[CLS] moreover , the values of red - to - green uorescence ratio were in a linear correlation with the relative concentrations of survivin ( fig . 4c ) , giving direct evidence of the potential of the rna sensor in quantifying mrna in living cells B-material . [SEP]
[CLS] the copy numbers of survivin in different cells B-material calculated according to the average values of the red - to - green uorescence ratio in rois also highly agreed with those as - determined by qrt - pcr , which further conrmed the ability of the rna sensor for quantitative imaging of mrna ( fig . s11 † ) . [SEP]
[CLS] we further demonstrated the rna sensor for quantitative imaging of survivin expression in drug - treated hela cells B-material . [SEP]
[CLS] the survivin expression in hela was altered by two small molecules of docetaxel and ym155 . [SEP]
[CLS] docetaxel treatment led to docetaxelinduced cell B-material apoptosis B-event with a concomitant upregulation of survivin , while ym155 was a survivin suppressant . [SEP]
[CLS] during plasmid transfection , hela cells B-material were co - incubated B-technique with docetaxel or ym155 of a given dose for 24 h . [SEP]
[CLS] we observed that red signals from the rna sensor were brighter in the cells B-material treated using docetaxel but darker in the ym155 - treated ones as compared to non - treated cells B-material ( fig . 5a ) , indicating changed survivin expression in cells B-material aer drug treatment . [SEP]
[CLS] moreover , a higher dose of the upregulating drug resulted in higher red uorescence , while a higher dose of the downregulating drug delivered lower red uorescence . [SEP]
[CLS] additionally , the ratiometric images displayed changing colors obviously depending on the doses of docetaxel and ym155 used for hela cell B-material treatment . [SEP]
[CLS] furthermore , we found the average ratio values in rois had a linear relationship with the amount of survivin in the treated cells B-material as determined by qrt - pcr ( fig . 5b and c ) . [SEP]
[CLS] these results conrmed the ability of the rna sensor for quantitative imaging of survivin in living cells B-material . [SEP]
[CLS] in summary , we have developed a novel genetically encoded rna sensor strategy based on a single - promoter system that coexpressed an rna sensor with gfp for quantitative imaging of mrnas in living tumor I-technique cells B-material . [SEP]
[CLS] we demonstrated that a direct fusion of the sr rna aptamer with gfp mrna in an individual transcript B-event using a single promoter distorted the correct folding the rna aptamer , which failed to show the light - up property in living cells B-material . [SEP]
[CLS] splitting of the rna aptamer from the fusion by incorporating a dicer substrate site could avoid the misfolding of the rna aptamer , retaining uorescence for both the rna aptamer and the gfp reference . [SEP]
[CLS] to our knowledge , this is the rst time that co - expression of light - up rna aptamers with uorescent proteins B-material using a single promoter has been realized in mammalian cells B-material . [SEP]
[CLS] moreover , we demonstrated that this single - promoter system enabled highly synchronous coexpression of the rna aptamer and gfp , affording a desirable platform for ratiometric imaging . [SEP]
[CLS] this system was successfully applied to ratiometric imaging of survivin mrna in tumor B-material cells B-material . [SEP]
[CLS] the results revealed that the ratiometric images were dynamically correlated with survivin mrna concentrations . [SEP]
[CLS] moreover , the values of the red - to green uorescence ratio in different cell B-material lines and drug - treated hela cells B-material exhibited a linear relationship with the concentration of survivin as determined by qrt - pcr . [SEP]
[CLS] our design may open the possibility for developing a genetically encoded ratiometric imaging platform for quantitative studies of living tumor B-material cells B-material . [SEP]
[CLS] 1 illustration of plasmid design ( a ) and the rna sensor from a splittable fusion transcript B-event with gfp for ratiometric mrna imaging ( b ) . [SEP]
[CLS] 1fluorescence images for hela cells B-material transfected using a twopromoter plasmid ( a ) and a single - promoter plasmid with no dicer substrate site ( b ) and a dicer substrate site ( c ) for co - expression of the sr aptamer and gfp mrna . [SEP]
[CLS] 3 fluorescence B-property images of survivin mrna in hela cells B-material ( a ) and c166 cells B-material ( b ) . [SEP]
[CLS] 4 ( a ) fluorescence B-property and ratiometric images for different cells B-material . [SEP]
[CLS] ( b ) qrt - pcr analysis for the cells B-material . [SEP]
[CLS] green : mda - mb - 435s . [SEP]
[CLS] blue : hela . [SEP]
[CLS] red : mcf - 7 . [SEP]
[CLS] black : mcf - 10a . [SEP]
[CLS] ( c ) relationship of average values of the red - to - green fluorescence B-property ratio in rois with relative concentrations of survivin mrna in different cells B-material . [SEP]
[CLS] 5 ( a ) fluorescence B-property and ratiometric images of hela cells B-material treated with 100 nm docetaxel ( a ) , 30 nm docetaxel ( b ) , 0 nm ( c ) , 5 nm ym155 ( d ) , and 20 nm ym155 ( e ) . [SEP]
[CLS] ( b ) qrt - pcr analysis for the treated hela cells B-material . [SEP]
[CLS] red : a , blue : b , green : c , black : d , and pink : e . [SEP]
[CLS] ( c ) relationship of average values of the red - to - green fluorescence B-property ratio in rois with survivin mrna concentrations in the treated hela cells B-material . [SEP]
[CLS] structure and luminescence B-property of dna - templated silver B-material clusters dna can template the formation of silver B-material clusters with bright , narrow - band fl uorescence spectra tuned by nucleobase sequence . [SEP]
[CLS] at the intersection of metal B-material cluster science and dna nanotechnology , these nanomaterials B-material are promising for applications ranging from sensing to bioimaging . [SEP]
[CLS] this review presents the current fundamental understanding of dnatemplated silver B-material clusters , with a particular focus on studies which employ purifi cation B-material prior to characterization . [SEP]
[CLS] topics include structure - property relations , the connection between nucleobase sequence and cluster fl uorescence , and strategies for higher - order assembly of silver B-material clusters into functional nanomaterials B-material . [SEP]
[CLS] metal B-material " nanoclusters " are the smallest of nanoparticles B-nanoparticle , consisting of only 2 to 10 2 metal B-material atoms B-material and possessing remarkable properties which are very nely tuned by cluster size , shape , and charge . [SEP]
[CLS] bare metal B-material clusters have been studied for decades in order to understand how single atoms B-material with quantized energy levels transition into the continuous properties of bulk anna gonzalez - rosell received her bachelor ' s degree in chemistry from institut quimic de sarria - universitat ramon llull ( barcelona , spain ) in 2019 . [SEP]
[CLS] she is currently pursuing a phd in materials science and engineering under the guidance of prof . stacy copp at the university of california , irvine . [SEP]
[CLS] her research focuses on the discovery and experimental characterization of nir - emitting dnatemplated silver B-material clusters . [SEP]
[CLS] anna is a recipient of the balsells graduate fellowship . [SEP]
[CLS] phd in chemistry at the university of copenhagen , denmark , in 2020 under the supervision of prof . tom vosch . [SEP]
[CLS] she worked on the synthesis , hplc purication , and photophysical characterization of dnastabilized silver B-material nanoclusters . [SEP]
[CLS] during her phd , she visited the group of prof . jiro kondo at sophia university , tokyo , where she gained knowledge about crystallization techniques of oligonucleotides . [SEP]
[CLS] she is currently a postdoctoral researcher in prof . vosch ' s group , where she has been working on the investigation of new dna - templated silver B-material nanoclusters and their applications . [SEP]
[CLS] cecilia cerretani received hermaterials [SEP]
[CLS] because the majority of unprotected metal B-material clusters are unstable at ambient conditions , fundamental studies of metal B-material clusters previously necessitated interrogation under ultra - high vacuum , 2 which limited practical applications of these nanomaterials B-material . [SEP]
[CLS] this challenge has been overcome by the use of stabilizing ligands and supporting surfaces to bring metal B-material clusters into the " real world " for applications such as catalysis , photonics , and electronics . [SEP]
[CLS] in the past two decades , advances in synthetic chemistry have produced a " zoo " of different stable metal B-material clusters passivated by molecular ligands , with cluster sizes that can even be tuned to atomic precision for especially ne control of their emergent properties . [SEP]
[CLS] this review concerns an especially unusual type of ligand - stabilized metal B-material cluster , the dna - templated silver B-material cluster ( ag n - dna ) , which combines the atomic precision of cluster science with the programmability of dna nanotechnology . [SEP]
[CLS] ag n - dnas are relatively new entrants into the diverse zoology of metal B-material clusters , with unique properties that arise from their polynucleic B-material acid I-material ligands . [SEP]
[CLS] following work by the dickson group on silver B-material clusters stabilized in dendrimers B-nanoparticle and silver B-material oxide I-material lms , [SEP]
[CLS] in 2004 , petty , dickson , and co - authors reported formation of uorescent silver B-material nanoclusters exhibiting 400 - 600 nm electronic transitions by chemically reducing an aqueous mixture of single - stranded cytosine - rich dna and agno 3 . [SEP]
[CLS] they then found that certain ag n - dnas exhibit very bright uorescence and signicant photostability and can be harnessed as biolabels . [SEP]
[CLS] 9 , gwinn , et al . , showed that the uorescence colors of ag n - dnas depend sensitively on nucleobase sequence and that ag n - dnas prefer to form on single - stranded ( ss ) dna rather than double - stranded ( ds ) dna , motivating the important role played by silver B-material - nucleobase interactions in ag n - dna formation . [SEP]
[CLS] in the next few years , ag n - dnas were shown to be effective sensors for toxic B-property metal B-material ions B-material , polynucleic B-material acids I-material , and other biomolecules [SEP]
[CLS] together , these and other early studies generated considerable interest in harnessing dna ' s sequence programmability for custom design of ag n - dna uorophores tailored for precise sensing , uorescence microscopy B-technique of cells B-material and tissues , and direct integration into dna nanotechnology schemes . [SEP]
[CLS] the most remarkable characteristic of ag n - dnas is their sequence - dependent uorescence . [SEP]
[CLS] by employing dna template strands with wide - ranging nucleobase sequences , a diverse color palette of ag n - dnas with uorescence emission colors of 450 nm to 1000 nm has been developed ( fig . 1a ) , with quantum yields as high as 93 % ( ref . 24 ) and stokes shis as large as 5893 cm a1 . [SEP]
[CLS] ag n - dna uorescence may be excited by at least two pathways , either directly at the cluster ' s size - , shape - , and charge - dependent excitation peak or universally via the dna bases ( fig . 1b ) . [SEP]
[CLS] ag n - dnas also exhibit unusual photophysics , intriguing dark states which can be harnessed for background - free uorescence microscopy B-technique , light - up or colorswitching behavior induced by various stimuli , and catalytic activity [SEP]
[CLS] most well - studied ligand - stabilized metal B-material clusters are protected by monolayers of small molecules such as thiolates and phosphines , with sizes smaller than or comparable to the metal B-material clusters themselves . [SEP]
[CLS] in contrast , ag n - dnas and their lessstudied counterparts , ag n - rnas , and properties of these and other metal B-material clusters stabilized by large macromolecular ligands , including proteins B-material and dendrimers B-nanoparticle , are less understood than for monolayer - protected clusters , in part because bulky ligands can obscure resolution of cluster ( s ) and challenge crystallization , a necessary step for " solving " structure by x - ray crystallography . [SEP]
[CLS] however , macromolecular ligands can also endow functionalities without the need for ligand exchange , adding a degree of versatility to applications of ag n - dnas and other macromolecule - stabilized nanoclusters . [SEP]
[CLS] ag n - dna synthesis is facile and is typically carried out by borohydride reduction of a solution of ag + and ssdna in neutral ph aqueous solution ( fig . 1c ) . [SEP]
[CLS] this method is robust to varying solution compositions , stoichiometries , and specic mixing / heating . [SEP]
[CLS] in contrast to the simplicity of synthesis , achieving compositionally pure solutions of ag n - dnas is more challenging because reduction forms a heterogeneous mixture of silver - bearing dna products containing varying numbers of silver B-material atoms I-material , n tot , and numbers of dna strands , n s . [SEP]
[CLS] the majority of these products are nonuorescent and include clusters , ag + - dna complexes , and larger silver B-nanoparticle nanoparticles I-nanoparticle . [SEP]
[CLS] it is also possible for a given dna template to stabilize multiple different emissive cluster species , as has been observed for up to 25 % of randomly selected dna template sequences . [SEP]
[CLS] due to characterization of as - synthesized ag n - dnas without purication and / or due to fragmentation during mass spectrometry ( ms ) , early reports underestimated ag n - dna sizes or found no correlation of uorescence color with silver B-material cluster size . [SEP]
[CLS] a lack of awareness of this heterogeneity continues to hinder accurate characterization of ag n - dnas , and the assumption that the composition of ag n - dnas is uncorrelated to the optical properties of these nanoclusters still persists . [SEP]
[CLS] the challenge of heterogeneity has been overcome by the use of reversed - phase high performance liquid B-technique chromatography I-technique ( hplc ) and size B-technique - I-technique exclusion I-technique chromatography I-technique ( sec ) to isolate a uorescent ag n - dna of interest prior to compositional and spectral characterization . [SEP]
[CLS] additionally , development of gentle electrospray ionization B-property ( esi ) ms now enables compositional analysis without fragmentation of the ag n - dna product . [SEP]
[CLS] using tandem hplc - ms with in - line uv / vis and uorescence spectroscopy B-technique , schultz , et al . determined the compositions of several uorescent ag n - dnas with uorescence emission wavelengths , l em , ranging from green to near infrared ( nir ) , nding that these clusters contained n tot ¼ 10 - 21 ag atoms B-material stabilized by n s ¼ 1 - 2 copies of the templating dna strand . [SEP]
[CLS] this ability to isolate and characterize compositionally pure solutions of ag n - dnas has enabled numerous future studies , leading to dramatic advances in our understanding of the structure - property relations of these nanoclusters , which we discuss in section 3 , and of their photophysical properties , which we discuss in section 4 . [SEP]
[CLS] this review focuses on the recent advances in fundamental understanding of ag n - dnas , with a particular emphasis on the recent detailed studies of compositionally pure ag n - dnas . [SEP]
[CLS] we note that this review is timely because previous reviews which primarily focused on fundamental structure and properties are several years old and do not discuss recent breakthroughs , including the rst reported ag n - dna crystal structures . [SEP]
[CLS] readers may also nd a comprehensive list of dna sequence / structure and optical properties for a large number of ag n - dnas by new , et al . , as well as previous reviews focused on the emerging applications of ag n - dnas as sensors and biolabels . [SEP]
[CLS] here , we summarize what is known about the connections among dna sequence , ag n structure , and photophysical properties . [SEP]
[CLS] we rst review current understanding of the ag + - mediated dna base paired structures that are the synthetic precursors of ag n - dnas ( section 2 ) . [SEP]
[CLS] next , we discuss current models for the structures of ag n - dnas , which have rapidly advanced due to detailed studies of compositionally pure ag n - dnas and a few breakthrough crystal structures ( section 3 ) . [SEP]
[CLS] then we review current understanding of the excited state processes which lead to uorescence in ag n - dnas and the unusual dark states exhibited by ag n - dnas ( section 4 ) . [SEP]
[CLS] we then discuss recent work to decode how dna sequence selects ag n - dna properties by combining high - throughput experimentation and machine learning ( section 5 ) . [SEP]
[CLS] finally , we review work on merging structural dna nanotechnology with ag n - dnas for ordered arrangement of these nanoclusters ( section 6 ) and comment on opportunities and challenges facing the eld of ag n - dna research ( section 7 ) . [SEP]
[CLS] it is our intent to provide a comprehensive and current picture of the properties of ag n - dnas which is accessible to researchers from many backgrounds , in order to aid others in developing applications of these unique nanoclusters and to inspire new experimental and computational studies of their fundamental properties . [SEP]
[CLS] a complete understanding of ag n - dna structure and sequencedependent properties naturally begins with an understanding of ag + - dna complexation . [SEP]
[CLS] this is because ( i ) ag n - dnas are formed by chemical reduction of ag + - dna complexes , ( ii ) high - resolution ms of hplc puried ag n - dnas shows that usually about half of the silver B-material atoms I-material within ag n - dnas remain cationic B-material , meaning that ag + - dna interactions play a key role in determining ag n - dna structure , and ( iii ) ag + - dna interactions are highly sequence - dependent , which may lead to the sequence dependence of ag n - dna size and uorescence properties . [SEP]
[CLS] here , we review recent advances in fundamental understanding of ag + - nucleobase interactions and secondary structures of ag + - dna complexes , with a focus on properties relevant to the formation and sequence - dependence of ag n - dnas . [SEP]
[CLS] we note that this topic is a small part of the rich eld of metal - mediated nucleobase pairing , an area of great interest as a route to expanded base - base interactions , dna - based electronics , and sensing . [SEP]
[CLS] we do not attempt to review this entire eld here and point to excellent comprehensive reviews elsewhere on metal - mediated pairing of both natural and articial bases and in the specic case of ag and au for natural dna . [SEP]
[CLS] the four natural nucleobases of dna are adenine ( a ) , cytosine ( c ) , guanine ( g ) , and thymine ( t ) . [SEP]
[CLS] in canonical watson - crick ( wc ) pairing of dsdna in b form , which is the most common structure of dna in vivo ( fig . 2a ) , two complementary dna strands join by hydrogen B-material bonds ( " h - bonds " ) between a and t and between c and g , forming the familiar antiparallel double helix . [SEP]
[CLS] c and g are held together by three h - bonds between the o2 , n3 and n4 positions of c and the o6 , n1 and n2 positions of g . in like manner , t and a are h - bonded through the o2 and n3 positions of t , and the n6 and n1 from a ( fig . 2c ) . [SEP]
[CLS] this difference in the number of h - bonds between nucleobase pairs results in a weaker a - t wc bond as compared to c - g . the wc b - form double helix is further stabilized by hydrophobic B-property stacking interactions between neighboring nucleobases . [SEP]
[CLS] additional less common dna structures also exist , including wc paired a - dna and z - dna and hoogsteen base pairing [SEP]
[CLS] the extensive scientic understanding of dna structure and thermodynamics has enabled the birth of dna nanotechnology , which exploits dna as a fundamental materials building block , engineering dna sequence to achieve self - assembled predened shapes , tuned colloidal interactions , and molecular computation . [SEP]
[CLS] 2 . 2 ag + - nucleobase interactions of homobase strands silver B-material cations B-material ( ag + ) are well - known to prefer binding B-event to I-event dna I-event nucleobases over the phosphate backbone at neutral ph . [SEP]
[CLS] hg 2 + possesses similar preference , 79 , 93 - 95 but its signicant toxicity B-property prohibits applicability ) . [SEP]
[CLS] this preference enables ag + intercalation into single base mismatches in wc - paired dsdna , typically by interactions with nucleobase ring nitrogens B-material . [SEP]
[CLS] cytosine ( c ) is especially well - known for affinity to ag + , and this has been harnessed to expand the interactions among dna oligomers , enabling ag + - paired c - c mismatches , ag + - folded imotif secondary structures in c - rich dna , ag + - crosslinked dna hydrogels , and dna nanotubes B-nanoparticle [SEP]
[CLS] more recently , the study of ag + - mediated nucleobase pairing has been extended to consider dna that is unconstrained by wc base pairs . [SEP]
[CLS] these studies show that silver B-material can completely rearrange canonical dna structures , as opposed to simply intercalating within base pair mismatches . [SEP]
[CLS] here , we review these recent advancements to provide context for the sequence - property connections that govern ag n - dnas ( section 5 ) . [SEP]
[CLS] to understand how ag + complexes with dna in the case where the dna does not form wc base pairs , swasey , et al . , investigated interactions of ag + with homobase dna strands . [SEP]
[CLS] aer solvent - exchanging dna oligomers to remove any residual salts B-material from oligomer synthesis , dna was mixed with agno 3 in an aqueous solution of ammonium acetate , followed by thermal annealing at 90 c . [SEP]
[CLS] resulting products were analyzed by highresolution negative ion B-material mode esi - ms to determine absolute composition by resolving the isotopic distribution ( discussed in section 3 . 1 ) . [SEP]
[CLS] 3a shows the compositions of all observed products for 11 - base homobase strands . [SEP]
[CLS] while c is best - known for affinity to ag + and was shown by ritchie , et al . , to form ag + mediated duplexes , g was actually found to associate the greatest number of ag + , with order of affinity : g > c > a > t . while the 4 types of natural nucleobases all formed ag + - bearing single homobase strands , ag + also mediates formation of homobase duplexes for c and g . when two different single homobase strands are mixed , ag + only mediates the heteroduplex a - ag + - t , completely replacing the wc a - t duplex . ag + also disrupts wc - paired c - g duplexes to instead form c - ag + - c and g - ag + - g homobase duplexes . [SEP]
[CLS] 3b summarizes all observed pairing between homobase strands . [SEP]
[CLS] c - ag + - c and g - ag + - g homoduplexes are remarkably stable , with c 6 - ag + - c 6 and g 6 - ag + - g 6 homoduplexes remaining intact at 90 c , while c 6 - g 6 wc duplexes melt below 20 c ( fig . 3c ) . [SEP]
[CLS] quantum chemical calculations support greater stability of ag + - mediated homoduplexes for c and g than for a and t . [SEP]
[CLS] in the absence of steric factors , ( base - ag + - base ) n duplexes have higher bond energies than ( base - ag + ) n structures . [SEP]
[CLS] because c - ag + - c and g - ag + - g are nearly coplanar , with dihedral angles of 171 . 9 and 181 . 2 respectively , while t - ag + - t and a - ag + - a are nonplanar , with dihedral angles of 140 and 101 . 6 , respectively , c - ag + - c and g - ag + - g homoduplexes are expected to be signicantly more stable ( fig . 3d ) . [SEP]
[CLS] the a - ag + - t bond is also non - coplanar , but its stability could be explained by the difference in size between a and t , which still allows adenine stacking interactions . [SEP]
[CLS] the nucleobase sites with which ag + interacts differ from wc pairing . [SEP]
[CLS] simulations by the lopez - acevedo group have determined that pyrimidines c and t interact with ag + at the n3 position , which is deprotonated for thymine , while purines a and g coordinate with ag + at the n7 position . [SEP]
[CLS] these binding sites correspond to the hoogsteen region ( fig . 2c ) . [SEP]
[CLS] however , these positions might change depending on the other nucleobase of the ag + - bridging bond , as is the case for the c - ag + - g bond , reported by kondo , et al . , where the interaction with the purine base is through the n1 position , which is deprotonated . [SEP]
[CLS] quantum chemical and hybrid quantum mechanics / molecular mechanics ( qm / mm ) calculations by the lopez - acevedo group predicted that , unlike the antiparallel strand orientation of natural wc duplexes where one 5 0 - 3 0 strand pairs to a complementary 3 0 - 5 0 strand , 82 c - ag + - c duplexes and g - ag + - g prefer a parallel orientation , with 5 0 ends aligned . [SEP]
[CLS] these helical duplexes align ag + along the helix axis and are stabilized not only by ag + - nucleobase interactions but also by novel interplanar h - bonds ( fig . 4a and b ) . [SEP]
[CLS] calculated electronic cd spectra of c 2 - ag + - c 2 tetramers agree well with experimentally measured cd spectra , further supporting a parallel arrangement . [SEP]
[CLS] however , other experimental studies report varying behavior . [SEP]
[CLS] one study of the conductivity of c - ag + - c duplexes achieves antiparallel duplex formation of strands conned at ends to a metal B-material surface and scanning probe tip . [SEP]
[CLS] as fig . 3 discussed in section 2 . 4 , both parallel 106 and antiparallel structures are reported [SEP]
[CLS] ag + mediates parallel strand orientation of highly stable homobase duplexesfor mixed base strands [SEP]
[CLS] recent study of unconstrained homobase strands conrms parallel duplex structure for c - ag + - c and g - ag + - g by utilizing forster resonance energy transfer ( fret ) experiments to determine dna strand orientation and ion B-material mobility B-property spectrometry ( ims ) ms coupled with density functional theory ( dft ) calculations to elucidate structure . [SEP]
[CLS] variations in fret efficiency between donor and acceptor dyes coupled to ends of two dna strands support parallel ag + - paired c homobase duplexes and g homobase duplexes ( fig . 4c ) . [SEP]
[CLS] this parallel orientation was further demonstrated by ims - ms experiments coupled with dft calculations of collision cross sections ( ccs ) , which support high aspect ratios for both guanine and cytosine duplexes , consistent with rigid , wire - like structures ( fig . 4d ) . [SEP]
[CLS] based on ccs values and their agreement to calculated values , the g - ag + - g duplex is found to be more rigid because nucleobases form additional h - bonds with the phosphate groups in the backbone , whereas the c - ag + - c duplex lacks these extra bonds and is more exible . [SEP]
[CLS] the vast majority of reported ag n - dna nanoclusters are stabilized by dna strands with mixed base sequences . [SEP]
[CLS] to understand how heterobase strands recruit ag + , swasey and gwinn examined ten noncomplementary 11 - base dna strands , determining composition of ag + - dna complexes by esi - ms ( hplc - ms was employed to analyze very heterogenous samples ) . [SEP]
[CLS] interestingly , strands with sequences formed by single - base " mutations " of c 11 increase the distribution of the number of ag + attached to duplexes , and inclusion of mutations in g 11 homobase strands can signicantly increase the average number of ag + by up to 7 or 8 ag + per duplex ( fig . 5a ) . [SEP]
[CLS] both homobase and heterobase ag + - mediated duplexes were found to be stable in various solution conditions , signicant mg 2 + concentrations , and high concentrations of urea ( a strong denaturant ) . [SEP]
[CLS] while the chemical structures adopted by these heterobase duplexes are not known , the differences in ag + recruitment have important implications for the origins of ag n - dna sequence dependence , which we discuss in section 5 . [SEP]
[CLS] kondo , et al . , recently developed remarkable uninterrupted ag + - dna " nanowires B-nanoparticle " and solved their 3d structure , determining formation of consecutive ag + - paired duplexes with antiparallel orientation . [SEP]
[CLS] the dna strand used to form the ag + wires , ggact ( br c ) gactcc , is a near - complement which forms a wc - paired homodimer with one c - c mismatch at room temperature in biologically relevant salt B-material concentrations ( determined using unafold soware ) . [SEP]
[CLS] c - ag + - c , g - ag + - g , t - ag + - t , and c - ag + - g bonds were observed in the nanowire B-nanoparticle , and interestingly , not all nucleobases in the strand participate in the principal linkage B-property between strands . [SEP]
[CLS] a ' s protrude outwards ( fig . 5b ) and contribute to crystal - packing through formation of at - ag + - a triplets and aa stacking interactions . [SEP]
[CLS] thanks to the near reversibility of the sequence used , and because a ' s do not participate in duplex bonding , most nucleobases are bonded to a like base in the partner strand , with only two c - ag + - g pairs fig . 4 ( a , b ) dft calculations predict the existence of novel inter - strand h - bonds in ( a ) c 2 - ag + - c 2 tetramers 101 and ( b ) ag + - paired g duplexes of varying lengths [SEP]
[CLS] these bonds add additional stability to ag + - paired dna duplexes . [SEP]
[CLS] ( a ) adapted from espinosa leal , et al . , ( ref . 101 ) with permission from the american chemical society . [SEP]
[CLS] copyright 2015 . [SEP]
[CLS] ( c ) emission spectra of ag + - mediated c 20 and g 15 duplexes labeled with donor ( green dot ) and acceptor ( red dot ) dyes at 5 0 end and 3 0 end , respectively ( orange curve ) or with both dyes at 3 0 ends ( blue ) , compared to emission of the donor - bearing strand alone ( blue dotted curve ) . [SEP]
[CLS] excitation is at 450 nm , which directly excites the donor only . [SEP]
[CLS] significant quenching of donor emission with concomitant acceptor emission ( high fret efficiency ) clearly demonstrates that ag + - mediated pairing of homo - duplexes arranges strands in a parallel orientation . [SEP]
[CLS] observed . pairing between the two strands occurs with a oneposition shi , enabling formation of nanowires B-nanoparticle up to 0 . 1 mm long . [SEP]
[CLS] despite the antiparallel orientation and the c - ag + - g pairs , in which the g is bonded through the n1 position , the system clearly does not obey wc pairing because the main interaction sites lie in the hoogsteen region . [SEP]
[CLS] furthermore , the propeller twist angles obtained are larger than in wc pairing , which can be explained by repulsions between amino and carbonyl groups of opposite bases . [SEP]
[CLS] liu , et al . solved the 3d structure of another ag + - paired mixed base strand , 5 0 - gcacgcgc - 3 0 , which forms curved dimers attached by one g - ag + - g bond and one c - ag + - c bond , with parallel strand orientation ( fig . 5c ) . [SEP]
[CLS] in this structure , it is only like bases which participate in ag + - mediated pairing , and these base pairs are less planar ( fig . 5c ) than the nearly coplanar angles predicted by previous dft calculations . [SEP]
[CLS] this suggests that mixed base strands can accommodate a wide range of ag + - mediated base interactions beyond just linear wires . [SEP]
[CLS] this 8 - base sequence was also uncovered in an unrelated study using machine learning methods to design templates for ag n - dnas with uorescence emission in the 600 nm < l em < 660 nm window . [SEP]
[CLS] this surprising coincidence suggests that some ag n - dnas are formed by chemical reduction of nontrivial ag + - dna complexes . [SEP]
[CLS] very recently , the kohler group reported evidence for a parallel oriented ag + - mediated duplex of c 20 with signicant " propeller " twist of the c - ag + - c base pairs , as has been reported in the studies above . [SEP]
[CLS] this evidence was based on strong agreement between experimentally measured and calculated cd spectra . [SEP]
[CLS] the authors note that such twisting has been associated with reduced exibility of dna , and this enhanced rigidity agrees with the past ims studies of c - ag + - c duplexes described in section 2 . 3 . [SEP]
[CLS] as synthetic precursors of ag n - dnas , ag + - dna complexes are the scaffolds that reorganize into the cluster - stabilizing cage of an ag n and , at least in part , provide the ag + " fuel " to grow the ag n upon reduction . early studies which found that ag n - dna do not form on completely dsdna templates have led to the false assumption that the ag n can always be conned within singlestranded regions of wc - paired dna structures such as hairpins or other dsdna structures with ssdna regions , based on the assumption that wc dna secondary structure is preserved in the presence of ag + . the dramatic rearrangement of dna homobase and heterobase strands by ag + , together with the signicant thermal and chemical stabilities of ag + - mediated dna duplexes , call into question whether this assumption is accurate . it is more likely that ag + can invade and unravel wc dsdna under appropriate conditions , rearranging secondary and tertiary structures which then further evolve upon chemical reduction . this has been suggested by several careful studies , and ag + has also been shown to rearrange the well - known g - quadruplex structure 108 and i - motif structure . [SEP]
[CLS] further studies will be needed to determine to what degree dna secondary structure is preserved aer ag n - dna synthesis , especially when ag n - dnas are incorporated into the larger dna structures discussed in section 6 . [SEP]
[CLS] the past several years have seen dramatic improvement in our understanding of ag n - dna chemical structures and their relation to optical properties , culminating in reports of the rst crystal structures of ag n - dnas . [SEP]
[CLS] nearly all of these advancements have been enabled by compositionally pure ag n - dnas isolated using hplc or sec [SEP]
[CLS] these techniques separate different dna complexes by exploiting variations in size and polarity that are induced by different silver B-material products on the dna template strands . [SEP]
[CLS] ( methods for isolating ag n - dnas using hplc have been reviewed in detail previously . [SEP]
[CLS] ) purication prior to characterization is crucial because assynthesized solutions contain multiple dark and uorescent products , including ag nanoparticles B-nanoparticle , ag n - dnas and ag + - dna complexes , as supported by lc - tandem ms . [SEP]
[CLS] even though one would naively expect ag n - dna properties to be similar in the as - synthesized and puried states , a recent report by gambucci , et al . , showed different rotational correlation times , indicating that synthesis fragments could be attached to the ag n - dnas , e . g . by ag + - mediated interactions . [SEP]
[CLS] compositional analysis methods that only infer average stoichiometry of the entire heterogeneous as - synthesized solutions may misjudge the number of silver B-material atoms I-material within an ag n - dna and cannot resolve the number of dna strands n s that stabilize a single cluster , and ms performed directly on as - synthesized samples makes it challenging to identify the uorescent ag n - dna of interest from the other products formed during synthesis . [SEP]
[CLS] here , we primarily review structural studies of hplc - isolated ag n - dnas with bright visible or nir uorescence , which have thus far been found to contain n tot ¼ 10 - 30 ag atoms B-material , as opposed to earlier reports of dimers or trimers of ag . [SEP]
[CLS] we then discuss other studies that focus on inference of the conformation of the dna template strand ( s ) around the ag n . [SEP]
[CLS] unless indicated , all ag n - dnas discussed are compositionally pure . [SEP]
[CLS] prior to the breakthrough crystallographic structures of ag n - dnas solved in 2019 , efforts to discern ag n - dna structure mainly employed correlations of experimentally measured absorption , excitation , and / or emission for ag n - dnas of known composition with computational studies or simple models . [SEP]
[CLS] these past studies do not provide the same level of structural detail as the recent crystal structures but do provide a more comprehensive picture of the structure - property relations of ag n - dnas in general , with detailed studies on about 20 different hplc - puried ag n - dnas as compared to the smaller number of crystal structures currently available . [SEP]
[CLS] since metal B-material cluster size , charge , and geometry strongly determine properties , accurate characterization of composition is a key step towards building a fundamental understanding of metal B-material clusters . [SEP]
[CLS] it is well - established that other ligandstabilized metal B-material clusters are only partially reduced because a fraction of the metal B-material atoms B-material in the cluster are bound to the surrounding ligands and that the number of remaining effective valence electrons in the cluster core B-material is a major determinant of the electronic properties of the cluster . [SEP]
[CLS] partially oxidized ag n - dnas were proposed by ritchie , et al . , based on the oxygen B-material and chloride B-material dependence of the uorescence of c 12 - stabilized clusters . [SEP]
[CLS] an experimental method to not only count the numbers of silver B-material atoms I-material n tot and dna strands n s in a puried sample of ag n - dna but also to separate n tot into neutral ( n 0 ) and cationic B-material ( n + ) silver B-material content can yield insights into how ag n clusters are ligated to the dna and enable computational studies of their electronic properties . [SEP]
[CLS] high resolution mass spectrometry ( hr - ms ) is an ideal tool to achieve this goal because hr - ms can be used to determine both ion B-material mass , m , and charge , z , rather than just the ratio m / z , by resolving the isotope B-material pattern that arises due to natural variation in isotopic abundances of elements . [SEP]
[CLS] koszinowski and ballweg determined the charge of an ag 6 4 + - dna by comparing the experimentally measured isotope B-material pattern to the calculated distribution of this cluster . [SEP]
[CLS] to characterize the properties of uorescent ag n - dnas , this approach has been developed in conjunction with chromatographic purication by the gwinn and petty groups . [SEP]
[CLS] because dna is easily deprotonated , negative ion B-material mode esi - ms is suitable to resolve weakly bound , noncovalent dna complexes and has been used to size a variety of silverbearing dna complexes . [SEP]
[CLS] ( while more sensitive , positive mode esi - ms can oxidize encapsulated clusters during electrospray , hindering full determination of composition . [SEP]
[CLS] ) the mass spectrum of an ag n - dna product may be collected either by tandem hplc - ms ( fig . 6a ) or by direct injection into the ms following previous purication . [SEP]
[CLS] determination of n 0 and n + for an ag n - dna from its mass spectrum is illustrated in fig . 6 . [SEP]
[CLS] first , the charge state z - of a m / z ( mass to charge ratio ) peak is determined by the spacing between adjacent peaks of the isotope B-material pattern : these peaks are spaced by 1 / z ( z is dened as a positive integer ) . [SEP]
[CLS] for example , fig . 6b shows the 7 - charge state ( z ¼ 7 , minus sign is due to negative ion B-material mode ms ) of an ag 30 - dna , with individual isotope B-material peaks separated by 1 / 7 . [SEP]
[CLS] ( the product shown in fig . 6b has a charge of - 7e , where e is the fundamental unit of charge ) . the total charge of the complex corresponding to this m / z peak is equal to the charge of the number of silver B-material cations B-material , en + , minus the charge of the number of protons removed from the dna , en pr , to reach the total charge of aez : [SEP]
[CLS] note that as number of silver B-material cations B-material , n + , increases , more protons must be removed from the complex to reach charge state z . [SEP]
[CLS] then , because n pr protons have been removed from the ag n - dna complex , the measured total mass m ( in amu ) is : [SEP]
[CLS] where m dna is the dna template strand mass , n s is the number of dna strands in the complex , and m ag is the silver B-material atom I-material mass ( the mass of a proton is treated here as 1 amu ) . [SEP]
[CLS] in the case of well - resolved patterns , n + and n 0 may be determined by calculating the isotope B-material distribution pattern for varying values of n + , and thus n pr , to determine the charge which best matches the isotope B-material pattern ( fig . 6b ) . [SEP]
[CLS] if signal is too low to precisely resolve the isotope B-material pattern , charge may be inferred by comparing gaussian ts of the calculated and experimentally measured isotope B-material patterns . [SEP]
[CLS] using this method , schultz , et al . , [SEP]
[CLS] determined that approximately half of the silver B-material atoms I-material within ag n - dna are cationic B-material in nature . [SEP]
[CLS] hr - ms is advantageous for determination of n s , n 0 , and n + without ambiguity , provided that gentle enough esi is applied . [SEP]
[CLS] inductively coupled plasma - atomic emission B-technique spectroscopy I-technique ( icp - aes ) has also been used to determine the composition of puried ag n - dnas , although n s cannot be determined by this method . [SEP]
[CLS] this has led to underestimates of the sizes of ag n - dnas with n s > 1 , which were later characterized by hr - ms . [SEP]
[CLS] the rst experimental evidence that ag n - dna cluster geometry differs from globular ( or quasi - spherical ) arose by comparing the absorption spectra of compositionally pure ag n - dnas ( whose n + and n 0 were determined by hplc - ms ) to the experimental and computed spectra of bare ag n in the gas phase which have similar numbers of effective valence electrons ( equal to n 0 ) . [SEP]
[CLS] the electronic properties of ligand - stabilized metal B-material clusters depend on the number of effective valence electrons in the cluster core B-material , not only the total number of atoms B-material n tot , and these valence electrons can delocalize to form " superatomic " orbitals . [SEP]
[CLS] thus , it is most appropriate to compare the properties of ag n - dnas with bare silver B-material clusters having like numbers of effective valence electrons . [SEP]
[CLS] due to ligation with the nucleobases , 24 not all ag atoms B-material in an ag n - dna will contribute to the valence electron count . [SEP]
[CLS] to determine the effective valence electron count of an ag n - dna , we subtract the charge of the cluster , n + , from the total number of atoms B-material in the cluster , n tot , nding that the number of effective valence electrons is n 0 ¼ n tot - n + . [SEP]
[CLS] schultz , et al . , found that the numbers and locations of peaks in the optical spectra of ag n - dnas differ markedly from their globular bare cluster counterparts . [SEP]
[CLS] naked ag n with cluster sizes n ¼ 2 to 20 exhibit globular geometries and absorption spectra with multiple uv transitions in the 3 to 5 ev spectral range . [SEP]
[CLS] in contrast , puried ag n - dnas have much simpler spectra with single dominant peaks in the visible to nir range < 3 ev , whose locations strongly depend on n 0 , and an additional uv absorption band due to the dna ligand ( fig . 1b ) . [SEP]
[CLS] the energies of the visible to nir absorbance peaks of ag n - dnas with varying n 0 can be described by quantum chemical calculations by guidez and aikens for linear atomic chains of silver B-material ( fig . 7a ) . [SEP]
[CLS] based on these results and on the signicant degree to which ag n - dna emission is polarized , as observed by spectroscopy B-technique of single ag n - dnas , a rod - like structure for ag n - dnas was proposed by the gwinn group . [SEP]
[CLS] following this model , ramazanov and kononov used dftcalculated electronic excitation spectra to argue that threadlike clusters show better agreement with experimental data than planar clusters . [SEP]
[CLS] a rod - like geometry is also supported by the magic n 0 numbers of ag n - dnas . [SEP]
[CLS] the energetic stability of many ligandstabilized metal B-material clusters can be described by the " superatom " we note that the chromatogram schematics are simplified for illustration ; [SEP]
[CLS] real chromatograms are more complex . [SEP]
[CLS] products of interest can either be sized by in - line negative - ion B-material mode esi - ms or collected for subsequent esi - ms . [SEP]
[CLS] a mass spectrum for a previously studied 30 - atom nir - emissive product is shown in the bottom right . [SEP]
[CLS] both monomeric and dimeric ( labeled " d " ) products are visible , with spacing of the isotopic peaks indicating the charge state of each product ( labeled as superscript of " d " ) for dimeric products . [SEP]
[CLS] ( b ) experimental mass spectrum of the ag 30 - dna product at the 7a charge state dimeric product ( labeled d a7 in ( a ) ) is shown in black , with the calculated mass distribution ( green bars ) for a product with 2 dna strands , n 0 ¼ 12 ag 0 , and n + ¼ 18 ag + . [SEP]
[CLS] inset : compares the experimental spectrum with the calculated distribution for a product with no charged silvers B-material ( 2 dna strands and 30 ag 0 ) , illustrating how the shift between the experimental and calculated isotopic finger distribution can be used to accurately determine the numbers of ag model , which states that the effective valence electrons in the cluster core B-material are characterized by an electronic shell B-material structure , similar to the shell structure of the atomic nuclei . [SEP]
[CLS] for spherical metal B-material clusters , closed shells B-material are expected for n 0 ¼ 2 , 8 , . , resulting in enhanced abundances of clusters of these sizes due to their signicantly enhanced stabilities ( the same behavior is observed for gas phase bare metal B-material clusters 2 ) . [SEP]
[CLS] copp , et al . , performed a large - scale study of ag n - dnas stabilized by $ 700 different dna templates , nding enhanced abundances of ag n - dnas with even numbers of neutral silver B-material atoms I-material : greenemissive ag n - dnas with n 0 ¼ 4 , red - emissive ag n - dnas with n 0 ¼ 6 , and larger nir - emissive n 0 ¼ 10 - 12 ag n - dnas ( fig . 7b ) ; the spherical magic numbers of 2 and 8 were not especially abundant ( fig . 7c ) . [SEP]
[CLS] this behavior is consistent with clusters that are signicantly aspherical , for which additional energy stability is primarily conferred by pairing of electron spins , resulting in enhanced stabilities for even values of n 0 . [SEP]
[CLS] chiroptical properties of ag n - dnas have been well - modeled by a thread - like cluster structure . because circular dichroism ( cd ) spectroscopy B-technique is extremely sensitive to specic geometrical structure and can be calculated using rst - principles methods , cd allows a direct interface with theory . [SEP]
[CLS] swasey , et al . , measured the cd spectra of four ag n - dnas spanning the visible to nir color palette . [SEP]
[CLS] quantum chemical calculations for bare atomic ag chains with a chiral twist agree well with the experimental spectra . [SEP]
[CLS] similarity between cd spectra of ag n - dnas and their unreduced ag + - dna precursors was also observed , pointing to the role played by the ag + - dna complex in dictating nal cluster structure ( we note that recent studies suggest the ag n itself is not the cause of the cd signal observed for ag n - dna but that the dna - silver B-material interaction of the intrinsically chiral dna plays a crucial role in generating chiroptical properties of these clusters ) . [SEP]
[CLS] past studies have found that classical theories which describe collective electronic excitations of colloids , 140 such as mie - gans theory , show surprising agreement with the optical properties of small metal B-material clusters , particularly for longitudinal plasmonic modes . [SEP]
[CLS] copp , et al . , examined whether ag n - dnas can also be described by classical models , applying mie - gans theory to hplc - puried ag n - dnas with 400 - 850 nm cluster excitation wavelengths and numbers of effective valence electrons , n 0 , determined by hr - ms in order to elucidate the aspect ratios of these clusters . [SEP]
[CLS] application of mie - gans theory to this experimental data predicted prolate cluster geometry , with aspect ratios of 1 . 5 for n 0 ¼ 4 up to $ 5 for n 0 ¼ 12 . [SEP]
[CLS] ( the currently reported crystallographic structures for ag n - dnas do not yet have determined charges , so these aspect ratios remain unconrmed by solved structures . [SEP]
[CLS] ) ag n - dnas with n 0 $ 6 displayed shis in peak excitation wavelength dependent on solvent dielectric , as is expected for a collective electronic excitation and observed for larger metal B-nanoparticle nanoparticles I-nanoparticle ; 146 such sensitivity may be useful for applications . [SEP]
[CLS] the increase in peak excitation wavelength and extinction coefficient with increasing cluster core B-material size n 0 ( ref . 147 ) is a characteristic shared by the longitudinal collective electronic excitation sustained by rod - like metal B-material clusters and larger metal B-nanoparticle nanoparticles I-nanoparticle [SEP]
[CLS] while the proper denition of a plasmon versus a collective electronic excitation at the cluster scale remains debated , other molecular - scale systems have also been shown to exhibit plasmon - like behavior . [SEP]
[CLS] the sanchez group simulated toy model ag n - dnas with magic number sizes , nding that a neutral silver B-material cluster rod surrounded by nucleobase - bound ag + is generated when a partial charge is placed on the cluster . [SEP]
[CLS] when excited , these clusters supported longitudinal plasmon - like modes . [SEP]
[CLS] intriguingly , single ag n - dnas studied at temperatures below 2 k exhibit surprisingly broad spectral linewidths . [SEP]
[CLS] for larger nanoparticles B-nanoparticle , surface plasmon resonance broadening is understood to arise from dephasing processes for multiple delocalized electrons , but such effects are less well understood at the cluster scale . [SEP]
[CLS] as silver B-material cluster rods , ag n - dnas may provide a unique platform to investigate these important questions . [SEP]
[CLS] it remains to be determined whether the optical transitions in ag n - dnas are collective or plasmonic - like , and further experimental and theoretical studies are needed to reveal which models are most suitable to represent the behavior of ag n - dnas . [SEP]
[CLS] 7 ( a ) peak absorbance energies for purified ag n - dnas characterized by ms as a function of the number of effective free electrons in the cluster ( red ) [SEP]
[CLS] experimental data are better described by simulations of silver B-material nanorods B-nanoparticle with 1 - atom B-material cross - sections ( green line ) than for thicker nanorods B-nanoparticle with 6 - atom B-material cross - sections ( gray line ) or spherical clusters ( blue band ) . [SEP]
[CLS] 3 . 3 x - ray and ir spectroscopy B-technique of solution - state ag n - dnas several groups have applied x - ray spectroscopy B-technique , nuclear magnetic B-property resonance ( nmr ) , and infrared ( ir ) spectroscopy B-technique to probe the structures and silver B-material - dna interaction in puried ag n - dnas . [SEP]
[CLS] to interrogate stoichiometry , oxidation state , ligand environment , and structure of a violet - absorbing ag n - dna , petty , et al . used esi - ms , x - ray absorption near edge structure ( xanes ) , and extended x - ray absorption fine structure ( exafs ) . [SEP]
[CLS] this dimly uorescent cluster has absorbance peaked at 400 nm but converts into a nir - emissive species upon perturbation of its dna template strand . [SEP]
[CLS] ms data ( fig . 8a ) and ag l 3 edge xanes spectra establish the sec - puried violet cluster to be an ag 10 6 + . [SEP]
[CLS] cd spectra of this ag 10 6 + remain stable above 70 c , pointing to the temperature stability of the dna - ag interaction ( fig . 8b ) . [SEP]
[CLS] ag k - edge exafs was used to probe organization of ag atoms B-material and ag - nucleobase interactions . [SEP]
[CLS] the experimental exafs trace ( black ) was tted to three individual scattering paths ( fig . 8c ) to infer specic bond lengths and coordination numbers . [SEP]
[CLS] based on these results , the authors proposed an octahedral cluster structure ( fig . 8d ) . [SEP]
[CLS] while creating an accurate model from exafs data is nontrivial given the vast number of possible geometries in such a complicated system , the model contains several structural elements later found in the crystal structure of an ag 16 investigated by cerretani , et al . , including an octahedral structural motif , limited to no classical wc pair interactions , and the fact that not all nucleobases interact with the ag n . [SEP]
[CLS] petty , et al . , later developed a different ag 10 6 + stabilized by an altered dna template which forms a hairpin at one terminus . [SEP]
[CLS] this altered cluster has the same oxidation state as the " violet " cluster above and can be reversibly converted by manipulation of the hairpin region . [SEP]
[CLS] the altered cluster is highly uorescent and has red - shied absorbance . [SEP]
[CLS] using differences in exafs data between the two clusters , the altered cluster is proposed to have a more extended and distinct metal B-material - like core B-material , presumably due to variations in coordination with the dna ligand . [SEP]
[CLS] these variations are supported by later studies using activated electron photodetachment ms . [SEP]
[CLS] volkov and co - workers used x - ray photoelectron spectroscopy B-technique ( xps ) to study an hplc - puried ag n - dna . [SEP]
[CLS] the oxygen B-material spectra are similar with and without ag + , supporting that ag + prefers to bind to nitrogen B-material when no reducing B-property agent I-property has been added . [SEP]
[CLS] for the puried ag n - dna , binding of silver B-material to oxygen B-material atoms I-material was present , suggesting that the interacting oxygens B-material belong to the sugar moiety and / or phosphodiester bond . [SEP]
[CLS] the crystal structures by cerretani , et al . , found ag atoms B-material bound to the phosphate group , conrming this observation . [SEP]
[CLS] in addition , ag 3d core B-material - level spectra were measured for various species containing both ag ( 0 ) and ag + . [SEP]
[CLS] the 3d 5 / 2 ag peak shis to higher binding energies ( x0 . 6 ev ) as one goes from ag ( 0 ) nanoparticles B-nanoparticle to ag n - dnas to ag + - dna complexes ( fig . 9 ) , supporting an ag n - dna with a positive charge which is neither purely cationic B-material nor fully reduced , in agreement with ms studies by others . [SEP]
[CLS] schultz , et al . , recently studied an hplc - puried ag n - dna 159 emissive at 670 nm with a previously measured high quantum yield of 0 . 75 . [SEP]
[CLS] by combining analytical centrifugation with nmr and ms , it became apparent that despite hplc isolation , the emissive product was a mixture of ag 15 and ag 16 . [SEP]
[CLS] thus , even rigorous chromatographic separation may not always fully separate ag n - dnas into compositionally pure solutions when two or more species have very similar compositions / conformations . [SEP]
[CLS] ir spectroscopy B-technique combined with md simulations provided insights into the dna B-event binding I-event sites of ag + . [SEP]
[CLS] the experimentally measured ir spectra of the ag n - dna and bare dna only show marked shis between 1350 - 1500 cm a1 aer cluster formation ( fig . 10a ) . [SEP]
[CLS] these shis correspond to the nucleobases ( fig . 10b ) , not phosphate backbone , conrming that the ag n ligates primarily to dna through ag - nucleobase interactions . [SEP]
[CLS] the kohler and petty groups very recently reported femtosecond time - resolved ir ( trir ) spectroscopic studies of two ag 10 6 + clusters stabilized by very similar 18 - base dna strands , c 4 ac 4 tc 3 xt 3 , where x represents either guanosine or inosine ( an articial nucleoside lacking the exocyclic c2 - nh 2 of natural guanosine ) . [SEP]
[CLS] these two dna strands stabilize products with nearly identical spectra but dramatically differing quantum yields and uorescence decay times , suggesting that the x nucleoside inuences the excited state processes of the ag 10 6 + . [SEP]
[CLS] following excitation of the clusters by a 490 nm femtosecond laser pulse , the trir spectra are collected in the 1400 - 1720 cm a1 range , corresponding to spectral features from the nucleobases . [SEP]
[CLS] while individual nucleobases are excited in the uv , trir spectra show that 490 nm excitation of the clusters results in bleaching of the vibrational modes of select nucleobases , most notably cytosine . [SEP]
[CLS] thymine appears unperturbed by cluster excitation , supporting the many past studies which show that silver B-material has low affinity for this nucleobase at neutral ph and suggesting that this base does not coordinate with the cluster . [SEP]
[CLS] slight differences in the trir spectra of x ¼ g and x ¼ i ag n - dnas suggest that this method may enable precise probing of the electronic coupling of the ag n and surrounding nucleobases , a topic which remains poorly understood for ag n - dnas . [SEP]
[CLS] many reports of transmission B-technique electron I-technique microscopy I-technique ( tem ) to characterize ag n - dnas report 2 - 20 nm particles , which have been attributed to the uorescent clusters of interest . [SEP]
[CLS] however , due to the much smaller sizes of ag n - dnas established by hplc - ms and recent crystallographic studies ( section 3 . 5 ) , it is highly likely that the particles observed in tem are silver B-nanoparticle nanoparticles I-nanoparticle formed as byproducts during chemical synthesis . [SEP]
[CLS] crystallographic studies have recently begun to yield important insights into the structures of monolayer - protected silver B-material clusters . [SEP]
[CLS] very recently , the rst crystal structures have been reported for ag n - dnas . [SEP]
[CLS] huard , et al . , reported the rst ag n - dna crystal structure for a cluster stabilized by two copies of a 6 - base strand , 5 0 - aacccc - 3 0 . [SEP]
[CLS] the asymmetric unit , shown in fig . 11a , reveals a " big dipper " shape of 8 ag atoms B-material with planar geometry , identied as an ag 8 - dna . [SEP]
[CLS] four additional silvers B-material not directly bound to the ag 8 promote crystal packing ( blue spheres in fig . 11 ) . [SEP]
[CLS] ag - ag distances in the pentameric core B-material are x2 . 9 a , comparable to ag - ag bond lengths in bulk silver B-material . [SEP]
[CLS] in this cluster core B-material , adenines interact with ag via n1 and n6 , whereas cytosines are coordinated through n3 and n4 ( fig . 11 ) . [SEP]
[CLS] the exocyclic nitrogens B-material ( n4 , n6 ) are hypothesized to be deprotonated . [SEP]
[CLS] the zipper region is characterized by c - ag + - c base pairs with parallel strand orientation and twisted base pairs , as observed elsewhere . [SEP]
[CLS] every ag interacts with the n3 site of one cytosine on each strand ( fig . 11c ) , as established previously for ag n - dnas stabilized by c 12 strands 52 and for c - ag + - c duplexes . [SEP]
[CLS] all base - ag interactions have distances of x2 . 1 a . [SEP]
[CLS] unlike the ag n - dna studied by schultz , et al . , signicant interactions between adenines and ag were found in the ag 8 - dna ( fig . 11d ) . [SEP]
[CLS] it is notable that one ag atom B-material of the pentamer portion of the ag 8 is stabilized by a neighboring strand ' s adenine ( ag atom B-material in orange in fig . 11d ) , which may explain why the cluster could not be formed in solution without modications to the dna template strand . [SEP]
[CLS] six crystal structures have also been reported by cerretani , et al . , for nir ag 16 - dnas stabilized by dna templates that differ by only one nucleobase . [SEP]
[CLS] the rst reported crystal structure is for an ag 16 - dna stabilized by two strands of a dna decamer , 5 0 - cacctagcga - 3 0 , previously identied by copp , et al . , [SEP]
[CLS] with an unusually large stokes shi . the second ag 16 - dna is stabilized by two copies of a 9 - base sequence corresponding to removal of the a 10 at the 3 0 - end of the decamer ( fig . 12a ) . [SEP]
[CLS] clusters formed on these two templates are nearly identical , and removal of the terminal a 10 has no discernable impact on the wavelength of the absorbance peak but causes a slight redshi in uorescence emission . [SEP]
[CLS] similarly , mutations of position 5 in the dna sequence allow one to produce and crystalize a similar nir emitter . [SEP]
[CLS] the latter study showed also that certain nucleotide positions in the dna sequence , while not relevant for binding to the ag n , could be mutated in order to promote or alter crystal packing interactions . [SEP]
[CLS] this concept could enable in the near future to re - engineer dna sequences to promote crystallization and determine the structure of the emissive ag n . [SEP]
[CLS] it also demonstrates that , especially when the ag n is stabilized by multiple strands , the 3d organization of the nucleotides is more relevant than the sequential 5 0 to 3 0 order . [SEP]
[CLS] unlike the ag 8 - dna investigated by huard , et al . , which did not perform nabh 4 reduction before the crystallization process , the ag 16 - dnas were synthesized in aqueous solution and then hplc - puried prior to crystallization . [SEP]
[CLS] the clusters comprise 16 ag atoms B-material with occupancy of 1 , along with additional silvers B-material with lower occupancy ( fig . 12a and b ) . [SEP]
[CLS] all bases , except thymine and one of the adenines in position 2 , interact with ag atoms B-material , with the thymine ensuring strand exibility and promoting crystal packing interactions ( fig . 12c - g ) . [SEP]
[CLS] most of the ag - ag distances are between 2 . 7 and 2 . 9 a , similar to or shorter than their metallic radius . [SEP]
[CLS] nevertheless , the cluster charge cannot be elucidated by these distances alone and as mentioned previously , ample hr - ms data suggests that ag n clusters are generally highly cationic B-material in nature . [SEP]
[CLS] similar to the crystal structure published by huard , et al . , ag atoms B-material interact with cytosines via n3 , and with adenines via n1 . [SEP]
[CLS] interestingly , additional interacting sites were discovered , consistent with schultz , et al . [SEP]
[CLS] silvers B-material coordinate o2 of cytosines , as well as n1 , n7 and o6 of guanines , and n7 and the oxygens B-material of the adenine phosphate group . [SEP]
[CLS] ag - n distances are 2 . 2 - 2 . 5 a , mostly shorter than the ag - o coordinate bond lengths 2 . 4 a to 2 . 9 a . [SEP]
[CLS] the ag - n bond lengths suggest that g 9 is deprotonated at n1 ( 2 . 3 - 2 . 4 a ) . [SEP]
[CLS] some ag n - dna crystal structures contain ag + which are not attached to the central cluster but do participate in non - wc base interactions and crystal packing . [SEP]
[CLS] it is possible that such " accessory " ag + also exist in solution - phase ag n - dnas , as recently suggested by gambucci , et al . if sufficiently tightly bound , these ag + would be counted by hr - ms as part of the ag n - dna but may not be part of the silver B-material cluster itself and , thus , may not contribute signicantly to the cluster ' s electronic properties . [SEP]
[CLS] hr - ms results have not been reported for the ag 8 reported by huard , et al . , nor the multiple ag 16 species reported by cerretani , et al . , so it remains unknown whether all of the accessory ag + are present in solution . [SEP]
[CLS] studies which compare the ms - determined sizes of ag n - dnas with their crystallographic sizes are needed in order to probe the existence and role ( s ) of accessory ag + in ag n - dnas and , more generally , to what degree hr - ms measurements of puried ag n - dna species can discern the size of the emissive cluster . [SEP]
[CLS] it will also be important to clearly state the assumptions made when assigning the cluster size n of an ag n - dna , particularly in light of the aforementioned evidence that observed optical properties are strongly correlated to the numbers of neutral silver B-material atoms I-material n 0 determined by hr - ms and not necessarily the total silver B-material atom I-material number n tot . [SEP]
[CLS] we have primarily reviewed compositional and structural studies of ag n - dnas which are synthesized by chemical reduction and , notably , are stable under hplc purication to enable accurate characterization . [SEP]
[CLS] smaller ag 2 and ag 3 clusters intercalated between base pairs of dsdna can be synthesized by electrochemical means and exhibit $ 300 nm uorescence emission , which supports smaller size and / or different cluster geometry than the hplc - puried ag n - dnas discussed here . [SEP]
[CLS] the versatility of macromolecular cluster ligands like dna may permit multiple classes of metal B-material clusters of the same metal B-material species , even for ag n - dnas synthesized by chemical reduction . [SEP]
[CLS] thus , it is likely that other cluster sizes and geometries than the hplc - puried ones discussed here may exist which may be unstable under the solvent and high - pressure conditions requisite for chromatographic separation . [SEP]
[CLS] in addition to cluster structure , the secondary / tertiary structures of the cluster ' s dna templates are of interest . [SEP]
[CLS] an understanding of this structure is also critical for schemes which integrate with dna nanotechnology . [SEP]
[CLS] as before , we primarily review studies of puried samples or which employ techniques which may lead to isolated ag n - dna species , including micro - uidic capillary B-technique electrophoresis I-technique , gel B-technique electrophoresis I-technique , and sec . [SEP]
[CLS] the petty group combined sec and other analytical methods to discern tertiary structures of their developed ag n - dna sensors , which signal binding B-event of I-event dna I-event analytes by transforming nonuorescent ag 11 clusters on ssdna templates into niremissive clusters of twice the size . [SEP]
[CLS] sec separates complexes by molecular size and shape , with larger products eluting more quickly . [SEP]
[CLS] a difference in retention time between two products indicates differences in molecular size . [SEP]
[CLS] to count the number of dna strands , n s , which scaffold the nir cluster , 10 - thymine tails were appended to one end of the target dna analyte . [SEP]
[CLS] sec shows that a 1 : 1 mixture of dna analytes with and without tails splits the chromatogram into three peaks . [SEP]
[CLS] this splitting can be interpreted as complexation of two dna probes to form the nir ag n - dna ( fig . 13a ) . [SEP]
[CLS] this was one of the rst demonstrations of formation of ag n - dnas stabilized by template strand dimers , [SEP]
[CLS] conformation of the dna template strandapart from hr - ms . [SEP]
[CLS] for a modied sensor scheme , alignment of thymine tails was further used to probe alignment of the two dna strands stabilizing the nir ag n - dna . [SEP]
[CLS] these clever experiments provide an alternate technique for inferring n s , which is especially useful for larger dna complexes hosting an ag n - dna , which may not be stable even under gentle negative mode esi - ms . [SEP]
[CLS] thymine tails were later used by del bonis - o ' donnell , et al . , to separate a set of ag n - dna - based probes for hepatitis a , b , and c in a single microcapillary electrophoresis B-technique protocol . [SEP]
[CLS] the petty and brodbelt groups recently determined and compared binding sites of two different ag 10 clusters to their dna templates using activated electron photodetachment ( a - epd ) ms . [SEP]
[CLS] one ag 10 was stabilized by a 20 - base strand which is single - stranded in the absence of silver B-material ( the subject of fig . 8 ) , and the other by a 28 - base strand which forms a hairpin in the absence of silver B-material ( ag n - dnas were studied without subsequent purication ) . [SEP]
[CLS] the dna templates with and without ag n were analyzed by a - epd , using 193 nm irradiation to induce dna fragmentation , followed by ms . [SEP]
[CLS] 13b - e compares mass spectra of the fragmented dna host strands with and without ag n , showing that certain fragments are suppressed in the presence of the ag n . [SEP]
[CLS] the suppression of fragmentation for certain regions of the dna templates was associated with binding of the nucleobases to the ag n in these suppressed regions . [SEP]
[CLS] for the ssdna template , a remarkably short 4 - base segment of cctt was suppressed ( fig . 13c ) ; in comparison to available crystal structures , it is reasonable that this segment represents only part of the silver - ligated nucleobases . [SEP]
[CLS] for the hairpin dna template , a much longer 13 - base segment was suppressed , most of which is in the wc paired hairpin stem in the absence of silver B-material ( fig . 13e ) . [SEP]
[CLS] this provides credence to the notion that ag + can signicantly reorganize dna secondary structure . [SEP]
[CLS] future a - epd studies could yield insights into other ag n - dnas . [SEP]
[CLS] compared to the current growing understanding of ag n - dna structure , the luminescence B-property process of ag n - dnas remains less understood . [SEP]
[CLS] ag n - dnas most certainly luminesce B-property through an allowed uorescence - like process , as supported by 1 - 4 ns uorescence decay times and quantum yields > 0 . 1 for most puried ag n - dnas . [SEP]
[CLS] in contrast , phosphorescencelike emission of other metal B-material clusters is characterized by much longer decay times and lower quantum yield values due to less allowed / forbidden transitions . [SEP]
[CLS] however , the ag n - dna uorescence process does differ from the simple jablonski diagram of organic uorophores . [SEP]
[CLS] ag n - dnas lack the characteristic vibronic shoulders of organic molecular uorophores , and their solvatochromic behavior is not well - described by onsager - based methods used to model many organic uorophores . [SEP]
[CLS] certain ag n - dnas retain surprisingly high quantum yields into the nir , while quantum yields of organic dyes diminish rapidly in this region . [SEP]
[CLS] ag n - dnas also have highly polarized excitation and emission due to well - dened transition dipole moments . [SEP]
[CLS] finally , the process of indirect uorescence excitation via the dna bases , which produces the same color of uorescence as direct excitation in the visible or nir excitation band of the ag n - dna ( fig . 1b ) , remains poorly understood . [SEP]
[CLS] here , we review spectroscopic studies of the photophysics of ag n - dnas , with a focus on puried ag n - dnas in more recent years . [SEP]
[CLS] in order to ensure that measured photophysical properties are not affected by the presence of byproducts , such as ag nanoparticles B-nanoparticle and nonuorescent ag n - dnas , purication is essential to preparation and analysis of these uorophores . [SEP]
[CLS] a limited number of experimental studies have probed the ultrafast dynamics that occur upon excitation of ag n - dnas to the initial excited state ( franck - condon state ) . [SEP]
[CLS] patel , et al . , proposed the rst phenomenological model describing the excitation process ( fig . 14 ) , based on ultrafast transient absorption experiments performed on three unpuried red and nir ag n - dnas ( fig . 15 ) . [SEP]
[CLS] it was observed that a fraction of the population in the franck - condon state returned to the ground state with a time constant in the hundreds of fs , as seen by the ground - state recovery ( fig . 15a ) . [SEP]
[CLS] additionally , a rise component of similar time scale was attributed to formation of the emissive state . [SEP]
[CLS] this emissive state then decays back to the ground state on a nanosecond timescale , as witnessed by similar time - scales of the ground - state recovery and the typical ns uorescence decay times measured by time - correlated single photon counting ( tcspc ) . [SEP]
[CLS] in addition , nanosecond transient absorption B-technique spectroscopy I-technique ( fig . 15b ) , together with single molecule blinking experiments , 8 also showed the presence of a dark state with a ms - scale decay time . [SEP]
[CLS] to date , no signicant emission from this dark state has been observed , indicating that it decays mainly nonradiatively back to the ground state . [SEP]
[CLS] whether the dark state originates directly from the franck - condon state or is formed from the emissive state can be determined by the rise time of the dark state formation itself . [SEP]
[CLS] nanosecond transient absorption experiments were performed to determine the rise time of the dark state . [SEP]
[CLS] these experiments concluded that dark state formation was limited by the instrument response function ( irf ) of 7 ns , prohibiting discernment of the state from which the dark state is formed . [SEP]
[CLS] recent single molecule results by krause , et al . , yielding secondary uorescence ( emission generated from oadf ) over primary uorescence intensity ratios higher than 1 , suggest that the dark state is formed from the initial frank - condon state . [SEP]
[CLS] recently , thyrhaug , et al . , performed 2d electronic spectroscopy B-technique experiments on a previously sized nir - emitting ag 20 - dna . [SEP]
[CLS] excitation into the franck - condon state led to ultrafast evolution of the franck - condon state into the emissive state , which then decayed on a nanosecond time - scale observed from tcspc measurements . [SEP]
[CLS] the transfer from the initially populated state to emissive state occurred in about 140 fs , in line with the order of magnitude reported by patel , et al . [SEP]
[CLS] additionally , the ag n - related absorption feature appeared to consist of two closely lying transitions , and a coherent excitation of both states occurred due to the short pulse width of the laser . [SEP]
[CLS] interestingly , for this particular ag n - dna , coherence was transferred to the emissive state and can be seen by oscillatory quantum beating features that dephased with a time constant of $ 800 fs . [SEP]
[CLS] thus , aer a few ps , all ultrafast processes were complete , and the only remaining process is the $ ns uorescence . [SEP]
[CLS] the dominant quantum beating mode frequency of 105 cm a1 is similar to ag - ag vibrational modes . [SEP]
[CLS] the presence of a ms - lived dark state in ag n - dnas was rst reported by vosch et al . [SEP]
[CLS] ag n - dnas were immobilized in a polyvinyl alcohol B-material ( pva ) lm and uorescence intensity was recorded as a function of time . [SEP]
[CLS] autocorrelations of the uorescence intensity trajectories revealed ms blinking . [SEP]
[CLS] a similar ms correlation time was observed by uorescence correlation spectroscopy I-technique ( fcs ) in solution . [SEP]
[CLS] fcs experiments are not only useful for determination of the decay time of the dark state and the quantum yield of dark state formation but also for estimation of the molar extinction coefficient by determining the number of emitters in a certain volume identied from a reference measurement . [SEP]
[CLS] while nontrivial to determine or suggest the exact nature of the dark state , dark states have been reported in other studies and may be common for most ag n - dnas . [SEP]
[CLS] the quantum yields of dark state formation have been estimated to range from a few up to 25 percent . [SEP]
[CLS] when removal of molecular oxygen B-material from the environment results in a lengthening of the dark state decay time , this is oen a good indicator B-property that the dark state is a triplet state . [SEP]
[CLS] for ag n - dnas , the dna scaffold around the silver B-material cluster might act as a physical barrier B-property for this type of dexter - type triplet state quenching , resulting in minimal or no effect of removal of oxygen B-material on the dark state decay time . [SEP]
[CLS] richards , et al . , demonstrated that the dark state formed by a primary excitation laser can be optically excited with a secondary nir laser , resulting in depletion of this long - lived state and an overall increase in uorescence intensity . [SEP]
[CLS] it was recently proven that optical excitation of the dark state can transition the ag n - dna to the emissive state , resulting in optically activated delayed uorescence ( oadf ) . [SEP]
[CLS] this process is similar to typical reverse - intersystem crossing processes observed in organic dyes . [SEP]
[CLS] oadf combined with timegating provides background - free signal because the delayed uorescence is on the anti - stokes side ( lower wavelength side ) of the secondary excitation laser , allowing any stokes - shied auto - uorescence from the secondary laser to be suppressed with a short - pass lter in the detection path . [SEP]
[CLS] 16 shows an example by krause , et al . , that demonstrates the oadf imaging concept . [SEP]
[CLS] additionally , krause , et al . , showed that the use of the secondary nir laser only ( blocking the primary excitation laser ) yielded similar uorescence which was linearly dependent on the excitation intensity . [SEP]
[CLS] this process is termed upconversion uorescence ( ucf ) , in analogy to the well - established upconversion processes in lanthanide based emitters . [SEP]
[CLS] while one would expect a single emissive species to exhibit mono - exponential uorescence decay , several hplc - puried ag n - dnas with long dna template strands ( 19 - 30 bases ) exhibit multi - exponential uorescence decay . [SEP]
[CLS] because solutions are puried prior to characterization and no shi is present in the steady - state emission as a function of excitation wavelength , a heterogeneous mixture of ag n - dna species can be excluded as the cause of this multi - exponential decay . [SEP]
[CLS] the multi - exponential decay behavior can instead be explained by relaxation of the emissive state on a time - scale similar to the uorescence decay . [SEP]
[CLS] this effect , termed " slow " spectral relaxation , can be conrmed by time - resolved emission spectra ( tres ) which show a gradual red - shi of the emission maximum on the nanosecond time scale ( fig . 17a ) . [SEP]
[CLS] we note that the " slow " spectral relaxation is a minor part of the overall stokes shi , with the majority of relaxation occurring on a timescale below the irf . [SEP]
[CLS] a consequence of " slow " spectral relaxation is that the average decay time increases as a function of emission wavelength ( fig . 17b ) . [SEP]
[CLS] furthermore , the decay associated spectra ( das ) usually lead to spectra where the fastest decay time component tends to have positive amplitudes at shorter wavelengths and negative amplitudes ( rise ) at longer wavelengths ( fig . 17c ) . [SEP]
[CLS] only two processes can cause this effect : energy transfer or " slow " spectral relaxation . [SEP]
[CLS] energy transfer can be excluded since , as stated above , there is no evidence for multiple independent emitters in the hplc - puried ag n - dna solutions . [SEP]
[CLS] unlike small solvent molecules which rearrange on picosecond timescales , the dna template and its structurally bound water B-material molecules require much longer , up to a few nanoseconds , to adapt to the new charge distribution of the ag n - dna in the emissive state . [SEP]
[CLS] a similar effect was observed when a coumarin dye was embedded in an abasic site of dsdna . [SEP]
[CLS] emission spectral shis could be observed from the femtosecond time scale up to tens of nanoseconds . [SEP]
[CLS] other parameters , e . g . changes to solvent viscosity B-property or temperature , also affect the " slow " spectral relaxation . [SEP]
[CLS] if spectral relaxation occurs entirely within the time - scale of the irf , the observed decay time will be mono - exponential . [SEP]
[CLS] this is the case for ag n - dnas stabilized by short , 9 - 10 base dna strands , whose " slow " spectral relaxation is negligible at room temperature and in low viscosity B-property solvents , most likely because multiple short strands are more exible and rearrange faster than one long oligomer . [SEP]
[CLS] spectral relaxation could be a useful tool to establish the rigidity of the dna scaffold and its effect on the excited state of ag n - dnas . [SEP]
[CLS] another interesting spectroscopic feature of ag n - dnas is their parallel excitation and emission transition dipole moments . [SEP]
[CLS] hooley , et al . , employed defocused wideeld microscopy B-technique to investigate the transition dipole moments of a c 24 - templated ag n - dna immobilized in pva . [SEP]
[CLS] by defocusing a common wideeld image , the emission of a single emitter displays a bilobed shape that depends on the orientation of its emission transition dipole . [SEP]
[CLS] in order to determine both excitation and emission transition dipoles simultaneously , defocused wide - eld microscopy B-technique was combined with rotating the polarized excitation light . [SEP]
[CLS] then , the intensity of each emitter is directly correlated to the excitation efficiency of the ag n - dna . [SEP]
[CLS] maximum emission intensity was observed when excitation light was aligned with the emission transition dipole , indicating that the excitation and emission transition dipole moments lie along a similar direction . [SEP]
[CLS] the vosch group has also observed further evidence of the alignment of excitation and emission transition dipole moments by time - resolved anisotropy measurements . [SEP]
[CLS] three different ag n - dnas , two nir emitting and one red , displayed limiting anisotropy values close to 0 . 4 , which indicates that the excitation and emission transition dipole moments are parallel ( one example in fig . 18 ) . [SEP]
[CLS] patel , et al . , rst reported two - photon excitation ( 800 to 1000 nm range ) of ag n - dnas in 2008 , in a study of four non - puried ag n - dnas with emission maxima at 620 nm , 660 nm , 680 nm and 710 nm . [SEP]
[CLS] for the 660 nm , 680 nm and 710 nm emitters , the two - photon emission exhibited quadratic dependency on excitation intensity , as expected , and one - photon and two - photon uorescence decay times were similar , indicating that emission occurred from the same emissive state . [SEP]
[CLS] the one versus twophoton excitation spectra of the 620 nm emitter indicated that cross - section maxima occurred at different wavelengths . [SEP]
[CLS] the reported two - photon cross - sections ranged from 33 900 to 50 000 gm , roughly two orders of magnitude higher than typical organic uorophores ( e . g . 210 gm at 840 nm for rhodamine b ) . [SEP]
[CLS] yau , et al . , reported a two - photon cross - section of $ 3000 gm at 800 nm and quadratic dependence of emission on excitation intensity for an unpuried 650 nm emitter . [SEP]
[CLS] this 650 nm emitter was made by rst creating an ag n - dna using ssdna , followed by addition of an excess of a complementary strand with a guanine - rich section . [SEP]
[CLS] because few studies have probed two - photon excitation of ag n - dnas , future investigations on puried ag n - dna could shed light on the origin of the very high two - photon cross - sections . [SEP]
[CLS] the fascinating sequence dependence of ag n - dnas results from the nucleobase - specic interactions of dna with silver B-material ( section 2 ) . [SEP]
[CLS] the ability of dna sequence to select for the sizes and optical properties of metal B-material nanoclusters has attracted great interest due to the promise of highly customized uorophores . [SEP]
[CLS] to date , it is likely that thousands of different dna template strands have been reported , corresponding to ag n - dnas with wideranging uorescence colors , stokes shis , quantum yields , chemical yields , photostabilities , and chemical stabilities . [SEP]
[CLS] yet the connection between dna sequence and ag n - dna properties has remained obscure . [SEP]
[CLS] most studies select ag n - dnas by experimentally testing small numbers of dna template strands rich in c or g . [SEP]
[CLS] one large - scale study by the dickson group used dna microarrays to identify uorescent ag n - dnas , but only a few of the dna template sequences were reported . [SEP]
[CLS] to fully realize ag n - dnas as programmable materials , it is crucial to " decode " the connection between dna sequence and ag n - dna properties . [SEP]
[CLS] rational design of ag n - dnas is especially challenging due to an astronomical number of possible dna template sequences and a complex connection between ag n - dna color and dna sequence . [SEP]
[CLS] ag n - dna templates are typically 10 - 30 base oligomers . [SEP]
[CLS] because a sequence of the four natural nucleobases can have 4 l distinct l - base sequences , ag n - dna templates must be chosen from 4 ( $ 10 18 ) possible sequences . [SEP]
[CLS] while in some cases subtle sequence changes can dramatically shi uorescence , in other cases different dna sequences can stabilize ag n - dnas with the same emission wavelength . [SEP]
[CLS] to make matters more complex , some dna sequences can stabilize different types of uorescent ag n clusters , with yields of each cluster species possibly depending on synthesis method and / or ag : dna stoichiometry . [SEP]
[CLS] first - principles computational methods [SEP]
[CLS] have not yet matured sufficiently to model the structures of realistic ag n - dnas , let alone their accurate electronic properties . [SEP]
[CLS] small - scale studies of dna sequences with constrained patterns have been useful for developing a few ag n - dnas with well - controlled properties but are limited in their applicability to the majority of reported ag n - dnas . [SEP]
[CLS] here , we review large - scale experimental studies of the ag n - dna sequence - color connection for 10 3 dna strands , in which machine learning enables predictive design and provides new physical insights . [SEP]
[CLS] the combinatorial nature of dna makes data science wellsuited to study how dna sequence selects ag n - dna properties . [SEP]
[CLS] copp , et al . , have pioneered high throughput experiments together with supervised machine learning ( ml ) to understand how dna sequence selects for ag n - dna uorescence emission and to predict new templates for optimized ag n - dnas . [SEP]
[CLS] the methods described here have uncovered ag n - dnas which are the subjects of later detailed studies . [SEP]
[CLS] for readers seeking to learn more about ml , we recommend a tutorial review by domingos and a review of ml for so matter by ferguson . [SEP]
[CLS] to train a ml algorithm to output ag n - dna uorescence properties ( or whether any uorescent product can be stabilized ) given an input dna template sequence , one must rst amass a data library connecting dna sequence to ag n - dna uorescence spectra for hundreds to thousands of sequences . [SEP]
[CLS] this data cannot be mined from the literature because ( i ) synthesis and characterization methods vary widely , prohibiting isolation of the effects of dna sequence from other experimental parameters , and ( ii ) while $ 75 % of dna sequences are unsuitable for templating uorescent ag n - dnas , these " negative " dna sequences are rarely reported . [SEP]
[CLS] the absence of negative sequences from the literature is problematic because to effectively learn what makes a suitable dna template for brightly uorescent ag n - dnas also requires knowledge of what does not make a suitable template . [SEP]
[CLS] to enable ml for ag n - dnas , copp , et al . , developed highthroughput ag n - dna synthesis and characterization in well plate format using robotic liquid handling followed by rapid uorimetry , 57 via universal uv excitation of all ag n - dna products through the nucleobases ( fig . 19 part i ) . [SEP]
[CLS] because uorimetry is performed one day , one week , and four weeks aer synthesis , this data set allows ml studies to focus only on timestable ag n - dnas . [SEP]
[CLS] experiments are normalized using a wellstudied ag n - dna control , for direct comparison of uorescence wavelength and intensity among all data in the library . [SEP]
[CLS] to date , we have reported on > 3000 distinct dna template sequences , most 10 bases long , for ag n - dna synthesis in 10 mm nh 4 oac aqueous solution of neutral ph . [SEP]
[CLS] effective ml requires appropriate choice of " feature vectors , " which are the parameterizations of training data provided as inputs to the ml classier ( s ) . [SEP]
[CLS] for ag n - dnas , feature vectors should represent the salient properties of a dna sequence which determine how sequence is mapped onto ag n - dna uorescence . [SEP]
[CLS] because these properties are not well - known ( otherwise ml would be unnecessary ) , this feature engineering process is a critical step in the ml workow and has led to new physical insights into ag n - dnas . [SEP]
[CLS] early work used training data for 684 randomly generated 10 - base dna sequences to learn to predict ag n - dna uorescence brightness given an input template strand sequence . [SEP]
[CLS] using integrated uorescence intensity , i int , as a metric of brightness , sequences with the top 30 % of i int values were dened as bright and the bottom 30 % of i int values dened as " dark . " [SEP]
[CLS] then , a ml algorithm called a support vector machine ( svm ) was trained to distinguish bright and dark sequences ( fig . 19 part ii ) . [SEP]
[CLS] it was found that the svm most accurately predicted a sequence ' s class if feature vectors were engineered to quantify the occurrence of certain dna subsequences called " motifs " which were identied by bioinformatics approaches to be correlated with one class but not the other . [SEP]
[CLS] the resulting trained svm ' s classication accuracy was 86 % , as determined by crossvalidation ( a process which trains on most of a training data set and reserves a small $ 10 % portion as a " test set " to assess svm performance on data which the ml classier has not yet encountered ) . [SEP]
[CLS] new dna templates for bright ag n - dnas were designed using the bright - correlated motifs as building blocks and then screened by the trained svm to choose those predicted as most likely to be bright . [SEP]
[CLS] 78 % of designed dna templates stabilized bright ag n - dnas , as compared to 30 % of the initial random sequences . [SEP]
[CLS] this early work pointed to the role of certain dna base motifs in stabilizing ag n - dnas , which agreed with later ndings that not all dna bases coordinate the ag n . [SEP]
[CLS] while predicting ag n - dna uorescence intensity increases the likelihood of selecting uorescent ag n - dnas by three - fold , this simple method also prefers red - uorescent ag n - dnas over green ag n - dnas . [SEP]
[CLS] it is ideal to instead predict both brightness and color from an input dna sequence . [SEP]
[CLS] to achieve this , copp , et al . , used physically motivated ag n - dna classication based on the known correlation between ag n - dna color and cluster size . [SEP]
[CLS] the multi - modal distribution of ag n - dna uorescence colors in the visible spectrum was shown to arise due to the magic numbers of these clusters : ag n - dnas in the 500 - 570 nm abundance have n 0 ¼ 4 neutral ag atoms B-material , while ag n - dnas in the 600 - 670 nm abundance have n 0 ¼ 6 ( fig . 20a ) . [SEP]
[CLS] because " green " and " red " ag n - dnas have distinct cluster sizes , there is likely a fundamental difference between template sequences for these two cluster sizes . [SEP]
[CLS] to learn to distinguish between dna sequences based on cluster structural differences , a training data set of $ 2000 10 - base dna sequences was separated into four color classes : the three shown in fig . 20a ( " very red " is dened as the high wavelength histogram shoulder , which may signal a different cluster structure ) and a " dark " class similar to the one previously dened . [SEP]
[CLS] because the numbers of sequences in these classes are unequal , with far more dark sequences than green sequences ( fig . 20b ) , it is critical to apply subsampling to balance classes prior to ml , ensuring training on equal numbers of sequences from each class . [SEP]
[CLS] feature vectors were then constructed using dna motif mining to identify color - correlated motifs , followed by feature selection to reduce the list of selected motifs to those most important for classication ; this critical step reduces problems which can arise from overtting . [SEP]
[CLS] we note that both data balancing and feature selection should generally be applied when using ml for real - world materials systems . [SEP]
[CLS] because svms are inherently binary classiers , a " one - versusone " approach was used to distinguish the four color classes . [SEP]
[CLS] six different svms were trained to discriminate between the six possible pairs of classes ( cross - validation scores , which represent the accuracy of classication , in fig . 20c ) . [SEP]
[CLS] to experimentally test the performance of the trained classiers , new dna template sequences were designed for the two least abundant classes , green and very red . [SEP]
[CLS] first , color - correlated dna motifs for the desired class were selected from a probability distribution weighted by intensity and placed into an initially empty dna sequence . [SEP]
[CLS] second , designed candidate dna templates were screened by the trained svms to estimate the probability of falling within the desired color class . [SEP]
[CLS] finally , templates corresponding to the top 180 probabilities were selected for experimental testing . [SEP]
[CLS] with this method , the likelihood of selecting a very red ag n - dna increased by nearly 330 % , and the likelihood of selecting a green ag n - dna was increased by > 80 % . [SEP]
[CLS] this method was later modied to enable design of ag n - dna templates of any strand length , and it was found that training data of only 10 - base sequences still enabled effective prediction of ag n - dna color for other lengths of dna templates , up to the maximum 16 - base length tested ( fig . 20d ) . [SEP]
[CLS] this suggests that there exist certain dna motifs which are selective of cluster type and thus color for a range of dna template lengths , making ml design approaches for ag n - dnas much more promising . [SEP]
[CLS] we note that thus far , all ag n - dnas stabilized by dna templates of < 19 bases have been found to be " strand dimers " which contain two template strands per cluster ; it is possible that longer dna templates , which have not been designed by ml , may have some different dna sequence rules for ag n - dna color selection . [SEP]
[CLS] in addition to improving design efficiency , ml provides key insights into how dna sequence selects silver B-material cluster size , and thus uorescence wavelength . [SEP]
[CLS] 21b shows average base composition of the motifs identied by feature selection to be most predictive of dark , green , red , and very red sequences . [SEP]
[CLS] to summarize , thymines are strongly correlated with no uorescence . [SEP]
[CLS] adenines show preference for smaller and green ag n - dnas , while guanines , particularly consecutive guanines , are correlated with long wavelength uorescence ( associated by ms with clusters containing more ag atoms B-material ) . [SEP]
[CLS] cytosines are strongly selective for uorescence brightness but less selective of color than a or g . [SEP]
[CLS] to better understand these correlations , we compare to hr - ms studies of dna - ag + complexes ( section 2 ) , which are the precursors of ag n - dnas prior to reduction by nabh 4 . [SEP]
[CLS] 3a shows the distribution of ag + attached to single dna homobase strands or pairs of strands , and fig . 5a shows the same distribution for ag + - mediated dimers of c or g strands with central base mutations . [SEP]
[CLS] because homo - thymine strands only weakly associate with ag + , thymine - rich dna sequences may be unsuitable ( at neutral ph ) to host uorescent ag n due to ( i ) too few ag atoms B-material recruited prior to reduction , resulting in insufficient silvers B-material to form a cluster and / or ( ii ) little to no coordination with the cluster . [SEP]
[CLS] the greater occurrence of t ' s in green ag n - dna templates further supports this notion , since these clusters are smaller in size and may require fewer nucleobase coordination sites . [SEP]
[CLS] adenine homobase strands bind to a few ag + , which may support formation of smaller n 0 ¼ 4 clusters with green emission . [SEP]
[CLS] in comparison to a and t , c - and g - rich homobase strands can form ag + - mediated duplexes with $ 1 ag + per base pair , providing more ag atoms B-material during cluster growth and supporting nucleobase - silver B-material binding in the ag n - dna . [SEP]
[CLS] interestingly , duplexes of g homobase strands with a single central a , c , or t base mutation can harbor $ 60 % more ag + than g homobase polymers B-material with no mutation . [SEP]
[CLS] this significant increase in ag + attachment as compared to c - rich strands , and the structural differences in the dna secondary / tertiary structures supported by ims - ms of these strands , could explain why consecutive g ' s are strongly associated with very red ag n - dnas . [SEP]
[CLS] 6 . supra - cluster assemblytowards applications in photonics and sensing [SEP]
[CLS] structural dna nanotechnology harnesses dna as a programmable building block for self - assembled nanostructures . [SEP]
[CLS] it is promising to combine sequence - controlled ag n - dnas with dna nanotechnology for realization of precise metal B-material cluster arrays , which could be envisioned as functional sensors and photonic devices . [SEP]
[CLS] these achievements will require robust strategies to effectively embed ag n - dnas into larger wc - paired architectures . [SEP]
[CLS] here , we review efforts to harness dna self - assembly for multi - ag n - dna organization ( many groups have incorporated single ag n - dnas within wc paired dna structures to build biomolecular sensors , which were recently reviewed elsewhere ) . [SEP]
[CLS] o ' neill , et al . , rst reported decoration of a dna nanostructure with multiple ag n - dnas . [SEP]
[CLS] a mixture of green and red clusters were synthesized onto ssdna hairpin protrusions programmed into dna nanotubes B-nanoparticle ( fig . 22a ) . without hairpins , nanotubes B-nanoparticle did not foster cluster growth , consistent with early ndings that dsdna is an unsuitable ag n - dna template . [SEP]
[CLS] the authors noted that tris buffers typical of dna self - assembly schemes were unsuitable for chemical synthesis of uorescent ag n - dnas ; this incompatibility is commonly faced in supracluster assembly of ag n - dnas . [SEP]
[CLS] orbach , et al . , demonstrated ag n - dna synthesis on mm - scale dna wires with hairpin protrusions . [SEP]
[CLS] resulting uorescence colors depended on salt B-material concentration , pointing to the complexity of controlled cluster synthesis on complex dna scaffolds . [SEP]
[CLS] the authors then incorporated ag n - dna - stabilizing hairpins into a hybridization chain reaction ( hcr ) , with wire formation only aer addition of an additional dna strand ( fig . 22b ) . [SEP]
[CLS] ag n - dnas have also been incorporated into dna hydrogels . [SEP]
[CLS] only two works have assembled puried ag n - dnas in order to approach atomic precision over cluster size in multi - cluster assemblies . [SEP]
[CLS] schultz , et al . , developed dna " clamps " for dualcolor ag n - dna pairs which exhibited forster resonance energy transfer ( fret ) between donor and acceptor ag n - dnas . [SEP]
[CLS] dna clamps were designed by appending complementary tails of a and t bases to templates for a green - emissive ag n - dna donor ( fig . 23a ) and a red - emissive ag n - dna acceptor ( fig . 23b ) , which have a 6 nm forster radius . [SEP]
[CLS] aer hplc purication of individual ag n - dnas , various geometries of clamps were formed by wc pairing . [SEP]
[CLS] for clamps where donor and acceptor were held within < 6 nm , donor excitation produced acceptor emission ( e . g . fig . 23c ) , with > 60 % fret efficiency estimated by donor quenching ( fig . 23d ) and assuming no isolated donor is present 186 ( use of excess of acceptor increased the likelihood of all donors in the paired state ) . [SEP]
[CLS] fret could be repeatedly cycled by heating and cooling , corresponding to cyclic melting and reforming of the dna clamp ( fig . 23e ) . [SEP]
[CLS] the clamp design is somewhat general and was demonstrated with a different acceptor cluster . [SEP]
[CLS] notably , schultz , et al . , found that hplc purication was essential to observing fret due to low chemical yield of ag n - dna synthesis ; without purication , very few clamps contain both donor and acceptor clusters . [SEP]
[CLS] recently , zhao , et al . , observed fret between donor and acceptor ag n - dnas without prior purication by synthesizing ag n - dnas within surfactant reverse micelles B-material with 5 - 10 nm diameters . [SEP]
[CLS] by this method , a fraction of the micelles B-material contained both donor and acceptor clusters conned together within a " nanocage B-nanoparticle " whose size is of the length scale of the forster radius of the pair . [SEP]
[CLS] this method also enabled spectroscopy - based measurement of micelle B-material diameter in agreement with more laborious cryoelectron microscopy B-technique , suggesting that ag n - dna - based fret may be a promising route to size measurement of biological " nanocage B-nanoparticle " structures . [SEP]
[CLS] copp , et al . presented a modular design strategy for multifunctional dna templates with distinct ag n - dna stabilizing regions and single - stranded " linker " regions . [SEP]
[CLS] this strategy exploits large data libraries 57 to identify 10 - base dna strands which do not foster uorescent ag n - dna growth . [SEP]
[CLS] these strands are candidate linkers to extend an ag n - dna template strand while leaving the cluster unchanged . [SEP]
[CLS] candidate linkers are appended to the dna sequence of an hplc - stable ag n - dna ( fig . 24a ) and experimentally screened to determine if linkers leave ag n - dna optical spectra unshied , signalling little to no change in cluster geometry . [SEP]
[CLS] a complementary " docker " site is then engineered onto a dna nanotube B-nanoparticle ( fig . 24a ) . [SEP]
[CLS] following hplc purication of the ag n - dna , multi - cluster assembly occurs by wc pairing of linker and docker strands ( fig . 24b ) . [SEP]
[CLS] ag n - dnas with atomically selected sizes are unperturbed aer binding to the nanotubes B-nanoparticle , as supported by unchanged spectral shapes aer assembly ( fig . 24c ) . [SEP]
[CLS] future studies are needed to conrm labelling efficiency . [SEP]
[CLS] the method is general to multiple sizes of ag n - dnas and linker sequences and could generalize to many types of dna scaffolds , for precise control over both cluster geometry and orientation . [SEP]
[CLS] recently , yourston , et al . , thoroughly studied ag n - dnas formed on rna nanorings with dna " arms . [SEP]
[CLS] " because in situ synthesis was used to decorate arms with ag n - dnas , it is uncertain how many ag n were harboured on a given nanoring . [SEP]
[CLS] interestingly , placement of the ssdna region on which ag n presumably formed affected not only uorescence spectra of the clusters , indicating variations in size / shape and possibly rigidity , but also time stability : clusters formed within the nanoring were much more time stable , perhaps due to enhanced protection from redox reactions which can blue - shi ag n - dna emission over time . [SEP]
[CLS] studies such as these will be important for assessing the practicality of dna - based ag n - dna arrays as functional materials . [SEP]
[CLS] the heterogeneous mixture of products and low chemical yield of ag n - dna synthesis can prohibit precise ag n - dna arrays by direct synthesis onto a dna nanostructure . [SEP]
[CLS] additionally , we pointed out in a previous section that one should also not a priori assume that the envisioned wc base pairing of the dna nanostructure will be maintained once silver B-material is introduced . [SEP]
[CLS] assembly methods which instead rely on wc pairing aer purication have their own limitations due to the limits of purity aer hplc and due to labelling efficiency of the dna nanostructure by binding of ag n - dna linkers to each docker site ; this may be overcome by adding an excess of ag n - dnas . [SEP]
[CLS] much more work is required to realize precise cluster arrays by either method . [SEP]
[CLS] signicant recent progress has been made in understanding the structure - property relations of ag n - dnas and achieving their rational design . [SEP]
[CLS] these advances were enabled by new experimental and computational strategies to purify and size ag n - dnas , to select new dna templates for especially uorescent ag n - dnas , and to crystallize ag n - dnas for structure determination , as discussed in this review . [SEP]
[CLS] here , we discuss outstanding challenges in this eld and areas of especial promise , which we hope will catalyze new research directions in this important eld . [SEP]
[CLS] nearly all reported ag n - dnas exhibit l em in the 500 - 750 nm range . [SEP]
[CLS] the most well - studied nir ag n - dnas have been developed by petty and coauthors . [SEP]
[CLS] high - throughput studies by copp , et al . , uncovered additional nir emissive clusters , and the vosch group has characterized several of these recently discovered nir ag n - dna , including one with an unusually large stokes shi and one with an impressively high 73 % quantum yield . [SEP]
[CLS] these quantum yields are competitive with organic uorophores , making ag n - dnas promising for development of biolabels in the nir tissue transparency windows . [SEP]
[CLS] until recently , only two ag n - dnas with l em > 800 nm were reported , and it was assumed that nir ag n - dnas are inherently rare compared to their visibly emissive counterparts . [SEP]
[CLS] however , because ag n - dna studies employ uv - vis optimized photodetectors commonly used for spectroscopy B-technique in the chemical and biological sciences , for which sensitivity is poor above $ 800 nm , it is possible that many nir - emissive ag n - dnas have simply gone undetected . [SEP]
[CLS] swasey and nicholson , et al . , developed a custom nir well plate reader equipped with an ingaas detector to search for nir uorophores in high - throughput ( fig . 25a ) . [SEP]
[CLS] using this tool to scan $ 750 ag n - dna samples , 161 previously unidentied nir - emissive ag n - dnas were uncovered ( fig . 25b ) . [SEP]
[CLS] this huge abundance of nir products was unexpected because the scanned ag n - dnas were stabilized by randomly selected dna template sequences or by oligomers previously designed for visible uorescence . [SEP]
[CLS] among the newly discovered ag n - dnas were found the longest wavelengthemissive ag n - dna to date , with 999 nm peak uorescence emission 23 ( fig . 25c ) and the largest ag n - dna to date , an ag 30 with 12 ag 0 and 18 ag + ( fig . 25d ) . [SEP]
[CLS] a directed search for nir ag n - dnas , using the informatics methods described in section 5 , is highly promising for discovery of new nir ag n - dnas . [SEP]
[CLS] both experimental and computational efforts are needed to further our understanding of the uorescence process in ag n - dnas , including the nature of the initial excited state , the relaxation process ( es ) leading to the origins of stokes shis for these emitters , and the roles of both the ag n and the surrounding nucleobases in governing excited state properties . [SEP]
[CLS] while a zoo of ag n clusters stabilized by different ligands have been described in literature , their optical properties largely differ from the distinctive features of ag n - dnas described in section 4 . [SEP]
[CLS] zeolite stabilized ag n clusters display mainly strong uv absorption bands , with emissive excited - state decay stretching from picoseconds to the microsecond range . [SEP]
[CLS] similarly , the mak group recently reported an intriguing octahedral silver B-material cluster with 95 % uorescence quantum yield and microsecond - scale uorescence decay times caused by thermally activated delayed uorescence . [SEP]
[CLS] such microsecond - scale uorescence decay times have not yet been observed for puried ag n - dnas . [SEP]
[CLS] only microseconds - lived dark states of ag n - dnas have been reported . [SEP]
[CLS] the unusual rod - like geometry of hplc - stable ag n - dnas , 24 which has been conrmed in recent crystal structures of nir ag n - dnas , makes ag n - dnas particularly interesting experimental systems for the study of collective electronic excitations in molecular - like materials . [SEP]
[CLS] with only n 0 ¼ 4 - 12 effective valence electrons in ag n - dnas characterized thus far by hr - ms , ag n - dnas lie well below the atomic size identied as the onset of plasmonic excitations in monolayerprotected gold B-material clusters . however , the high aspect ratios of some identied ag n - dnas 147 may make certain ag n - dnas better approximated as atomic B-material silver I-material rods , which computational studies have shown to exhibit plasmonic - like excitations . [SEP]
[CLS] future studies probing the ultrafast excited state dynamics of ag n - dnas are needed to better understand whether , or to what degree , collective electronic excitations are involved in the luminescence B-property process of ag n - dnas . [SEP]
[CLS] another feature of ag n - dna photophysics which remains poorly understood is the exact nature of the uv excitation process , which for the case of pure ag n - dna solutions leads to the same uorescence spectral shapes as visible / nir excitation ( fig . 1b ) . [SEP]
[CLS] due to dna ' s complex and elegant excited state dynamics , it is possible that dna imbues ag n with similar properties . [SEP]
[CLS] berkadin , et al . , have computationally examined the uv excitation process in ag n - dnas using molecular dynamics ( md ) to simulate thread - like silver B-material clusters in a dna duplex , followed by dft - based tight binding to calculate the electronic dynamics of the relaxed structure . [SEP]
[CLS] interesting , uv excitation results in a net negative charge transfer to the cluster , due to promotion of electrons from the localized p state of the dna to the cluster . [SEP]
[CLS] such simulations performed on the recently reported crystal structures would be of great interest . [SEP]
[CLS] furthermore , recent experiments by the kohler group on ag + nucleobase complexes are also promising for enhancing our understanding of this aspect of ag n - dna photophysics , with their very recent study nding evidence for an extremely long - lived , $ 10 ns excited state in a c 20 - ag + - c 20 duplex . [SEP]
[CLS] many chemical and biomolecular sensing schemes employing ag n - dnas have been developed , such as nanocluster beacons , 32 , 39 ratiometric sensors , and microrna sensors ( more complete list in a past review ) . [SEP]
[CLS] designing these sensors is extremely challenging , and designs may not generalize because silver B-material clusters are not conned only to the expected regions of a probe . [SEP]
[CLS] further , the mechanisms underlying the function of these sensors remain uncertain in most cases , although color and brightness changes are likely due to restructuring of ag n - dnas . [SEP]
[CLS] recent efforts have focused on strategies to improve sensitivity and selectivity of ag n - dna sensors , such as by addressing the low chemical yield of these clusters . [SEP]
[CLS] due to the complexity of designing ag n - dna sensors , we propose that high - throughput experimentation combined with machine learning approaches may be a useful path forward . [SEP]
[CLS] the yeh group has recently pioneered highthroughput screening of ag n - dna sensors , which may signicantly expedite progress in this area . [SEP]
[CLS] puried ag n - dnas could also serve as sensitive nanophotonic sensors . [SEP]
[CLS] the photophysical properties of pure ag n - dnas have also been shown to exhibit sensitivity to temperature , refractive B-property index I-property , and viscosity B-property . [SEP]
[CLS] combined with advancement in the ability to pattern dna nanostructures with ag n - dnas , it may be possible to design sensors which colocalize ag n - dnas with analyses of interest for nanoscale measurements . [SEP]
[CLS] while dna has been well - studied as a template for silver B-material clusters , and rna to a lesser extent , 46 much less is known about the suitability of non - natural polynucleic acids to template silver B-material clusters . [SEP]
[CLS] because rna is less exible than dna , it has been noted that rna may be less suitable as a scaffold for ag n if signicant exibility of oligomer ligands is required for a given cluster geometry . [SEP]
[CLS] synthetic polynucleic acids could expand cluster structures and geometries , enhance stabilities , and imbue added functionalities . [SEP]
[CLS] in addition to the four natural nucleobases , numerous articial nucleobases have well - studied affinity for silver B-material and other metals B-material . [SEP]
[CLS] a large range of uorescent nucleotide analogues are currently available , with more being actively developed continuously . [SEP]
[CLS] these bases could shi the universal uv excitation peak into the blue region of the visible spectrum , and fret experiments could help elucidate the energy transfer processes in ag n - dnas and even unravel distances and proximity of selected nucleobases to the ag n cluster . [SEP]
[CLS] also of interest are chemical modications developed for therapeutics to reduce enzymatic nucleotide digestion , which have been reported to template ag n - dnas , and other backbone modications which would inuence ligand conformation and , therefore , possible stabilized cluster geometries . [SEP]
[CLS] future studies are needed in this promising area . [SEP]
[CLS] ag n - dnas are oen touted as nontoxic and biocompatible B-property uorophores , but very few studies have established these properties . [SEP]
[CLS] while ag + is certainly less toxic B-property than other heavy metals B-material that compose luminescent B-property nanoparticles B-nanoparticle such as quantum B-nanoparticle dots I-nanoparticle , 249 it is also a common environmental metal B-material pollutant . [SEP]
[CLS] ag nanoparticles B-nanoparticle can break down in the body in a range of different ways , resulting in toxicities B-property due to either the nanoparticles B-nanoparticle themselves or to ag + and silver B-material salts B-material . [SEP]
[CLS] ag nanoparticles B-nanoparticle are also nding use as anti - cancer therapeutics , adding further complexity to our understanding of the toxicity B-property of ag n - dnas . [SEP]
[CLS] in - depth studies of the toxicities B-property of ag n - dnas and their uptake and possible clearing from tissues and organisms are needed to advance their applications in the biomedical sciences and to ensure environmentally responsible use and disposal . [SEP]
[CLS] while ag n - dnas are oen touted as extremely photostable and / or chemically stable , degradation of ag n - dnas in biologically relevant solutions and in the presence of living cells B-material is a significant hindrance to their practical use in bioimaging . [SEP]
[CLS] to overcome this challenge , jeon , et al . , encapsulated ag n - dnas within silica B-nanoparticle nanoparticles I-nanoparticle , signicantly increasing cluster chemical stability . [SEP]
[CLS] the encapsulated ag n - dnas can also be used to monitor the stability of their silica B-nanoparticle nanoparticle I-nanoparticle hosts in various biological media . [SEP]
[CLS] in situ synthesis of ag n - dnas within dna hydrogels can improve photostability , likely by shielding clusters from oxidative degradation . [SEP]
[CLS] lyu , et al . , reported signicantly enhanced chemical stability and increased cellular uptake of ag n - dnas modied by cationic B-material polyelectrolytes . [SEP]
[CLS] ag n - dnas lie at the unique intersection of metal B-material cluster science and dna nanotechnology , combining the atomic precision of ligand - stabilized metal B-material clusters with the sequence programmability of dna nanomaterials B-material . [SEP]
[CLS] their photophysical properties also provide a window into the regime between behavior associated with single small molecules and behavior associated with nanoparticles B-nanoparticle . [SEP]
[CLS] for these reasons , the study and engineering of ag n - dnas are both extremely challenging and extremely promising . [SEP]
[CLS] here , we have reviewed recent advances in the fundamental understanding of these nanoclusters , with a focus on studies of puried ag n - dnas with chromatographically selected sizes . [SEP]
[CLS] the latest ( and evolving ) ndings of ag n - dna structure and the nature of the dna - silver B-material interaction have been discussed . [SEP]
[CLS] photophysical studies , particularly of puried ag n - dnas , have been summarized . [SEP]
[CLS] the current understanding of how dna sequence selects for cluster size and optical properties has been reviewed , as have emerging methods for predictive design of ag n - dnas and their larger organization into multi - cluster arrays . [SEP]
[CLS] we provide perspectives on emerging areas of interest and signicant unanswered questions related to these uorescent clusters in the hopes of stimulating researchers to explore these fascinating nanomaterials B-material . [SEP]
[CLS] are protected by bulky polynucleic B-material acids much larger than the silver B-material cluster . [SEP]
[CLS] the structure peter mastracco received his bachelor ' s degree in physics from binghamton university in 2019 . [SEP]
[CLS] he is currently working towards his phd in materials science and engineering at the university of california , irvine in the group of prof . stacy copp . [SEP]
[CLS] his research focuses on the use of machine learning to predict the physical and chemical properties of various materials systems , with a particular focus on biomaterials . [SEP]
[CLS] tom vosch is an associate professor in the department of chemistry at the university of copenhagen ( ucph ) , denmark . [SEP]
[CLS] he received his phd in 2003 from k . u . leuven , belgium where he was also a postdoctoral researcher , within the groups of prof . f . c . de schryver and prof . j . hoens . [SEP]
[CLS] he did a postdoctoral stay of two years in the group of prof . r . m . dickson at georgia tech , usa , where he was introduced to the intriguing properties of dnatemplated silver B-material nanoclusters . [SEP]
[CLS] at ucph since 2010 , he leads the nanospectroscopy group , which focuses on development of new emissive materials and new microscopy B-technique imaging modalities . [SEP]
[CLS] stacy copp is an assistant professor of materials science and engineering and of physics at the university of california , irvine ( uci ) . [SEP]
[CLS] she received her phd in physics from uc santa barbara in 2016 with prof . e . gwinn and was then a hoffman distinguished postdoctoral fellow at los alamos national laboratory until 2019 . [SEP]
[CLS] she now leads the so matter photonics research group at uci , harnessing self - assembly of biopolymers B-material and synthetic polymers B-material to create novel optical materials . [SEP]
[CLS] prof . copp has received numerous awards , such as the l ' oreal usa for women in science fellowship and air force office of science young investigator award . [SEP]
[CLS] 1 ( a ) the fluorescence B-property colors of ag n - dnas , which are selected by dna sequence , span a large spectral range from visible to nir wavelengths and are correlated with cluster size . [SEP]
[CLS] 20 ( b ) ag n - dna excitation spectra exhibit a dominant peak in the visible to nir spectral range as well as a uv excitation band corresponding exactly to the dna template strand . [SEP]
[CLS] fluorescence B-property spectra excited via the dna bases ( inset , purple ) have the same lineshapes as spectra excited at the cluster ' s unique visible to nir transition . [SEP]
[CLS] 21 adapted from o ' neill , et al . , ( ref . 21 ) with permission from the american chemical society . [SEP]
[CLS] copyright 2011 . [SEP]
[CLS] ( c ) ag n - dnas are chemically synthesized in aqueous solution by mixing ssdna with a silver B-material salt B-material , followed by reduction with sodium B-material borohydride . [SEP]
[CLS] 2 ( a ) illustration of the double helix structure of natural watson - crick - paired b form dna . 82 adapted from bandy , et al . ( ref . 82 ) with permission from the royal society of chemistry . [SEP]
[CLS] ( b ) chemical structure of the natural nucleotides and ( c ) the h - bonded configurations of the nucleobases . [SEP]
[CLS] green regions represent the bonding positions of the nucleobases to the backbone . [SEP]
[CLS] 3 ( a ) percentages of integrated counts ( ic ) for each product detected by esi - ms for mixtures of ag and 11 - base homobase strands , at two different stoichiometries . [SEP]
[CLS] pink boxes and " d " represent ag + - paired duplexes . [SEP]
[CLS] ( b ) summary of wc h - bonded base pairs and observed ag + mediated base pairs for duplexes of homobase strands . [SEP]
[CLS] ( c ) circular dichroism ( cd ) spectra for c 6 - ag + - c 6 and g 6 - ag + - g 6 show the significant thermal stabilities of these homobase - ag + duplexes . [SEP]
[CLS] ( d ) calculations of ground state geometries for ag + - mediated homobase pairs finds planar geometries for g and c and nonplanar geometries for a and t , with binding energies of the trend g > c > a > t . 54 ( a , c and d ) adapted from swasey , et al . , ( ref . 54 ) with permission from springer nature . [SEP]
[CLS] copyright 2015 [SEP]
[CLS] 4 ( a , b ) dft calculations predict the existence of novel inter - strand h - bonds in ( a ) c 2 - ag + - c 2 tetramers 101 and ( b ) ag + - paired g duplexes of varying lengths . [SEP]
[CLS] 108 these bonds add additional stability to ag + - paired dna duplexes . [SEP]
[CLS] ( a ) adapted from espinosa leal , et al . , ( ref . 101 ) with permission from the american chemical society . [SEP]
[CLS] copyright 2015 . [SEP]
[CLS] ( c ) emission spectra of ag + - mediated c 20 and g 15 duplexes labeled with donor ( green dot ) and acceptor ( red dot ) dyes at 5 0 end and 3 0 end , respectively ( orange curve ) or with both dyes at 3 0 ends ( blue ) , compared to emission of the donor - bearing strand alone ( blue dotted curve ) . [SEP]
[CLS] excitation is at 450 nm , which directly excites the donor only . [SEP]
[CLS] significant quenching of donor emission with concomitant acceptor emission ( high fret efficiency ) clearly demonstrates that ag + - mediated pairing of homo - duplexes arranges strands in a parallel orientation . 108 ( d ) dft - optimized structures of ag + - dna duplexes of g 20 and c 20 compared to wc duplexes of a mixed base ( gc ) 11 show that ag + mediates formation of highly rigid duplexes of g homo - base strands and less rigid c homo - base duplexes . [SEP]
[CLS] ag + - dna nanowires B-nanoparticle have parallel duplex strand orientation , as compared to canonical antiparallel strand orientation of wc duplexes . [SEP]
[CLS] 108 ( b - d ) adapted from swasey , et al . , ( ref . 108 ) with permission from the american chemical society . [SEP]
[CLS] copyright 2018 [SEP]
[CLS] relevance of ag + - mediated base pairing for ag n - dnas [SEP]
[CLS] 5 ( a ) ms - determined distributions of the numbers of ag + attached to dna oligomers ( sequences indicated on each graph ) determined by relative integrated intensity of individual mass peaks relative to all silver B-material - bearing duplexes . [SEP]
[CLS] single - base mutations in g - rich oligomers enable attachment of many more ag + . [SEP]
[CLS] 55 adapted with permission from swasey , et al . ( ref . 55 ) with permission from the institute of physics . [SEP]
[CLS] ( b ) crystal structure of an ag + - paired dna duplex with antiparallel orientation . [SEP]
[CLS] end - to - end assembly of these duplexes forms uninterrupted nanowires B-nanoparticle . [SEP]
[CLS] protruding adenines foster assembly of multiple wires into 3d lattices . [SEP]
[CLS] 102 silver B-material atoms I-material are shown in gray and potassium B-material atoms B-material in purple . [SEP]
[CLS] image created from pdb id 5ix7 with ngl viewer . 112 ( c ) structure of a dimer of 5 0 - gcacgcgc - 3 0 ( orange , green ) paired by two ag + ( grey ) . [SEP]
[CLS] the third ag + ( bottom right of structure ) supports supramolecular assembly of the structure during crystallization . [SEP]
[CLS] 106 image created from pdb id 5xjz with pymol . [SEP]
[CLS] 6 tutorial schematic of tandem hplc - ms with in - line uv / vis and fluorescence B-technique spectroscopy I-technique , developed by schultz and gwinn . 53 ( a ) in this illustration , the initial sample ( yellow tube ) is a mixture of products including multiple dark ag - dna complexes , one green - fluorescent B-property ag n - dna species , and one red - fluorescent B-property ag n - dna species . [SEP]
[CLS] the as - synthesized ag n - dna solution is injected into an hplc outfitted with a core - shell c 18 column for reverse - phase , ion - pair ( ip ) hplc . [SEP]
[CLS] products are separated due to slight variations in column affinity with a water - methanol gradient and a triethyl ammonium acetate ( teaa ) ip agent . [SEP]
[CLS] by monitoring both absorbance at $ 260 nm , which correlates to the absorbance of dna , and fluorescence B-property emission ( e . g . uv - excited fluorescence B-property 21 ) , correlation of absorbance and fluorescence B-property chromatogram peaks indicate elution of a fluorescent B-property ag n - dna species . [SEP]
[CLS] we note that the chromatogram schematics are simplified for illustration ; real chromatograms are more complex . [SEP]
[CLS] products of interest can either be sized by in - line negative - ion B-material mode esi - ms or collected for subsequent esi - ms . [SEP]
[CLS] a mass spectrum for a previously studied 30 - atom nir - emissive product is shown in the bottom right . [SEP]
[CLS] both monomeric and dimeric ( labeled " d " ) products are visible , with spacing of the isotopic peaks indicating the charge state of each product ( labeled as superscript of " d " ) for dimeric products . [SEP]
[CLS] ( b ) experimental mass spectrum of the ag 30 - dna product at the 7a charge state dimeric product ( labeled d a7 in ( a ) ) is shown in black , with the calculated mass distribution ( green bars ) for a product with 2 dna strands , n 0 ¼ 12 ag 0 , and n + ¼ 18 ag + . [SEP]
[CLS] inset : compares the experimental spectrum with the calculated distribution for a product with no charged silvers B-material ( 2 dna strands and 30 ag 0 ) , illustrating how the shift between the experimental and calculated isotopic finger distribution can be used to accurately determine the numbers of ag 0 and ag + in an ag n - dna product . [SEP]
[CLS] mass spectra are adapted fromswasey , et al . , ( ref . 23 ) with permission from the royal society of chemistry . [SEP]
[CLS] 6 tutorial schematic of tandem hplc - ms with in - line uv / vis and fluorescence B-technique spectroscopy I-technique , developed by schultz and gwinn . 53 ( a ) in this illustration , the initial sample ( yellow tube ) is a mixture of products including multiple dark ag - dna complexes , one green - fluorescent B-property ag n - dna species , and one red - fluorescent B-property ag n - dna species . [SEP]
[CLS] the as - synthesized ag n - dna solution is injected into an hplc outfitted with a core - shell c 18 column for reverse - phase , ion - pair ( ip ) hplc . [SEP]
[CLS] products are separated due to slight variations in column affinity with a water - methanol gradient and a triethyl ammonium acetate ( teaa ) ip agent . [SEP]
[CLS] by monitoring both absorbance at $ 260 nm , which correlates to the absorbance of dna , and fluorescence B-property emission ( e . g . uv - excited fluorescence B-property 21 ) , correlation of absorbance and fluorescence B-property chromatogram peaks indicate elution of a fluorescent B-property ag n - dna species . [SEP]
[CLS] we note that the chromatogram schematics are simplified for illustration ; real chromatograms are more complex . [SEP]
[CLS] products of interest can either be sized by in - line negative - ion B-material mode esi - ms or collected for subsequent esi - ms . [SEP]
[CLS] a mass spectrum for a previously studied 30 - atom nir - emissive product is shown in the bottom right . [SEP]
[CLS] both monomeric and dimeric ( labeled " d " ) products are visible , with spacing of the isotopic peaks indicating the charge state of each product ( labeled as superscript of " d " ) for dimeric products . [SEP]
[CLS] ( b ) experimental mass spectrum of the ag 30 - dna product at the 7a charge state dimeric product ( labeled d a7 in ( a ) ) is shown in black , with the calculated mass distribution ( green bars ) for a product with 2 dna strands , n 0 ¼ 12 ag 0 , and n + ¼ 18 ag + . [SEP]
[CLS] inset : compares the experimental spectrum with the calculated distribution for a product with no charged silvers B-material ( 2 dna strands and 30 ag 0 ) , illustrating how the shift between the experimental and calculated isotopic finger distribution can be used to accurately determine the numbers of ag 0 and ag + in an ag n - dna product . [SEP]
[CLS] mass spectra are adapted fromswasey , et al . , ( ref . 23 ) with permission from the royal society of chemistry . [SEP]
[CLS] 7 ( a ) peak absorbance energies for purified ag n - dnas characterized by ms as a function of the number of effective free electrons in the cluster ( red ) [SEP]
[CLS] experimental data are better described by simulations of silver B-material nanorods B-nanoparticle with 1 - atom B-material cross - sections ( green line ) than for thicker nanorods B-nanoparticle with 6 - atom B-material cross - sections ( gray line ) or spherical clusters ( blue band ) . [SEP]
[CLS] adapted from schultz , et al . , ( ref . 24 ) with permission from john wiley and sons . [SEP]
[CLS] copyright 2013 . [SEP]
[CLS] ( b ) neutral ag atom B-material numbers , n 0 , as determined by hr - ms for hplc - purifiable ag n - dnas , including brightly fluorescent B-property ag n - dnas ( colored dots with rgb color matching fluorescence B-property wavelength ( nir ¼ gray ) ) and ag n - dnas without measurable fluorescence B-property ( black ) . [SEP]
[CLS] ( c ) histogram of n 0 values show abundances of clusters with even n 0 as compared to magic numbers 2 and 8 predicted by the spherical " superatom " model . 57 ( b and c ) adapted from copp , et al . , ( ref . 57 ) with permission from the american chemical society . [SEP]
[CLS] copyright 2014 [SEP]
[CLS] 8 ( a ) mass spectrum of 20 - base dna - templated ag n . [SEP]
[CLS] the peaks labeled a10 to a5 correspond to the ions B-material of the ag n - dna , while the peaks labeled a8 dna to a6 dna are ascribed to the ions B-material of the bare dna strand . [SEP]
[CLS] the inset shows the zero - charge spectrum that identifies the native dna at 5878 amu and the dna with 10 ag at 6951 amu . [SEP]
[CLS] ( b ) cd spectra of ag n - dnas at different temperatures . [SEP]
[CLS] ( c ) ag k - edge exafs trace of the solution state ag n - dnas . [SEP]
[CLS] the experimental data ( black ) was fitted ( red ) with three individual scattering paths ( magenta , purple and blue ) displayed separately . [SEP]
[CLS] ( d ) suggested ag n - dna structure after combining all information from ms and exafs measurements . [SEP]
[CLS] 130 adapted from petty , et al . , ( ref . 130 ) with permission from the american chemical society . [SEP]
[CLS] copyright 2016 [SEP]
[CLS] 9 ag 3d peak doublet for ( a ) ag + - dna complexes , ( b ) agno 3 salt B-material , ( c ) hplc - purified fluorescent B-property ag n - dnas ( which combine both neutral and cationic B-material ag ) and ( d ) metallic ag nanoparticles B-nanoparticle . [SEP]
[CLS] adapted from volkov , et al . , ( ref . 158 ) with permission from the american chemical society . [SEP]
[CLS] copyright 2017 . [SEP]
[CLS] 11 asymmetric unit of the ag 8 - dna reported by huard , et al . , 22 with the 8 - atom cluster ( as defined in the authors ' report ) indicated by the gray spheres and additional silvers B-material associated with crystal packing indicated by blue spheres . [SEP]
[CLS] ( b ) illustration of silver B-material atoms I-material only , for various crystal sections . [SEP]
[CLS] ( c and d ) structure of the ag 8 - dna in ( c ) sections 0 to 2 and ( d ) sections 3 to 5 as defined in ( b ) . [SEP]
[CLS] one silver B-material atom I-material ( orange ) is stabilized by an adenine from a neighboring dna strand . [SEP]
[CLS] details on the structure can be found at the pdb database using accession code 6niz . [SEP]
[CLS] 12 ( a ) subunit structure of the ag 16 - dna ( 5 0 - cacctagcg - 3 0 ) . [SEP]
[CLS] silvers B-material with occupancy of 1 are gray , while lower occupancy silvers B-material ( $ 0 . 3 ) are magenta . [SEP]
[CLS] ( b ) cluster structure with dna removed , with sections numbered . [SEP]
[CLS] ( c ) sections 0 and 1 , ( d ) section 1 and a part of section 2 , and ( e ) section 2 of the ag 16 - dna subunit . [SEP]
[CLS] ( f ) section 3 , and ( g ) sections 4 and 5 of the ag 16 - dna . [SEP]
[CLS] red dashed lines indicate ag - ag interactions , and black lines represent coordination bonds . [SEP]
[CLS] details on the structure of the nir emissive ag 16 - dna can be found at the pdb database using accession code 6m2p . [SEP]
[CLS] adapted from cerretani , et al . , ( ref . 25 ) with permission from the royal society of chemistry . [SEP]
[CLS] 13 ( a ) schematic of a sensor formed by a $ 11 ag atom B-material cluster with violet absorption , which converts into a nir emissive cluster of twice the size upon hybridization with a target strand ( green ) . [SEP]
[CLS] bottom right : size exclusion chromatogram shows three separate peaks when a 10 - thymine tail is appended to the target strand , indicating that the nir ag n - dna forms by complexation of two dna sensors . [SEP]
[CLS] 38 adapted from petty , et al . , ( ref . 38 ) with permission from the american chemical society . [SEP]
[CLS] copyright 2013 . [SEP]
[CLS] ( b - d ) deconvoluted a - epd spectra and sequence coverage maps for a ssdna template ( b ) without and ( c ) with an ag 10 and for a " hairpin " dna template ( d ) without and ( e ) with an ag 10 . [SEP]
[CLS] comparison of spectra with and without the ag 10 shows suppression of fragmentation for certain subregions of the dna templates , which are correlated to regions where the dna templates interact with their ag 10 clusters . [SEP]
[CLS] 121 adapted from blevins , et al . , ( ref . 121 ) with permission from the american chemical society . [SEP]
[CLS] copyright 2019 [SEP]
[CLS] 14 general phenomenological model for ag n - dnas . [SEP]
[CLS] s0 and s1 represent the ground and emissive states , respectively , fc indicates the initially populated franck - condon state , and d1 is the dark state . [SEP]
[CLS] the dashed blue line stands for the absorption process , wavy lines represent non - radiative pathways , and the straight line defines the emissive decay . [SEP]
[CLS] adapted from cerretani , et al . , ( ref . 185 ) with permission from the royal society of chemistry . [SEP]
[CLS] 15 ( a ) femtosecond transient absorption kinetic traces for 680 nm emissive ( " ag680 " ) ag n - dnas [SEP]
[CLS] the wavelengths shown for this emitter reflect the transient absorption ( black ) and the ground - state depletion ( red ) . [SEP]
[CLS] the depletion appears at negative dod , but is plotted in its absolute value . [SEP]
[CLS] it has been corrected for the spectral overlap by subtracting the contribution from the transient absorption , which is based on the kinetics at 775 nm calibrated to the expected value based on the peak curve fittings . [SEP]
[CLS] the data was collected by exciting with a 100 fs ti - sapphire laser at 1 khz , then probing with a white light continuum generated from the same laser . [SEP]
[CLS] the excitation wavelength was tuned to the peak of the ground state absorption . [SEP]
[CLS] ( b ) normalized femtosecond and nanosecond transient absorption spectra for ag680 . [SEP]
[CLS] the sample was excited by 100 fs pulsed excitation , except for the long delay time curve , which was generated from excitation by a 7 ns pulsed laser . [SEP]
[CLS] the dip in the spectrum around 800 nm is an instrumental artifact . [SEP]
[CLS] 193 adapted from patel , et al . , ( ref . 193 ) with permission from the american chemical society . [SEP]
[CLS] copyright 2009 [SEP]
[CLS] 16 oadf microscopy B-technique . [SEP]
[CLS] ( a ) energy diagram for oadf of a red emissive ag n - dna . [SEP]
[CLS] vertical colored arrows indicate absorption of a photon from primary ( 560 nm ) and secondary ( 765 - 850 nm ) excitation lasers and fluorescence B-property emission at 630 nm , respectively . [SEP]
[CLS] ( b ) primary fluorescence B-property decay curve ( first decay after excitation with 560 nm at 7 ns ) and oadf decay ( second decay after illumination with 765 - 850 nm at 46 ns ) for red ag n - dna embedded in pva . [SEP]
[CLS] ( c - e ) fluorescence B-property images of a heterogeneous sample of fluorescentlylabeled polystyrene microspheres , which are auto - fluorescent B-property to simulate undesired background , and red - emitting ag n - dnas within pva film ( the signal of interest ) . [SEP]
[CLS] images were constructed using ( c ) all detected photons ( 0 - 65 ns ) , ( d ) primary fluorescence B-property ( 7 - 17 ns ) and ( e ) oadf signal ( 46 - 55 ns ) . [SEP]
[CLS] scale bar corresponds to 10 mm . [SEP]
[CLS] the time gates used to construct images ( d ) and ( e ) are shown in ( b ) with the same colors . [SEP]
[CLS] images acquired with 3 . 7 kw cm a2 primary excitation power . [SEP]
[CLS] 30 adapted from krause , et al . , ( ref . 30 ) with permission from the royal society of chemistry . [SEP]
[CLS] 18 anisotropy decays of a nir ag n - dna in 10 mm nh 4 oac aqueous solution at 5 c , 25 c , and 40 c . data was fitted assuming a single rotational correlation time . [SEP]
[CLS] 176 adapted from bogh , et al . , ( ref . 176 ) with permission from the institute of physics [SEP]
[CLS] 17 ( a ) tres , ( b ) average decay time as a function of emission wavelength , and ( c ) das of red emissive ag n - dnas at 25 c , excited at 561 nm . [SEP]
[CLS] the gray line in ( c ) indicates the zero line . [SEP]
[CLS] in order to construct tres and das , the intensity decays were acquired from 575 nm to 725 nm , in steps of 5 nm . [SEP]
[CLS] the three decay time values were globally linked in the fit . [SEP]
[CLS] 185 adapted from cerretani , et al . , ( ref . 185 ) with permission from the royal society of chemistry . [SEP]
[CLS] 17 ( a ) tres , ( b ) average decay time as a function of emission wavelength , and ( c ) das of red emissive ag n - dnas at 25 c , excited at 561 nm . [SEP]
[CLS] the gray line in ( c ) indicates the zero line . [SEP]
[CLS] in order to construct tres and das , the intensity decays were acquired from 575 nm to 725 nm , in steps of 5 nm . [SEP]
[CLS] the three decay time values were globally linked in the fit . [SEP]
[CLS] 185 adapted from cerretani , et al . , ( ref . 185 ) with permission from the royal society of chemistry . [SEP]
[CLS] 19 schematic of the workflow for supervised learning applied to prediction of dna template sequences for brightly fluorescent B-property ag n - dnas . 215 adapted from copp , et al . , ( ref . 215 ) with permission from john wiley and sons . [SEP]
[CLS] copyright 2014 [SEP]
[CLS] 20 ( a ) distribution of peak fluorescence B-property emission wavelength for ag n - dnas stabilized by $ 2000 different 10 - base dna templates , with arrows and colors indicating the color classes defined in the text . [SEP]
[CLS] ( b ) numbers of dna sequences in color classes from ( a ) , corresponding to samples with bright spectral peaks in only one class ( gray ) . [SEP]
[CLS] other sequences exhibited secondary bright peaks in a different color class ( checkered blue ) but were omitted from the training data in order to best learn the features of dna sequences suitable for only one size of fluorescent B-property ag n - dna . [SEP]
[CLS] ( c ) cross - validation scores for trained one - versus - one svms . [SEP]
[CLS] the color bar indicates score value , which is also indicated by text on each pixel . [SEP]
[CLS] 175 [SEP]
[CLS] ( a - c ) adapted from copp , et al . , ( ref . 175 ) with permission from the american chemical society . [SEP]
[CLS] copyright 2018 . [SEP]
[CLS] ( d ) distributions of observed fluorescence B-property peaks for 10 - base ag n - dna training data ( black ) and ag n - dnas designed by ml ( colored bars ) for red 600 - 660 nm fluorescence B-property ( target color band indicated by orange brackets ) . [SEP]
[CLS] designed dna template strand lengths vary : 8 bases ( purple ) , 10 bases ( blue ) , 12 bases ( green ) , and 16 bases ( red ) . [SEP]
[CLS] 111 adapted from copp , et al . , ( ref . 111 ) with permission from the american chemical society . [SEP]
[CLS] copyright 2020 . [SEP]
[CLS] 21 average numbers of ( a ) single bases and ( b ) two - base patterns in motifs identified by feature selection to be correlated to ag n - dna color ( bar color indicates sequence class : gray ¼ dark , green ¼ green , red ¼ red , dark red ¼ very red ) . [SEP]
[CLS] in ( b ) , two - base patterns are ordered along the horizonal axis by selectivity , defined to be the standard deviation of the heights of the four bars for each base pattern . [SEP]
[CLS] 175 adapted from copp , et al . , ( ref . 175 ) with permission from the american chemical society . [SEP]
[CLS] copyright 2018 [SEP]
[CLS] 22 ( a ) schematic and fluorescence B-property micrograph of an ag n - dnalabeled dna nanotube B-nanoparticle , with clusters templated by hairpin protrusions on select dna tiles ( red asterisk ) . [SEP]
[CLS] 100 adapted from o ' neill , et al . , ( ref . 100 ) , with permission from the american chemical society . [SEP]
[CLS] copyright 2012 . [SEP]
[CLS] ( b ) schematic of hybridization chain reaction ( hcr ) forming wire scaffolds for ag n - dna synthesis , and fluorescence B-property micrograph of synthesized ag n - dna wire . [SEP]
[CLS] 118 adapted from orbach , et al . , ( ref . 118 ) with permission from the american chemical society . [SEP]
[CLS] copyright 2013 [SEP]
[CLS] 23 ( a ) excitation - emission maps ( eems ) for an hplc - purified green - emissive donor ag n - dna , ( b ) red - emissive acceptor ag n - dna , ( c ) and the wc - paired clamp . [SEP]
[CLS] the eem of the duplex is not simply an addition of ( a ) and ( b ) because fret causes emission of the acceptor via excitation of the donor , evidenced by two peaks along the black dashed line in ( c ) . [SEP]
[CLS] ( d ) emission spectra for 490 nm excitation of donor ( green ) , acceptor ( red ) , and wc pair ( black ) . [SEP]
[CLS] ( e ) fret is cycled ( intensity : green and red lines ) by thermal melting ( temperature : dashed black line ) and reformation of the fret pair . [SEP]
[CLS] 222 adapted from schultz , et al . , ( ref . 222 ) with permission from the american chemical society [SEP]
[CLS] copyright 2013 [SEP]
[CLS] 25 ( a ) schematic of well plate reader for nir rapid screening of candidate fluorophores , 226 using an ingaas pin - type femtowatt photodetector . [SEP]
[CLS] adapted from swasey , et al . , ( ref . 226 ) with permission from aip publishing . [SEP]
[CLS] ( b ) colormaps of well plates containing ag n - dnas scanned using the modified plate reader , with box colors indicating peak emission wavelength and box size indicating relative fluorescence B-property intensity . [SEP]
[CLS] ( c ) absorbance and emission spectra for three nir - ag n - dnas identified in ( b ) and purified by hplc , including the longest - wavelength emitting ag n - dna identified to date ( bottom panel ) . [SEP]
[CLS] ( d ) mass spectrum of the ag n - dna associated with the top panel of ( c ) , as measured using esi - ms . [SEP]
[CLS] 23 ( b - d ) adapted from swasey , et al . , ( ref . 23 ) with permission from the royal society of chemistry [SEP]